U.S. patent application number 11/395925 was filed with the patent office on 2007-10-04 for abeta-heme peroxidase.
This patent application is currently assigned to Children's Hospital Oakland Research Institute. Invention is credited to Bruce Ames, Hani Atamna.
Application Number | 20070231848 11/395925 |
Document ID | / |
Family ID | 38559582 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070231848 |
Kind Code |
A1 |
Atamna; Hani ; et
al. |
October 4, 2007 |
Abeta-heme peroxidase
Abstract
A .beta.-heme peroxidase activity is selectively measured and
inhibited, packaged as a pharmaceutical composition with heme
biosynthetic cofactors or as an industrial enzyme, and used to
oxidize target substrates.
Inventors: |
Atamna; Hani; (Albany,
CA) ; Ames; Bruce; (Berkeley, CA) |
Correspondence
Address: |
RICHARD ARON OSMAN
4070 CALLE ISABELLA
SAN CLEMEMTE
CA
92672
US
|
Assignee: |
Children's Hospital Oakland
Research Institute
|
Family ID: |
38559582 |
Appl. No.: |
11/395925 |
Filed: |
March 30, 2006 |
Current U.S.
Class: |
435/28 ; 514/185;
514/224.8; 514/575 |
Current CPC
Class: |
C12Q 1/28 20130101 |
Class at
Publication: |
435/028 ;
514/185; 514/575; 514/224.8 |
International
Class: |
C12Q 1/28 20060101
C12Q001/28; A61K 31/555 20060101 A61K031/555; A61K 31/5415 20060101
A61K031/5415; A61K 31/19 20060101 A61K031/19 |
Claims
1. A method of detecting A.beta.-heme peroxidase activity, the
method comprising a step of selectively measuring peroxidase
activity of A.beta.-heme to obtain a measurement of A.beta.-heme
peroxidase activity.
2. The method of claim 1, wherein the method comprises steps of
contacting the A.beta.-heme with an inhibitor of A.beta.-heme
peroxidase activity, and detecting a resultant inhibition of the
A.beta.-heme peroxidase activity.
3. The method of claim 1, wherein the method comprises steps of
contacting the A.beta.-heme with an inhibitor of A.beta.-heme
peroxidase activity, and detecting a resultant inhibition of the
A.beta.-heme peroxidase activity, and the contacting step is
effected by administering the inhibitor to an individual determined
to be in need thereof.
4. The method of claim 1, wherein the method comprises steps of
contacting the A.beta.-heme with an inhibitor of A.beta.-heme
peroxidase activity, and detecting a resultant inhibition of the
A.beta.-heme peroxidase activity, and the contacting step is
effected by administering the inhibitor to an individual determined
to be in need thereof, and the inhibitor is selected from the group
consisting of a A.beta.-heme peroxidase-inhibiting hydroxylamine,
diaminophenothiazine, and metalated porphyrin.
5. The method of claim 1, wherein the method comprises steps of
contacting the A.beta.-heme with an inhibitor of A.beta.-heme
peroxidase activity, and detecting a resultant inhibition of the
A.beta.-heme peroxidase activity, and the contacting step is
effected by administering the inhibitor to an individual determined
to be in need thereof, and the inhibitor is a A.beta.-heme
peroxidase-inhibiting metalated porphyrin.
6. The method of claim 5, wherein the contacting step further
comprises coadministering the inhibitor and a cofactor of heme
biosynthesis, wherein the cofactor is selected from the group
consisting of iron, copper, zinc and biotin.
7. (canceled)
8. The method of claim 1, wherein the method comprises a step of
contacting the A.beta.-heme with a candidate agent under conditions
wherein but for the presence of the agent, the A.beta.-heme
presents a reference A.beta.-heme peroxidase activity, and wherein
the detecting step measures an agent-biased A.beta.-heme peroxidase
activity, wherein a reduced agent-biased A.beta.-heme peroxidase
activity as compared with the reference A.beta.-heme peroxidase
activity indicates that the agent inhibits A.beta.-heme peroxidase
activity.
9-12. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] The field of the invention is a novel peroxidase.
[0002] Intracellular amyloid-.beta. peptide (A.beta.) and its
aggregates are thought to be the neurotoxic agents in Alzheimer's
disease (AD) (1-3). Important cytopathologies in AD include loss of
iron homeostasis; mitochondrial complex IV dysfunction; oxidative
stress (4-7); and cholinergic, dopaminergic, and serotonergic
dysfunctions. These cytopathologies play a role in subsequent
cognitive impairment (8). Although the molecular link between
A.beta. and the important cytopathologies seen in AD is not fully
understood, our earlier work produced strong evidence that heme may
play a key role in the molecular link between A.beta. and these
cytopathologies. We found that heme metabolism is altered in AD
brain (9); that the complex phenotype of heme deficiency (10, 11)
is associate with important cytopathologies in AD (10, 11); and
that heme readily binds with A.beta., forming an A.beta.-heme
complex (9). We proposed a model in which excessive A.beta. in AD
brain binds to regulatory heme, creating a functional heme
deficiency (9). Frey and coworkers (12) demonstrated the
inactivation of muscarinic acetylcholine receptors by oxidative
damage catalyzed by a low-molecular weight substance enriched from
AD brain; heme was proposed to be involved in this inactivation. It
has been previously demonstrated that heme prevented aggregation of
A.beta..sub.(1-40) and A.beta..sub.(1-42) (13). We have published
aspects of the present invention (Atamna H & Boyle, K (2006)
Proc Nat'l Acad Sci USA 103, 3381-3386).
BRIEF SUMMARY OF THE INVENTION
[0003] The invention provides for using the peroxidase formed by
amyloid-.beta. peptide (A.beta.) and heme as a measure of the
pathology of Alzheimer's Disease. It provides for using this
peroxidase in the diagnosis, prevention, and/or treatment of
chronic neurodegenerative diseases, especially, but not
exclusively, Alzheimer's Disease. This invention can also serve as
an assay for the risk of developing such chronic neurodegenerative
diseases, especially but not exclusively, Alzheimer's Disease, for
determining the progression of said diseases, especially but not
exclusively, Alzheimer's Disease, and the effectiveness of other
agents in treating or preventing such diseases, especially, but not
exclusively, Alzheimer's Disease.
[0004] The invention provides methods and compositions for
detecting, measuring, modulating and packaging A.beta.-heme
peroxidase activity. The invention includes ways to measure the
peroxidase activity of A.beta.-heme, inhibitors of A.beta.-heme
peroxidase activity, and methods to detect inhibition of
A.beta.-heme peroxidase activity. Another embodiment is
incorporating the method of measuring peroxidase activity of
A.beta.-heme into screening assays, wherein A.beta.-heme peroxidase
activity is first used as a reference, a potential inhibiting agent
is then contacted with the A.beta.-heme, and wherein a reduction of
A.beta.-heme peroxidase activity indicates that the agent inhibits
A.beta.-heme peroxidase activity.
[0005] In one embodiment, the methods comprise selectively
measuring peroxidase activity of A.beta.-heme to obtain a
measurement of A.beta.-heme peroxidase activity, wherein prior to
the measuring step, the method may further comprise the step of
contacting the A.beta.-heme with an inhibitor of A.beta.-heme
peroxidase activity, and then detecting a resultant inhibition of
the A.beta.-heme peroxidase activity.
[0006] The disclosed methods for measuring peroxidase activity of
A.beta.-heme may also be incorporated into screening assays,
wherein prior to the measuring step the method further comprises
the step of contacting the A.beta.-heme with a candidate agent
under conditions wherein but for the presence of the agent, the
A.beta.-heme presents a reference A.beta.-heme peroxidase activity,
and wherein the measuring step detects an agent-biased A.beta.-heme
peroxidase activity, wherein a reduced agent-biased A.beta.-heme
peroxidase activity as compared with the reference A.beta.-heme
peroxidase activity indicates that the agent inhibits A.beta.-heme
peroxidase activity.
[0007] The invention also provides methods of inhibiting
A.beta.-heme peroxidase activity comprising the step of contacting
a mixture comprising A.beta.-heme peroxidase with an A.beta.-heme
peroxidase inhibitor such as hydroxylamine, a diaminophenothiazine,
ferulic acid and ferulic acid derivatives, and curcumin, and
combinations thereof, and selectively detecting a resultant
inhibition in peroxidase activity of the A.beta.-heme peroxidase.
The contacting step may be effected by coadministering the
inhibitor and a cofactor of heme biosynthesis to an individual
determined to be in need thereof, particularly wherein the cofactor
is iron, copper, zinc or biotin.
[0008] The invention also provides related pharmaceutical
compositions comprising an inhibitor of A.beta.-heme peroxidase
activity, and a cofactor of heme biosynthesis, particularly wherein
the inhibitor is a hydroxylamine, a diaminophenothiazine or
curcumin, and the cofactor is iron, copper, zinc or biotin.
[0009] The invention also provides methods of inducing oxidation of
a substrate by contacting a substrate determined to be in need of
oxidation with A.beta.-heme under conditions wherein the
A.beta.-heme induces oxidation of the substrate, and detecting a
resultant oxidation of the substrate, particularly wherein the
method further comprises the prior step of determining the
substrate is in need of oxidation.
[0010] The invention also provides compositions comprising an
A.beta.-heme complex having predetermined, measured and stabilized
peroxidase activity and packaged as an industrial enzyme in a
predetermined, measured amount in a sealed and labeled package,
particularly solvated in an organic solvent such as ethanol,
methanol, and acetone.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The inventors have characterized a novel peroxidase formed
from A.beta.-heme complexes. The invention provides A.beta.-heme
peroxidase activity as a marker for age-related diseases involving
A.beta.. The invention provides methods and compositions for
detecting, measuring, inhibiting and packaging A.beta.-heme
peroxidase activity. In one embodiment, the methods comprise
selectively measuring peroxidase activity of A.beta.-heme to obtain
a measurement of A.beta.-heme peroxidase activity.
[0012] Peroxidase activity of the A.beta.-heme complex may be
measured or determined using a wide variety of applicable
peroxidase assays, including commercially-available assays (e.g.
ImmunoPure TMB kit; Pierce Biotechnology, Rockford, Ill.). In one
example, the A.beta.-heme complex is contacted with an oxidizable
substrate under conditions wherein the A.beta.-heme catalyzes
oxidation of the substrate, and resulting peroxidase activity is
measured by detecting oxidation product(s) of the substrate (e.g.
see Example 2). In specific embodiments, the substrate is selected
from TMB, ABTS, AEC, NC, DAB, Guaiacol, OPD, PPHP, etc.
[0013] Selective measurement may be achieved, for example, by using
isolated or labeled A.beta.-heme peroxidase, an A.beta.-heme
peroxidase selective substrate, A.beta.-heme peroxidase selective
reaction conditions such as organic solvents, temperature, pH,
ionic strength, cofactors, etc. Selective measurement may also be
effected by examining tissue samples that do not present
conflicting peroxidase activities, such as blood plasma,
cerebral-spinal fluid, etc. A wide variety of noninvasive imaging
technologies may be used for selective measurement of A.beta.-heme
peroxidase, including MRI, ESR, etc. Alternatively tissue samples
may be collected, and A.beta.-heme peroxidase activity selectively
measured therefrom (e.g. using A.beta.-heme peroxidase-specific
immunoselection or specific ELISA). Increased peroxidase activity
relative to normal tissue identifies patient suitability for
A.beta.-heme peroxidase inhibitor therapy.
[0014] In particular embodiments, the methods comprise the step of
contacting the A.beta.-heme with an inhibitor of A.beta.-heme
peroxidase activity, and then detecting a resultant inhibition of
the A.beta.-heme peroxidase activity. Preferred target populations
for detecting and/or inhibiting A.beta.-heme peroxidase activity
are determined to be associated with or at risk for age-related
disorders such as Alzheimer's disease and pathologies associated
with Down's syndrome. The disclosed methods of selectively
measuring peroxidase activity of A.beta.-heme can also be used to
identify patients with (elevated) A.beta.-heme peroxidase activity
as suitable for treatment with an A.beta.-heme peroxidase
inhibitor.
[0015] The invention provides methods of inhibiting A.beta.-heme
peroxidase activity comprising the step of contacting a mixture
comprising A.beta.-heme peroxidase with an A.beta.-heme peroxidase
inhibitor such as hydroxylamine (e.g. PCT/US00/29634;
PCT/US05/16580), a diaminophenothiazine (e.g. PCT/US06/06320), or
ferulic acid, including dimers (e.g. US Pat Pub No. 20040236148),
or ferulic acid derivatives, particularly esterified derivatives
such gamma-oryzanol and cycloartenyl ferulate, and detecting a
resultant inhibition in peroxidase activity of the A.beta.-heme
peroxidase. The inhibitor can function in different ways, e.g. by
directly interfering with peroxidase activity of the active enzyme,
or by interfering with active complex formation, such as with
metalated porphyrins (e.g. PCT/US06/03315), such as heme with
non-iron metals (e.g. Cu, Zn, Sn, etc.).
[0016] In particular embodiments, A.beta.-heme peroxidase-specific
antibodies are used to inhibit activity and/or formation of
A.beta.-heme peroxidase. The antibodies may be exogenous to the
patient, or elicited with A.beta.-heme complexes, or A.beta.-heme
complex-specific immunogens, which are readily produced by
screening antibodies elicited by candidate A.beta.-heme
complex-derived immunogens in animal models against native
A.beta.-heme.
[0017] In some embodiments, particularly wherein the inhibition
depletes or depends in part on endogenous heme or heme precursors,
the contacting step may be advantageouly effected by
coadministering the inhibitor and a cofactor of heme biosynthesis
to an individual determined to be in need thereof, particularly
wherein the cofactor is iron, copper, zinc or biotin.
[0018] The invention also provides related pharmaceutical
compositions comprising an inhibitor of A.beta.-heme peroxidase
activity, and a cofactor of heme biosynthesis, particularly wherein
the inhibitor is a hydroxylamine, a diaminophenothiazine or
curcumin, and the cofactor is iron, copper, zinc or biotin. Dosages
are effective yet physiological, typically from 0.1 to 10, or from
0.2 to 5, or from 0.5-1.5, or about 1 times governmental
recommended daily allowances (RDA).
[0019] The disclosed methods for measuring peroxidase activity of
A.beta.-heme may also be incorporated into screening assays,
wherein prior to the measuring step the method further comprises
the step of contacting the A.beta.-heme with a candidate agent
under conditions wherein but for the presence of the agent, the
A.beta.-heme presents a reference A.beta.-heme peroxidase activity,
and wherein the measuring step detects an agent-biased A.beta.-heme
peroxidase activity, wherein a reduced agent-biased A.beta.-heme
peroxidase activity as compared with the reference A.beta.-heme
peroxidase activity indicates that the agent inhibits A,-heme
peroxidase activity. These screening assays identify agents that
modulate (i.e. increase or inhibit) A.beta.-heme peroxidase
activity, and may be performed as a high-throughput screening assay
to identify A.beta.-heme peroxidase activity inhibitors in a
chemical library.
[0020] The invention also provides methods of inducing oxidation of
a substrate comprising the step of contacting a substrate
determined to be in need of oxidation with A.beta.-heme under
conditions wherein the A.beta.-heme induces oxidation of the
substrate, and detecting a resultant oxidation of the substrate. In
one embodiment, the method further comprises the prior step of
determining that the substrate is in need of oxidation. The
oxidation of various substrates by A.beta.-heme is described herein
(e.g. Example 2).
[0021] A.beta.-heme peroxidase can be isolated from biological
tissues (e.g. using conventional techniques like
immunoprecipitation, chromatography, etc.); alternatively,
A.beta.-heme can be readily prepared using commercially available,
synthetic or recombinant A.beta. and heme. Solutions of A.beta. and
heme are prepared and mixed together; the ratio of A.beta. to heme
is 100:1 to 1:100, preferably 10:1 to 1:10, preferably 2:1, 1:2, or
1:1. The complex self-assembles, and can be visually detected by a
change in heme color, e.g. from a greenish-brown to a golden brown
color in aqueous solution. The composition can comprise any
suitable diluent such as water, a buffer, an organic solvent (e.g.
ethanol, methanol, and acetone), etc.
[0022] The invention also provides compositions comprising an
A.beta.-heme complex having predetermined, measured and stabilized
peroxidase activity and packaged as an industrial enzyme in a
predetermined, measured amount in a sealed and labeled package,
particularly solvated in an organic solvent such as ethanol,
methanol, and acetone. Packaging of industrial enzymes is pursuant
to standards in the art, and involves containing stabilized enzyme
in discrete volume, weight or activity unit amounts, in saleable,
sealed, labeled packaging. Stabilization can be effected by freeze
drying (lyophilization), protein stabilizer reagents, etc. Discrete
amounts of the A.beta.-heme complex are packaged and labeled with
applicable information regarding amount, units of peroxidase
activity, etc., and instructions for use (e.g. as an industrial
enzyme, in drug screening assays, etc.)
EXAMPLE 1
Preparation of A.beta.-Heme Complex
[0023] Heme is prepared by dissolving hemin (Frontier Scientific,
Logan, Utah) in 0.1 N NaOH to make 5 mM solution. Unless otherwise
contraindicated, we use the term "heme" to refer to heme and salts
thereof, including hemin, hematine, etc. The heme solution is then
diluted in 50 mM Hepes to make 600 .mu.M (pH about 7.4).
Amyloid-.beta. (A.beta.) peptide (American Peptide, Sunnyvale,
Calif.) is dissolved in distilled deionized water to make a 600
.mu.M solution. A.beta. is mixed with heme at a 1:1 ratio to make a
300 .mu.M solution of each (this is a 300 .mu.M stock A.beta.-heme
complex) and incubated for 10-30 minutes in the dark at room
temperature to insure complete complexation and removal of possible
aggregates. Binding is instantaneous and can be visually detected
by the change to hemin's color or by measuring the red shift in the
absorbance spectrum of heme. The color changes from a
greenish-brown to a golden brown when the heme complexes with
A.beta..
EXAMPLE 2
Peroxidase Activity of A.beta.-Heme Complex
[0024] The peroxidase activity of A.beta.-heme was measured by the
oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H.sub.2O.sub.2
by following the increase in absorbance at 652 nm, which allows
continuous monitoring of the TMB oxidation product. Alternatively,
the reaction was terminated by 4M sulfuric acid, which shifts the
absorbance peak of the TMB oxidation product to 450 nm. The
peroxidase activity of .beta.-heme was tested in the concentration
range of 100-700 nM of the complex for 15-30 min at room
temperature.
[0025] Several types of organic solvents are known to inactivate
enzymes including peroxidases. We tested the effect of ethanol,
methanol, and acetone on A.beta.-heme peroxidase. A.beta.-heme was
incubated in the each organic solvent for 10 min. The final
percentage of the organic solvent was maintained at 99%. A 50 .mu.l
aliquot of A.beta.-heme/organic solvent solution was added to 200
.mu.l TMP peroxidase assay kit, and activity of A.beta.-heme was
monitored by following TMB oxidation at 652 nm. The rate of TMB
oxidation was calculated from the first linear 100 seconds of the
reaction. The final concentration of A.beta.-heme was 0.15 .mu.M.
The peroxidase activity of A.beta.-heme was not affected by organic
solvents, reflecting the stability of the A.beta.-heme complex.
More than two folds increase in the peroxidase activity of
A.beta.-heme was seen upon treatment with acetone.
[0026] The effect of potential substrates (serotonin and DOPA) on
A.beta.-heme peroxidase activity was measured by the TMB assay or
by HPLC. For the HPLC assay, 100 .mu.M serotonin was incubated with
3 mM H.sub.2O.sub.2 and 500 nM A.beta.-heme in 50mM Hepes, pH 7.2.
At specific time intervals, the oxidation products of serotonin
were analyzed by injecting 23.5 .mu.l from the reaction mixture
into RP-HPLC on a 300-.times.3.9-mm column of Bond-Clone-C18
(Phenomenex) by using as mobile-phase 10% methanol, pH 3. Serotonin
or oxidation products were analyzed with a UV-online detector set
at 280 nm. The retention time of serotonin was 4.7 minutes. Adding
A.beta.-heme to serotonin plus H.sub.2O.sub.2 decreased the
serotonin peak after a 60-min reaction and led to the formation of
two oxidation products having retention times of 3.3 and 4.0
min.
EXAMPLE 3
A.beta. Induces Heme Synthesis And Iron Uptake
[0027] The effect of A.beta. on heme synthesis was used to test the
biological effect of the A.beta. and heme interaction. We expected
an increase in heme synthesis due to depletion of regulatory heme
if the binding between heme and A.beta. occurs in the cell. A.beta.
at concentrations of 0.1, 1, and 10 .mu.M significantly increased
the synthesis of heme in human neuroblastoma (SHSY5Y) cells. A 40%
increase in heme synthesis was seen after 2 h incubation with 0.1
.mu.M A.beta.. A 4-fold and 13-fold increase in heme synthesis was
seen after 1 h of incubation with 1 and 10 .mu.M A.beta.,
respectively. A 2.5-fold and 18-fold increase in heme synthesis was
seen after 2 h of incubation with 1 and 10 .mu.M A.beta.,
respectively. An increase in the uptake of cofactors of heme
biosynthesis is necessary to support heme synthesis. For example,
A.beta. at concentrations of 0.1, 1, and 10 .mu.M significantly
increased the intracellular iron. A 55% increase in iron was seen
after 2 h of incubation with 0.1 .mu.M A.beta.. A 4-fold and
12.5-fold increase in iron uptake was seen after 1 h of incubation
with 1 and 10 .mu.M A.beta., respectively. A 2-fold and 8-fold
increase in iron uptake was seen after 2 h of incubation with 1 and
10 .mu.M A.beta., respectively. The exposure of SHSY5Y cells to
A.beta. was not toxic as measured by trypan blue exclusion tests
and compared with controls.
EXAMPLE 4
Inhibition of Peroxidase Activity of A.beta.-Heme Complex;
Inhibitor Screen
[0028] The effect of potential inhibitors of A.beta.-heme
peroxidase activity was measured by the TMB assay (supra) or by
HPLC. The peroxidase activity of A.beta.-heme was significantly
inhibited by 1 .mu.M curcumin, 100 .mu.M N-benzylhydroxylamine
(NBHA), and 10 .mu.M methylene blue. Synthetic N-hydroxylamine and
diaminophenothiazine libraries are similarly screened for
inhibitors of A.beta.-heme peroxidase activity in the same TMB
assay, identifying numerous inhibitors including N-hydroxylamines:
N-tert-butylhydroxylamine, N-cyclohexylhydroxylamine,
N-isopropylhydroxylamine, N-(4-hydroxybutyl)hydroxylamine,
N-(2-carboxyethyl)hydroxylamine,
N-(benzylcarbonyl)methylhydroxylamine, and N-(acetyl)hydroxylamine,
N-(3-nitropropyl)hydroxylamine; diaminophenothiazines: azure A,
azure B, azure C, thionine, toluidine blue, methylene blue, new
methylene blue, and 1-9-dimethyl methylene blue; and metalated
porphyrins: uroporphyrin III, uroporphyrin I, coproporphyrin III,
and coproporphyrin I.
EXAMPLES 5-7
Inhibition of Oxidative Damage In Animal Model of Alzheimer's
Disease By Administration A.beta.-Heme Peroxidase Inhibitors
[0029] The Tg2576 APPSw transgenic mouse is an animal model for
Alzheimer's disease. Methodology for these examples was adapted
from a previously reported study that showed reduced oxidative
damage in the curcumin-treated Tg2576 APPSw mouse (Lim et al, J.
Neurosci (2001) 21:8370-8377). In these examples we compare
curcumin-treated mice with mice treated with the A.beta.-heme
peroxidase inhibitory hydroxylamines and diaminophenothiazines, and
metalated porphyrins identified in Example 4 (supra), and
combinations of the identified metalated porphyrins and cofactors
of heme biosynthesis ((a) iron, (b) biotin, and (c) iron and
biotin). The hydroxylamines are administered to achieve 1-2
mg/kg/day dosage; diaminophenothiazines are administered (in
drinking water; 100 nM final concentration) to achieve a 1-2
.mu.g/kg/d dosage; and metalated porphyrins are administered to
achieve a 25-50 mg/kg/d dosage. Iron and biotin supplementation is
provided per kg food at 600 mg/kg and 0.1 mg/kg, respectively. All
treatment groups demonstrate reduced oxidative damage, reduced
soluble A.beta., and reduced plaque burden compared with control
animals.
EXAMPLE 5
Oxidative Damage Is Reduced In Mice Treated With A.beta.-Heme
Peroxidase Inhibitors
[0030] Oxidative damage is assessed in Tg- untreated, Tg+
untreated, and Tg+ treated groups using Western blot analysis, in
which carbonyl groups on oxidized proteins are derivitized with
DNPH and detected using an anti-DNP antibody. Statistical analysis
reveals a significant transgene effect in oxidized protein levels.
A combined regional analysis of four brain regions reveals that
animals treated with our A.beta.-heme peroxidase inhibitors have
lower levels of oxidized proteins compared with cofactor only, and
untreated animals.
EXAMPLE 6
A.beta.-Heme Peroxidase Inhibitors Reduce TBS-Soluble A.beta. And
SDS-Insoluble A.beta. (Amyloid)
[0031] Previous studies showed that a chronic dose of curcumin
significantly reduces insoluble and soluble A.beta. in APPSw mice
(Lim 2001, supra). To demonstrate this effect in animals treated
with our A.beta.-heme peroxidase inhibitors, levels of
SDS-insoluble A.beta. are measured by ELISA in entorhinal cortex,
hippocampus, and residual cortex regions. Statistical analysis
reveals a significantly enhanced reduction in insoluble A.beta., as
compared with control animals.
EXAMPLE 7
Low Doses of A.beta.-Heme Peroxidase Inhibitors Reduce Plaque
Burden In APPSw Brains
[0032] To evaluate whether treatment with our A.beta.-heme
peroxidase inhibitors reduces plaque pathology, cryostat hemibrain
sections from Tg+ control and Tg+ treated mice are immunostained
with an antibody against A.beta.1-13. Statistical analysis reveals
a reduction in plaque burden in treated animals.
EXAMPLE 8
A.beta.-Heme Peroxidase Inhibitors Reduce Memory Loss And Cognitive
Decline
[0033] A.beta.-heme peroxidase inhibitors reduce memory loss and
cognitive decline in 3.times.Tg-AD mice (Oddo et al., Neuron 39
(2003), 409-421) as determined in established spatial reference and
contextual learning paradigms (1).
[0034] A.beta.-heme peroxidase inhibitors (group I hydroxylamines:
N-tert-butylhydroxylamine, N-cyclohexylhydroxylamine,
N-isopropylhydroxylamine, N-(4-hydroxybutyl)hydroxylamine,
N-(2-carboxyethyl)hydroxylamine,
N-(benzylcarbonyl)methylhydroxylamine, and N-(acetyl)hydroxylamine,
N-(3-nitropropyl)hydroxylamine; group II diaminophenothiazines:
azure A, azure B, azure C, thionine, toluidine blue, methylene
blue, new methylene blue, and 1-9-dimethyl methylene blue; and
group III metalated porphyrins: uroporphyrin III, uroporphyrin I,
coproporphyrin III, and coproporphyrin I were characterized in
Example 4 above. Hydroxylamines are provided in food pellets to
achieve 1-2 mg/kg/day dosage; diaminophenothiazines are
administered (in drinking water; 100 nM final concentration) to
achieve a 1-2 .mu.g/kg/day dosage; and metalated porphyrins are
provided in food pellets to achieve a 25-50 mg/kg/day dosage.
[0035] Morris water maze (MWM). The apparatus used for water maze
tasks is a circular aluminum tank (1.5 m diameter) painted white
and filled with water maintained at 26.degree. C.-29.degree. C. The
maze is located in a room containing several simple visual,
extramaze cues. To reduce stress, mice are placed on the platform
in both the hidden and cued versions of the task for 10 s prior to
the first training trial.
[0036] Spatial Reference MWM Training. Mice are trained to swim to
a 14 cm diameter circular clear Plexiglas platform submerged 1.5 cm
beneath the surface of the water and invisible to the mice while
swimming. The platform location is selected randomly for each
mouse, but kept constant for each individual mouse throughout
training. On each trial, the mouse is placed into the tank at one
of four designated start points in a pseudorandom order. Mice are
allowed to find and escape onto the submerged platform. If a mouse
fails to find the platform within 60 s, it is manually guided to
the platform and allowed to remain there for 10 s. After this, each
mouse is placed into a holding cage under a warming lamp for 25 s
until the start of the next trial. To ensure that memory
differences are not due to lack of task learning, mice are given
four trials a day for as many days as were required to train the
3.times.Tg-AD-H and 3.times.Tg-AD-h mice to criterion (<20 s
mean escape latency before the first probe trial was run). To
control for overtraining, probe trials are run for each group, both
as soon as they reach group criterion and after all groups reach
criterion.
[0037] Retention of the spatial training is assessed 1.5 hr and
again 24 hr after the last training trial. Both probe trials
consist of a 60 s free swim in the pool without the platform. Mice
are monitored by a camera mounted in the ceiling directly above the
pool, and all trials are stored on videotape for subsequent
analysis. The parameters measured during the probe trial include
(1) initial latency to cross the platform location, (2) number of
platform location crosses, and (3) time spent in the quadrant
opposite to the one containing the platform during training. For
the 6 month training, the target quadrant is changed to avoid
savings from previous water maze experience. Target quadrants vary
between mice within a group to control for potential differences in
the salience of extramaze cues.
[0038] Cued MWM Training. The cued task training consists of four
consecutive 60 s trials per day (40 s ITI), and begin the first day
after the spatial testing ends. Each trial consists of the mouse
starting from one of five positions along the side of the water
tank. The mouse is given 60 s to find the visible platform
(indicated by a striped flag above the water level). If the mouse
does not mount the platform within the 60 s, it is manually guided
to the platform. A group mean of <20 s escape latency is the
required criterion. After mounting the platform, the mouse is
immediately removed to a holding cage. The visible platform is
moved to different locations between each trial, so that the
mouse's starting position and the platform location are unique
between trials.
[0039] Inhibitory avoidance (IA). For IA, testing begins with a
training trial in which a mouse is placed in a lighted chamber;
when the mouse crosses over to the dark chamber, it receives a mild
(0.25 mA/1 s) footshock. This initial latency to enter the dark
(shock) compartment serves as the baseline measure. During the
probe trials, 1.5 or 24 hr after training, the mouse is again
placed in the light compartment, and the latency to return to the
dark compartment (previously associated with shock) measured as an
index of passive fear avoidance.
[0040] A.beta.-heme peroxidase inhibitor treated and untreated
3.times.Tg-AD mice are evaluated with the foregoing spatial
reference and contextual learning paradigms. All treatment groups
show reduced impairment during acquisition on the MWM task;
improved short- and long-term memory as measured by latencies to
cross the platform location, number of platform location crosses,
and time spent in the opposite quadrant as compared to untreated
mice. Treated mice also demonstrate significant improvements on the
visible platform (cued) training in the MWM, and improved retention
for contextual fear.
[0041] The foregoing examples and detailed description are offered
by way of illustration and not by way of limitation. All
publications and patent applications cited in this specification
are herein incorporated by reference as if each individual
publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims. As used herein, the
singular forms "a," "an," and "the," refer to both the singular as
well as plural, unless the context clearly indicates otherwise.
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