Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMBINATION OF ANTIOXIDANT SUBSTANCES FOR THE
TREATMENT OF ALZHEIMER'S DISEASE
FIELD OF THE INVENTION
The present invention relates to the use of a combination of antioxidant
substances for the treatment of cognitive disorders such as Alzheimer's
disease
(AD).
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is the most common of dementias. Its frequency, in
later
years, has dramatically increased due to the rise of life expectancy. This
disease
represents, according to different statistical data, from 50 to 75% of all
dementias.
Etioiogical hypotheses of AD such as those based on amyloid hypothesis,
cholinergic hypothesis, oxidative hypothesis, genetic hypothesis and
immunologic
hypothesis are numerous and complex. It is considered that the trigger of the
disease is a combination of them, and particularly of genetic and
neurobiologic
processes that take place simultaneously.
Amyloid and AD
The patient's brain with Alzheimer's disease is invaded by a protein known as
beta- amyloid peptide (beta A4). Beta A4 is part of the senile plaques that
together
with the neurofibrillary degeneration constitute the histopathology of AD, of
other
dementias and of the aging process depending on the density of the injury.
The Amyloid Precursor Protein (APP), codified in chromosome 21, has 695 to 770
amino-acid deposits, being a normal constituent of the neuronal membranes.
The formation of protein beta A4 requires the cleavage of the APP, which is
expressed by numerous types of cells. The beta-secretase cleaves an end of the
APP, while a second secretase, known as gama secretase, cleaves the other end
of A beta. It is a very difficult task to identify these divisory enzymes
(beta and
gama secretase) since the cells contain hundreds of these protease types.
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Therefore, the applied strategy to find these enzymes was based on identifying
their genes.
The beta-secretase in isolation has been called Beta-site APP-Cleaving Enzyme
(RACE). It is an enzyme that can cleave the APP, and it diminishes the
production
of A beta in cell cultures. Its levels are higher in the neurons than in the
glia,
supporting the hypothesis that the neurons are the first extracellular source
of beta
A4 deposited in amyloid plaques.
According to Dennis Selkoe et al, the preseniline 1, involved in some
hereditary
forms of AD, could be identified with the gama-secretase. Nowadays, it is
considered that this enzyme plays a secondary role.
The beta A4 would cause cell death via apoptosis, for disruption in the
homeostasis of calcium, activating calcium channels and second messengers like
the protein kinases which provoke nerve cell death.
Oxidative stress and AD
The oxygen free radicals have been involved in the etiology and the
consequences of different diseases, including AD. The aging processes, brain
injury and ischemia are related to the production of free radicals.
There are several hypotheses regarding the mechanisms that trigger oxidative
stress in AD, that is to say, the damage produced by an increase of the free
radicals activity on the biomolecules and, in the case of the central nervous
system, predominantly on the lipids. The excess of free oxygen alters the
ionic
balance of calcium between the neurons and its mitochondrias.
The beta amyloid protein seems to be involved in the oxidative stress
mechanism.
The increased presence of this peptide is related to an important production
of free
radicals and subsequent cell damage. At the same time, the lipid peroxidation
of
the membrane may increase the vulnerability of the APP transmembrane to the
abnormal cleavage by the proteases related to the deposit of BA4, and for this
reason the BA4 increases.
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Postmortem exams of cerebral cortex of patients with AD were compared to brain
tissue of patients that died without having antecedents of the
neurodegenerative
disease. In the first ones there was a significant increase of lipid
peroxidation
measuring dialdehyde (TEARS and malondialdehyde).
Some authors have determined the oxidative stress by chemiluminescence (a
technique by means of which some components of lipid peroxidation are detected
as they have the peculiarity of liberating energy by means of photons and
whose
quantification determines the magnitude of the damage made to the lipids).
In a study made by Barkats M, et al in the Hospital de la Pitie Salpetriere,
in
France, published in Neurochemist,(4J, the authors utilized a strategy of gene
transfer to increase the antioxidant potential of nerve cells before exposing
them to
toxic fragments of A-beta. More precisely, they evaluated if the intracellular
overexpression of glutathione peroxydase (GPx) could increase the resistance
of
cells PC12 of pheochromocytoma and of the embryonic neurons of the cortical
area of rats against the toxicity of the A-beta. As the adenovirus is an
efficient
vector for the transfer of genes on postmiotic cells both in vitro as on alive
cells, it
has been used to increase intracellular expression of GPx which is the main
captor
at brain cell level of the peroxide of hydrogen radical. The cells infected
this way
were then exposed to toxic concentrations of A-beta. Both PC12 cells and the
cortical ones infected with Ad-GPx have been significantly more resistant to A-
beta
exposure. This information strengthens the hypothesis of the role of the
hydrogen
peroxide in the toxicity mechanism by A-beta injury.
The increase of glutamate, which is present in dementias, produces an
excessive
opening of the calcium channels and this produces a slow, toxic increase of
the
intraneural calcium. This factor activates enzymatic and metabolic processes
with
lipid peroxidation and formation of free oxygen radicals. The free radicals
wreck
the organelles and the neuron membranes, contributing in this way to the
neurodegenerative process.
Other reports have pointed out possible alterations in the availability or
quality of
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the antioxidant enzymes. The results have not been conclusive, or rather they
suggest that the neuronal death mechanism, if it is produced by oxidative
stress, is
not due to deficiency of these enzymes, at least in the cerebral cortex.
Schippling et al measured the lipoprotein oxidation in the cerebrospinal fluid
and in
the plasma of 29 patients with AD. They found that such oxidation was
significantly
high in comparison with strict controls. In the same way, the ascorbic levels
were
low in patients with AD, but not those of alpha-tocopherol.
The presence of oxidative stress has not only been detected by the increase of
lipid peroxidation through the different mentioned techniques, but also by the
decrease in the activity of antioxidant enzymes, particularly the superoxide
dismutase (SOD).
Immunoloqical activity and AD
In Alzheimer's disease there is an immunological hyperactivity. The cytokines
(interleukins 1 and 6), produced by the increase of the microglial cell
activity when
amyloid is deposited in the neurons, increase in plasma. Alpha-
antichymotrypsin
and alfa-2 macrogtobulin also increase.
It is believed that cytokines would influence in the regulation of the gene
expression of the APP and/or in the proteolytic process, contributing to the
production of oxygen free radicals. That is the reason of the importance of
some
studies carried out with non-steroidal anti-inflammatory drugs.
The inflammatory response makes that the astrocytes become part of the
plaques.
Recently, it has been established that there is a relationship between the
apolipoprotein E4 and the TEARS levels (products of lipid lipoperoxidation).
This
protein that degrades the amyloid substance is made based on an anomaly
codified in chromosome 19. This anomaly makes the amyloid not to degrade
appropriately and to deposit it in the neurons.
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Apolipoprotein E plays an important role in the transport, generation and
cleareance of lipids. It has to do with the myelinization and with the neuro-
plasticity. The anomaly of apolipoprotein E-4 (APOE-4) is codified in
chromosome
19, related with the AD of late beginning.
5
APOE-4 is a physiological transporter of cholesterol that also intervenes in
the
myelinization, in the neuroplasticity and in the deposit of amyloid. It is
present in
the senile plaques, in the neurotibrillary degeneration and in the
cerebrovascular
amyloid. There are studies that evidence the neurotrophic, immunomodulatory
and
antioxidant functions of the APOE. The APOE-4 is nowadays considered as one of
the recognized risk factors for the development of the Alzheimer's disease.
Genetic hypothesis of AD
Other biomolecules like proteins and the DNA nucleotides suffer damage in
their
molecular structure in the brain of patients with AP. The carbonyl protein,
one of
the components of protein oxidation, is significantly increased in the
hippocampus
and in the inferior parietal lobe of these patients.
One of the methods most commonly used to evaluate the oxidative alteration of
the molecular structure is the measurement of an oxidative component of one of
its bases: the 8-hydroxy-2-deoxyguanosine. This substance increases in the
brain
in the aging process, particularly the one coming from mitochondria) DNA. In
patients suffering from AD, this increase triples, indicating an oxidative
stress that
also affects the DNA. The oxidative stress with hemi-oxygenase 1 is superior
in
neurons and astrocytes of cortex and hippocampus.
Another theory is the presence of an increase in the iron deposits in the
cerebral
cortex of these patients. As it is known, iron is an important catalyst of
free radicals
and it activates the lipid peroxidative processes.
Another evidence of the presence of an important amount of oxidative stress in
these patients is the so called Products of Advanced Glycosylation (PAG),
formed
by non enzymatic reactions of glucose with earlier deposits of proteins which
are
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potentially toxic for the cells. The PAG are usually produced by an
accelerated
oxidation of the glycoproteins and it has been observed that they are
increased in
the senile plaques of the patients with AD.
SUMMARY OF THE INVENTION
A first object of the present invention concerns a combination of specific and
known antioxydants used for the treatment of cognitive symptoms caused by the
Alzheimer's disease.
More precisely, the combination comprises vitamin E, quercitin, caffeic acid
nicotinic acid and derivatives and/or analogs thereof.
A second object of the present invention concerns a pharmaceutical composition
comprising the above-mentioned combination of antioxidants.
A third object of the invention concerns a method for treating Alzheimer's
disease
by the administration of the pharmaceutical composition comprising the
combination of antioxidants of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of the oxidative stress pattern in AD.
Figure 2 shows the behavior of the average scores during the entire time
period
for the different groups for the ADAS Test.
Figure 3 shows the behavior of the average scores during the entire time
period
for the different groups for the MMSE Test.
Figure 4 shows the behavior of the average scores during the entire time
period
for the different groups for the Hamilton Test.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a pharmaceutical composition which comprises
a
combination of antioxidants selected from the group consisting of vitamin E,
quercitin, nicotinic acid and caffeic acid.
Other antioxidants such as selegiline, idebenone, oestrogens, antlinflammatory
products, ginkgo bilova, ascorbic acid, beta carotene, melatonin, coenzyme Q
and
phenolic compounds can also be used in the pharmaceutical composition.
According to the present invention, it is possible to use from 0.1 to 100 mg
of
vitamin E, quercitin, nicotinic acid and caffeic acid and from 500 to 2500 Ul
of
vitamin E. In a preferred embodiment, the following dose is used: 50 mg of
quercitin; 50 mg of caffeic acid; 50 mg nicotinic acid and 1000 UI of vitamin
E.
Preferably, RRR-a-tocopherol (d-alpha tocopherol) is used as the vitamin E;
quercitin dehydrate (3,3',4'5,7 pentahydroxyflavone.is used as the quercitin;
and
(3,4-dihydroxycinnamic) acid is used as the caffeic acid. No preferred form of
nicotinic acid is used.
The pharmaceutical composition of the present invention is best administered
to a
patient in the solid form such as in a capsule. In the solid form, vitamin E
is more
stable (to temperature and light). In liquid form, vitamin E is less stable
and its life
time is shorter. Furthermore, in this later form, the efficacy of vitamin E
cannot be
guaranteed for a long period. furthermore, in the liquid form, vitamin E may
be
insoluble when mixed with other antioxydants or chemical compounds.
Vitamin E
Vitamin E is an essential nutrient that functions as an antioxidant in the
human
body. It is essential, by definition, because the body cannot manufacture its
own
vitamin E and thus it must be provided by foods and supplements.
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Vitamin E is a generic term that includes all entities that exhibit the
biological
activity of a-Tocopherol. In nature, eight substances have been found to have
vitamin E activity. They are defined by four tocopherols and four
tocotrienols. The
tocopherols are alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-
tocopherol. The tocotrienols are the alpha-tocotrienol, beta-tocotrienol,
gamma-
tocotrienol and delta-tocotrienol.
The vitamin E derivatives or related compound and analogs are potent
antioxidants that block the lipid peroxidation by donation of hydrogen to the
peroxidated lipids. The central nervous system is especially vulnerable to
lipid
peroxidation.
It has further been observed that vitamin E blocks the neurotoxic effects of
the free
radicals produced by excitotoxicity and it improves the performance altered by
old
age.
Thus according to the present invention, any chemical entities exhibiting the
biological activity of a-tocopherol is meant to encompassed by the definition
of
vitamin E.
Alternate names for vitamin E include Alpha Tocopherol, D-Alpha- Tocopherol, D-
Beta- Tocopherol, D-Delta- Tocopherol, D-Gamma- Tocopherol, D- Tocopherol,
DL-Alpha- Tocopherol, DL- Tocopherol, Mixed Tocopherols, Tocopheryl Acetate,
Tocopheryl Succinate.
Other vitamin E related compounds may also be used in the context of the
present
invention. They may be selected from the group consisting of vitamin E
acetate,
DL-a-Tocopherol succinate, d-alpha-Tocopherol acetate, (+)-alpha- Tocopherol,
mixed isomers thereof, (+)-alpha- Tocopherol and mixed isomers, (+)-alpha-
Tocopherol acid succinate, (+)-alpha- Tocopherol acetate, (+)-alpha-
Tocopherol,
(t)-alpha- Tocopherol nicotinate, (f)-alpha- Tocopherol acetate, (t)-alpha-
Tocopherol, DL-a-Tocopherol acetate, (t)-alpha- Tocopherol phosphate Disodium
Salt, (+)-a-Tocopherol, (~)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
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acid, (R)-TroloxTM methyl ether, (S)-Trolox'~'M methyl ether, 2-(alpha-D-
glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol, 2,2,5,7,8-pentamethyl-6-
chromanol (PMC), 2,3-dihydroxy-3,3-enono-1,4-lactone and chromone residues,
a-Tocophenol hydroquinone, a-Tocophenol quinone, Vitamin CE(5a-tocopheryl
ascorbate) and 2-(a-D-glucopyranosyl) methyl-2,5,7,8-tetramethyl chroman-6-OI.
The vifamin E analogs may be selected from the group consisting of: Ascorbic
acid, Beta-Caroten, Butylated-Hydroxy-Toluene, Butyated-Hydroxy-Anisol,
Calcium Citrate, Canthaxanthin, Melatorin, Nordihydroguaiaretic acid, Propyl
Gallate, Selenium, Silymarin, Sulfur Dioxide, Thioctic Acid.
The acetate and succinate derivatives of the natural Tocopherols have vitamin
E
activity, as do synthetic tocopherols and their acetate and succinate
derivatives. Of
these, d-alpha-tocopherol (RRR-alpha-tocopherol) has the highest
bioavailability
and is the standard against which all the others must be compared.
Natural and synthetic vitamins E are not equivalent in composition, structure
and/or bioavailability. Natural vitamin E (RRR-alpha-tocopherol or di-alpha-
tocopherol) is a single entity. Synthetic vitamin E (all-rac-alpha-tocopherol
or di-
alpha tocopherol) is a mixture of eight stereoisomers in equal amounts.
As mentioned above, vitamin E is recognized to be the major antioxidant in
lipid
body tissues and the primary defence against lipid peroxidation-neutralizing
free
radicals, terminating chain reactions and limiting free radicalloxidative
damage.
Vitamin E is particularly important in tissues that contain relatively high
levels of
polyunsaturated fatty acid (brain and central nervous system) and in those
that are
in contact with oxygen (lung), providing protection for microsomes and
mitochondria.
In rats and in cell cultures it slows down damage and neuronal death even in
cells
with amyloid deposit.
In cell cultures and in animals, it reduces neurotoxicity and neuronal death.
The
hydrogen peroxide and the free radicals produced by beta A4 to cultured
neurons
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and endotelial cells is blocked by vitamin E and other antioxidants. Thus,
vitamin E
reduces beta A4 induced cell death in hippocampal cell cultures. Vitamin E
also
improves cognitive performance in aged animals and reduces degeneration of
hippocampal cells following cerebral ischemia.
5
Vitamin E status in EA is controversial. A study found increased vitamin E
levels
suggesting a possible compensatory response to oxidative stress, another found
no difference with controls, and another found decrease of vitamin E in AD.
The range of daily dose goes between 200 and 3,000 UI, with an average range
of
10 40o to l,ooo u1.
Adverse symptoms are uncommon: cataract, haemorrhage risk in patient with
vitamin K deficiency, and syncope. It would have protective effects on the
immunologic response and cardiac diseases. With high doses (3000 IU), appear
indigestion, gastric distress, diarrhea and severe cramps.
In a multicenter comparative double blind study of 2 years of duration with
selegiline (10 mg daily}, vitamin E and their combination, with 341 patients,
significantly positive results were obtained with the vitamin E (2,000
UI/daily) and
the selegiline in single-agent therapy; but not with their combination. A
retarded
cognitive deterioration of 25% was observed for both drugs, as well as the
delay in
institutionalisation of the patients due to an improvement of the global
function.
Caffeic acid
Caffeic acid also known as 3-(3,4-Dihydroxyphenyl)-2-propenoic acid, found in
many fruits, vegetables, seasonings and beverages consumed by humans,
principally in conjugated forms such as chlorogenic acid.
Its action mechanism is not completely known, but the caffeic acid would have
a
beneficial effect in atherosclerosis, inflammation, neurodegenerative
dysfunctions
and acquired immunodeficiency syndromes.
Equivalents of such a compound may be selected from the group consisting of
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10
5(4)-(2-Carboxyethenyl)-1 ,2-dihydroxybenzene; 4-(2'-Carbcxyvinyl)-1 2-
dihydroxybenzene; 3,4-Dihydroxybenzeneacrylic acid; 3,4 Dihydroxycinnamic
acid; 3-(3,4-Dihydroxyphenyl)propenoic acid; 3,4-Dihydroxyhydrocinnamic acid;
o-
Coumaric acid; p-Coumaric acid; and Caffeic acid phenethy! ester.
The effects of phenolic compounds like the caffeic acid, present in the olive
oil,
over the oxidation of the low density protein (LDL) have been investigated.
This
process plays an important role in atherosclerosis, through the alteration of
intra-
cell signals in the vessel wall. The antioxidant effect of the caffeic acid is
protective. When 0.025-0.3 mg /I of caffeic acid is added to isolated LDL, its
time
of oxidation is prolonged in a dose dependant mechanism.
Recently the action of the caffeic acid has been studied compared with that of
the
N-acetylcystein, that of the acetate d-alpha-tocopherol, and that of the
ascorbic
acid in the modulation of signal transduction related with the apoptosis
induced by
the ceramide. The ceramide acts as a second messenger in signal transduction
produced by the stress or extracellular agents. It was observed that the
caffeic
acid inhibits significantly, in comparison with the other compounds, the
activity of
the NF-kappa B binding induced by ceramide and the apoptotic response by its
antioxidant effects.
Another hypothesis on its action mechanism, apart from its antioxidant effect,
is
the inhibition of the protein tyrosine kinase activity, and this would inhibit
the
apoptosis.
Nicotinic acid
Niacin and niacinamide function in the biochemistry of humans and other
organisms as components of the two coenzymes: nicotinamide adenine
dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP).
These operate in many enzyme-catalysed oxidation and reduction reactions. The
deficiency state in humans causes skin disease, diarrhea, dementia, and
ultimately death. Lean meats, peanuts and other legumes, and whole-grain or
enriched bread and cereal products are among the best sources of niacin.
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Nicotinic acid and nicotinamide, commonly called niacin, are the dietary
precursors
for NAD(+) (nicotinamide adenine dinucleotide), which is required for DNA
synthesis, as well as for the activity of the enzyme poly (ADP-ribose)
polymerase-
1 for which NAD(+) is the sole substrate. This enzyme is highly activated by
DNA
strand breaks during the cellular genotoxic stress response and is involved in
base
excision repair.
In vitro as well as animal studies indicate that niacin deficiency increases
genomic
instability especially in combination with genotoxic and oxidative stress.
Studies suggest that nicotinamide can be considered as a potent antioxidant
capable of protecting the cellular membranes in brain, which is highly
susceptible
to prooxidants, against oxidative damage induced by reactive oxygen species
(ROS).
In animal studies, it had been observed that nicotinamide showed significant
inhibition of oxidative damage induced by R05 in rat brain mitochondria. In a
study with the tertiary butylhydroperoxide-(t-Bu00H) treated mouse was used as
a model to study the oxidative stress that is associated with various
neurodegenerative diseases. The results directly implicate DNA damage in
apoptosis and necrosis. Nicotinamide was able to prevent DNA fragmentation
induced by low-dose t-Bu00H.
Other authors observed that nicotinamide is a robust neuroprotective agent
against ischemialreperfusion-induced brain injury in rats, even when
administered
up to 2 hours after the onset of stroke: nicotinamide improved both anatomic
and
functional indices of brain damage.
Nicotinic acid also known as pyridine 3-carboxylic acid and analogs thereof
may
be used in the combination of the present invention for the treatment of AD.
To this day, more than 233 different compounds of nicotinic acid are known.
Each
of them may be used in the context of the present invention. More
particularly, the
nicotinic acid compounds may be selected from the group consisting of:
Nicotinic
acid, Isonicotinic acid, 3-Pyridinecarbonitrile, 2,4,5,6-Tetrachloro-3-
pyridinecarboxylic acid, 2,6,-Dichoro-5-fluoro-3-pyridinecarboxylic acid, 2-
Chloro-
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6-methylnicotinic acid, 5-Bromonicontinic acid, Arecoline Hydrobromide, NO-711
Hydrochloride and Nicotinic acid ethyl ester, nicotinic acid (1-4-bromo-
phenyl)-
propylidene)-hydrazide, nicotinic acid (1 -(4-pentyl-phenyl-ethylidene)-
hydrazide,
nicotinic acid (1 -benzyl-propylidene)-hydrazide, nicotinic acid (1 -methyl-5-
vitro-2-
oxo-1 ,2-dihydro-indol-3-ylidene)-hydrazide, nicotinic acid (1 -thiophen-2-yl-
ethylidene)-hydrazide, nicotinic acid (2,4-dichloro-benzylidene)-hydrazide,
nicotinic
acid (2-chloro-benzylidene)-hydrazide, nicotinic acid (2,4-dihydroxy-
benzylidene)-
hydrazide, nicotinic acid (2,4-dimethoxy-benzylidene)-hydrazide, nicotinic
acid (2-
bromo-3-phenyl-allylidene)-hydrazide, nicotinic acid (2-hydroxy-benzylidene)-
hydrazide, nicotinic acid (2-methyl-3-phenyl-allylidene)-hydrazide, nicotinic
acid (2-
trifluoromethyl-benzylidene)-hydrazide, nicotinic acid (3,4,5-
trimiethoxybenzylidene)-hydrazide, nicotinic acid (3,5-di-tert-butyl-4-hydroxy-
benzylidene)-hydrazide, nicotinic acid (3,5-dibromo-2-hydroxy-benzylidene)-
hydrazide, nicotinic acid (3-ethoxy-hydroxy-benzylidene)-hydrazide, nicotinic
acid
(3-vitro-benzylidene)-hydrazide, nicotinic acid (3-phenyl-allylidene-
hydrazide,
nicotinic acid (4-bromo-benzylidene)-hydrazide, nicotinic acid (4-chloro-
benzylidene)-hydrazide, nicotinic acid (4-dimethylamino-benzylidene)-
hydrazide,
nicotinic acid (4-hydroxy-benzylidene)-hydrazide, nicotinic acid (4-isopropyl-
benzylidene)-hydrazide, nicotinic acid (4-methyl-benzylidene)-hydrazide,
nicotinic
acid (4-vitro-benzylidene)-hydrazide, nicotinic acid (5-indanylrnethylene)-
hydrazide, nicotinic acid n'- (1,1 -dioxo-tetrahydro-thiophen-3-ylrn'-phenyl-
hydrazide, nicotinic acid n'- (4-methoxy-benzoyl)-hydrazide, nicotinic acid n'-
phenoxyacetyi-hydrazide, nicotinic acid naphthalen-1-ylmethylene-hydrazide,
nicotinic acid 0-tolyl ester, nicotinic acid p-tolyl ester, nicotinic acid
pyridin-3-
ylmethylene-hydrazide, nicotinic acid thiophen-2-ylmethylene-hydrazide, 1-
bicyclo
(2.2.1) hept-2-yl-ethyiamine, nicotinic acid benzo (1,3)dioxol-5-ylmethylene-
hydrazide, nicotinic acid benzylidene-hydrazide, nicotinic acid fluoren-9-
ylidene
hydrazide, nicotinic acid (4-diethylamino-benzylidene)-hydrazide, nicotinic
acid, 1
,2-diphenylethylammonium salt, nicotinic acrd, 2-amino 1 - (4-vitro-phenyl)-
ethanol, nicotinic acid, octadecylamine salt, nicotinic acid (2,4-dimethoxy-
benzylidene)-hydrazide, nicotinic acid (2-bromo 3-phenyl-allylidene)-
hydrazide,
nicotinic acid (2-chloro-benzylidene) hydrazide, nicotinic acid hydroxamate,
nicotinic acid mononucleotide, nicotinic acid n-oxide, 1-methylnicotinamide
chloride salt, 1-methylnicotinamide iodide salt, and nicotinic acid adenine
CA 02401452 2002-09-04
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dinucleotide sodium salt.
Isonicotinic acid salts may also be used in the combination of the present
invention
for the treatment of AD. To this day, 86 different compounds of isonicotinic
acid
salts are known. More particularly, these may be selected from the group
consisting of: Isonicotinic acid ((4-vitro-phenyl)-phenyl-methylene)-
hydrazide;
Isonicotinic acid (1-allyl-2-oxo-1,2-dihydro-indol-3-ylidene) hydrazide;
Isonicotinic
acid (1-benyl-2-oxo-1,2-dihydro-indol-3-ylidene)hydrazide; Isonicotinic acid
(1-
benzyl-propylidene)-hydrazide; Isonicotinic acid (1-methyl-1 H-pyrrol-2-
ylmethylene)-hydrazide; lsonicotinic acid (1-methyl-2-oxo-1,2,dihydro-indol-3-
ylidene) hydrazide; Isonicotinic acid (1-methyl-2-oxopropylidene)-hydrazide;
Isonicotinic acid (1-P-tolyl-ethylidene)-hydrazide; Isonicotinic acid (1-
phenyl-
ethylidene)-hydrazide; Isonicotinic acid (1 H-indol-2-ylmethylene)-hydrazide;
Isonicotinic acid (2,3,4,6-tetramethyl-benzylidene)-hydrazide; Isonicotinic
acid
(2,4-dihydroxy-benzylidene)-hydrazide; Isonicotinic acid (2,5-dimethoxy-
benzylidene)-hydrazide; Isonicotinic acid (2-chloro-benzylidene)-hydrazide;
and 1-
oxy-isonicotic acid methyl ester.
Quercetin
Quercetin is a member of a group of naturally occurring compounds, the
flavonoids, which have common flavone nucleus composed of two benzene rings
linked through a heterocyclicpyrone ring.
Quercetin is found in various plants, food products, and dyes of natural
origin. The
estimated average daily intake of quercetin by an individual in the United
States is
25 mg.
The Food and Drug Administration nominated quercetin for toxicity and
carcinogenicity studies in the rat because it is a chemical that is widely
distributed
in foods. Quercetin was administered to rats by dosed feed since human
exposure
is by dietary consumption.
The Quercetin derivatives and/or analogs that may also be used in the
combination of the present invention may be selected from the group consisting
of
CA 02401452 2002-09-04
flavone,3,3',4',5,7-pentahydroxy-4H-1-benzopyran-4-one;.2-(3,4,-
dihydroxypehnyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one; 3,3',4' ,5,7,-
pentahydroxyflavone; 3,5,7,3',4'-pentahydroxyflavone; 2-(3,4-dihydroxyphenyl)-
3,5,7-trihydroxy-4H-1-benzopyran-4-one; 3,5,7,3',4'-pentahydroxyflavone;
3',4',5,7-
5 tetrahydroxyflavan-3-OL; 2-(3,4-dihydroxyphenyl)-4H-1-benzopyran-4-one;
cyanidelonon 1522; C.I. natural yellow 10; C.I. natural yellow 10 & 13; C.I.
natural
red 1; C.l. 75670; meletin; quercetine; quercetol; quercitin; quertine;
sophoretin; T-
Gelb BZW. Grun lXanthaurine; and NCI-C60106.
10 Quercetin dehydrate analogs may be selected from the group consisting of:
Quercetin dehydrate; (t)-Taxifolin; Fisetin; Quercetin-3-rhamnoside; Quercetin-
3-D-
xyloside; Quercetin-3-D-galactoside; Quercitrin; Rutin trihydrate; Morin; and
Morin
hydrate.
As mentioned earlier other antioxydant substances may be used in the context
of
15 the present invention. For instance, Ginkgo biloba is known for its
antioxydant
caracteristic for reducing oxidative stress or damage in the brain of AD
patients.
Ginkgo biloba
The Ginkgo biloba extract, obtained from the leaf of a tree of the same name,
is a
vegetable extract used in Europe to alleviate the symptoms associated with
cognositive disorders.
Recently its use has been approved in Germany for the treatment of dementia.
Its
action mechanism on the central nervous system is only partially known, but
its
main effects would be related with its antioxidant capacity, diminishing the
oxidative stress or damage detected in the brain of patients with Alzheimer's
disease. For that purpose, its active principle, characterized as Egb 761,
would
develop a synergistic action of flavonoids, terpenoids and organic acids that
constitute it, that would act synergistically in various processes such as the
inflammation homeostasis and the oxidative stress, protecting the membrane and
the modulation of the neurotransmission.
CA 02401452 2002-09-04
16
Hofferberth et al. studied 36 patients with typical symptoms of psychotic
organic
syndromes. These patients were divided in two groups, one treated with placebo
and the other with EGb 761, 120 mglday, during a period of 8 weeks.
At the beginning and at the end of the treatment, the patients were subjected
to a
quantitative electroencephalogram, saccadic eye movements and a psychometric
test. Four weeks after the beginning of the treatment, a significant
difference could
already be observed that persisted until the end of it.
Kanowski et al. performed a multicenter study with 216 patients during 24
weeks
divided in a placebo group and a group treated with 240 mg of EGb 761.
The analyzed variables were:
- clinic global impression for phyisiopathologic evaluation;
- evaluation test of attention and memory; and
- evaluation test of daily activities.
Of the total enrolled patients, 156 completed the protocol and, according to
Fisher's statistical test, significant improvement was achieved (p < 0.005) in
the
treated group.
LeBars et al. have recently made an important study taking into account the
number of patients enrolled and the duration of the treatment. It was a
multicenter
study with 309 patients of both sexes over 45 years with duration of 52 weeks.
In
the selection of the cases, dementia was mild to moderate with a score of 9 to
26
carried out according to Mini-mental-Test-Examination (MMSE) and 3 to 6 points
according to the Clinical Global Impression of Change, CGIC scale.
The patients received placebo or 120 mg of EGb 761.
CA 02401452 2002-09-04
17
The treatment did not show detectable differences in the CGIC, but there was a
cognositive significant improvement in the Alzheimer's Disease Assessment
Scale
[ADAS].
A meta-analysis comparing the cholinesterase inhibitors of the tacrine,
rivastigmine and metrifonate and the Egb 761, based on published double blind
studies of at least 6 months of duration, shared similar effectiveness of both
types
of compounds evaluated with the ADAS in AD from mild to moderate.
Future investigations are required to specify these mechanisms in connection
with
the EGb 761, in order to explore the potential of this vegetable extract.
PROCEDURE
2.1. Objective
The effectiveness of the following combination referred to as CP08T was
evaluated:
- nicotinic acid:50 mg;
- caffeic acid :50 mg;
- Quercitin:50 mg;
- vitamin E:1,000 UI.
It was compared to placebo, according to the following design:
A double blind comparative trial with cross-over design was used.
Patients were divided in 4 groups:
Group 1: patients 1 to 5: they took CP01 P (which corresponds to a placebo
cocktail) during 180 days.
Group 2: patients 6 to 9 took CP01 P during 90 days and CP08T during 90 days.
Group 3: patients 10 to 18: they took CP08T during 180 days.
Group 4: patients 19 to 22: they took CP08T during 90 days and CP01 P during
90 days.
2.2. Inclusion criteria
22 patients with probable Alzheimer's disease were included according to the
criteria of the DSM iV (Diagnostic and Statistical Manual Mental of Disorders)
and
CA 02401452 2002-09-04
18
of the NINCDS-ARDRA National Institute of Communicative Disorders and Stroke-
Alzheimer's Disease and Related Disorder Association). The selected patients
were of both sexes, with ages between 50 and 90 years.
They presented a mild to moderate degree of the disease, which was evaluated
with the Mental Mini State of Folstein: they had a score from 10 to 24 points.
The
Computed Axial Tomography (CAT) or the Nuclear Magnetic Resonance (NMR}
did not show infection evidence, infarct or other focal lesions previous to
the 12
months of the beginning of the study.
The patients who presented any of the following items were excluded:
1- Knowable hypersensitivity to any of the drugs
2- Not controlled systematic illnesses, such as
- cancer in a stage of radio or chemiotherapic treatment
- acute myocardal infarctus anger
- chronic obstructive lung disease or severe asthma
- active gastroduodenal ulcer or recent digestive hemorrhage
- severe -anemia (Hb < 11 ), decresase of platelets, acute leukemia
- decompensated diabetes
- not controlled hypertension
- antecedents of severe hepathic illness, acute or chronic hepatopathy
- coagulopathy or vitamin K deficit within two years
Neurological diseases such as:
- Parkinson disease
- Multiinfarct dementia
- Huntington disease
- Hydrocephalus normotensive
- Cerebral -tumor
- Progressive supranuclear paralysis
- Convulsive -illnesses
- Subdural haematoma
- Multiple-sclerosis
- antecedents of cranial trauma followed by neurological dysfunctions
Psychotic dysfunctions
CA 02401452 2002-09-04
19
Patients treated with:
- Cholinesterase inhibitors, vasodilators, calcium antagonists, antioxidants
or nootropic drugs.
- beta blockers, metildope, clonidine
- typical neuroleptic sedatives or clozapine
- opioids
- benzodiazepine: allowed alprazolam up to 1 mg/d
- begining of a treatment with antidepressants within 4 weeks
- cholinergics or anticholinergics (tricyclic antidepressants included)
- corticosteroids
- anticonvulsivants
- warfarin, and
- multivitamins.
All patients and their relatives gave their formal written consent to
participate in the
study.
The cocktail was administered to the patients. The cocktail was given in
person to
them, and they were reminded by telephone to take the compounds 3 times a
week during the 180 days of the study.
The cognitive subscale of the scale ADAS (Alzheimer's Disease Assesment Scale)
was taken previous to the administration of the drug, and 3 and 6 months after
the
beginning of its administration to evaluate the changes of the cognitive
symptoms.
The cognitive subscale was then taken again at 9 months, that is to say, 3
months
after the interruption of the administration of the compounds.
Evaluation instruments
1. Mini mental State (Folstein et al, 1975):evaluates orientation, memory,
attention,
concentration, possibility to name objects, repetition, understanding, ability
to
make a sentence and to copy two polygons in intersection. The lesser cognitive
performance, the lower score. The maximum performance is 30.
CA 02401452 2002-09-04
2. Alzheimer's Disease Assessment Scale (ADAS) cognitive subscale (Rosen,
Mohs et al, 1984):evaluates memory, attention, reasoning, language,
orientation
and praxis. The lesser cognitive performance the higher score. Maximum
severity
is 70.
5
3. Hamilton depression scale of 21 items: evaluates mood, feeling of guilt,
suicide,
insomnia, work and activities, inhibition, agitation, anxiety, somatic
symptoms,
perspicacity, diurne variation, paranoid symptoms and obsessive-compulsive
symptoms.
The higher the score, the greater is the severity. Maximum is 57.
Clinical interviews were carried out previous to the beginning of the study,
and
then quarterly during the following 9 months.
Complete blood and urine tests were made at the beginning of the study and
then
quarterly during the following 9 months.
25
CA 02401452 2002-09-04
21
!ll. Results
STATISTICAL ANALYSIS
Patient division according to medication distribution
GROUP CP01 PICP01 P n=5
1:
GROUP CP01 PICP08T n=4
2:
GROUP CP08T/CP08T n=9
3:
GROUP CP08T/CP01 P n=4
4:
Evaluation at different periods
T OM: Before the treatment
T 3M: 3 months
T 6M 6 months
T 9M 9 months (3 months after end
of treatment)
Patients that did not present results from any tests were eliminated from the
research. Because of this, the groups of treated individuals are not balanced
and
therefore the means of the groups are used instead of using individual patent
date.
The statistical study used analysis of variance (ANOVA) using Randomised Block
Design (RBD). The RBD allowed to distinguish possible differences between
blocks and columns. The block is the random factor. Each block is an
individual, or
in this case in working with means, a homogeneous group of individuals, who
undergo different treatments or different methods or evaluation. Therefore,
the
research consisted in determining if significant differences existed between
the
means obtained in the different groups under different treatments and between
different time periods of evaluation.
CA 02401452 2002-09-04
22
T OM T 3M T 6M T 9M
GROUP 1 yij y1.
GROUP 2
GROUP 3
GROUP 4
yj. y..
yij is each data, in the case working with means it is considered the mean of
each
group in each period of time (box)
yi. is the mean of each block
yj. is the mean of each column
y.. is the general mean
,1 is the number of trials or evaluations
I is the number of blocks or groups or treatments
The following is the ANOVA table for RBD
S of V DF SS MS f
F
Between the blocks I-1 J.SUMyI. -Ny.,' SCEb~IGLEbI CMEb~CMD
FI-1,GLD,0,05
Between columns J-1 I.SUMyjz-Ny..z
SCE°°~IGLE°°~ CME~,ICMD
FJ-'I,GLD,0.05
Error or inside (I-1 )(J-1 ) SC~-(SCEb~+SCEcoI) SCD/G(_D
Total N-1 SUMyij2-Ny..z
S of V: Source of variation
DF: Degree of freedom, SS: sum of the squares, MS: mean of the squares, SUM:
summation.
The result is significant with 95% certainty when f > statistical result of F
or, what is
the same when p < 0,05.
CA 02401452 2002-09-04
23
The RBD is an additive mode( as long as there is no interaction between the
blocks and the columns. To make sure of this, each case was tested for non
additivity of Tukey.
On the other hand, when significant differences are obtained between blocks or
between columns in the ANOVA, contract studies must be carried out to
determine
between which, of the blocks or the columns, these differences are produced.
The
contrasts oppose the blocks together or the columns together, and establish
the
origin of the differences. Contrasts of Scheffe, Newman Keuls and LSD were
used.
Statistical Analysis for the ADAS Test
Table of Means
T OM T 3M T 6M T 9M Mean y1.
GROUP 41.000 44.000 42.500 45.750 43.313
1
GROUP 40.800 36.800 37.800 41.800 39.300
2
GROUP 40.444 36.667 39.889 40.444 39.361
3
GROUP 40.250 36.750 38.750 39.750 38.125
4
Mean y.j 40.624 38.304 39.235 41.936 40.025
I ' I ~ I
RBD (Randomized Block Design) - ANOVA (Analysis of Variance)
S of V DF SS MS f F
Groups 3 61.536123620.512047.81933039>3.88 S (p<0,05)
(Blocks)
12
Periods 3 30.365933610.128643.88110857-3.66 (p-0,05)
45
9 23.60923012.623247
79
Total 15 115.531287
S: Significant differences
CA 02401452 2002-09-04
24
NS: Non-significant differences.
The ANOVA reveals significant differences (p<0,05) between the groups or
blocks.
That is to say between the different treatments as for the average scores
obtained
for the ADAS test. However, the differences were not as significant for the
different
times of evaluation.
Contrasts
The different groups were compared to evaluate how their averages
differentiate in
the ADAS test
Scheffe Test
GROUP 1 GROUP 2 GROUP 3 GROUP 4
GROUP 0.120102320.12742966 0.0348689
1
GROUP 0.120102316 0.99998283 0.89121008
2
GROUP 0.1274296640.99998283 0.8767038
3
GROUP 0.0348688960.891210080.8767038
4
GROUP 1 vs 2: NS
GROUP 1 vs 3: NS
GROUP 1 vs 4: S p<0.05
GROUP 2 vs 3: NS
GROUP 2 vs 4: NS
GROUP 3 vs 4: NS
CA 02401452 2002-09-04
Newman-Keuls Test
GROUP 1 GROUP 2 GROUP 3 GROUP 4
GROUP 1 0.12010232 0.127429660.0348689
GROUP 2 0.049438715 0.969412150.44869167
GROUP 3 0.0218878390.96941215 0.69663686
GROUP 4 0.0213646890.44869167 0,69663886
, I I
GROUP 1 vs 2: S p<0.05
5 GROUP 1 vs 3: S p<0.05
GROUP 1 vs 4: S p<0.05
GROUP 2 vs 3: NS
GROUP 2 vs 4: NS
GROUP 3 vs 4: NS
It can be inferred from observing the graph of the means and the contrasts
that the
existing significant differences between the treatments are mainly found
between
groups 1 and 4. Group 1 obtained average scores of ADAS greater than the rest
of
the groups during the entire time period and moreover, demonstrated a tendency
to increase, which indicates a worsening during the treatment. Instead, the
other
groups average scores decreased during the first 3 months of the treatment but
started to increase again from that point on until reaching, in general, the
values
obtained at the beginning of the study. The Scheffe Test reveals less (is less
sensitive) and, for that reason, only demonstrates this difference between
groups
1 and 4. On the contrary, the Newman Test demonstrates significant differences
between the scores of the groups 1 and 2, 1 and 3, and 1 and 4, resembling the
values obtained by the patients of groups 2, 3 and 4 with similar behaviors of
their
averages during the entire time.
Statistical Analysis for the MMSE test
CA 02401452 2002-09-04
26
Table of Means +8287
T OM T 3M T 6M T 9M Mean y1.
GROUP 18.000 17.750 18.750 17.750 18.063
1
GROUP 19.000 19.200 20.000 18.000 19.050
2
GROUP 19.000 20.333 19.222 18.778 19.333
3
GROUP 19.250 20.750 20.750 18.250 19.750
4
Mean y.j 18.813 19.508 19.681 18.194 19.049
y..
Behavior of the average scores during the entire time period for the different
groups (treatments)+B333
S of V DF SS MS F F
Groups 3 6.161358822.0804526.08060482>3.66 S (p<0,05)
(Blocks)
87
Periods 3 5.583733871.8612445.49272112<3.88 S (p<0,05)
82
9 3.04970910.338858
57
Total 15 14.8148016
S: Significant differences
NS: Non-significant differences.
In this case, the ANOVA reveals significant differences (p<0,05) between the
groups or treatments in their average scores obtained in the MMSE test and
also
in their different evaluation periods.
CONTRASTS
On the one hand, the different groups were compared together and, on the other
hand, the different periods of evaluation were compared together, using ANOVA,
to evaluate how their averages differentiate in the MMSE test. The following
tables
CA 02401452 2002-09-04
27
illustrate the p values of those comparisons:
Scheffe Test
GROUP1 GROUP2 GROUPS GROUP4
GROUP 1 0,46793833 0.26549053 0.09729582
GROUP 2 0.46793833 0.97262728 0.71923995
GROUP 3 0.26549353 0.97262728 0.92056388
GROUP 4 0.09729852 0.71923995 0.92056388
GROUP 1 vs 2: NS
GROUP 1 vs 3: NS
GROUP 1 vs 4: NS
GROUP 2 vs 3: NS
GROUP 2 vs 4: NS
GROUP 3 vs 4: NS
Newman-Keuls Test
GROUP 1 GROUP 2 GROUP 3 GROUP 4
GROUP+B254 0.12574399 0.127661110.06535465
1
GROUP 2 0.12574399 0.645358320.49389362
GROUP 3 0.12766111 0.64535832 0.50054574
GROUP 4 0.06535465 0.49389362 0.50054574
GROUP 1 vs 2: NS
GROUP 1 vs 3: NS
GROUP 1 vs 4: NS
GROUP 2 vs 3: NS
GROUP 2 vs 4: NS
GROUP 3 vs 4: NS
CA 02401452 2002-09-04
28
LSD Test
GROUP 1 GROUP 2 GROUP 3 GROUP 4
GROUP 1 0.46793833 0.05565747 0.01564395
GROUP 2 0.12558138 0.6452055 0.26582634
GROUP 3 0.055657470.6452055 0.50037628
GROUP 4 0.015643950.26582634 0.50037628
GROUP 1 vs 2: NS
GROUP 1 vs 3: NS
GROUP 1 vs 4: S p<0.05
GROUP 2 vs 3: NS
GROUP 2 vs 4: NS
GROUP 3 vs 4: NS
Scheffe Test
T OM T 3M T 6M T 9M
T OM 0.74233615 0.59628534 0.80315143
T 3M 0.74233615 0.99412596 0.26565802
T 6M 0.596285340.99412596 0.1812412
T 9M 0.803151430.26565802 0.1812412
T OM vs T 3M: NS
T OM vs T 6M: NS
T OM vs T 9M: NS
T 3M vs T 6M: NS
T 3M vs T 9M: NS
T 6M vs T 9M: NS
CA 02401452 2002-09-04
29
Newman-Keuls Test
T OM T 3M T 6M T 9M
T OM 0.28400975 0.37189716 0.33882374
T 3M 0.28400975 0.78607857 0.12776077
T 6M 0.371897160.78607857 0.13094723
T 9M 0.338823740,12776077 0.13094723
T OM vs T 3M: NS
T OM vs T 6M: NS
T OM vs T 9M: NS
T 3M vs T 6M: NS
T 3M vs T 9M: NS
T 6M vs T 9M: NS
LSD Test
TOM T3M T6M T9M
T OM 0.28388375 0.18696201 0.33869615
T 3M 0.28388375 0.78594285 0.05570481
T 6M 0.186962010.76594285 0.03376284
T 9M 0.338696150.05570481 0.03376284
T OM vs T 3M: NS
T OM vs T 6M: NS
T OM vs T 9M: NS
T 3M vs T 6M: NS
T 3M vs T 9M: NS
T 6M vs T 9M: S p<0,05
In regards to the contrasts between the groups of treatments, the Scheffe and
Newman-Keuls tests do not succeed in finding differences between those, except
in groups 1 vs 4, which demonstrate the smallest value of p, although it is
not
significant (p<0,05) in either case. However, with a third test (LSD Test),
one can
CA 02401452 2002-09-04
see how groups 1 and 4 present this significant difference amongst each other,
which was starting to be seen with prior contrasts. On the other hand, one can
also see that a p value very close to 0,05 appears when comparing groups 1 vs
3,
which also would have markedly different averages between each other.
5 Observing the graph, one can clearly note these differences but also make
out the
tendencies and the behavior of the means for each of the treatments. Note how
the means of the Groups 2, 3 and 4 increase during the first 3 months of the
treatment while in Group 1 one can observe a certain decrease during this same
period. Then, during the next 3 months, only group 3 shows a decrease in its
10 values and therefore, in the cognitive performance of the patients, while
the other
groups maintain or increase their scores.
It must be emphasized however that the mean of the initial score for group 1
was
less than that of the other groups of the study, fact that must be taken into
15 consideration and without which the behaviour of this group would not
differ as
much from those of the groups 2 and 4. Finally, all the curves start
decreasing
from a critical point, marked by the end of the treatments, reaching MMSE
score
values similar or even less than those obtained at the beginning of the
experiment.
In regards so the comparison between the different evaluated time periods,
again
20 the Scheffe and Newman-Keuls tests do not show differences between those
although they present lesser p values at times T 3M vs T 9M and T 6M vs T 9M.
Then the LSD test confirms one of the significant differences with p<0,05 and
the
other with p=0.0557. This means that the values of the averages of the MMSE
are
significantly different between 6 and 9 months, and, with a somewhat lower
25 significance, between 3 and 9 months.
The graph clearly demonstrates these differences, mostly those which appear
between the last two evaluations.
CA 02401452 2002-09-04
31
Statistical Analysis for the Hamilton Test
Table of Means
T OM T 3M T 6M T 9M Mean y1.
GROUP 13.75 18,25 24.75 22.5 19.813
1
GROUP 17.39996 15 20 22.799999218.800
2
GROUP 17.555555318.111110724.444444720.44444720.139
3
GROUP 17.250 18.5 22.25 22.25 20.063
4
Mean y.j 16.489 17.465 22.861 21.999 19.703
y..
S of V DF SS MS f F
Groups 3 4.586562811.5288500.43474727>3.88NS
(Blocks) 94
Periods 3 122.3256340.7752011.5948245>3.66S (p-0,05)
98
9 31.64978693.516642
99
Total 15 158.581969
In this case, the ANOVA reveals significant differences (p<0,05) in the
average
values of the Hamilton test for depression between the different periods of
evaluation but not between the groups or treatments.
Contrasts
The different time periods were compared, given the signification of the ANOVA
to
evaluate how their averages differentiate in the Hamilton test. The following
tables
demonstrate the p values of these results.
20
CA 02401452 2002-09-04
32
Scheffe Test
T OM T 3M T 6M T 9M
T OM 0.8872587 0.00216968 0.0065899
T 3M 0.88725287 0.0076508 0.02397896
T 6M 0.0021169680.0076508 0.91840172
T 9M 0.0065899 0.02397896 0.91840172
' I
T OM vs T 3M: NS
T OM vs T 6M: S p<0,05
T OM vs T 9M: S p<0,05
T 3M vs T 6M: S p<0,05
T3MvsT9M:S
T 6M vs T 9M: NS
Newman-Keuls Test
T OM T 3M T 6M T 9M
T OM 0.44252211 0.00127918 0.0021444
T 3M 0.00127918 0.00248516 0.00324839
T 6M 0.001279180.00248516 0.49628913
T 9M 0.0021444 0.00324839 0.49628913
T OM vs T 3M: NS
T OM vs T 6M: S p<0,05
T OM vs T 9M: S p<0,05
T 3M vs T 6M: S p<0,05
T3MvsT9M:S
T 6M vs T 9M: NS
It can be inferred from the contrasts that there are significant differences
in the
means between the time periods of T OM vs T 6M, T OM vs T 9M, T 3M vs T 6M
and T 3M vs T 9M. Observing the graph of the means, we see how they vary in
CA 02401452 2002-09-04
33
the time periods mentioned but not between the different groups for each
particular period. One can observe how similar the curves are between the
groups
(ANOVA NS), the means increasing in all cases during the entire time period
until
the end of the treatment. From that point on, only one of the groups
demonstrates
an increase while the values of the other groups start to decrease.
Finally, in regards to the ADAS test, the results of group 1 increased during
the
entire treatment, which would indicate an increase of the severity of the
disease.
The rest of the groups demonstrated a marked decrease in the three frist
months
and then increased, in a small or large measure, but without reaching the
severity
of group 1 (group 4 < group 2 < group 3). As for the results of the MMSE, once
more, group 9 demonstrates a failure to respond to the treatment because weak
values are always obtained, while group 4 obtained the best scores, remaining
stable in time during the entire treatment. Group 3 improved its scores during
the
first three months and then they decreased. As for group 2, the scores
increased
lightly during the 6 months of
the experiment.
IV. Intercurrences
- Urinary infection
Meningitis
- Sudden deaths
- Headache
DISCUSSION
In this double-blind, controlled study of patients with Alzheimer's disease,
treatment with Nicotinic acid, Caffeic Acid, Quercitin, and Vitamin E
combination
has proved to be beneficial in delaying disease progression. Disease
progression
was primarily studied using the standardized tests ADAS and MMSE where longer
median time reflects the worsening of symptoms. The median time to the primary
outcome was longer with each treatment than with placebo alone. There was a
trend toward a delay in reaching each of the individual end points making up
the
primary outcome, with a significant delay in Alzheimer's Disease Assessment
Scale (ADAS) in the treatment Group # 4. There were also significant delays in
the
CA 02401452 2002-09-04
34
deterioration of the performance of activities of daily living and the need
for care.
These findings should be of interest since, to date, no treatment for
Alzheimer's
disease has shown similar benefits with respect to these outcomes. The
possibility
that our i:fndings reflect aberrations in the placebo Group # 1 is unlikely,
since the
patients in this group reached the end points at the same rate of significance
delay
in ADAS in the treatment Groups 2 and 3. The Newman Test, demonstrates
significant differences between the ADAS score of the Groups 1 and 2, 1 and 3,
1
and 4 resembling the values obtained by the patients of the Groups 2, 3 and 4
with
similar behaviors of their averages during the entire period.
There were no demonstrable differences between the results in the group
receiving treatment followed by placebo versus the groups receiving treatment
throughout the course of the study (Group 2 vs Group 3, Group 2 vs Group 4,
and
Group 3 vs Group 4).
On the other hand, the ANOVA Test reveals significant differences (p<0,05)
between the groups or treatments in their average scores obtained in the MMSE
test and also in their different evaluation periods.
The HAMILTON test, which is a measurement of depression, demonstrates that
there are significant differences in the means ADAS score between the time
periods of T OM Vs T 6M, T OM vs T 9M, T 3M vs T 6M and T 3m VS T 9M. The
results of group 1 increased during the entire treatment, which indicate an
increase in the severity of the disease (demonstrate a failure to respond to
the
treatment), while group 4 obtained the best ADAS scores, remaining stable in
the
time during the entire treatment. Group 3 improved its scores during the first
three
months and then they decreased. As for Group2, the scores increased lightly
during the 6 months of the trial.
The above findings suggest that the use of Nicotinic acid, Caffeic Acid,
Quercitin,
and Vitamin E combination may improve the ADAS scores and delay clinically
important functional deterioration in patients with Alzheimer's disease.
Nicotinic acid, Caffeic Acid, Quercitin, and Vitamin E combination may have
enhanced the functioning of nigral neurons or enhanced their survival by
inhibiting
oxidative stress.
CA 02401452 2002-09-04
In the above study, there was improvement in cognitive portion of the
Alzheimer's
Disease Assessment Scale and the Mini-Mental State Examination scores in the
treatment groups.
The role of Nicotinic acid, Caffeic Acid, Quercitin, and Vitamin E combination
in the
5 treatment of neurodegenerative diseases is currently of great interest.
Previous
trials of alpha-tocopherol at much higher doses than used here, have
demonstrated benefit in patients with AD. The neuronal populations involved in
Alzheimer's disease are more sensitive to oxidative stress than those in other
neurodegenerative diseases. Also, in elderly populations it has been suggested
10 that antioxidants improve cardiovascular function and the immune response
and
also reduce the risk of cancer. Although it has been found that there is no
differences in the frequency of these other types of disease in our study
groups,
we have no biologic data to evaluate additional possible benefits.
Nicotinic acid, Caffeic Acid, Quercitin, and Vitamin E combination delay
functional
15 deterioration, particularly as reflected by the need for
institutionalization, and
should be considered for use in patients with moderate dementia. Statistically
significant results were seen in our model that included adjustment for the
base-
line differences among the groups in the score on the ADAS test, Mini-Mental
State Examination.
20 Further studies are needed to evaluate patients with more advanced forms of
Alzheimer's Disease, and longer observation period to observe any changes in
the
cognitive scores, behavioral disturbances and functional impairments.
CA 02401452 2002-09-04
36
Bibliographical References
1. Barkats M, et al. Hopital de la Pitie Salpetriere, France. J Neurochemistry
75:
1438-46; 2000.
2. Behl C, Davis J, Lesley R, et al.: Hydrogen peroxide mediates amyloid b
protein
toxicity. Cell; 77:817-827; 1994.
3. Behl C, Davis J, Cole GM et al: Vitamin E protects nerve cells fron amyloid
beta
protein toxicity; Biochem Bi6phis Res Commun, 186:944-950;1992. S.Benzi
G,Moretti A:Are reactive oxygen species involved in Alzheimer's disease;
Neurobiology of aging; 16:661-674; 1995.
4. Benzi G., Moretti A:Are reactive oxygen species involved in Alzheimer's
disease?; Neurobiology of aging; 16:661-674; 1995
5. Cott J. Natural product formulations available in Europe for psychotropic
indication. Psychopharmacol Bull ,31 :745-51; 1995.
6. Ehman WD, Ma rkesbery WR, Alauddin M, et al.: Brain trace elements studies
in Alzheimer's disease. Neurotoxicology ;7:197-206;1986.
7. Fito M, Covas MI,Lamuela-Raventos RM et al: Protective effect of olive oil
and
its phenolic compounds against low density lipoprotein oxidation; Lipids,
35(6):633-8;Jun 2000.
8. Harris ME, Hensley K, Buttefield DA, et al.: Direct evidence of oxidative
injury
produced by the Alzheimer's b amyloid peptide in cultured hippocampal neurons.
Exp Neurol ; 131:193-202; 1995.
9. Hensley K, Hail N, Subramanian R, et al.: Brain regional correspondence
between Alzheimer1 s disease histopatology and biomarkers of protein
oxidation; J
Neurochem; 65:2146-2156; 1995.
10. Huguet F, Tarrade T. Aipha-adrenoreceptor change during cerebral aging:
the
effect of Ginkgo biloba extract. J Pharma Pharmacol ;44:24-27;1992.
11. Kanowski S, et al. Proof of efficacy of the ginkgo biloba special extract
EGb
761 in outpatients suffering from mild to moderate primary degenerative
dementia
of the Alzheimer type or multi-infarct dementia. Pharmacopsychiatry; 29:47-56;
1996.
12. Kappus H,Diplock AT:Tolerance and safety of vitamin E:a toxicological
position
report; Free Radic Biol Med;13:55-74;1992.
CA 02401452 2002-09-04
37
13. Kilander L,Ohrvall M:Alpha tocopherol and Alzheimer's disease;N Engl J
Med;337:572-573; 1997.
14. Le Bars PL, Katz, M, German N, et aL A placebo-controlled, double-blind,
randomized trial of an extract of Ginkgo biloba for dementia. JAMA; 278:1327-
32;
1997.
15. Llesuy S: EI proceso de lipoperoxidaci6n. Antioxidantes y Calidad de
Vida;1:
18-19;1994.
16. Lucesoli F, Fraga GC: Evaluaci6n de! estre's oxidativo. Antioxidantes y
Calidad de Vida ;1:8-13;1995.
17. Marcus DL, Rodirguez TC, Simberkoff K, et al.: Increased peroxidation and
reduced antioxidant enzyme activity in Alzheimer--s disease. Exp Neurol 150:40-
44;1998.
18. Mclntosh U, Trush MA, Troncoso JC. Increased susceptibility of Alzheimer's
disease temporal cortex to oxygen free radical- mediated processes. Free Radic
Biol Med; 23: 183-190; 1997.
19. Mecocci P, MacGarvey U, Beau MF: Oxidative damage to mitochondrial DNA
is increased in Alzheimer's disease. Ann Neurol 1994;36:747-751.
20.Moizeszowicz J, Monczor M: Psicofarmacologia Psicogeria'tnca; in
Psicofarmacologia dinamica IV; Buenos Aires;ed Paid6s;1998.
21. Packer L, Haramaki N, Kawabata T, et al. Gingko biloba extract (Egb 761 ).
In
Christen Y, Courtois Y, Droy-Lefaix MT, eds. Effect of Gingko biloba Extract
(Egb
761 ) on aging and age-related disorders. Paris. France: Editions
Scientifiques
Elsevier Paris;23-47; 1995.
22. Ramassamy C, Avenil D, Theroux L. Oxidative insults are associated with
apolipoprotein E genotype in Alzheimer's disease brain. Neurobiol Dis ;7:23
37;2000.
23. Rezvani AH,Levin ED:Cognitive effects of nicotine;Biol
Psychiatry,49(3):258-
67;Feb 2001.
24.Sano M, Ernesto C, Thomas R et al: A controlled trial of selegiline, alpha
tocoferol or both as treatment for Alzheimer's disease ; N Engl J Med;336:1216-
1222;1997.
25.Schrattenholz A,Pereira EF,Roth U et al:Agonist responses of neuronal
nicotinic acetylcholine receptors are potentiated by a novel class of
allosterically
acting Iigands;Mol Pharmacol;49: 1-6; 1996.
CA 02401452 2002-09-04
38
26. Schippl!ng S, Kontush A, Buhmann C, et al. Increased lipoprotein oxidation
in
Alzheimerts disease. Free Radic Biol Med;28:351-360;2000. 33.Schipper H,Sisse
S,Stopa E:Expression of heme oxigenase 1 in the senescent and Alzheimer
disease ;Ann Neuro!,37:758-768;1995.
27.Wettstein A:Cholinesterase inhibitors and ginkgo extracts - are they
comparable in the treatment of dementia? Comparison of published placebo-
controlled efficacy studies of at least six months' duration; Phytomedicine;
6(6):393-401; Jan 2000.
28.Wevers A,Witter B,Moser N et al:Classical Alzheimer features and
cholinergic
dysfunctinaowards a unifying hypothesis?;Acta neurol scand Suppi; 176:42
8;2000.
29.Zamani MR,A!len YS:Nicotine and its interaction with beta-amyloid protein:
a
short review ;Biol Psychiatry,49(3) :221-32 ;Feb 2001.
