U.S. patent application number 15/669443 was filed with the patent office on 2019-08-08 for compositions and methods for the treatment of plaques and tangles in humans and animals.
This patent application is currently assigned to Cognitive Clarity Inc.. The applicant listed for this patent is Cognitive Clarity Inc.. Invention is credited to Judy Cam, Joel Cummings, Qubai Hu, Thomas Lake, Alan D Snow.
Application Number | 20190240279 15/669443 |
Document ID | / |
Family ID | 56789214 |
Filed Date | 2019-08-08 |
United States Patent
Application |
20190240279 |
Kind Code |
A9 |
Cam; Judy ; et al. |
August 8, 2019 |
COMPOSITIONS AND METHODS FOR THE TREATMENT OF PLAQUES AND TANGLES
IN HUMANS AND ANIMALS
Abstract
Methods of treating the formation, deposition, accumulation, or
persistence of beta-amyloid containing plaques and tau protein
containing tangles including the step of treating the plaques or
tangles with an effective amount of a composition comprising an
Uncaria tomentosa extract and an oolong tea extract Compositions
containing an Uncaria tomentosa extract in combination with an
oolong tea extract.
Inventors: |
Cam; Judy; (Bellevue,
WA) ; Lake; Thomas; (Snohomish, WA) ; Hu;
Qubai; (Kirkland, WA) ; Cummings; Joel;
(Seattle, WA) ; Snow; Alan D; (Lynnwood,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cognitive Clarity Inc. |
Lynnwood |
WA |
US |
|
|
Assignee: |
Cognitive Clarity Inc.
Lynnwood
WA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170333512 A1 |
November 23, 2017 |
|
|
Family ID: |
56789214 |
Appl. No.: |
15/669443 |
Filed: |
August 4, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15053572 |
Feb 25, 2016 |
10307454 |
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15669443 |
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62126026 |
Feb 27, 2015 |
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62170822 |
Jun 4, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/74 20130101;
A61P 35/00 20180101; A61K 36/82 20130101; A61P 43/00 20180101; A61P
25/16 20180101; A61P 21/00 20180101; A61P 25/28 20180101; A61P 9/00
20180101; A61P 25/00 20180101; A61K 36/74 20130101; A61K 2300/00
20130101; A61K 36/82 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 36/82 20060101
A61K036/82; A61K 36/74 20060101 A61K036/74 |
Claims
1. A method of treating the formation, deposition, accumulation, or
persistence of beta-amyloid containing "plaques", comprising
treating the plaques with an effective amount of a composition
comprising an Uncaria tomentosa extract and an oolong tea
extract.
2. The method of claim 1 wherein the extracts are formulated for
administration by one of the delivery routes selected from the
group of delivery routes consisting of pills, tablets, caplets,
soft or hard gelatin capsules, lozenges, sachets, cachets,
vegicaps, liquid drops, elixirs, suspensions, emulsions, solutions,
beverage preparations, cold or hot tea beverages, syrups, tea bags,
aerosols, suppositories, sterile injectable solutions, and sterile
packaged powders.
3. The method of claim 2 wherein the selected delivery route is
capsules.
4. The method of claim 1 wherein the amount of Uncaria tomentosa
extract and an oolong tea extract administered is between about
100-500 mg of Uncaria tomentosa extract aod about 100-500 mg oolong
tea extract.
5. The method of claim 1 wherein 100-500 mg of Uncaria tomentosa
extract is combined with 100-500 mg oolong tea extract and
formulated into a 200-1000 mg capsule.
6. A method of treating the formation, deposition, accumulation, or
persistence of tau protein containing tangles, comprising treating
the tangles with an effective amount of a composition comprising an
Uncaria tomentosa extract and an oolong tea extract.
7. The method of claim 6 wherein the extracts are formulated for
administration by one of the delivery routes selected from the
group of delivery routes consisting of pills, tablets, caplets,
soft or hard gelatin capsules, lozenges, sachets, cachets,
vegicaps, liquid drops, elixirs, suspensions, emulsions, solutions,
beverage preparations, cold or hot tea beverages, syrups, tea bags,
aerosols, suppositories, sterile injectable solutions, and sterile
packaged powders.
8. The method of claim 7 wherein the selected delivery route is
capsules.
9. The method of claim 6 wherein the amount of Uncaria tomentosa
extract and an oolong tea extract administered is between about
100-500 mg of Uncaria tomentosa extract aod about 100-500 mg oolong
tea extract.
10. The method of claim 6 wherein 100-500 mg of Uncaria tomentosa
extract is combined with 100-500 mg oolong tea extract and
formulated into a 200-1000 mg capsule.
11. A composition comprising a therapeutically effective amount of
an Uncaria tomentosa extract in combination with an oolong tea
extract.
12. The composition of claim 11 formulated for administration by
one of the delivery routes selected from the group of delivery
routes consisting of pills, tablets, caplets, soft or hard gelatin
capsules, lozenges, sachets, cachets, vegicaps, liquid drops,
elixirs, suspensions, emulsions, solutions, beverage preparations,
cold or hot tea beverages, syrups, tea bags, aerosols,
suppositories, sterile injectable solutions, and sterile packaged
powders.
13. The composition of claim 12 wherein the selected delivery route
is capsules.
14. The composition of claim 11 wherein 100-500 mg of Uncaria
tomentosa extract is combined with 100-500 mg oolong tea
extract.
15. The composition of claim 11 wherein 100-500 mg of Uncaria
tomentosa extract is combined with 100-500 mg oolong tea extract
and formulated into a 200-1000 mg capsule.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application in a Continuation of U.S. patent
application Ser. No. 15/053,572 filed Feb 25, 2016 which claimed
priority to U.S. PA 62/126,026 filed Feb. 27, 2015, and to U.S. PA
62/170,822 filed Jun. 4, 2016, each of which is hereby incorporated
by reference.
TECHNICAL FIELD
[0002] The invention relates to blended compositions of plant
extracts of Uncaria tomentosa and Oolong tea and methods for
treating "plagues and tangles" that accumulate in the aging brain
in amyloidosis and tauopathies in humans and animals (i.e. such as
aged dogs and cats). In addition, the invention relates to the
development of a nutraceutical blended composition consisting of a
combination of a plant extract of Uncaria tomentosa and a specific
Oolong tea, to prevent and treat traumatic brain injury (TBI),
concussions (as observed in most athletes and the
military/soldiers), single and repeated blows to the head, and
chronic traumatic encephalopathy (CTE).
BACKGROUND
Amyloidosis and the Accumulation of Beta-Amyloid Plaques in the
Brain of a Variety of Disorders
[0003] Alzheimer's disease is characterized by the accumulation of
a 39-43 amino acid peptide termed the beta-amyloid protein or AP,
in a fibrillary form, existing as extracellular amyloid plaques and
as amyloid within the walls of cerebral blood vessels. Fibrillar
A13 amyloid deposition in Alzheimer's disease is believed to be
detrimental to the patient and eventually leads to toxicity and
neuronal cell death, a characteristic hallmark of Alzheimer's
disease. Accumulating evidence implicates amyloid, and more
specifically, the formation, deposition, accumulation and/or
persistence of AP fibrils, as a major causative factor of
Alzheimer's disease pathogenesis. In addition, besides Alzheimer's
disease, a number of other amyloid diseases involve formation,
deposition, accumulation and persistence of AP fibrils, including
Down's syndrome, disorders involving congophilic angiopathy, such
as but not limited to, hereditary cerebral hemorrhage of the Dutch
type, inclusion body myositosis, dementia pugilistica, cerebral
(3-amyloid angiopathy, dementia associated with progressive
supranuclear palsy, dementia associated with cortical basal
degeneration and mild cognitive impairment.
[0004] The amyloid diseases (amyloidosis) are classified according
to the type of amyloid protein as well as the underlying disease.
Amyloid diseases have a number of common characteristics including
each amyloid consisting of a unique type of amyloid protein. The
amyloid diseases include, but are not limited to, the amyloid
associated with Alzheimer's disease, Down's syndrome, Canine
Dysfunction syndrome (CDS), Canine Cognitive Dysfunction (CCD), as
seen in aged animals such as dogs and cats, hereditary cerebral
hemorrhage with amyloidosis of the Dutch type, dementia
pugilistica, inclusion body myositosis (Askanas et al., Ann.
Neurol. 43:521-560, 1993) and mild cognitive impairment (where the
specific amyloid is referred to as beta-amyloid protein or
A.beta.), the amyloid associated with chronic inflammation, various
forms of malignancy and Familial Mediterranean Fever (where the
specific amyloid is referred to as AA amyloid or
inflammation-associated amyloidosis), the amyloid associated with
multiple myeloma and other B-cell dyscrasias (where the specific
amyloid is referred to as AL amyloid), the amyloid associated with
type 2 diabetes (where the specific amyloid is referred to as
amylin or islet amyloid polypeptide or IAPP), the amyloid
associated with the prion diseases including Creutzfeld-Jakob
disease, Gerstamann-Straussler syndrome, kuru and animal scrapie
(where the specific amyloid is referred to as PrP amyloid), the
amyloid associated with long-term hemodialysis and carpal tunnel
syndrome (where the specific amyloid is referred to as
.alpha.2-microglobulin amyloid), the amyloid associated with senile
cardiac amyloidosis and Familial Amyloidotic Polyneuropathy (where
the specific amyloid is referred to as transthyretin or
prealbumin), and the amyloid associated with endocrine tumors such
as medullary carcinoma of the thyroid (where the specific amyloid
is referred to as variants of procalcitonin). In addition, the
.alpha.-synuclein protein which forms amyloid-like fibrils, and is
Congo red and Thioflavin S positive (specific stains used to detect
amyloid fibrillary deposits), is found as part of Lewy bodies in
the brains of patients with Parkinson's disease, Lewy body disease
(Lewy in Handbuch der Neurologie, M. Lewandowsld, ed., Springer,
Berlin pp. 920-933, 1912; Pollanen et al, J. Neuropath, Exp.
Neurol. 52:183-191, 1993; Spillantini et al, Proc. Natl. Acad. Sci.
USA 95:6469-6473, 1998; Arai et. al, Neurosc, Lett. 259:83-86,
1999), multiple system atrophy (Wakabayashi et al, Acta Neuropath.
96:445-452, 1998), dementia with Lewy bodies, and the Lewy body
variant of Alzheimer's disease. For purposes of this disclosure,
Parkinson's disease, due to the fact that fibrils develop in the
brains of patients with this disease (which are Congo red and
Thioflavin S positive, and which contain predominant beta-pleated
sheet secondary structure), is now regarded as a disease that also
displays the characteristics of an amyloid-disease.
Amyloid as a Therapeutic Target for Alzheimer's Disease
[0005] Alzheimer's disease is characterized by the deposition and
accumulation of a 39-43 amino acid peptide termed the beta-amyloid
protein, A.beta. or .beta./A4 (Glenner and Wong, Biochem. Biophys,
Res, Comm. 120:885-890, 1984; Masters et al, Proc. Natl. Acad. Sci.
USA 82:4245-4249, 1985; Husby et al, Bull, WHO 71:105-108, 1993),
A.beta. is derived by protease cleavage from larger precursor
proteins termed .beta.-amyloid precursor proteins. (APPs) of which
there are several alternatively spliced variants. The most abundant
forms of the APPs include proteins consisting of 696, 751 and 770
amino acids (Tanzi et al. Nature 31:528-530, 19980.
[0006] The small A.beta. peptide is a major component that makes up
the amyloid deposits or "plaques" in the brains of patients with
Alzheimer's disease. In addition, Alzheimer's disease is
characterized by the presence of numerous neurofibrillary
"tangles", consisting of paired helical filaments which abnormally
accumulate in the neuronal cytoplasm (Grunddke-Iqbal et al, Proc.
Natl. Acad. Sci. USA. 83:4913-4917, 1986; Kosik et al, Proc. Natl.
Acad. Sci. USA 83:4044-4048, 1986; Lee et al, Science 251:675-678,
1991). The pathological hallmark of Alzheimer's disease is
therefore the presence of both "plaques" and "tangles", with
amyloid being deposited in the central core of the plaques. The
other major type of lesion found in the Alzheimer's disease brain
is the accumulation of amyloid in the walls of blood vessels, both
within the brain parenchyma and in the walls of meningeal vessels
that lie outside the brain. The amyloid deposits localized to the
walls of blood vessels are referred to as cerebrovascular amyloid
or congophilic angiopathy (Mandybur, J. Neuropath. Exp. Neurol.
45:79-90, 1986; Pardridge et al., J. Neurochem, 49:1394-1401,
1987).
[0007] For many years there has been an ongoing scientific debate
as to the importance of "amyloid" in Alzheimer's disease, and
whether the "plaques" and "tangles" characteristic of this disease
were a cause or merely a consequence of the disease. Within the
last few years, studies now indicate that amyloid is indeed a
causative factor for Alzheimer's disease and should not be regarded
as merely an innocent bystander. The Alzheimer's A.beta. protein in
cell culture has been shown to cause degeneration of nerve cells
within short periods of time (Pike et al, Br. Res. 563:311-314,
1991; J. Neurochem. 64:253-265, 1995). Studies show that it is the
fibrillary structure (consisting of a predominant .beta.-pleated
sheet secondary structure) characteristic of all amyloids that is
responsible for the neurotoxic effects, A.beta. has also been found
to be neurotoxic in slice cultures of hippocampus (Harrigan et al,
Neurobiol. Aging 16:779-789, 1995) and to induce nerve cell death
in transgenic mice (Games et. al, Nature 373:523-527, 1995; Hsiao
et al, Science 272:99-102, 1996). Injection of the Alzheimer's
A.beta. into rat brain also causes memory impairment and neuronal
dysfunction (Flood et al, Proc. Natl. Acad. Sci. USA 88:3363-3366,
1991; Br. Res. 663:271-276, 1994).
[0008] Probably, the most convincing evidence that A.beta. amyloid
is directly involved in the pathogenesis of Alzheimer's disease
comes from genetic studies. It was discovered that the production
of A.beta. can result from mutations in the gene encoding its
precursor, .beta.-amyloid precursor protein (Van Broeckhoven et al,
Science 248:1120-1122, 1990; Murrell et al, Science 254:97-99,
1991; Haass et al, Nature Med. 1:1291-1296, 1995). The
identification of mutations in the beta-amyloid precursor protein
gene that cause early onset familial Alzheimer's disease is the
strongest argument that amyloid is central to the pathogenic
process underlying this diseases. Several reported disease-causing
mutations have been discovered which demonstrate the importance of
A.beta. in causing familial Alzheimer's disease (reviewed in Hardy,
Nature Genet. 1:223-234, 1992). All of these studies suggest that
providing a therapy, drug or supplement to reduce, eliminate and/or
prevent fibrillary A.beta. formation, deposition, accumulation
and/or persistence in the brains of humans and animals, well serve
as an effective therapeutic.
The Accumulation of "Plaques and Tangles" in the Aging Human and
Animal Brain
[0009] The human brain is the most complex organ in the universe.
It weighs only 3 pounds, or about 2% of body weight. Yet is uses
20-30% of the calories consumed, 20% of the oxygen breathed, and
25% of the blood flow in the body; it consists of 85% water (Daniel
G. Amen, M.D. 12 prescriptions for creating a brain healthy life.
Source:
www.amenclinics.com/cybcyb/12-prescriptions-for-creating-a-brain-healthy--
life/). There are approximately 100 billion nerve cells (i.e.
neurons) in the brain, and up to a quadrillion connections called
synapses (ibid). The human brain as it ages, loses about 85,000
cortical neurons per day, or about one every second (Deepak Chopra,
M.D. and Rudolph Tanzi, Ph.D. Super Brain. Unleashing the Explosive
Power of Your Mind to Maximize Health, Happiness, and Spiritual
Well Being.
See///www.chopra.com/super-brain-by-deepak-chopra-rudolph-tanzi.)
As the brain ages, starting in the 20's there is a slow but
deliberate accumulation of two neurotoxic proteins. The first is
the brain accumulation of an insoluble (aggregated) specific
neurotoxic protein known as the "beta-amyloid protein" or A.beta..
Beta-amyloid protein deposits in the form of "plaques" (looking
like "meatballs" in the brain under a microscope), have been shown
to be instrumental in killing healthy neurons that lead to a
decline in hippocampus-dependent memory and cognition. Dr. Alan
Snow and co-inventors developed patented methods to produce
"plaques in a test-tube" (identical to what is seen in the human
brain) and used these methods to screen for and identify natural
"plaque-reducing" nutraceutical ingredients (U.S. Pat. No.
7,148,001, which is incorporated herein by reference in its
entirety).
[0010] The second neurotoxic protein that accumulates in the aging
brain is known as the "tau protein" and forms twisted paired
helical filaments known as "tangles." Neurofibrillary tangles
accumulate inside neurons that causes them to die, and look like
"dried spaghetti strands" under a microscope. Dr. Snow's
laboratories developed proprietary methods to form "tangles" in a
test-tube, and then used these assays to identify
"tangle-inhibiting" nutraceutical ingredients (see examples below).
Thus, in the aging brain, both "plaques and tangles" accumulate,
causing neurons to die; connections between nerve cells (called
synapses) to disintegrate; and memory and cognition to
progressively decline. Compounds or agents able to disaggregate and
reduce the accumulation of "plaques and tangles" have been shown to
lead to memory improvement and a reduction in memory decline
(Karlnoski et al. Suppression of amyloid deposition leads to
long-term reductions in Alzheimer's pathologies in Tg2576mice. J.
Neurosc. 29:4964-4971, 2009; Vellas et al. Long-term follow-up of
patients immunized with AN1792: Reduced functional decline in
antibody responders. Current Alz. Res. 6:144-151, 2009; Morgan et
al, A.beta. peptide vaccination prevents memory loss in an animal
model of Alzheimer's disease. Nature 408:982-985, 2000; Chen et al.
A. learning deficit related to age and .beta.-amyloid plaques in a
mouse model of Alzheimer's disease. Nature 408:975-979, 2000; Janus
et al. A.beta. peptide immunization reduces behavioral impairment
and plaques in a model of Alzheimer's disease. Nature 408:979-985,
2000; Schenk et al. Immunization with amyloid-.beta. attenuates
Alzheimer-disease like pathology in PDAPP mouse. Nature
400:173-177, 1999; Yanamandra et al. Anti-tau antibodies that block
tau aggregate seeding in vitro markedly decreases pathology and
improves cognition in vivo. Neuron 80:402-414, 2013; Dumont et al.
Bezafibrate administration improves behavioral deficits and tau
pathology in P3015 mice. Human Molecular Genetics 21:5091-5105,
2012; Oddo et al. Reduction of soluble Abeta and tau, but not
soluble Abeta alone, ameliorates cognitive decline in transgenic
mice with plaques and tangles. J. Biol. Chem. 281:39413-39423,
2006; Santacruz et al. Tau suppression in a neurodegenerative mouse
model improves memory function. Science 309:476-481, 2005.)
[0011] The only difference between an aging brain that could lead
to age-associated memory impairment (AAMI), then to mild-cognitive
impairment (MCI), and potentially to Alzheimer's disease, and a
brain that does not, is the number of "plaques and tangles" in the
brain. Alzheimer's diseased brains are loaded with tens to hundreds
of thousands of "plaques and tangles," per square millimeter,
causing a marked increase in the death of neurons, leading to a
loss of synapses (connections between neurons), and concurrent
memory loss and cognitive decline.
[0012] Therefore, beta-amyloid and tau are two key proteins in the
aging brain that accumulate as insoluble "plaques and tangles" that
have been shown to be directly linked to memory loss and cognitive
decline. There is currently no pharmaceutical drug that has been
approved for reducing and removing both beta-amyloid protein
"plaques" and tau protein-containing "tangles" in the brain.
The Accumulation of "Plaques" in the Aging Dog and Cat Brain
[0013] Dogs and cats also accumulate "plaques" (and, to a lesser
extent, "tangles") in their brains as they age that are believed to
contribute to memory decline and cognitive impairment. The same
beta-amyloid protein (i.e. "plaques") and tau protein ("tangles")
that accumulate in the human brain also accumulate in aged dogs
(Papoiannou et al, Immunohistochemical investigation of the brain
of aged dogs. I. Detection of neurofibrillary tangles and of
4-hydroxynonenal protein, an oxidative damage product, in senile
plaques. Amyloid 8:11-21, 2001; Uchida et al, Amyloid angiopathy
with cerebral hemorrhage and senile plaque in aged dogs. Nihon
Juigaku Zasshi 52: 605-11, 1990) and cats (Gunn-Moore et al,
Cognitive dysfunction and the neurobiology of ageing in cats. J
Small Anim. Pract. 48: 546-53, 2007; Nakamura et al. Senile plaques
in very aged cats. Acta Neuropath. 91:437-9, 1996).
[0014] Canine Cognitive Dysfunction (CCD) (also known as Cognitive
Dysfunction Syndrome or CDS) is a disease prevalent in dogs (and
cats) that exhibit symptoms of dementia or Alzheimer's disease as
seen in humans. CCD creates pathological changes in the brain that
slow the mental functioning of dogs (and cats) resulting in loss of
memory, motor function and learned behaviors from training early in
life. In the dog's and cat's brain, the culprit is again is the
beta-amyloid protein or A.beta. that forms "plaques" in the brain.
As the dog ages, more and more "plaques" accumulate and nerve cells
die. Although the initial symptoms of the disorders are mild, they
gradually worsen over time, also known as "cognitive decline".
Amyloid "plaques" occur in aged dogs at about five to seven years
of age, and in cats of about ten years of age (which is
proportional to their average lifespan of 15-20 years). In fact,
clinical signs of cognitive dysfunction syndrome are found in 50%
of dogs over the age of 11, and by the age of 15, 68% of dogs
display at least one sign. Dogs will often find themselves confused
in familiar places of the home, spending long periods of time in
one area of the home, not responding to calls or commands, and
experiencing abnormal sleeping patterns.
[0015] Beta-amyloid protein containing "plaques" also have been
identified in the brains of other higher mammals including monkeys,
bears, camels, and horses, (Nakamura et al, Histopathological
studies of senile plaques and cerebral amyloidosis in cynomolgus
monkeys. J Med Primatol. 27:244-52, 1998; Capucchio et al studies.
J Comp Pathol 142:61-73, 2010; Nakamura et al, Senile plaques in an
aged two-humped (Bactrian) camel (Camelus bactrianus), Acta
Neuropathol 90: 415-8, 1995; Uchida et al. Senile plaques and other
senile changes in the brain of an aged American black bear, Vet.
Pathol. 32:412-4, 1995).
Tauopathies and "Tangles"
[0016] Tau was discovered in the mid-1970s as a microtubule
associated protein (Weingarten, 1975). Besides being a stabilizer
of microtubules in neurons and other cells, it has since been found
to play important roles in cell differentiation, polarization and
axonal transport. Normal tau is a soluble protein bound to
microtubules, but in a series of neurodegenerative diseases, now
known as tauopathies, tau accumulates as a pathogenic insoluble,
fibrillar protein. These tau inclusions appear to modulate the
severity of dementia and clinical features of these
neurodegenerative diseases. Tauopathies include diseases such as
Alzheimer's disease. frontotemporal lobar degeneration with tau
inclusions (FTLD-tau) such as Pick's disease, progressive
supranuclear palsy, and corticobasal degeneration, agyropbillic
grain disease, some prion diseases, amyotrophic lateral
sclerosis/parkinsonism-dementia complex, chrome traumatic
encephalopathy, and some genetic forms of Parkinson's disease (V.M.
Lee et al., Ann. Rev. Neurosci. 24:1121-1159, 2001; B. Omalu et
al., Neurosurgery 69(1): 173-83, 2011; A. Rajput et al., Neurology
67:1506-1508, 2006; G. Santpere and I. Ferrer, Acta Neuropathol.
117: 227-246, 2009).
[0017] One of the most notable effects of increasing fibrillar tau
in the brain is the gradual deterioration of short term memory;
that is, the ability to recall immediately those memories only
recently stored (P. Giannakopoulos et al., Neurology 60:1495-1500,
2003). As there is no treatment for these disorders, it is
important to find a novel invention that could target this
pathogenic protein and improve memory deficits.
"Tangles" Accumulate in Brain in Traumatic Brain Injury (TBI),
Concussions, Head Trauma and Chronic Traumatic Encephalopathy
(CTE)
[0018] Brain "tangles" consisting of tau protein also accumulate
progressively in the brain following blows to the head and include
concussions, head injury, post-traumatic stress disorder (PTSD),
and blast-induced traumatic brain injury (seen in soldiers and
military personnel who have traumatic head injuries induced by a
single blast). The movie "concussion" and the NFL Players
Association all discuss the dementia-type behavior that has been
seen in athletes following repealed concussions and/or blows to the
head (known as traumatic brain injury or TBI). Loss of
consciousness is a clinical hallmark of concussion but is not
required to make the diagnosis. Other symptoms include confusion,
disorientation, unsteadiness, dizziness, headache, and visual
disturbances.
[0019] The long-term consequences to traumatic brain injury is
referred to as Chronic Traumatic Encephalopathy (CTE), which is
form of tauopathy (i.e. tau protein containing "tangles" in the
brain). CTE is a progressive degenerative disease found in people
who have suffered repeated brain trauma including sub-concussive
hits to the head that do not cause immediate symptoms. The disease
was previously called dementia pugilistica (DP), i.e. "punch-drunk"
as it was initially found in those with a history of boxing. CTE
has now been found in the brains of professional athletes including
NFL athletes who play football, athletes prone to head injury
including those that play ice hockey, rugby, skiing, skateboarding,
stunt performing, bull riding, rodeo, and all other contact sports
where participants experience repeated brain trauma. Individuals
with CTE show many signs of dementia such as memory loss,
aggression, confusion, and depression, which may appear years or
many decades after the trauma. In September 2015, researchers with
the Department of Veterans Affairs and Boston University announced
they had identified CTE in 96% of NFL football players that they
had examined and in 79% of all football players (Jason Breslow,
New; 87 deceased NFL players test positive for brain disease,
Frontline Jan. 9, 2016).
[0020] The neuropathology under a microscope is clear--there is
primarily an accumulation of "tangles" that consist of tau protein,
similar to the "tangles" seen in the brains of Alzheimer's disease
patients. There is also some beta-amyloid protein deposition (i.e.
"plaques"), but this is usually uncommon and less of a feature then
the "tangle" accumulation in brain. These findings suggest that
blows to the head can lead to near immediate brain "tangle"
accumulation that then leads to dementia-like symptoms including
memory loss and cognitive decline. Identification of a
nutraceutieal that can help in the reduction and/or clearance of
brain "tangles" would be an extraordinary supplement to take every
day by all kinds of athletes, NFL players, the military and its
soldiers.
DISCLOSURES
[0021] An object of the present invention is to provide the
combination of plant extracts from Uncaria tomentosa and an oolong
tea extract for the development of a cognition and memory
supplement, to prevent, reduce and/or clear brain "plaques and
tangles" in humans. This invention is predicated on the surprising
discovery that various plant extracts, even from the same plant
source have differing effectiveness for dissolving plaques and
tangles, and that oolong tea extract, in particular LOTE, in
combination with Uncaria tomentosa extract has a surprising and
unexpected activity compared other plant extracts or with each
extract alone.
[0022] Another object of the present invention is to provide the
combination of plant extracts from Uncaria tomentosa and an oolong
tea extract to produce a pet food supplement for aged dogs that
reduce brain amyloid "plaques" and improve cognition, memory,
short-term memory, focus and concentration.
[0023] Another object of the present invention is to provide the
combination of plant extracts from Uncaria tomentosa and an oolong
tea extract to produce a pet food supplement for aged cats that
reduce brain amyloid "plaques" and improve cognition, memory,
short-term memory, focus and concentration.
[0024] Although some health care providers have suggested that
Uncaria tomentosa may be used to treat a variety of ailments,
nowhere has there been any use, or suggestion of use, of this
compound for the treatment of tau fibril formation, deposition,
accumulation and/or persistence, such as that which occurs in the
tauopathies. Furthermore, nowhere is it suggested that certain
other compounds might have synergistic or supplemental efficacy in
combination with Uncaria tomentosa in treating tauopathies to
prevent and/or treat, for example, traumatic brain injury (TBI),
concussions (as observed in most athletes and the
military/soldiers), post-traumatic stress disorders, head trauma,
blows to the head (either single or repeated), and chronic
traumatic encephalopathy (CTE).
[0025] Another object of the present invention is to provide the
combination of plant extracts from Uncaria tomentosa and an oolong
tea extract to produce an effective preventer and/or reducer of
brain "tangles," such as those found in people with traumatic brain
injury (TBI), concussions (as observed in most athletes and the
military/soldiers), post-traumatic stress disorders, head trauma,
blows to the head (either single or repeated), and chronic
traumatic encephalopathy (CTE).
[0026] The present invention clearly demonstrates the effectiveness
of plant extracts of Uncaria tomentosa (cat's claw) in specific
combination with an Oolong tea plant extract for 1) inhibition of
tau fibril formation (important for patients in early- to mid-stage
tauopathy), 2) inhibition of tau fibril growth (important for
patients in early- to mid-stage tauopathy), and 3) causing the
dissolution/disruption of preformed tau fibrils (important for
late-stage tauopathy).
[0027] An object of the present invention is to use the inner bark
and/or roots from Uncaria tomentosa (also referred to as Una de
Gato or Cat's claw) for the treatment/inhibition of tau deposition,
accumulation and/or persistence in tauopathies, in conjunction with
an oolong tea extract, disclosed below, to achieve a beneficial
therapeutic effect. Uncaria tomentosa or Cat's claw is also
referred to as, but not limited to, Paraguayo, Garabato, Garbato
casha, Tambor huasca, Una de gavilan, Hawk's claw, Nail of Cat, and
Nail of Cat Schuler.
[0028] Another object of the present invention is to provide the
use of Uncaria tomentosa with an oolong tea extract (regardless of
commercial source and regardless of final form for consumption by
humans, i.e. pills, tablets, caplets, soft and hard gelatin
capsules, lozenges, sachets, cachets, vegicaps, liquid drops,
elixirs, suspensions, emulsions, solutions, syrups, tea bags,
aerosols (as a solid or in a liquid medium), suppositories, sterile
injectable solutions, sterile packaged powders, bark bundles and/or
bark powder) for inhibition of tau fibril formation, deposition,
accumulation, and/or persistence, regardless of its clinical
setting.
[0029] These and such other objects of the invention as will become
evident from the disclosure below are met by the invention
disclosed herein.
APPLICATIONS
[0030] Application of the invention provides for use of plant
extracts from Uncaria tomentosa and a specific oolong tea extract
to benefits aged dogs and cats, who develop brain "plaques" (and to
a lesser extent, "tangles"), and have dementia, as defined in
Canine Cognitive Dysfunction (CCD).
[0031] We have earlier discovered and disclosed a naturally
occurring plant product, the inner bark and/or roots from the plant
Uncaria tomentosa, or Cat's Claw, that we call PTI-00703.RTM., in
WIPO International publication number W098/51302 entitled
`Composition and Methods for Treating Alzheimer's Disease and other
Amyloidoses` dated Nov. 19, 1998, which is incorporated herein by
reference in its entirety. As disclosed therein, this plant
compound alone has surprising efficacy in disrupting and/or
dissolving amyloid deposits and other accumulations, and is
believed to be a potent inhibitor of amyloid formation in
Alzheimer's disease. Type II Diabetes, and other amyloidosis.
[0032] However, our data now demonstrates that the formulation of
PTI-00703.RTM. with a specific oolong tea extract (referred to as
`LOTE`) disclosed herein has surprising and hitherto unsuspected
potent efficacy in treating Tauopathies and diseases in which
"tangles" are found in the brain (such as in TBI, CTE in humans
with concussions, head injury and/or head, trauma), and diseases
that contain an overabundance of "plaques in brain" such as in aged
dogs and cats (in Canine Cognitive Dysfunction).
[0033] The invention relates to the use of mixed compositions
including one or more of the following: oolong tea extract and
Uncaria tomentosa extract PTI-00703.RTM. referred to as `703` or
`PTI-703` for the therapeutic intervention of amyloidosis. Use of a
mixed composition of an oolong tea extract, and/or PTI-00703.RTM.,
contained within different commercial preparations show unexpected
effects on the inhibition of amyloid fibril formation and
disaggregation of amyloid fibrils.
[0034] Application of the invention to these needs is especially
beneficial in that the invention is the only system that
effectively provides for use of extracts from the inner bark and
root parts of Uncaria tomentosa, together with an oolong tea
extract, to benefit human patients with Tauopathies, amyloid
diseases, "plaques and/or tangles" in the brain, and those with
traumatic brain injury (TBI), concussions (as observed in most
athletes and the military/soldiers), post-traumatic stress
disorders, head trauma, blows to the head (either single or
repeated), and chronic traumatic encephalopathy (CTE).
[0035] Application of the invention to these needs is especially
beneficial in that the invention is the only system that
effectively provides for use of extracts from the inner bark and
root parts of Uncaria tomentosa, together with an Oolong tea
extract, to benefit human patients with tauopathies, due to the
newly discovered ability of Uncaria tomentosa in combination with
an oolong tea extract, to effectively inhibit tau fibril formation,
inhibit tau fibril growth, inhibit fibrillar tau-proteoglycan
interactions, fibrillar tau-glycosaminoglycan interactions, and
cause dissolution and/or disruption of preformed tau fibrils.
[0036] Over 25 extracts of teas, along with extracts of coffee and
yerba mate, were screened for amyloid fibril aggregation inhibition
and disaggregation in vitro using a variety of assays. One lead
oolong tea extract was selected and tested in combination with
PTI-00703.RTM.. It was found that the combination of this specific
lead oolong tea extract (LOTE) and PTI-00703.RTM. inhibited amyloid
fibril formation more than the tea extract or PTI-00703.RTM. alone,
and the combination of LOTE and PTI-00703.RTM. also had the ability
to rapidly disaggregate (within 15 minutes) preformed amyloid
fibrils.
[0037] The invention also relates to the use of mixed compositions
including one or more of the following: oolong tea extract and
Uncaria tomentosa extract PTI-00703 referred to as `703` or
`PTI-703` for the therapeutic intervention of tauopathies. Use of a
mixed composition of oolong tea extract, and/or PTI-00703,
contained within different commercial preparations show unexpected
effects on the inhibition of tau fibril formation and
disaggregation of tau fibrils.
[0038] Over 25 extracts of teas along with extracts of coffee and
yerba mate were screened for tau fibril aggregation inhibition and
disaggregation in vitro. One lead oolong tea extract was selected
and tested in combination with PTI-00703.RTM.. It was found that
the combination of this specific lead oolong tea extract (LOTE) and
PTI-00703.RTM. inhibited tau fibril formation more than the tea
extract or PTI-00703.RTM. alone, and also has the ability to
rapidly disaggregate preformed tau fibrils.
[0039] It was also found that the combination of this specific lead
oolong tea extract (LOTE) and PTI-00703.RTM. inhibited beta-amyloid
protein fibril formation more than the tea extract or
PTI-00703.RTM. alone, and also has the ability to rapidly
disaggregate preformed amyloid fibrils.
[0040] A preferred pharmacological agent preferably has a
therapeutically effective amount of Uncaria tomentosa in a dosage
in the range of from about 10 to about 1,000 mg/kg of body weight
of the patient, and more preferably in the range of from about 10
to about 100 mg/kg of body weight of the patient.
[0041] The compositions preferably have a therapeutically effective
amount of the mixed composition lead oolong tea extract (LOTE) and
PTI-00703.RTM., in a dosage in the range of from about 0.1 to about
500 mg/kg of body weight of the patient, and more preferably in the
range from about 1.0 to about 100 mg/kg of body weight of the
patient.
[0042] Preferred pharmaceutical agents may also have a
pharmaceutically acceptable carrier, diluent or excipient. The
pharmaceutical agent preferably has a fibril inhibitory activity or
efficacy greater than 50%.
[0043] The plant matter is preferably comprised of commercially
obtained pills, tablets, caplets, soft and hard gelatin capsules,
lozenges, sachets, cachets, vegicaps, liquid drops, elixirs,
suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a
solid or in a liquid medium), suppositories, sterile injectable
solutions, sterile packaged powders, bark bundles and/or bark
powder, which contain Uncaria tomentosa, extracts or derivatives
thereof (and may be taken from commercially available
gelatin-coated capsules which contain dried powder of Uncaria
tomentosa, extracts or derivatives thereof), in combination with a
specific oolong tea extract.
[0044] An object of the present invention is to use the inner bark
and/or roots from Uncaria tomentosa (also referred to as Una de
Gato or Cat's claw) for the treatment/inhibition of amyloid
deposition, accumulation and/or persistence in amyloidosis, in
conjunction with an oolong tea extract, disclosed below, to achieve
a beneficial therapeutic effect. Uncaria tomentosa or cat's claw is
also referred to as, but not limited to, Paraguiayo, Garabato,
Garbato casha, Tambor huasca, Una de gavilan, Hawk's claw, Nail of
Cat, and Nail of Cat Schuler.
[0045] Another object of the present invention is to provide the
use of Uncaria tomentosa with an oolong tea extract (regardless of
commercial source and regardless of final form for consumption by
humans, i.e. pills, tablets, caplets, soft and hard gelatin
capsules, lozenges, sachets, cachets, vegicaps, liquid drops,
elixirs suspensions, emulsions, solutions, syrups, tea bags,
beverage preparations, aerosals, as a solid or in a liquid medium,
suppositories, sterile injectable solutions, sterile packaged
powders, bark bundles and/or bark powder, for inhibition of amyloid
fibril formation, deposition, accumulation and/or persistence,
regardless of its clinical setting.
[0046] Application of the invention to these needs is especially
beneficial in that the invention is the only system that
effectively provides the use of extracts from the inner bark and
root parts of Uncaria tomentosa, together with a specific Oolong
tea extract, to benefit human patients with amyloidosis, and aged
mammals (such as dogs and cats that develop amyloid "plaques" in
the brain as they age), due to the newly discovered ability of
Uncaria tomentosa in combination with an Oolong tea extract, to
most remarkably and effectively inhibit amyloid fibril formation,
inhibit amyloid fibril growth, and cause dissolution and/or
disruption of preformed amyloid fibrils.
[0047] A method is also disclosed for treating tauopathy in a
patient, comprising the step of administering to the patient a
therapeutically effective amount of plant matter from a plant of
the genus Uncaria, species tomentosa, in combination with an Oolong
tea extract. The plant matter is preferably administered orally
such as in oral capsules, beverage preparations, or any other
method, or by aerosol spray or in a parenterally injectable or
infusible form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] This paragraph intentionally left blank.
[0049] FIG. 1 A graph of A.beta. 1-42 aggregation measured by
Thioflavin T fluorometry and Congo red binding after incubation
with lead Oolong tea extract (LOTE) and PTI-00703.RTM.
[0050] FIG. 2A-C Representative images of Congo red, Thioflavin S,
and electron micrographs of A.beta. fibrils +/- LOTE and
PTI-00703.RTM.
[0051] FIG. 3 A graph of A.beta. 1-40 aggregation measured by
Thioflavin T fluorometry and Congo red binding after incubation
with LOTE and PTI-00703.RTM.
[0052] FIG. 4 Circular dichroism spectra of A.beta. 1-40 after
treatment with increasing concentrations of LOTE and
PTI-00703.RTM.
[0053] FIG. 5A-G Data from various internal tests to characterize
TauRD fibrils for screening of tau aggregation inhibitors.
[0054] FIG. 6 A graph of tau aggregation measured by Thioflavin S
fluorometry after incubation with tea extracts.
[0055] FIG. 7 A graph of tau aggregation measured by Thioflavin S
fluorometry after incubation with LOTE and PTI-00703.
[0056] FIG. 8 A graph of tau secondary structure measured by
circular dichroism spectroscopy after treatment with LOTE.
[0057] FIG. 9 Electron micrographs of tau fibril formation with
PTI-0073 and LOTE.
[0058] FIG. 10 Electron micrographs of preformed tau fibrils
treated with PTI-00703 and LOTE.
DETAILED DESCRIPTION
[0059] A "therapeutically effective amount" in general means the
amount that, when administered to a subject or animal for treating
a disease, is sufficient to affect the desired degree of treatment
for the disease. A "therapeutically effective amount" or a
"therapeutically effective dosage" preferably inhibits, reduces,
disrupts, disassembles tau fibril formation, deposition,
accumulation and/or persistence, or treats a disease associated
with these conditions, such as a tauopathy, by at least 20%, more
preferably by at least 40%, even more preferably by at least 60%,
and still more preferably by at least 80%, relative to an untreated
subject. Effective amounts of a compound of this invention or
composition thereof for treatment of a mammalian subject are about
0.1 to about 1000 mg/Kg of body weight of the subject/day, such as
from about 1 to about 100 mg/Kg/day, especially from about 10 to
about 100 mg/Kg/day. A broad range of disclosed composition dosages
are believed to be both safe and effective.
[0060] "Amyloid diseases" or "amyloidosis" suitable for treatment
with the compositions of this inventions are diseases associated
with the formation, deposition, accumulation, and/or persistence of
amyloid fibrils, especi ally the fibrils of an amyloid protein
selected from the group consisting of beta-amyloid protein or
A.beta., AA amyloid, AL amyloid, IAPP amyloid, PrP amyloid,
.alpha.2-microglobilin amyloid, transthyretin, prealbumin, and
procalcitonin, especially A.beta. and IAPP amyloid, Suitable such
diseases include Alzheimer's disease, Down's syndrome, Mild
cognitive impairment (MCI), Cognitive Canine Dysfunction (CDD),
traumatic brain injury (TBI), chronic traumatic encephalopathy
(CTE), concussions, hear trauma, single- and multiple blows to the
head, post-traumatic stress disorders, dementia pugilistica,
multiple system atrophy, inclusion body myositosis, hereditary
cerebral hemorrhage with amyloidosis of the Dutch type,
Niewman-Pick disease type C, cerebral .beta.-amyloid angiopathy,
dementia associated with cortical basal degeneration, the
amyloidosis of type 2 diabetes, the amyloidosis of chronic
inflammation, the amyloidosis of malignancy and Familial
Mediterranean Fever, the amyloidosis of multiple myeloma and B-cell
dyscrasias, the amyloidosis of prion diseases, Creutzfeldt-Jakob
disease, Gertsmann-Straussler syndrome, kuru, scrapie, the
amyloidosis associated with carpal tunnel syndrome, senile cardiac
amyloidosis, familial amyloidotic polyneuropathy, and the
amyloidosis associated with endocrine tumors.
[0061] "Fibrillogenesis" refers to the formation, deposition,
accumulation and/or persistence of tau fibrils, filaments,
inclusions, deposits, inclusions, or the like.
[0062] "Inhibition of fibrillogenesis" refers to the inhibition of
formation, deposition, accumulation and/or persistence of such
amyloid "plaque" or tau "tangle" fibril-like deposits.
[0063] "Disruption of fibrils or fibrillogenesis" refers to the
disruption of preformed beta-amyloid or tau fibrils that usually
exist in a pre-dominant .beta.-sheet, secondary structure. Such
disruption by compounds of the invention may involve marked
reduction or disassembly of beta-amyloid or tau fibrils as assessed
by various methods such as circular dichroism spectroscopy,
Thioflavin S fluorometry, SDS-PAGE/Western blotting, or negative
stain electron microscopy, as demonstrated by the Examples
presented in this application.
[0064] "Mammal" includes both humans and non-human mammals, such as
companion animals (dogs, cats and the like), laboratory animals
(such as mice, rats, guinea pigs, and the like) and farm animals
(cattle, horses, sheep, goats, swine, and the like).
[0065] "Plaques" refer to the meatball looking "amyloid deposits"
consisting of beta-amyloid protein or A.beta. found in various
regions of brain (including hippocampus, cerebral cortex, frontal
cortex etc. ) that is a pathological hallmark of brain aging,
mild-cognitive impairment (MCI), Alzheimer's disease, and is found
in aging mammals such as dogs, cats (referred to as Cognitive
Canine Dysfunction or CDD), monkeys, polar bears, horses and the
like. Accumulation of amyloid "plaques" in brain is believed to
lead to neurodegeneration, loss of synapses and connections between
neurons, cognitive decline, memory decline and loss, and loss of
focus and concentration.
[0066] "Tangles" refer to the "dried-up" spaghetti looking
"tangle-deposits" consisting of tau protein found in various
regions of brain (including hippocampus, cerebral cortex, frontal
cortex etc.) that is a pathological hallmark of brain aging,
mild-cognitive impairment, Alzheimer's disease, concussions,
traumatic brain injury (TBI), single and repeated blows to the
head, post-traumatic stress disorders, chronic traumatic
encephalopathy (CTE) and the like.
[0067] "Tauopathies" suitable for treatment with the compounds of
this invention are also diseases associated with the formation,
deposition, accumulation, or persistence of tau fibrils. Suitable
diseases include Alzheimer's disease, frontotemporal lobar
degeneration with tau inclusions (FTLD-lau) such as Pick's disease,
progressive supranuclear palsy, and corticobasal degeneration,
agyrophillic grain disease, some prion diseases, amyotrophic
lateral sclerosis/parkinsonism-dementia complex of Guam (also
called Lytico Bodig disease), dementia pugilistica, chronic
traumatic encephalopathy, Parkinson's disease and particularly some
genetic forms of Parkinson's disease, tangle-predominant dementia
(with neurofibrillary tangles similar to Alzheimer's disease, but
without amyloid plaques).
[0068] Tau fibrils is a generic term referring to a group of
diverse, but specific intracellular or extracellular protein
deposits which all have common morphological properties, staining
characteristics, and x-ray diffraction spectra.
[0069] "Treating" or "treatment" of a disease includes preventing
the disease from occurring in a mammal that may be predisposed to
the disease but does not yet experience or exhibit symptoms of the
disease (prophylactic treatment). Treatment can also mean
inhibiting the disease (slowing or arresting its development),
providing relief from the symptoms or side-effects of the disease
(including palliative treatment), and relieving the disease
(causing regression of the disease), such as by disruption of
preformed tau fibrils. Treatment does not need be absolute. One
such preventive treatment may be use of the disclosed compounds for
the treatment of Mild Cognitive impairment (MCI).
[0070] The plant Uncaria tomentosa, also known as "Una de Gato" (in
Spanish) or "Cat's claw" (in English) refers to a woody vine which
grows within the Peruvian Amazon rain forest. This slow-growing
vine takes 20 years to reach maturity, and can grow over 100 feet
in length as it attaches and wraps itself around the native trees.
It is found abundantly in the foothills, at elevations of two to
eight thousand feet. The vine is referred to as "cat's claw"
because of its distinctive curved claw-like thorns that prefect
from the base of its leaves. Uncaria tomentosa is expected to have
immune-supporting, anti-inflammatory, anti-viral, anti-mutagemic
and antioxidant properties. The anti-inflammatory properties, for
example, are expected to be beneficial for the treatment of
arthritis, rheumatism, bursitis and gout. Without being bound by
theory, it is believed that is beneficial effects in treating
arthritis pain can be due, in part, to its ability to cleanse the
digestive tract and aid in removing toxins from the body.
Furthermore, Uncaria tomentosa or cat's claw is expected to
alleviate pain, and is expected to be helpful in reducing pain
associated with, for example, chemotherapy, radiation treatment and
AZT use.
[0071] Uncaria tomentosa or cat's claw is also expected to be
useful in stopping viral infections in stopping viral infections in
the early stages, fighting opportunistic infections in AIDs
patients and decreasing the viable size of some skin tumors and
cysts. Uncaria tomentosa can also be used to treat a variety of
ailments, including cancer, AIDs, Crohn's disease, respiratory
infections, allergies, herpes, prostrate problems, lupus, Epstein
Barr virus, chronic fatigue syndrome, and a variety of stomach and
bowl disorders.
[0072] For additional and further information and background on
Uncaria tomentosa, the reader is also referred to the inventor's
WIPO International publication number W098/51302, which is
incorporated herein by reference in its entirety.
Further Aspects and Utilizations of the Invention
[0073] Another embodiment of the present invention is to formulate,
prior to administration in a patient, a pharmaceutical blend
comprising Uncaria tomentosa and an oolong tea extract in one or
more pharmaceutical acceptable carriers, diluents or excipents.
[0074] In another embodiment Uncaria tomentosa obtained
commercially in any form could be further modulated using suitable
carroers, excipients and diluents including lactose, dextrose,
sucrose, sorbitol, mannitol, starches, gum acacia, calcium
phosphate, alginates, tragacanth, gelatin, calcium silicate,
microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water
syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc,
magnesium stearate and mineral oil. The formulations can
additionally include lubricating agents, wetting agents,
emulsifying and suspending agents, preserving agents, sweetening
agents or flavoring agents. The compositions of the invention may
be formulated so as to provide quick, sustained or delayed response
of the active ingredient after administration to the patient. The
compositions are preferably formulated in a unit dosage form, each
dosage containing from about 1 to about 10,000 mg of Uncaria
tomentosa (or its active ingredients), more usually about 500 to
about 2,000 mg of Uncaria tomentosa (or its active
ingredients).
[0075] However, it will be understood that the therapeutic dosage
administered will be determined by the physician in the light of
the relevant circumstances including the clinical condition to be
treated, the organ or tissues affected or suspected to be affected
with tau fibril accumulation, and the chosen route of
administration. Therefore, the above dosage ranges are not intended
to limit the scope of the invention in any way.
[0076] The term "unit dosage form" refers to physically discrete
units suitable as unitary dosages for human subjects and other
mammals, each unit containing a predetermined quantity of active
material calculated to produce the desired therapeutic effect, in
association with a suitable pharmaceutical carrier.
[0077] Use of extracts from the inner bark and root parts of
Uncaria tomentosa, and its blends benefit human patients with
tauopathies due to the newly discovered ability of Uncaria
tomentosa in combination with an Oolong tea extract to inhibit tau
fibril formation, inhibit tau fibril growth, inhibit tau
fibril-proteoglycan interactions, inhibit tau
fibril-glycosaminoglycan interactions, and cause dissolution and/or
disruption of preformed tau fibrils.
Compositions and Administration
[0078] In general, isolated, and/or purified Uncaria tomentosa and
oolong tea plant extracts are administered in therapeutically
effective amounts by any of the usual modes known in the art,
either singly or in combination with at least one of the extracts
of the disclosure. Administration will be by one of the following
routes: oral, topical, and synthetic (e.g. transdermal, intranasal
or by suppository), or parenteral (e.g. intramuscular,
subcutaneous, or intravenous injection). Compositions can take the
form of tablets, pills, capsules, semisolids, powders, sustained
release formulations, solutions, suspensions, elixers, aerosals, or
any other appropriate compositions, and comprise at least one
pharmaceutical acceptable excipient. Suitable excipients are well
known to persons of ordinary skill in the art, and they, and the
methods of formulating the compositions, can be found in standard
references as Alfonso A R: Remington's Pharmaceutical Sciences,
17.sup.th ed., Mack Publishing Company, Easton, Pa., 1985. Suitable
liquid carriers, especially for injectable solutions, include
water, aqueous saline solution, aqueous dextrose solution, and
glycols.
[0079] In particular, the compound(s) can be administered orally,
for example, as tablets, trouches, lozenges, aqueous or oily
suspensions, dispersible powders or granules, emulsions, hard or
soft capsules, syrups or elixers. In one embodiment, only one such
compound is administered in any particular dosage form.
Compositions intended for oral use can be prepared to any method
known in the art for the manufacture of nutraceutical compositions
and such compositions can contain one or more agents selected from
the group consisting of sweetening agents, flavoring agents,
coloring agents and preserving agents in order to provide
nutraceutically elegant and palatable preparations.
[0080] Tablets contain the plant extracts in admixture with
non-toxic pharmaceutically acceptable excipients that are suitable
for the manufacture of tablets. These excipients include, for
example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, maize starch or
alginic acid; binding agents, for example, maize starch, gelatin or
acacia; and lubricating agents, for example magnesium stearate or
stearic acid or talc. The tablets can be uncoated or they can be
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glycerol monostearate or glycerol distearate can be
employed. Formulations for oral use can also be prepared as hard
gelatin capsules wherein the compounds are mixed with an inert
solid diluent, for example, calcium carbonate, calcium phosphate or
kaolin, or as soft gelatin capsules, wherein the active ingredient
is mixed with water or an oil medium, for example, peanut oil,
liquid paraffin or olive oil.
[0081] Aqueous suspensions contain the compound in admixture with
excipients suitable for the manufacture of aqueous suspensions.
Such excipients include, for example, suspending agents, for
example, sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethyl cellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; and dispersing
and wetting agents that are naturally occurring phosphatides, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids; for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethyleneoxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids such as hexitol, for example polyoxyethylene sorbitol
monooleate, or condensation products of ethylene oxide with partial
esters from fatty acids and a hexitol annyhydride, for example
polyethylene sorbitan monooleate. The aqueous suspensions can also
contain one or more preservatives, for example ethyl or n-propyl
p-hydroxybenzoate, one or more coloring agents, one or more
flavoring agents, and/or one or more sweetening agents, such as
sucrose or saccharin.
[0082] Oily suspensions can be formulated by suspending the
extracts in a vegetable oil, for example arachs oil, olive oil,
sesame oil, or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions can contain a thickening agent for
example beeswax, hand paraffin or cetyl alcohol. Sweetening agents,
such as those set forth below, and flavoring agents can be added to
provide a palatable oral preparation. These compositions can be
preserved by the addition of an antioxidant such as ascorbic
acid.
[0083] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredients in admixture with a dispersing or wetting agent, a
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already described above. Additional excipients, for example
sweetening, flavoring and agents, can also be present.
[0084] The plant extracts can also be in the form of oil-in-water
emulsions. The oily phase of a vegetable oil, for example, olive
oil or arachis oils, or a mineral oil, for example liquid paraffin,
or mixtures thereof. Suitable emulsifying agents can be
naturally-occurring gums, for example gum acacia or gum tragacanth,
naturally occurring phosphatides, for example soy bean, lecithin,
and esters or partial esters derived from fatty acids and hexitol
anhydrides, for example sorbitan esters with ethylene oxide, for
example polyoxyethylene sorbitan monooleate, and condensation
products of the said partial esters with ethylene oxide, for
example polyethylene sorbitan monooleate. The emulsion can also
contain sweetening and flavoring agents. Syrups and elixers can be
formulated with sweetening agents, for example, glycerol, sorbitol
or sucrose. Such formulations can also contain a demulcent, a
preservative and flavoring and coloring agents.
[0085] The following non-limiting Examples are given by way of
illustration only and are not considered a limitation of this
disclosure, many apparent variations of which are possible without
departing from the spirit or scope thereof.
EXAMPLES
Composition Preparation
[0086] For these studies, commercially available tea leaves or
extracts were obtained from various vendors. To make tea extracts
from tea leaves, 2 g of tea leaves were extracted into 100.degree.
C. deionized water for 20 min with occasional mixing. Tea extracts
were filtered through a >10 .mu.m cut-off fitter to remove large
particulates. Extracts were snap frozen in dry ice/ethanol then
lyophilized to obtain a dried, concentrated powder. Dried powder
was weighed and resuspended in DMSO to make concentrated 100 mg/ml
stock solutions. Stock solutions were diluted into aggregation
reactions such that DMSO concentration was less than 0.28% in the
final reaction.
[0087] LOTE was prepared from oolong tea (AuNutra Industries Inc.,
Chino, Calif.). PTI-00703.RTM. is a powder extract made from water
extraction of the inner bark of Peruvian Uncaria tomentosa (RFI
Ingredients, Blauvelt, N.Y.). Methods for preparing PTI-00703.RTM.
have been described, for example in WIPO International Pub, No.
W098/51302. Ethanol extracts of oolong tea and/or Uncaria tomentosa
are within the scope of the current invention.
Example 1
Compositions of this Invention are Potent Disrupters/Inhibitors of
Alzheimer's A.beta. Fibrils or Aggregates
[0088] The compositions were found to be potent
disrupter/inhibitors of A.beta. protein fibrils or aggregates. In a
set of studies, the efficacy of the compositions to cause
disassembly/disruption/disaggregation of pre-formed amyloid fibrils
of Alzheimer s disease (i.e. consisting of A.beta. 1-42 or A.beta.
1-40) was analyzed.
Part A: Thioflavin T Fluorometry
[0089] In this study, Thioflavin T fluorometry was used to
determine the effects of the compositions. Thioflavin T
specifically binds to fibrillary amyloid, and this binding produces
a fluorescence enhancement at 485 nm that is directly proportional
to the amount of amyloid fibrils formed. The higher the
fluorescence, the greater the amount of amyloid fibrils formed
(Nakai et al., Lab. Invest, 65:104-110, 1991; Levine III, Protein
Sci. 2:404-410, 1993; Amyloid:Int. J. Exp. Clin. Invest. 2:1-6,
1995).
[0090] In this study, 40 .mu.l of a 1 mg/ml solution (in distilled
water) or pre-fibrillized human A.beta. 1-42 (rPeptide) was
incubated at 37.degree. C. for 3 days either alone (control), or in
the presence of LOTE+PTI-00703.RTM. (at test composition: A.beta.
weight ratios of 1:1 and 1:0.1) (referred to as "Cognitive
Clarity.TM."). The final concentration of A.beta. in the reaction
was 0.4 mg/ml (88 .mu.M) in phosphate-buffered saline (PBS), pH
7.4+0.02% sodium azide in 100 .mu.l final volume. Following 3-days
of co-incubation, 12.5 .mu.l of each incubation mixture was
transferred into a 96-well microtiter plate containing 37.5 .mu.l
of PBS and 200 .mu.l of a Thioflavin T solution (i.e. 125 .mu.M
Thioflavin T in 62.5 mM phosphate buffer, pH 6.8). The emission
fluorescence was read at 485 nm (444 nm excitation wavelength)
using an ELISA plate fluorometer after subtraction with buffer
alone or composition alone, as blank.
[0091] The results of the 3-day incubations are presented in FIG.
1. Incubation of A.beta. 1-42 with "Cognitive Clarity" (i.e.
combination of LOTE+PTI-00703.RTM.) caused a dose-dependent
disruption
[0092] disassembly/disaggregation of preformed A.beta. 1-42
fibrils. At a test composition. A.beta. weight ratio of 0.1:1
LOTE+PTI-00703 inhibited fibrils 62.8%. At equal weight equivalents
(test composition: A.beta. weight radio of 1:1) there was a 93.6%
inhibition of Thioflavin T fluorescence. This study indicated that
this unique combination is a potent disruptor/inhibitor of
Alzheimer's disease type A.beta. fibrils, and exerts its effect in
a dose-dependent manner.
Part B: Congo Red
[0093] In the Congo red binding assay the ability of a test
composition to alter .beta.-amyloid binding to Congo red is
quantified. In this assay, A.beta. 1-42 (as prepared for the
Thioflavin T assay) was incubated for 3 days either alone
(control), or with increasing amounts of test compositions and then
vacuum filtered through a 0.2 .mu.m filter. The amount of
A.beta.1-42 retained in the filter was then quantitated following
staining of the filter with Congo red (125 .mu.M Congo red, 100 mM
Tris, 50 mM NaCl, pH 7). After appropriate washing of the filter,
any lowering of the Congo red color on the filter in the presence
of the test composition (compared to the Congo red staining of the
amyloid protein in the absence of the test composition) was
indicative of the test composition's ability to diminish/alter the
amount of aggregated and congophilic A.beta..
[0094] In one study, the ability of A.beta. fibrils to bind Congo
red in the absence or presence of increasing amounts of
LOTE+PTI-007003.RTM. (at test composition: A.beta. weight ratios of
1:1, and 0.1:1) was determined. The results of 3-day incubations
are presented in FIG. 1 LOTE+PTI-00703.RTM. caused a dose-dependent
inhibition of A.beta. binding to Congo red. At a test
composition:A.beta. weight ratio of 0.1:1, LOTE+PTI-00703.RTM.
inhibited Congo red binding 20.9% (p<0.001). At equal weight
equivalents (test composition:A.beta. weight ratio of 1:1) there
was a 58.6% (p<0.001) inhibition of Congo red binding.
[0095] Similar to the results for the Congo red binding assay, this
study also indicated that this combination of a specific lead
oolong tea extract (LOTE) and PTI-00703.RTM. are potent
disrupters/inhibitors of A.beta. fibrils as assessed by a
Thioflavin T fluorometry assay, and exerted its effects in a
dose-dependent manner (FIG. 1). The combination of
LOTE+PTI-00703.RTM. caused a dose dependent reduction of Thioflavin
T binding (indicating disruption/reduction of A.beta. fibrils) by
70% (p<0.001) at a test composition: A.beta. weight ratio of
0.1:1 (FIG. 1), and by 95% at a test composition:A.beta. weight
ratio of 1:1 (FIG. 1).
Part C: Slide-Based Congo Red Binding, Thioflavin S and Electron
Microscopy
[0096] In the slide-based Congo red assay, Congo red dye is
incubated with A.beta.1-42, dotted on a slide and imaged under
polarized light. Amyloid fibrils bound by Congo red emits a
characteristic "apple-green birefringence" under polarized light.
In this study, pre-fibrillarized 0.4 mg/ml A.beta.1-42 (as prepared
for the Thioflavin T assay) with or without test compositions was
incubated for 3 days. 10 .mu.l Congo red solution (250 mg Congo red
dye (54% pure; Sigma) dissolved in 1 L dH20) was added to 10 .mu.l
of A.beta.1-42 -/+ test compositions and mixed by vortexing for 30
seconds. Samples were incubated 10 minutes at room temperature with
Congo red solution. Samples were then centrifuged at 2000 g for 3
minutes and 10 .mu.l of supernatant was removed. 2 .mu.l glycerol
was added to the pellet and mixed by pipetting up and down 15
times. Sample was vortexed then 10 .mu.l of stained protein was
dotted onto 18-well 5 MM HTC(R) autoclavable blue slides. Samples
were covered with small circular coverslips and then immediately
imaged under polarized light. Images were captured with a Zeiss
Axioscope 2 Plus microscope with HBO 100 illuminator equipped with
a Q-Imaging Retiga 1300 digital camera.
[0097] In FIG. 2A (left panel), a representative image shows that
untreated A.beta.1-42 has characteristic apple-green birefringence
and abundant fibrillar protein uniformly distributed across the
viewing field. In FIG. 2A (right panel) treatment with
LOTE+PTI-00703 (0.1:1 weight ratio with A.beta.42) resulted in
substantially less Congo red stained fibrils, demonstrating that
the plant extract combination can reduce and disaggregate/dissolve
pre-formed A.beta. 1-42 pre-formed fibrils.
[0098] Similar to Thioflavin T, Thioflavin S is a related anionic
dye that binds to fibrillar amyloid proteins and can be used to
detect fibrillar proteins bound to a glass microscope slide. In
this study, prefibrillarized A.beta.1-42 (as prepared for the
Thioflavin T assay) with or without test compositions was incubated
for 3 days. 4 .mu.l of 0.4 mg/ml A.beta. 1-42 -/+ test compositions
were dotted onto 18-well 5 MM HTC(R) autoclavable blue slides.
Samples were allowed to air dry for 2 h. 10 .mu.l of Thioflavin S
solution (31 mg of Thioflavin S dissolved in 50 mL dH20) was gently
applied to dried protein on slides. Protein was stained for 1
minute, then Thioflavin solution was removed by pipette. 40 .mu.l
of a 70% ethanol solution was gently pipetted onto stained protein
for 1 minute to rinse. This solution was gently removed by pipette.
2 .mu.l of Vectashield (Vector) mounting media was applied to the
stained protein and then covered with circular coverslips. Images
were viewed under fluorescent light and images captured with a
Zeiss Axioscope 2 Plus microscope with HBO 100 illuminator equipped
with a Q-Imaging Retiga 1300 digital camera.
[0099] In FIG. 2B (left panel), a representative image of untreated
A.beta. 1-42 shows abundant Thioflavin S fluorescent fibrils
uniformly distributed across the viewing field. Preformed A.beta.
1-42 fibrils incubated with LOTE+PTI-00703.RTM. (at 0.1:1 weight
ratio with A.beta.1-42) for 72 hr showed a substantial reduction in
fluorescent fibrils (FIG. 2B, right panel).
[0100] In FIG. 2B (right panel), representative images show that
with LOTE+PTI-00703 treatment, the Thioflavin S-fluorescent fibrils
disappeared.
[0101] Negative stain electron microscopy (EM) analysis was used to
independently monitor the effectiveness of different compositions
to disrupt pre-formed A.beta. fibrils. In these experiments,
pre-formed A.beta. 1-42 fibrils (as prepared for the Thioflavin T
assay) were incubated in the absence (control) or presence of
increasing concentrations of test compositions. After the 3-day
incubation, 10 .mu.l samples were spotted onto grids, stained with
2% uranyl acetate, and visualized at 8,000.times. to 30,000.times.
magnifications with a JEOL 1010 transmission electron
microscope.
[0102] In FIG. 2C (bottom panel), EM analysis confirmed formation
of A.beta. fibrils in the absence of treatment (i.e. FIG. 2C,
Control). Without treatment (2C, left panel), A.beta. formed large
clumped fibrils that uniformly covered the field. These samples
were also tested by the Thioflavin T assay and confirmed to be
Thioflavin T-fluorescence positive fibrils. In the presence of
LOTE+PTI-00703.RTM. (FIG. 2C, right panel), the number of clumped
A.beta. fibrils was significantly reduced and dissolved. These
results correlate well with the Thioflavin T fluorometry date that
showed a reduction in Thioflavin T fluorescence with treatment and
Congo red binding data that showed a reduction in Congo red binding
with treatment. Using these independent methodologies, we have
identified and validated the lead oolong tea extract (LOTE) in
combination with PTI-00703.RTM. (i.e. LOTE+PTI-00703.RTM.) as
potent A.beta. disrupters/inhibitors.
Example 2
Compositions of this Invention Directly Inhibit/Disrupt the In
Vitro Conversion of A.beta. to .beta.-Sheet Containing Fibril
Structures
Part A: Thioflavin T Fluorometry
[0103] To test whether LOTE+PTI-00703.RTM. can inhibit the
.beta.-sheet formation of A.beta., the same Thioflavin T assay as
described in Example 1 was utilized, but with A.beta. 1-40 as a
substrate instead. Similar to A.beta.1-42, A.beta. 1-40 forms
Thioflavin T positive aggregates but requires >24 h incubation
at 37.degree. C. shaking to become fully fibrillarized. Since
A.beta. 1-40 is in a non-fibrillar state at the start of the assay,
this protein can be aggregated in the presence of compositions to
measure aggregation inhibition. Lyophilized human A.beta. 1-40
(rPeptide) was dissolved to 1 mg/mL (220 .mu.M) in dH20. In
separate test tubes, test composition stocks were prepared in PBS
at various concentrations such that final reactions containing
equal volumes of the test composition stocks and the A.beta.
solution would result in a final A.beta. concentration of 0.5 mg/mL
(110 .mu.M) with test composition:A.beta. weight ratios of 1:1,
0.5:1, 1:1, and 0.2:1. The reactions containing A.beta.+test
compositions (or A.beta.+PBS as a control for A.beta. aggregation)
were then incubated for 2 days. The incubation mixtures were
diluted 1:10 to 0.05 mg/ml. A.beta. and 50 .mu.L of each diluted
incubation mixture was transferred into a 96-well microliter plate
containing 200 .mu.L of Thioflavin T solution (i.e. 125 .mu.M
Thioflavin T in 62.5 mM phosphate buffer, pH 6.8). The fluorescence
was read at 485 nm (444 nm excitation wavelength) using an ELISA
plate fluorometer after subtraction with PBS buffer alone or
composition alone, as blank.
[0104] The results of this study presented in FIG. 3 indicated that
LOTE+PTI-00703.RTM. of this invention interfered with A.beta.
aggregation as indicated by its ability to prevent the formation of
fibrils as assessed by Thioflavin T fluorometry. At a test
composition:A.beta. weight ratio of 0.2:1 LOTE+PTI-00703.RTM.
inhibited fibrils by 14.1% and at 0.5:1 LOTE+PTI-00703.RTM.
inhibited fibrils at 73% (p<0.001). At equal weight equivalents
(test composition:A.beta. weight ratio of 1:1) there was an 89.3%
inhibition (p<0.001) of Thioflavin T fluorescence. This study
indicated that LOTE+PTI-00703.RTM. is a potent inhibitor of
.beta.-sheet rich-A.beta. fibril formation as assessed by
Thioflavin T fluorometry, and this combination exerts its effect in
a dose-dependent manner.
Part B: Congo Red
[0105] To test whether LOTE+PTI-00703.RTM. can inhibit .beta.-sheet
formation of A.beta. the same Congo red assay as described in
Example 1 was utilized, but with A.beta. 1-40 as a substrate
instead. In this assay, A.beta. 1-40(as prepared for the Thio T
assay) and test compositions were incubated for 2 days and then
vacuum filtered through a 0.2 .mu.m filter. The amount of A.beta.
1-40 retained in the filter was then quantitated following staining
of the filter with Congo red (125 .mu.M Congo red, 100 mM Tris, 50
mM NaCl, pH 7). After appropriate washing of the filter, any
lowering of the Congo red color on the filter in the presence of
the test composition (compared to the Congo red staining of the
amyloid protein in the absence of the test composition) was
indicative of the test composition's ability to diminish/alter the
amount of aggregated and congophilic A.beta..
[0106] In one study, the ability of A.beta. fibrils to bind Congo
red in the absence or presence of increasing amounts of
LOTE+PTI-00703.RTM. (at test composition:A.beta. weight ratios of
1:1, 0.5:1, and 0.2:1) was determined. The results of 2-day
incubations are presented in FIG. 3. LOTE+PTI-00703.RTM. caused a
dose-dependent inhibition of A.beta. binding to Congo red. At a
test composition:A.beta. weight ratio of 0.2:1 LOTE+PTI-00703
inhibited Congo red binding 41.7% and 0.5:1 LOTE+PTI-00703.RTM.
inhibited Congo Red binding 62.5% (p<0.001). At equal weight
equivalents (test composition: A.beta. weight ratio of 1:1) there
was an 83.3% inhibition (p<0.001) of Congo red binding. Similar
to the results for the Thioflavin T fluorometry assay, this study
also indicated that LOTE+PTI-00703.RTM. is a potent inhibitor of
A.beta. fibrils as assessed by A.beta. fibril binding to Congo red,
and exerts its effect in a dose-dependent manner.
Part C: CD Spectroscopy
[0107] CD spectroscopy was performed to determine
LOTE+PTI-00703.RTM.'s potency to inhibit formation of A.beta. 1-40
.beta.-sheet secondary structure under aggregation-prone
conditions. Since .beta.-sheet structure is characteristic of
A.beta. fibrils, monitoring secondary structure of protein can
provide additional proof of a compositions' effectiveness at
inhibiting aggregation. CD spectra of A.beta. 1-40 samples with
increasing -/+ concentrations of compositions were analyzed at
25.degree. C. on a JASCO Model J-810 Spectropolarimeter. CD
spectroscopy and ThioT assays were analyzed in parallel from the
same sample preparation in order to correlate the results from two
independent assays.
[0108] In FIG. 4, the CD spectra of A.beta. 1-40 treated with
LOTE+PTI-00703.RTM. indicated a dose-dependent inhibition of
.beta.-sheet containing fibrils. A minima at 218 nm indicates the
presence of .beta.-sheet structure. A positive shift of ellipticity
at 218 m indicates less .beta.-sheet structure. At a test
composition: A.beta. weight ratio of 0.2:1 LOTE+PTI-00703.RTM.
demonstrated 30.9% less .beta.-sheet structure from untreated 0.5:1
LOTE+-PTI-00703.RTM. showed 53.9% less .beta.-sheet structure than
control. At equal weight equivalents (test composition: A.beta.
weight ratio of 1:1) there was 64.8% less .beta.-sheet structure
compared to control.
[0109] These data verified LOTE-PTI-00703.RTM. has a signigicant
ability to inhibit abnormal assembly of A.beta. into fibrillar,
.beta.-sheet assemblies and maintains A.beta. 1-40 in a less
pathogenic form.
Use of Recombinant Tau Repeat Domain for In Vitro Screening of Tau
Aggregation Inhibitors
[0110] During in vitro screening for identification of tau
aggregation inhibitors, we found that under the same experimental
conditions, formation of paired helical filaments (PHFs) from
commercially-purchased full-length tau protein (Tau441; from
rPeptide) was much slower (>11 days; data not shown) than that
from the tau repeat domain (TauRD; containing Q244-E372 of Tau441)
(.gtoreq.24 hr) (S. Barghorn et al, Methods Mol Biol, 299:35-51,
2005).
[0111] Because of the remarkably short turn-around time and common
aggregation properties, we used TauRD for in vitro screening to
identify tau aggregation inhibitors in our current invention. Since
the TauRD protein is not commercially available, we produced our
own protein for this project. A cDNA fragment coding for the human
TauRD was cloned into a bacterial expression vector and the
construct was then expressed in E. coli. Bacterial clones
demonstrating high levels of expression of TauRD were then selected
for protein purification. The recombinant TauRD protein was then
purified by heat-stability treatment and cation exchange
chromatography as described with minor modifications (S. Barghorn
et al, Methods Mol Biol, 299:35-51, 2005). Using this method, we
achieved a protein yield of 20 mg per liter of bacterial culture).
Aggregation and PHF formation of purified TauRD were evaluated and
validated by independent assays including Thioflavin S (ThioS)
fluorometry, a dye that fluorescences after binding to fibrils
(FIG. 5D), circular dichroism (CD) spectroscopy, a method that
detects changes in secondary structure of proteins (FIGS. 5B and
5C) and electron microscopy (FIGS. 5E-G). The results consistently
demonstrate that TauRD (10 .mu.M) is able to form ThioS-positive,
.beta.-sheet-containing PHFs when incubated with equimolar heparin
(Sigma-Aldrich, St. Louis, Mo.) at 37.degree. C., with shaking at
800-1000 rpm for .gtoreq.1 day.
[0112] In FIG. 5A, TauRD (15 kDa) protein purified from E coli was
evaluated by SDS-PAGE/silver stain with typical purity of >95%.
In FIG. 5B-C are examples of CD spectroscopy of non-aggregated and
aggregated TauRD proteins. TauRD aggregates were prepared in the
presence of equimolar (10 .mu.M) ratios of TauRD and heparin in 20
mM sodium phosphate buffer (pH7.4), incubated at 37.degree. C. with
shaking for 0-120 h. In the absence of heparin, CD spectra of
non-aggregated TauRD was random coil with ellipticity minima at 195
nm (FIG. 5B). In the presence of heparin, CD spectra showed
time-dependent conformational changes of TauRD protein from random
coil (minima at 195 nm) at time 0 to .beta.-sheet (minima at 218
nm) at 20-120 h (FIG. 5C). FIG. 5D is an example of TauRD
aggregation monitored by ThioS fluorometry over time. The results
in FIG. 5D, show formation of ThioS-positive TauRD fibrils after 20
h incubation. In the absence of heparin without fibril formation,
tauRD ThioS signals were <200 arbitrary units (A.U.) of
fluorescence at all time points. Tau fibril formation was confirmed
by negative stain EM in FIG. 5E-G. Representative images show
formation of Tau fibrils after 48 h incubation with heparin. TauRD
monomers at time 0 are shown in FIG. 5E (Bar=200 nm). Formation of
TauRD fibrils at 48 hr are shown in FIGS. 5F-G (Bar=50 nm). Both
straight and paired helical filaments were found.
Example 3
Identification of Novel Tau Aggregation Inhibitors by Thioflavin S
Fluoremetry Screening
[0113] A well-known method for measuring fibril formation is
Thioflavin T (ThioT) fluorometry (H. Naiki et al, Lab. Invest
65:104-110, 1991; H. Levine III, Protein Sci. 2:404-410, 1993; H.
Levine III, Amyloid 2:1-6, 1995; H. Naiki and K. Nakakuki, Lab.
Invest. 74:374-383, 1996). ThioT is known to bind to fibrillar
proteins, and an increase in fluorescence correlates with an
increase in fibril formation, whereas a decrease in fluorescence
correlates with a decrease in fibrils due to disassembly and/or
disruption. We modified the assay by replacing ThioT with
Thioflavin S (ThioS), a related anionic dye with similar
properties, because the latter has been shown to be more sensitive,
and reproducible for quantifying Tau PHFs (data not shown; P.
Friedhoff et. al, Biochemistry, 37(28): 10223-30, 1998). ThioS
fluorometry was employed to assess whether the above described
mixed compositions were capable of causing a disassembly/disruption
of fibrils.
[0114] Aggregated tau fibrils were prepared in the presence of
equimolar ratios of TauRD and heparin (10 .mu.M each) in 20 mM
sodium phosphate buffer, pH7.4. The reaction mixture was incubated
at 37.degree. C. with shaking (800-1000 rpm) for 24 h to 72 h. Test
compositions were tested at varying weight to weight concentrations
with 0.14 mg/ml TauRD with heparin. The same reaction mixtures
(+increasing concentrations of test compositions) but without TauRD
were also set up in parallel to serve as background controls. For
all test compounds background ThioS fluorescence readings were very
low, usually <5% of those of the TauRD-containing wells.
[0115] Following 24-72 h of co-incubation, 50 .mu.l of each
incubation mixture was transferred into a black 96-well microtiter
plate (Santa Cruz Biotechnology, Inc., Dallas, Tex.) with 50 .mu.l
of phosphate buffered saline (PBS; Sigma-Aldrich, St Louis, Mo.)
and 25 .mu.l of Thioflavin S solution (500 mM Thioflavin S;
Sigma-Aldrich, St. Louis, Mo. in PBS, pH 7.4). Using an ELISA plate
fluorometer, fluorescence was read at 485 nm (444 nm excitation
wavelength) after subtraction with buffer alone or composition
alone, as blank.
[0116] Inhibition of tau aggregation by 50%, IC.sub.50, was
calculated using Prism version 5 software (GraphPad Software) by
nonlinear regression [(Log [inhibitor] vs, normalized response;
variable slope)]. In initial screenings, the test compounds
demonstrated a broad range of activities for inhibiting tau protein
fibril formation: IC.sub.50 values ranged from <0.14 mg/ml (less
or equal to the concentration of tauRD in the assay) to infinity
(i.e. no activity at all). The results suggested that the
inhibitory activities were test subject specific. A few samples
were also tested for their ability to disrupt preformed tau fibrils
(disruption assay).
[0117] Over 25 tea extracts were tested for inhibition of
ThioS-positive tau aggregates. A majority of teas were oolong tea
extracts. FIG. 6, is an example of ThioS fluorescence of tau
aggregated in the presence of a few oolong tea extracts, chamomile
tea and green tea. Increasing concentrations of extracts were
incubated with 0.14 mg/ml (10 .mu.M) tauRD and equimolar heparin
for 24-72 h, shaking at 1000 rpm, 37.degree. C. The lead oolong tea
extract (LOTE) inhibited tau aggregates more than other oolong
teas, chamomile tea or green tea extract LOTE inhibited tau
aggregation 50% (IC.sub.50) with a ratio of 0.23:1, weight:weight,
of composition to tau. This experiment demonstrates LOTE has a
superior ability aver other compositions to inhibit abnormal
assembly of tau into fibrillar, pathogenic assemblies.
[0118] After identifying a tea extract that has excellent tau
aggregation inhibition, lead oolong tea extract (LOTE) was
incubated alone or in combination with PTI-00703.RTM. in the tau
aggregation inhibition assay. A combination of specific oolong tea
and PTI-00703.RTM. inhibited ThioS-positive tau aggregates more
than LOTE or PTI-00703.RTM. alone (*significant by paired t-test,
p<0.05) (FIG. 7). This combination of lead oolong tea extract
and PTI-00703.RTM. is a potent inhibitor of tau
aggregation/fibrillogenesis that could be used as a novel
therapeutic for tauopathies.
Example 4
Tau Aggregation Inhibition by Lead Oolong Tea Extract Confirmed by
Analysis of Protein Secondary Structure by Circular Dichroism (CD)
Spectroscopy
[0119] CD is a powerful method that provides dye-independent
confirmation that the structure of a protein has changed. CD
measures the differential absorption between left and right handed
circularly polarized light. Proteins contain elements of asymmetry
that exhibit distinct CD signals measured in units of ellipticity.
Thioflavin S fluorescent, tau aggregates have an ellipticity minima
of 218 nm, which is characteristic of .beta.-sheet containing
proteins. Non-aggregated tau has an ellipticity with a
characteristic minima at 195 nm indicating random coil structure.
CD spectroscopy was performed to determine each composition's
potency to inhibit formats of .beta.-sheet secondary structure in
TauRD under aggregation-prone conditions. CD spectra were taken
from samples containing +/- TauRD with increasing concentrations of
compounds, and analyzed at 25.degree. C. on a JASCO Model J-810
Spectropolarimeter. CD spectroscopy and ThioS assays were analysed
in parallel from the same sample preparation in order to correlate
the results from two independent assays.
[0120] In FIG. 8, tau was treated with lead oolong tea extract
(LOTE) or chamomile tea extract for 48 h. Tau treated with LOTE is
dose-dependently inhibited from converting to .beta.-sheet
containing fibrils. At the highest concentration of LOTE treatment,
tau remains in soluble, random coil form with a minima .about.195
nm. Conversely, at the highest concentration of treatment with
chamomile tea extract, tau still changes to an aggregated,
.beta.-sheet structure similar to the untreated control. These data
confirm LOTE has a significant ability over other compositions to
inhibit abnormal assembly of tau into fibrillar, .beta.-sheet
assemblies and maintains tau in a non-pathogenic soluble, random
coil form.
Example 5
Inhibition of Tau Protein Fibrillogenesis and Disaggregation of
Preformed Tau Fibrils by PTI-00703+LOTE Determined by Negative
Stain Electron Microscopy (EM)
[0121] EM analysis was used to independently monitor the
effectiveness of compositions to inhibit tau fibrillogenesis. In
these experiments, tau fibrils were assembled by incubating
equimolar ratios of TauRD protein and heparin (10 .mu.M each) in
the absence (control) or presence of increasing concentrations of
test compositions. After the 2-day incubation, samples were spotted
onto grids, stained with 2% uranyl acetate, and visualized at
8,000.times. to 30,000.times. magnifications with a JEOL 1010
transmission electron microscope.
[0122] In FIG. 9, left panel, EM analysis confirmed formation of
tau fibrils in the absence of treatment. Without treatment, tau
formed a mixture of paired straight and helical filaments similar
to those found in human tauopathies (V. M. Lee et al., Ann. Rev.
Neurosci. 24:1121-159, 2001). These samples were also tested by the
ThioS assay and CD and confirmed to be ThioS-fluorescence positive
and .beta.-sheet in structure. In the presence of PTI-00703.RTM.
and PTI-00703.RTM.+LOTE (FIG. 9, middle and right panel), tau
fibrils become shortened and sparse, indicating an inhibition of
tau fibril formation. Even less fibrils are apparent in tau samples
treated with both PTI-00703.RTM. and LOTE. These results correlate
well with ThioS fluorometry and CD analysis, which showed a
reduction in ThioS fluorescence and decrease in .beta.-sheet
structure. Using three independent methodologies, we have
identified and validated PTI-00703+lead oolong tea extract as a
potent inhibitor of tau aggregation/fibrillogenesis.
[0123] Utilizing EM analysis, preformed tau fibrils were shown to
rapidly disaggregate in the presence of both PTI-00703.RTM. and
LOTE. TauRD was incubated with equimolar heparin to form fibrils as
described in the previous assay. TauRD was diluted with or without
test compositions and incubated, shaking at 37.degree. C. for
various time points. At each time point tauRD+/- test compounds was
assayed for ThioS fluorescence and snap frozen for EM analysis. In
FIG. 10 (left panel) without treatment, pre-fibrillarized tau
remained in long filaments. In the next panels, pre-fibrillarized
tau was found to rapidly disaggregate in the presence of
PTI-00703.RTM.+LOTE. As early as 15 minutes incubation with
PTI-00703.RTM.+LOTE, tau fibrils are shorter and sparser than
without treatment. Disruption of tau fibrils was also confirmed by
ThioS assay (data not shown). These data indicate the composition
of PTI-00703.RTM.+LOTE not only can inhibit tau aggregation but can
disaggregate preformed tau fibrils.
Example 6
Further In Vivo Testing of the PTI-00703.RTM.+LOTE for Improvement
of Cognition and Memory
[0124] Further in vivo studies are used to test the
PTI-00703.RTM.+LOTE plant extract combination for their
effectiveness in the reduction of brain "plaque and tangle" load
and improvement of cognition and memory. 40-60 men and women are
selected for a clinical study. Subjects have age-associated memory
impairment (AAMI), and expecting to have worsening symptoms of
memory loss within the 6-month study period, but are in good
general health otherwise.
[0125] This study includes a placebo group, i.e. the subjects are
divided into two groups, one of which receives the
PTI-00703.RTM.+oolong tea extract, combination capsules (two 390 mg
capsules containing the PTI-00703.RTM.+oolong tea extract in a 1:1
weight/weight combination with a meal; preferably lunch) of this
disclosure, and the other receives a placebo (two capsules
containing capsules without the study product active ingredients).
The patients are benchmarked as to memory, cognition, focus,
concentration, reasoning and other symptoms associated with
mild-cognitive impairment (MCI). Subjects in the test groups
receive a therapeutic dose of the combination study product extract
or placebo for 6 months, with analysis for short-term memory,
cognition, focus and concentration to be examined at 0, 1, 3, and
6-months of treatmen. Accurate records with regards to memory,
focus and concentration are kept as to the benchmarked symptoms in
both groups and at the end of the study these results are compared.
The results also are compared between members of each group. In
addition, the results for each patient are compared to the symptoms
reported by each patient before the study began. Activity of the
combination PTI-00703.RTM.+oolong tea extract study product is
illustrated by the attenuation of the typical cognitive decline,
decline in short-term memory, cognition, focus and concentration,
and/or associated behavioral disruptions associated with
age-associated memory impairment (AAMI)
[0126] All references and patent publications provided herein are
hereby incorporated by reference in their entireties.
* * * * *
References