Note: Descriptions are shown in the official language in which they were submitted.
METHODS AND COMPOSITIONS WITH PURIFIED BOMBYX MORI COCOON SILK PEPTIDE FIBER
AND
REFINED BUGLOSSOIDE5 ARVENSI5 SEED OIL PROVIDING ANTI-INFLAMMATORY EFFECTS AND
NEUROPROTECTION FOR DISEASE STATES
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application is a continuation-in-part application claiming the
benefit of US Patent
Application No. 17/067,489, filed October 9, 2020, which claims the benefit of
US Provisional Patent
Application No. 62/912,956, filed October 9, 2019. Each of the applications
identified above is
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[002] The present invention relates to compositions comprising a synergistic
combination of purified
Bombyx mori cocoon silk peptide fiber, refilled Buglossoides arvensis seed
oil, and optionally Blueberry
extract, and related methods for decreasing inflammation and providing
neuroprotection. The compositions
provide synergistic effects and may be used to treat relevant diseases and
disorders.
BACKGROUND
[003] In a rapidly advancing society, interest in brain health and memory as
well as a demand for related
health products is increasing continuously along with the aim of obtaining
additional information about
neuro-protective and neuro-restorative dietary interventions, given the recent
rise in aging population
demographics, in the levels of information-intensive jobs, and in the far
greater learning and information
management tasks they require. Although the focus of the demand historically
has been patient-centric,
typically restricted to prescription medicines aiding in the reduction of
partial or early-onset memory
decline, such as with age-related dementia, nowadays healthy and normal people
who wish to strengthen
their memory and cognitive performance along with patients facing mild memory
decline are actively
searching for nutritional supplements and "neurotropic"/"nootropic" health
foods or functional foods that
are able to act as memory boosters. Therefore, many related products have been
released and sold in various
forms following indications of in vitro neural cell protection/enhancement or
in vivo memory improvement.
Neuroscience (Pathology of brain memory)
[004] Memory does not work through discrete fragmentary mechanisms. Instead,
memory appears to
result from highly complex interactions between many physiological functions
and neurotransmitters acting
in concert. For example, the following neurotransmitters which are either
directly or indirectly related to
memory exist:
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Date Recue/Date Received 2022-04-14
Neurotransmitters Representative Lack Causes Excess Causes
Functions
__________________________________________________________________________
m====
Acetylcholine Learning Memory problem, Violent muscle
Dementia, contractions
Alzheimer's disease
Dopamine Pleasure Parkinson's disease, Schizophrenia
ADHD (Attention-
Deficit/Hyperactivity
Disorder)
Seroton in Mood Rumination, Depression Serotonin
Syndrome
Norepinephrine Concentration Hypotension, ADHD Increase heart
rate &
(noradrenaline) pressure.
GABA Calming Anxiety Paradoxical
Anxiety
Among such neurotransmitters, acetylcholine is of particular interest, given
that significant memory
improvement effects can be anticipated when related functions are locally
controlled. Acetylcholine
deficiency is known to occur frequently with aging. Maintaining an appropriate
level of acetylcholine, as
opposed to other neurotransmitters, is particularly crucial for the
maintenance of memory.
[005] Existing health supplements supporting brain health and memory are
mostly simple, made from
purportedly effective Active Dietary Ingredients (ADIs).
[006] The majority of commercialized products are made either from a sole
nootropic ingredient, which
affects cognitive abilities and memory, or simple combinations of many natural
ingredients. Although some
beneficial effects can be expected from many of these products, such products
created without underlying
scientific designs may result in substantial differences in effectiveness and
in various side effects among
different patients.
[007] To successfully activate effective neuro-protective and/or neuro-
restorative actions of a candidate
medicinal ingredient that will avoid any negative side effects in virtually
all patients, makers of the product
2
Date Recue/Date Received 2022-04-14
must consider several factors including the activity of the medicinal
ingredient, the underlying
mechanism(s) of actions, patients' pathophysiology, and the absorption of the
medicinal ingredient.
Corresponding characteristics of consumers
[008] Consumers of such health products can be divided into two main groups,
namely: those who require
memory improvement due to aging; and students and workers who wish to learn,
process, and retain new
knowledge more efficiently and with greater cognitive performance.
[009] Following an examination of older people's pathophysiology, digestive
functions (e.g., secretion of
digestive fluids and gastrointestinal movement) and ADME (absorption,
distribution, metabolism,
elimination) are found generally to diminish with increasing age. Although
health product types differ
depending on the medicinal ingredients involved, their administration in
smaller more frequent dosages is
needed given that their speed of metabolism is often slower. Atrophic
gastritis, also known as achlorhydria,
occurs in 30% of the elderly population, while many cases of incomplete
absorption despite taking in
sufficient nutrition are commonly reported.
[010] Students who wish to increase their learning and workers who must
complete various complex
cognitive tasks commonly experience indigestion and abdominal pain due to
stress related to more intensive
learning requirements. Further, nutrient absorption and metabolism are
sometimes reduced indirectly as
well. Also, homeostenosis can commonly occur in these consumers, especially in
seniors or in those with
chronic stress. A nutritional imbalance in this condition can increase the
possibility of either permanent
functional damage or vulnerability to other diseases, given that the ability
to return to the original state is
compromised, even when proper dietary supplementation or other corrections are
performed later.
Designing health products which sufficiently consider neuro-protective and/or
neuro-restorative effects
related to memory as well as points as mentioned earlier would be
advantageous.
[011] There is a need for compositions and methods that may decrease
inflammation and oxidative stress
in the body, and may provide for instance neuroprotection and other protection
from inflammation and
oxidative stress. See for instance Degan et al., The Role of Inflammation in
Neurological Disorders, Curr.
Pharm. Des. 24(14):1485-1501 (2018). See also for instance Morris et al.,
Leaky brain in neurological and
psychiatric disorders: Drivers and consequences, Austr. New Zeal. J. Psychiat.
52(10):924-948 (2018);
Enache et at., Markers of central inflammation in major depressive disorder: A
systematic review and meta-
analysis of studies examining cerebrospinal fluid, positron emission
tomography and post-mortem brain
tissue. Brain Behay. Immun. 81:24-40 (2019); Salter et al., Microglia emerge
as central players in brain
disease. Nat. Med. 23(9):1018-1027 (2017); Kwon et al., Neuroinflammation in
neurodegenerative
disorders: the roles of microglia and astrocytes. Transl. Neurodegen. 9:42
(2020; 12 pages); Kaur et al.,
3
Date Recue/Date Received 2022-04-14
Neuroinflammation Mechanisms and Phytotherapeutic Intervention: A Systematic
Review. ACS Chem.
Neurosci. 11:3707-3731(2020). These documents are hereby incorporated by
reference for the purpose
of identifying disorders, diseases, conditions, and the like that may be
treated, prevented, protected from,
or otherwise assisted with the invention described below, to the extent
allowed by law.
SUMMARY OF THE INVENTION
[012] The present invention is directed to compositions comprising purified
Bombyx mori cocoon silk
peptide fiber, refmed Buglossoides arvensis seed oil (e.g. NeurXcele), and
preferably Blueberry extract
powder. Also, the invention is directed to methods for reducing inflammation
and/or oxidative stress,
providing neuroprotection, and/or treating/preventing diseases and disorders
associated with inflammation
and oxidative stress, including for instance those associated with sugar.
[013] A composition of this invention, in an embodiment, comprises 20-2000 mg
purified Bombyx mori
cocoon silk peptide fiber and 20-10,000 mg refined Buglossoides arvensis seed
oil. Other compositions of
this invention comprise 200-400mg, for instance about 400mg, of purified
Bombyx mori cocoon silk
peptide fiber and/or 200-2500 mg, for instance 250 mg, refined Buglossoides
arvensis micro-encapsulated
seed oil. Optionally, a composition of the invention comprises 25-500 mg
Blueberry Extract, or in another
embodiment, 50-100 mg Blueberry Extract. Other compositions of this invention
are also included in this
invention.
[014] Compositions of the invention are a dietary supplement, in an
embodiment. Also, compositions of
the present invention may include or be combined with further ingredients such
as Acetyl-L-Camitine, L-
theanine, L-serine, Zinc (as zinc glycinate, zinc citrate, or zinc
picolonate), Huperzine A extracted from
clubmoss (Huperzia chinensis), Bacopa monnieri extract, Ginseng Extracts,
Zingiber officinalis Extracts,
Citicoline, Ginkgo biloba extract, Folic acid, and Haskap blue honeysuckle
berry (Lonicera caerulea),
Vitamin B12, Vitamin B6, Vitamin Bl, Vitamin D3 (cholecalciferol), Greek
Mountain Tea (Sideritis
spp.), Lion's Mane mushrooms. The ingredients may be added to said
compositions individually or
according to groupings, such as those discussed below and throughout this
application.
[015] The present invention is also directed to a method for making the above-
described compositions.
The present invention is also directed to methods for using the present
compositions to improve memory
and/or cognitive performance in human subjects or other subjects, to provide
neuroprotection, and/or to
treat e.g. neurodegenerative diseases or disorders, and/or other neurological
diseases, disorders, or pre-
clinical conditions. Such methods generally comprise orally administering
compositions according to the
present invention to a human subject. Other methods are described or apparent
from the data and
throughout the application.
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Date Recue/Date Received 2022-04-14
BRIEF DESCRIPTION OF THE DRAWINGS
[016] Fig. 1 is a graph showing in vitro cell viability in the presence of
purified Bombyx mori cocoon silk
peptide fiber alone (A) or after administration of compositions of the present
invention (B-E).
[017] Fig. 2 is a graph showing synergistic in vitro neuroprotective effects
of compositions of the present
invention (B-E), as compared with purified Bombyx mori cocoon silk peptide
fiber alone (A), following
hydrogen peroxide (H202)-induced damage to neurons.
[018] Fig. 3 is a graph showing synergistic in vitro neuroprotective effects
of compositions of the present
invention (B-E), as compared with purified Bombyx mori cocoon silk peptide
fiber alone (A), following
iron sulfate (FeSO4)-induced damage to neurons.
[019] Fig. 4 is a graph showing the synergistic in vitro inhibitory effect of
compositions of the present
invention (Formulas B-E), as compared with purified Bombyx mori cocoon silk
peptide fiber alone
(Formula A), on the generation of reactive oxygen species (ROS) following
exposure to H2O
[020] Fig. 5 is a graph showing the synergistic in vitro inhibitory effect of
compositions of the present
invention (Formulas B-E), as compared with purified Bombyx mori cocoon silk
peptide fiber alone
(Formula A), on the generation of reactive oxygen species (ROS) following
exposure to FeSO4
[021] Fig. 6 is a graph showing in vitro results of H202-induced cell death
inhibition by compositions of
the present invention and various controls.
[022] Fig. 7 is a graph showing in vitro results of FeSO4-induced cell death
inhibition by compositions
of the present invention and various controls.
[023] Fig. 8 is a graph showing in vitro inhibition of H202-induced Reactive
Oxygen Species (ROS)
generation by compositions of the present invention and various controls.
[024] Fig. 9 is a graph showing in vitro inhibition of FeSO4-induced Reactive
Oxygen Species (ROS)
generation by compositions of the present invention and various controls.
[025] Fig. 10 is a graph showing in vitro cell viability tests of various
compositions.
[026] Fig. 11 is a graph showing in vitro cell viability tests of various
compositions.
[027] Fig. 12 is a graph showing synergistic in vitro neuroprotective effects
by compositions of the
present invention after H202 challenge.
[028] Fig. 13 is a graph showing synergistic in vitro inhibition of Reactive
Oxygen Species generation
by compositions of the present invention after H202 challenge.
5
Date Recue/Date Received 2022-04-14
[029] FIG. 14 shows synergistic CNS Vital Signs results in vivo in healthy
adult seniors after
administration of Braini , a synergistic composition of this invention.
[030] FIG. 15 shows synergistic CNS Vital Signs results in vivo in an adult
subject with Multiple
Sclerosis after administration of Braini , a synergistic composition of this
invention.
[031] FIG. 16A shows synergistic CNS Vital Signs results in vivo in adult
subjects with dyslexia after
administration of Braini , a synergistic composition of this invention. FIG.
16B shows physician's
observational data after Braini administration in dyslexic subjects.
[032] Fig. 17 shows synergistic CNS Vital Signs results in vivo in adult
subjects first treated with
Peptylin , and then Braini , a synergistic composition of this invention.
[033] Fig. 18 is a table showing improvements in CNS Vital Signs Psychomotor
Speed in human
subjects after Braini administration.
[034] Fig. 19 is a table showing improvements in CNS Vital Signs Reaction Time
in human subjects
after Braini administration.
[035] Fig. 20 is a table showing improvements in CNS Vital Signs Cognitive
Flexibility in human
subjects after Braini administration.
[036] Fig. 21 is a table showing improvements in CNS Vital Signs Processing
Speed in human subjects
after Braini0 administration.
[037] Fig. 22 is a table showing improvements in CNS Vital Signs Executive
Function in human
subjects after Braini administration.
[038] Fig. 23 is a table showing improvements in CNS Vital Signs Motor Speed
in human subjects after
Braini administration.
[039] Fig. 24 is a series of graphs including a Cambridge Brain Sciences
DoubleTrouble graph,
showing improved concentration in subjects administered Braini .
[040] Fig. 25 is a non-parametric model showing statistically significant
improvement in Executive
Function in healthy young adult human subjects administered Braini (A)
compositions of this invention,
as compared with subjects administered a Placebo (P) composition.
[041] Fig. 26 is a parametric model showing statistically significant
improvement in Executive
Function in healthy young adult human subjects administered Braini (A)
compositions of this invention,
as compared with subjects administered a Placebo (P) composition.
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Date Recue/Date Received 2022-04-14
[042] Fig. 27 is a multiple regression model showing improvement in Executive
Function in healthy
young adult human subjects administered Braini (A) compositions of this
invention, as compared with
subjects administered a Placebo (P) composition, based on baseline analyses.
[043] Fig. 28 is a multiple regression model showing improvement in Executive
Function in healthy
young adult human subjects administered Braini (A) compositions of this
invention, as compared with
subjects administered a Placebo (P) composition, based on analysis of absolute
deviation from the scaled
mean of 100.
[044] Fig. 29 is a scanning electron micrograph showing NeurXcel powder in
discrete, 10-100 um
spheres. (Scale: top: 100 urn; bottom: 10 urn).
[045] Fig. 30 is a scanning electron micrograph showing Peptylin powder as an
angular crystal-like
material. (Scale top: 100 um; bottom: 10 urn).
[046] Fig. 31 is a scanning electron micrograph showing Wild blueberry powder
particles ranging from
10-50 um across. (Scale top: 100 um; bottom: 10um).
[047] Fig. 32 is a scanning electron micrograph showing Braini powder as an
agglomerate of
NeurXcel powder, Peptylin powder, and Wild blueberry powder. (Scale top:
100um; bottom: 20 um).
[048] Fig. 33 is a scanning electron micrograph showing Braini powder as an
agglomerate of
NeurXcel powder, Peptylin powder, and Wild blueberry powder. (Scale: 20
urn).
[049] Fig. 34 is a scanning electron micrograph showing Braini powder as an
agglomerate of
NeurXcel powder, Peptylin powder, and Wild blueberry powder. (Scale: 20
urn).
[050] Fig. 35 is a scanning electron micrograph showing Braini powder as an
agglomerate of
NeurXcel powder, Peptylin powder, and Wild blueberry powder. (Scale: 20 um).
[051] Fig. 36 is a scanning electron micrograph showing Braini Lex powder as
an agglomerate of
NeurXcel powder, Peptylin powder, and Wild blueberry powder. (Scale top: 100
urn, bottom: 10
urn).
[052] Fig. 37 is a scanning electron micrograph showing powder from Braini
capsules as an
agglomerate of NeurXcel powder, Peptylin powder, and Wild blueberry powder.
(Scale top: 100 um,
bottom: 10 urn).
[053] Fig. 38 is a scanning electron micrograph showing powder from Braini
capsules as an
agglomerate of NeurXcel powder, Peptylin powder, and Wild blueberry powder.
(Scale top, middle,
bottom: 100 um).
7
Date Recue/Date Received 2022-04-14
[054] Fig. 39 is a scanning electron micrograph showing powder from Braini Lex
capsules as an
agglomerate of NeurXcel powder, Peptylin powder, and Wild blueberry powder.
(Scale top: 100 urn,
bottom: 10 urn).
[055] Fig. 40 is a scanning electron micrograph showing powder from Braini
capsules as an
agglomerate of NeurXcel powder, Peptylin powder, and Wild blueberry powder.
(Scale top, middle,
bottom: 100 urn).
[056] Fig. 41 show HPLC mass spectra of (top to bottom) Braini capsule,
Braini powder, Peptylin
powder, NeurXcel powder, Wild Blueberry Extract powder.
[066] Fig. 42 is a graph showing WST-8 cell viability assay results after
exposure of RAW264.7 cells
to Peptylin and NeurXcel .
[067] Fig. 43 is a graph showing a standard curve generated from ELISA assay
results and used to
calculate TNF-a production.
[068] Fig. 44 is a graph showing amounts of TNF-a produced in RAW264.7 cells
in the presence of
Peptylin and NeurXcel .
[0069] Fig. 45 is a graph showing WST-8 cell viability assay results after
exposure of RAW264.7 cells
to Peptylin and NeurXcel .
[0070] Fig. 46 is a graph showing a standard curve generated from ELISA assay
results and used to
calculate IL-6 production.
[0071] Fig. 47 is a graph showing amounts of IL-6 produced in RAW264.7 cells
in the presence of
Peptylin and NeurXcel .
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a composition that improves memory and/or
cognitive
performance, provides neuroprotection, and/or can be used to treat diseases,
disorders, and/or clinical
conditions such as neurodegenerative disorders and neurological conditions
such as dyslexia.
A composition of the present invention comprises purified Bombyx mori cocoon
silk peptide
fiber, refined Buglossoides arvensis seed oil, and preferably Blueberry
extract. A composition in one
embodiment is a simple blend of Peptylin powder (purified Bombyx mori cocoon
silk peptide fiber in
powder form), refined Buglossoides arvensis seed oil (tradename: Ahiflower or
NeurXcel , and for
8
Date Recue/Date Received 2022-04-14
instance as NeurXcel Seed Oil modified powder), and BLUEd'OR Blueben-y
Extract powder. As
discussed below, through careful research, the present application shows that
the present compositions
provide synergistic effects over their individual ingredients and demonstrates
chemical modifications of
the compositions of this invention.
Compositions used in exemplary in vitro and in vivo trials are described below
in the section
"Examples". Compositions used in exemplary in vivo (human) empirical and
controlled clinical trials
were formulated as a homogenous blend of micronized and/or microencapsulated
powdered forms of the
referenced active ingredients described throughout this application and for
instance in the section
"Definitions".
The present invention is also directed to a method for preparing compositions
of the present
invention. In one embodiment, a method for making a composition of the present
invention comprises the
step(s) of combining Peptylin powder, refined Buglossoides arvensis seed oil
in the form of modified
powder such as NeurXcel seed oil modified powder, and optionally BLUEd'OR
blueberry extract
powder in specified amounts together, for instance to prepare a simple blend.
In another embodiment, a method of preparing a composition of the present
invention comprises
the step(s) of adding powdered ingredients to an inner capsule and refined
Buglossoides arvensis seed oil
to an outer capsule. For instance, Lonza/Capsugel's DuoCap system provides
inner and outer capsules
which together may be administered to a subject as a single dose unit.
In another embodiment, a method of preparing a composition of the present
invention comprises
the steps of combining at least B. mori fiber, B. arvensis oil, and Blueberry
Extract into a bulk powdered
blend. In an embodiment, said combining is by blending the ingredients
together in a V-mixer; in an
embodiment, all ingredients are blended to homogeneity. In an embodiment, the
particle size of a
composition of this invention is set by the particle sizes of the original
ingredients. In an embodiment, the
particle size is of a powdered composition of this invention is such more than
90% of the blended powder
will pass through an 80 mesh screen. In an embodiment, the blend may be
ingested as is, added to food,
or mixed with water, milk (for instance seed, nut, dairy), juice (fruit and/or
vegetable, or the like), and/or
other beverages (for instance, as a frozen or non-frozen shake) for direct
consumption. In an embodiment,
the blended powder composition may be added to a capsule for ingestion in
capsule form. Compositions
such as those described above, in an embodiment, are dietary supplements of
this invention. In an
embodiment, the active ingredients may be formulated into a gummi blend or
other oral dosage form.
The present invention is also directed to methods for using the present
compositions to improve
memory and/or cognitive performance in human subjects, provide
neuroprotection, and/or to treat
9
Date Recue/Date Received 2022-04-14
neurodegenerative diseases or disorders or neurological conditions (for
instance dyslexia), and/or other
diseases, disorders, or pre-clinical conditions. The methods generally
comprise orally administering the
compositions in an effective amount to a human subject, for instance up to the
maximum daily intake
levels allowed by federal regulatory bodies.
Silk proteins
Silk proteins and silk peptides refer to protein from the cocoon of the Bombyx
mori L. silkworm,
that may be partially hydrolyzed into peptides. The cocoons are frequently
characterized as having two
proteins, namely fibroin (-75 %) and sericin (-25 %).
Recently, silk peptide has been hydrolyzed and used in new ways compared to
traditional
applications. The silk peptide family is known to increase the concentration
of acetylcholine in the body
and to decrease acidic stress condition, providing a neural protection effect.
Moreover, it is recognized to
provide superb memory improvement and brain protection effects through the
promotion of brain blood
flow and the selective inhibition of catecholamine enzyme activity.
Silk peptide has been well documented by many clinical research studies to
improve memory.
Furthermore, the absence of side effects was reported several times across age
groups. Clinical trials on
healthy adults, children, young college students, young children, and seniors
all confirmed high long-term
and short-teun memory improvements without side effects. They also revealed
the silk peptide to be a
safe and effective ingredient to use without distinction of either age or
gender.
Vegetable Omega-3 oil fatty acids
Omega-3 fatty acids are a type of essential unsaturated fatty acid which are
actively used in the
brain's metabolic activities. Omega-3 essential fatty acids are not
synthesized in the body and must be
consumed from dietary sources. Omega-3 fatty acids include: alpha-linolenic
acid (ALA), stearidonic acid
(SDA), eicosatetracnoic acid (ETA), eicosapentacnoic acid (EPA),
docosapentacnoic acid (DPA), and
docosahexaenoic acid (DHA).
Omega-3 fatty acids are the main ingredients composing neural cell membranes
in the brain. In
particular, while DHA is dominant in brain cell structures, other unsaturated
omega-3 fatty acids are shorter
carbon-chain metabolic precursors that are elongated and synthesized by the
body and are converted into
DHA when required, thus promoting an increase in acetylcholine. Specifically,
ALA (a vegetable Omega-
3) has acetylcholinesterase activity and the ability to increase the efficient
use of acetylcholine.
Date Recue/Date Received 2022-04-14
While previous clinical studies described vegetable omega-3 oils to be
essential for brain
development, optimal cellular membrane functioning, and physical health, their
concentration in the
blood was reported to be related to both comprehension ability and memory
capacity. Moreover, a
previous investigation identified memory to be supported by a balance between
omega-3 ALA, SDA, and
omega-6 GLA, maintaining older adults' fluid intelligence and protecting
frontal neocortex structure and
Fornix white matter microstructure. Of note, refined Buglossoides arvensis
seed oil is believed to be a
rich available dietary source of combined ALA, SDA, and GLA from a single non-
genetically modified
plant. Without being bound by theory, this unique omega-3 and omega-6 fatty
acid composition is a
reason for using this dietary oil in the present invention.
Blueberry extract (standard)
Blueberry is a member of the Vaccinium genus and it exists in various species
worldwide.
Additionally, it contains much of the Polyphenol compounds (i.e., anthocyanin
and flavonoids constituents
with the highest-level content) and has potent antioxidant and anti-
inflammatory effects.
The mechanisms behind these blueberries are known to include the prevention of
age-induced
oxidation of the brain cells, the ability to activate the neurotransmitter
pathway of the brain cells, and blood
vessel promotion effects that can latently induce neural cell growth.
Also, blueberry extracts containing such anthocyanin and flavonoids were shown
to have an
effect on memory improvement in paired-associate learning and word list recall
in adults. A recent study
on seniors also showed it to improve temporary memory.
Definitions
The below definitions and discussion are intended to guide understanding but
are not intended to
be limiting with regard to other disclosures in this application. References
to percentage (%) in
compositions of the present invention refers to the % by weight of a given
component to the total weight
of the composition or preparation being discussed, also signified by "w/w",
unless stated otherwise.
According to the present invention, "administration", "administering", and the
like refer to
providing a composition of the present invention to a subject so that the
active ingredients (purified
Bombyx mori cocoon silk peptide fiber, refined Buglossoides arvensis seed oil,
and optionally blueberry
extract) reach the subject's bloodstream and/or tissues, cells, or other
bodily components and act to for
instance to support cellular membrane functions, including barrier properties,
porosity, permeability, ion
transfer, and structural integrity, and to protect, generate, regenerate,
upregulate, and/or reinforce
neurophysiological pathways, neurotransmitter compounds, and/or naturally-
occurring neuroprotective
11
Date Recue/Date Received 2022-04-14
factors including microglial cells and other neuro-immune modulatory cells. In
an embodiment, such
actions are in particular in the brain and central nervous system.
Administration may be by the subject or
by another. Administration may be to a healthy subject or to a subject having
a neurological disease or
disorder. As discussed throughout this application, administration to the
subject may be oral, for instance
in the folin of a dietary supplement, and/or in a solid dosage form, for
instance as a discrete dose unit.
Administration may also be through parenteral, intramuscular, transderrnal,
topical, nasal, sublingual,
intravenous, and other physiologically acceptable routes. Administration of
the present invention may be,
for instance, in keeping with daily doses described throughout this
application, for at least 1 day, any
number of 1-7 days, 7-14 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, 6 weeks, 7 weeks,
1 month, 2 months, 3 months, 4 months, and any number of days, weeks or months
up to 1 year. In an
embodiment, administration of the present invention may be daily for 1 year, 2
years, 3 years, 4 years or
longer, as desired by the subject or a health care provider. In an embodiment,
a composition of this
invention is administered every day during a period of treatment (i.e. on
consecutive days); in an
embodiment, the composition is administered for a period of treatment
including at least 50% consecutive
days. In an embodiment, administration of a composition of this invention is
for at least 10 consecutive
days.
"Co-administration" refers to administering a composition of the present
invention with another
known drug or substance. Compositions of the present invention may be
administered with other drugs
and/or substances, whether to further aid in improving neurocognitive
functions or treating neurological
diseases, disorders, or conditions, or in keeping with other needs of a given
subject. Subjects taking
statins (e.g. Rosuvastatin), blood thinners (e.g. Eliquis), levothyroxine,
losarten, progesterone, estrogen,
anti-histamines (e.g. Claritin), guaifenesin (e.g. Mucinex), acetaminophen
(e.g. Tylenol), ibuprofen (e.g.
Advil), naproxen (e.g. Aleve), antidepressants, antibiotics, Remdesevir ,
steroids, plasma with COVID19
antibodies, and other medications, with a composition of the present invention
(Braini capsules) reported
no side effects or adverse events.
According to the present invention, an "effective amount" is an amount of
active ingredient
administered in a sufficient amount to reach bodily cells and tissues and act
on the bodily cells and tissues
to effectively improve memory and/or cognitive performance, and/or provide
neuroprotection, and/or
support the body's natural defense against neurodegenerative states or
diseases and/or a neurological
condition such as dyslexia, or treats such diseases or conditions such as
dyslexia, when administered to
the human. Such may be achieved for instance by protection against oxidation
and generation of reactive
oxygen species, as shown in the Examples. In an embodiment, an active
ingredient of this invention is
12
Date Recue/Date Received 2022-04-14
optionally taken in combination with another active ingredient or ingredients
of the present invention; in
an embodiment, action at cells and tissues may include action by metabolites
or other modifications of the
active ingredient(s) by the body.
For instance, as shown in in vivo Examples below, an effective amount of
active ingredients for
synergistically improving CNS Vital Signs test results in adult humans is the
composition Braini of the
present invention, comprising the following effective amounts of active
ingredients of this invention: 400
mg/day Peptylin , 500 mg/day NeurXcel microencapsulated powder, and 100
mg/day Blueberry extract
powder. 500 mg of NeurXcel microencapsulated powder includes 250 mg NeurXcel
oil according to
this invention.
In another embodiment, a composition of the present invention is a capsule of
about 500 mg,
having about 375 mg total by weight of the composition of a combination,
preferably a synergistic
combination, of purified Bombyx mori cocoon silk peptide fiber (e.g.
Peptylie), refined Buglossoides
arvensis seed oil (e.g. NeurXcel ), and Wild Canadian (Vaccinium
cmgustifolium) blueberry extract. In
addition, other ingredients in the capsule include one or more of non-GMO
modified food starch, non-
GMO corn syrup solids, rice starch, vegetable cellulose (capsule shell),
rosemary extract (anti-oxidant),
natural tocopherols (anti-oxidant), ascorbyl palmitate (anti-oxidant), and
natural flavors. In another
embodiment, such a capsule would contain an effective amount of the active
ingredients, or for instance 2
capsules would contain an effective amount of the active ingredients, and
would be administered to a
human daily.
In another embodiment, the compositions of this invention are for
administration to humans. In
an embodiment, compositions of the present invention may be administered to a
subject such as a human
or other animal, including for instance a companion animal, including for
instance a mammal, such as a
dog, cat, horse, pig, mouse, rat, or for instance a non-human primate such as
a monkey, gorilla, orangutan,
and so forth.
Using a small volume rather than a large dose may be beneficial, along with
inducing synergistic
effects through different and various mechanisms, rather than through merely
additive effects through a
single mechanism. For example, in the in vitro cell challenge trials described
in this application, refined
Buglossoides arvensis seed oil administered by itself did not exhibit any
notable cytoprotective benefits,
but in complex with the other active ingredients of the present invention, as
in Formulas D and E, the
cytoprotective effect substantially improved and ROS generation was
substantially lowered.
13
Date Recue/Date Received 2022-04-14
A "composition" according to this invention comprises purified Bombyx mori
cocoon silk peptide
fiber and refined Buglossoides arvensis seed oil. The composition may comprise
the fiber and oil so they
are combined and taken together e.g. as a single discrete dose unit like a
pill or measured amount of
powder or a liquid/suspension, or e.g. two different formulations to be taken
together e.g. on the same
day. Optionally, said composition also comprises a Blueberry Extract and one
or more other ingredients,
as discussed throughout this application. A composition of the present
invention may comprise, consist
essentially of, or consist of, purified Bombyx mori cocoon silk peptide fiber
and refined Buglossoides
arvensis seed oil, optionally blueberry extract, and optionally other
ingredients including for instance
those expressly named in this application.
Administration of a composition of this invention to a subject is in an
effective amount of the
active ingredient, to effectively improve memory and/or cognitive performance,
and/or provide
neuroprotection, and/or support the body's natural defense against
neurodegenerative diseases or a
neurological condition such as dyslexia, when administered to the human. Other
ingredients of
compositions of this invention, and forms and routes of administration and the
like, are discussed
throughout this application.
An "extract" according to this invention refers to a natural substance (such
as B. arvensis seeds or
blueberries) that has been disrupted from its natural state (for instance
chopped or ground or crushed or
pressed) and steeped with water or other solvent(s) (e.g. oil, ethanol) and
for instance specific salt, pH,
and/or additional chemical components and/or exposed to elevated temperatures
and pressures to form the
extract. A "standardized" extract of this invention identifies specific
components to characterize an
extract in a specified amount or range, including for instance defined by
minimum or maximum amount,
so as to render the extract consistent at least with regard to those
components from one batch to the next.
A "standardized aqueous extract" refers for instance to an extract prepared
with water as the primary
solvent.
A "dietary supplement" of this invention is an addition to the human diet,
which is not a natural
or conventional food, which is administered orally such that the B. mori
fiber, B. arvensis seed oil,
optionally Blueberry Extract of this invention, including metabolites or other
modifications of such by the
body, reach bodily cells and tissues and other components and act on the cells
and tissues and other
components to effectively improve memory and/or cognitive performance, and/or
provide
neuroprotection, and/or support the body's natural defense against
neurodegenerative diseases or a
neurological condition such as dyslexia, or treat such disease or condition
such as dyslexia, when
administered to the human. A composition of the present invention may be a
dietary supplement.
14
Date Recue/Date Received 2022-04-14
In the present invention, "treatment" and the like refers to improving a
subject's neurological
status, said improvement shown for instance by improved test scores (e.g. one
or more of Psychomotor
Speed, Reaction Time, Cognitive Flexibility, Processing Speed, Executive
Function, and/or Motor Speed
as tested by CNS Vital Signs) after administration of a composition of this
invention, or for instance by
subjective improvement as reported by the subject or for instance a guardian
of the subject after
administration of a composition of this invention. In an embodiment, a disease
or disorder that may be
treated according to this invention includes dyslexia, multiple sclerosis,
memory impairment, attention
deficit, forgetfulness, Alzheimer's disease, Alzheimer's dementia, vascular
dementia, dementia,
Parkinson's disease, depression, a sleep disorder, dysgraphia, Anxiety
Disorder, ADD, ADHD, autism
spectrum disorder, Asperger's, strabismus, depression, brain fog from cancer
chemotherapy treatment,
brain fog from COVID-19 infection, concussion, or a demyelination disease or
disorder. In addition,
administration of a composition of the present invention may be administered
to a subject to aid recovery
from a cardiovascular incident or a stroke, or from calcification of the heart
and myocardial infarction.
"Purified Bombyx mori cocoon silk peptide fiber" according to the present
invention refers to a
preparation of fibroin peptides and optionally also fibroin amino acids from
Bombyx mori L. cocoon, in
one embodiment having at least 85% purified Bombyx mori cocoon silk peptide
fiber, in one embodiment
having at least 95% purified Bombyx mori cocoon silk peptide fiber, by weight
of the preparation.
Purified Bombyx mori cocoon silk peptide fiber is made by hydrolyzing and
purifying fibroin protein
and/or peptides from Bombyx mori L. cocoon, via enzymatic hydrolysis in an
embodiment. Purified
Bombyx mori cocoon silk peptide fiber in an embodiment of the present
invention includes less than 10%
by weight sericin, less than 5% by weight in an embodiment, and less than 1%
by weight sericin in an
embodiment. Purified Bombyx mori cocoon silk peptide fiber in an embodiment of
this invention is at
least 89% by weight fibroin, and may be present in amounts of about 90%, about
91%, about 92%, about
93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or
about 100% by weight.
hi an embodiment, enzyme hydrolysis is used to prepare purified Bombyx mori
cocoon silk peptide fiber.
The hydrolyzed fibroin preparation is digestible and absorbed easily, in an
embodiment. In an
embodiment, purified Bombyx mori cocoon silk peptide fiber of the present
invention is safe for human
consumption.
Purified Bombyx mori cocoon silk peptide fiber according to an embodiment of
the present
invention is Peptylin (purified Bombyx mori cocoon silk peptide fiber; BF-7 ,
Famenity Co., Ltd.,
Korea, sold as Peptylin in the United States, Europe, South America, and
Canada; also, BioGrand Co.
Ltd., Korea). The preferred molecular weight range for purified Bombyx mori
cocoon silk peptide fiber
Date Recue/Date Received 2022-04-14
(e.g. Peptylin ) is 800-1500 daltons. Purified Bombyx mori cocoon silk peptide
fiber (e.g. Peptylin ) is a
water-soluble preparation of silk fibroin peptides and amino acids, easily
digestible and absorbable.
Peptylin is a registered trademark of Famenity Co. Ltd. and pure silk fibroin
peptide is
manufactured exclusively by Famenity Co. Ltd. In an embodiment, purified
Bombyx mori cocoon silk
peptide fiber such as Peptylin is made through the following procedure:
Silk cocoons from Bombyx mori are composed primarily of two proteins; fibroin,
an insoluble
fiber which constitutes the structural center of the silk, and sericin, a
gummy protein that covers the
fibroin fibers. For the manufacture of Peptylin , the fibroin is harvested by
removing the sericin coating.
This process is accomplished by a series of steps that utilize heat, pressure
and calcium chloride salt. This
process is followed by an enzymatic digestion, that results in a minimum 95%
purity of the fibroin
protein. Other processes may also be used for preparing purified Bombyx mori
cocoon silk peptide fiber
according to the present invention.
In an embodiment, purified Bombyx mori cocoon silk peptide fiber according to
the present
invention may be prepared as follows: The cocoons are weighed and cut into
lengths of 1-2 cm. Purified,
reverse-osmosis deionized water (DI) is added to a stainless-steel reactor,
equipped with mechanical
stirrer and water jacket, along with the processed cocoons at a 1:10 w/v
ratio. Temperature initiates at
C, latm and is then raised to 100-105 C and boiled for 3 hours with moderate
stirring. The
temperature is then cooled to 50 C, and water is removed using pressure
filtration until the refined
cocoons are left with ¨2% water content. The weight of refined cocoons is
measured, along with the
20 assessed water content, to determine the ratio of calcium chloride and
DI water for the subsequent step.
The refined cocoons are then diluted with water and calcium chloride into the
reactor at a 9:55:45
(w/w/w), cocoon:CaC12:DI water. When the refined cocoons are completely
immersed, the temperature is
raised to 100 C and the solution is stirred moderately for one hour. The
reactor temperature is then raised
to 120-125"C with steam and stirred for 6 to 7 hours, resulting in a viscous
slurry. Once the cocoon
25 material has completely solubilized, the solution is cooled down to 80-
90 C. The solution is then further
diluted with DI water, ¨1.3 times the initial amount, and then filtered with a
10um microfilter. The
filtered solution is desalinated using electric desalting equipment until the
salinity is less than 0.3%. The
refined protein is then sterilized at 95+3 C for 30 min, then cooled to 53-55
C.
The pH is adjusted to 6.5-7.0 by addition of either --6% Na0H(Aq) or acetic
acid. A 1% food grade
enzyme mixture of Aminopeptidases and Cellulase (w/w) is added to conduct the
enzymatic hydrolysis.
This solution is then held at a temperature of 53-55 C for 24 hours. The
resulting product is then sterilized
16
Date Recue/Date Received 2022-04-14
at 95+3 C for 30 min, which stops the enzymatic activity. The solution is then
cooled to 50+3 C. The
product is then concentrated through decompression, until the slurry viscosity
reaches a 25+2 brix% at
45+5 C, 650-700mmHg. The slurry is then sterilized again, to ensure enzyme
deactivation, at 95+3 C for
30 min and then allowed to cool to 60+3 C. The slurry is frozen at -18 C for
18 hours, and then
immediately moved to the dryer and allowed to dry at -40 C, 0.2-0.8 mmHg. Upon
completion of the
drying process, the dried product is milled and passed through a 40-mesh
screen to yield the powdered
purified Bombyx mori cocoon silk peptide fiber. Peptylin is then packed in
various sizes (lkg, 5kg, 10kg
or 20kg) and stored at room temperature in air tight containers.
Peptylin , in an embodiment, is a free-flowing light green to brown powder.
For Peptylin , in an
embodiment, a minimum of 95% fibroin content is confirmed via analysis of the
amino acid profile of the
product. In some batches tested after the above preparation, mercury and
cadmium were no more than
0.2ppm, and lead and arsenic were no more than 1.0ppm. During manufacture, a
small amount of free
amino acids may be released from the peptides, which include alanine and
tyrosine. As these free amino
acids present in higher concentrations compared to other amino acids, in an
embodiment, they may be
used as markers for chemistry and manufacturing controls. In an embodiment,
purified Bombyx mori
cocoon silk peptide fiber according to an embodiment of the present invention
has at least one or more of
the following characteristics; in an embodiment all of the following
characteristics: a light green to
brown powder by visual determination; not less than 90% crude protein, for
instance at least 95% or 99%;
not more than 10% moisture, for instance, not more than 3% or not more than 2%
or not more than 1%;
with heavy metals (Mercury (Hg), Lead (Pb), Cadmium (Cd), Arsenic (As)) for
instance each
undetectable by USP 261 or 233, or for instance each not more than 1 part per
million; tyrosine present in
amounts of about 2.0-12.0 mg/g (for instance, about 7 mg/g) and alanine
present in amounts of about 3.0-
15.0 mg/g (for instance, about 8 mg/g) as assayed by HPLC; and with a total
aerobic microbial count not
more than 1,000 cfu/g for instance as determined by USP 61 plate-count
methods; yeas and molds not
more than 100 cfu/g for instance as determined by USP 61 plate count methods;
with tests for salmonella,
E. coli, and S. aureus providing a negative result (not detectable), for
instance per USP 62 protocols for
each; and with aflatoxin summative amounts (B1, B2, Gl, G2) not more than 20
parts per billion.
Purified Bombyx mori cocoon silk peptide fiber according to an embodiment of
the present
invention is orally administered to a human as a dietary supplement in an
amount of about 200-600 mg
per day, with about 20mg as a lower range limit for daily administration, and
up to about 5000mg as a
daily upper range limit. Amounts greater than 5000mg, for instance 5000mg to
10,000mg, may be
included in a composition of this invention. Compositions prepared as a
discrete dose unit according to an
17
Date Recue/Date Received 2022-04-14
embodiment of the present invention may include about 50-800 mg purified
Bombyx rnori cocoon silk
peptide fiber; in an embodiment, about 20-2000 mg or about 20-40 mg of
purified Bombyx mori cocoon
silk peptide fiber; about 40-80 mg, about 50-100 mg, about 60-150mg, about 100-
200 mg, about 150-300
mg, about 200-600 mg, about 300-500 mg, about 400-800 mg, about 200-1000mg,
about 200-2000mg,
about 50-5000 mg, about 350-400 mg, about 100-1600 mg, about 3000-4000mg, and
the like of purified
Bombyx mori cocoon silk peptide fiber. In an embodiment, 400 mg of purified B.
mori cocoon silk
peptide fiber is included in a composition of this invention, and/or
administered as a daily dose. In an
embodiment, the above amounts are for daily administration of a composition of
the present invention.
"Refined Buglossoides arvensis Seed Oil" according to the present invention
refers to oil extracted
from Buglossoides arvensis seeds. In an embodiment, oil from Buglossoides
arvensis seeds may be
prepared by pressing and/or grinding and then extracting oil from the seeds,
for instance as known for such
and similar seeds in the art. In an embodiment, refined Buglossoides arvensis
seed oil is available for
instance under the tradenames Ahiflower or NeurXcel . The seeds are from
patented or patent-pending
varieties of the plant species Buglossoides arvensis which have uniquely
higher oil content, fatty acid
.. composition, and disease resistance than wild-type varieties of the same
species. The seeds are refined
according to proprietary manufacturing methods by Nature's Crops International
Ltd. (Kensington, PE,
Canada) or its authorized agents. While there are many rich sources of plant-
based omega-3 content
(notably flax, chia, perilla, sacha inchi oils), and while there are even
single-plant sources of omega-3-6-9
(notably hemp and echium seed oils), without being bound by theory, the
uniquely rich chemical content
of NeurXcel seed oil including its biologically advanced omega-3-6-9 content
contributes to NeurXcel
seed oil's unique and necessary contribution to the present invention.
NeurXcel seed oil according to an
embodiment of the present invention is standardized to contain about 17-24% of
stearidonic acid (c18:4, n-
3) and about 40-48% of alpha-linolenic acid (c18:3, n-3) and about 4% to about
8% gamma-linolenic acid
(c18:3, n-6).
In an embodiment of compositions of the present invention, NeurXcel Seed Oil
Modified ¨ Starch
SDA Powder (Nature's Crops International, Kensington, PEI, Canada) is used.
The powder contains
microencapsulated oil from the seeds of the Buglossoides arvensis plant in an
amount of 500 mg NeurXcel
seed oil per gram. The powder is described as white to off-white powder having
a pleasant aroma and flavor
and a particle size of 600um or less (100% of particles passing through US
Standard Sieve No. 30 (600um)).
The oil is described as rich in the essential fatty acids stearidonic acid
(SDA) and gamma linolenic acid
(GLA), and as having the following minimum fatty acid content: 18 mg/g
palmitic acid; 28 mg/g oleic acid;
42 mg/g linoleic acid; 21 mg/g Gamma-linolenic acid (GLA); 198 mg/g alpha-
linolenic acid (ALA); 80
18
Date Recue/Date Received 2022-04-14
mg/g stearidonic acid (SDA). In addition, the powder is described as having
less than 5% moisture, cold
water dispersible, less than 1000 cfu microbial total plate count/gram, less
than 100 cfu yeast/mold per
gram, less than 10 cfu coliforms/gram, and negative for E. coli and Salmonella
spp. in 10 grams of the
powder. In an embodiment, refined Buglossoides arvensis seed oil of this
invention includes more than
80% of combined alpha-linoleic acid (c18:3, n-3), stearidonic acid (c18:4, n-
3), and gamma-linolenic acid
(c18:3, n-6) as part of the total ingredients comprising omega 3-6-9 fatty
acids in the composition.
In another preferred embodiment, NeurXcel Seed Oil (Nature's Crops
International, Kensington,
Canada) is used. The fatty acid profile disclosed for the oil is as follows:
Stearidonic Acid C18:4 (SDA):
17-21%; Gamma Linolenic Acid C18:3 (GLA): 4.5-8%; Alpha-Linolenic Acid C 18:3
(ALA): 42-48%;
Palmitic Acid C16:0: 4-7%; Oleic Acid C18:1: 6-14%; Linoleic Acid C18:2: 9-
15%.
In an embodiment, refined Buglossoides arvensis seed oil powder (e.g. NeurXcel
seed oil powder
CWD, the Wright Group, Crowley LA) according to the present invention includes
one or more, and
preferably all, of the following characteristics: a fine white to off-white
powder having a particle size in
which 100% of particles pass through a U.S. Standard Sieve No. 30 (600um),
dispersible in cold water,
having 50% B. arvensis seed oil including 25% alpha linolenic acid (ALA), 8%
stearidonic acid (SDA),
and 3% gamma linolenic acid (GLA). Additional ingredients may include modified
starch, corn syrup
solids, antioxidants, and natural flavors; moisture is less than 5%.
In the present invention, NeurXcel Seed Oil is preferably orally administered
as a dietary
supplement in an amount of about 20mg -10,000 mg oil per day, preferably about
100-10,000 or about 100-
2500 mg per day, with about 100 mg as a preferred lower range limit for daily
administration, and about
10,000 mg as a daily preferred upper range limit. In an embodiment,
compositions prepared as a discrete
dose unit according to the present invention may include for instance about
500-5,000 mg NeurXcel Seed
Oil; about 20-250 mg of NeurXcel Seed Oil; about 40-500 mg, about 100-600 mg,
about 60-750mg, about
100-1250 mg, about 150-1900 mg, about 200-2500 mg, about 400-600 mg, about 300-
700mg, or about
400-5,000 mg of NeurXcel Seed Oil. In an embodiment, a composition of the
present invention includes
about 500 mg B. arvensis oil, or about 500 mg B. arvensis oil in powdered form
(for instance having about
250 mg oil per 500 mg powder). The above references to B. arvensis oil may
also be applied to oil in
powdered form, for instance as discussed throughout this application. In
embodiment, the above amounts
are for daily administration of a composition of the present invention.
"Blueberry extract" according to the present invention refers to an extract
obtained from blueberries
(Vaccinium spp.; i.e. from species of the genus Vaccinium). In an embodiment,
blueberries may be disrupted
19
Date Recue/Date Received 2022-04-14
for instance by crushing or pureeing and then water or another liquid applied
to extract substances from the
blueberries, for instance as known for blueberries and other berries in the
art. In an embodiment, a
36:1 standardized blueberry extract (36 grams of blueberries correspond to 1
gram of blueberry extract) is
used. In another embodiment, a 75:1 standardized blueberry extract (75 grams
of blueberries correspond
to 1 gram of blueberry extract) is used. (BLUE D'oR, Villeroy, Canada).
Another Blueberry extract of this
invention is the American Blueberry (Vaccinium corymbosum) extract
(VitaBlue8), a powder from
Futureceuticals.
In an embodiment, a composition of the present invention includes about 25-
2000 mg Blueberry
extract, including for instance about 50 mg to about 150 mg or about 50 mg to
about 2000mg, or about 100
mg to about 500 mg Blueberry extract. Similarly, a Blueberry extract of this
invention is administered in
an amount of about 50 to about 500 mg per day, for instance in an amount of
about 50 mg ¨ about 2000
mg, or about 50-500mg, or about 100 to about 150 mg per day.
Recent work to develop Haskap blue honeysuckle (Lonicera caerulea) berries in
North America
and Northeastern Asia, containing levels of bioavailable anthocyanins
considerably higher than typical
blueberries, and with indications of memory enhancement properties, includes
this species of berries and
preparations thereof as a potential replacement for blueberry extract in the
present invention, or as an
additional preferred ingredient. However, this inclusion is not intended to
indicate that anthocyanins alone
provide the beneficial, synergistic effects seen with blueberry extract in
this application. Rather, as
discussed in the Examples, concentrated wild Canadian blueberry powder
performed very well in tests of
the present invention as compared with concentrated American blueberry,
although the Canadian blueberry
powder contains approximately 120x fewer anthocyanins than the American
blueberry powder.
Other ingredients
Compositions of the present invention, in addition to purified Bombyx mori
cocoon silk peptide
fiber, refined Buglossoides arvensis seed oil, and optionally blueberry
extract, in other embodiments may
further comprise one or more of the following ingredients: Acetyl-L-Carnitine,
L-theanine, L-serine, Zinc
(as zinc glycinate, zinc gluconate, zinc citrate, or zinc picolonate),
Huperzine A extracted from Huperzia
chinensis, Bacopa monnieri extract, Ginseng Extracts, Citicoline, Ginger
(Zingiber officinalis) Extracts,
Ginkgo biloba extract, Folic acid, Vitamin B12, Vitamin B6, Vitamin Bl, and/or
Vitamin D3
(cholecalciferol), Green Mountain Tea (Sideritis app.), Lion's Mane mushrooms.
Each of these is briefly
.. discussed below, some in preferred groupings. Other ingredients are also
noted throughout the application.
Multipath Memory Grouping
Date Recue/Date Received 2022-04-14
Each of the ingredients discussed below ¨ Acetyl-L-Camitine, Huperzine A
extracted from
Huperzia chinensis, Bacopa monnieri extract, Ginseng Extracts, Citicoline,
Ginkgo biloba extract ¨ may
be added to a composition according to the present invention as a preferred
embodiment. However, in
another preferred embodiment, all of these ingredients would be administered
together as part of the
composition of the present invention, for instance in the same discrete dose
unit, or for instance in separate
dose units on the same day, in the preferred dosages provided below.
Acetyl-L-Carnitine
Acetyl-L-carnitine is a substance which is naturally produced and used in the
body. Supplements
are therefore taken when it is deficient. This substance maximizes the
generation of neural energy in the
neural cells and, as an antioxidant, it plays various roles, including
cellular recycling and respiration of the
mitochondria. Also, acetyl-L-carnitine increases nerve growth factor (NGF)
value, which is an important
brain recovery complex. NGF protects cholinergic neurons in the central
nervous system and helps to
provide the appropriate level of choline acetyltransferase (ChAT).
Furthermore, it has been used to counter
neurodegenerative diseases as a medical product in several countries,
including Korea. Acetyl-L-camitine
showed a beneficial effect on both cognitive impairment and early-stage
Alzheimer's Disease in double-
blind, placebo-controlled clinical studies. Useful results were seen in the
Logical Memory, Trail Making
Test, Hooper test, and brain waves in a study on patients (severely epileptic
patients) compared to the
placebo group.
In the present invention, acetyl-L-carnitine is preferably administered in an
amount of about 200-
2000 mg per day; more preferably, about 300-800 mg per day. Compositions of
the present invention
preferably include this dosage.
Bacopa monnieri extract
Bacopa is one of the most frequently used health/functional food ingredients
for memory
improvement. It has previously been employed in India and Southeast Asia. The
main active ingredients of
the Bacopa extract are bacosides A and B, which have a variety of memory-
related mechanisms. They are
known to inhibit acetylcholinesterase (AchE), activate choline
acetyltransferase (ChAT), and promote
antioxidant activity and brain blood flow. The functions of the bacopa extract
are supported by extensive
clinical evidence. For example, while a previous study on the memory
improvement of healthy elderly
people reported it to significantly improve memory gain and maintenance,
another on seniors suggested it
inhibits plasma AchE activity, resulting in improvements in attention,
cognitive processing, and working
memory. However, some studies mention its low solubility and emphasize the
need for further research to
21
Date Recue/Date Received 2022-04-14
improve its bioavailability. In a composition of the present invention, the
standardized bacopa extract
product obtained from the Bacopa monnieri root and that contains 20-60%
Bacosides content, is
appropriate.
In the present invention, Bacopa monnieri extract is preferably administered
in an amount of about
50-300 mg per day; more preferably, about 100-200 mg per day. Compositions of
the present invention
preferably include this dosage.
Ginseng Extracts
Ginseng extract is traditionally used in the East and is mainly comprised of
saponin glycosides. It
is reported to have a variety of pharmacological roles, including anti-
fatigue, work performance
enhancement, and hypoglycemic agent functions. Ginsenosides, such as Rb 1, are
generally contained in
Panax ginseng extract and are recognized to promote the choline
acetyltransferase (ChAT) activity, which
is important for acetylcholine synthesis. A major clinical study on memory
improvement associated with
the ginseng comprises Ginkgo biloba extract, which may be included in
compositions of the present
invention, which was successful in 2506 subjects. In concordance, it was also
found to help working
memory. Similarly, American ginseng was described to improve working memory
and calmness in people.
In this composition, Panax ginseng extract obtained from either the Panax
ginseng or the Panax
quinquefolius roots is appropriate.
In the present invention, ginseng extract is preferably administered in an
amount of about 100-500
mg per day; more preferably, about 100-200 mg per day. Compositions of the
present invention preferably
include this dosage. Also, Ginkgo biloba extract may be used in an amount for
instance as in the major
clinical study mentioned above.
Citicoline
Citicoline has been widely used as a nutritional supplement ingredient for
memory improvement
as a substance that naturally occurs within the cells in the body. Citicoline
is known to help memory in
various ways, such as through its neural protection effects, increases in the
amount of choline used in
acetylcholine synthesis, and in improvement in cellular communication by
increasing the utility of human
neurotransmitters. Previous clinical studies showed it to improve memory in
elderly people. Specifically,
participants who consumed drinks including citicoline were found to have a
much faster maze learning
time, error reduction, and a higher information processing speed.
22
Date Recue/Date Received 2022-04-14
In the present invention, citicoline is preferably administered in an amount
of about 250-2000 mg
per day; more preferably, about 300-1000 mg per day. Compositions of the
present invention preferably
include this dosage.
Ginkgo biloba extract
Ginkgo biloba extract is a material that is obtained from the leaves of the
Ginkgo tree, then
processed and standardized. It has been widely used for decades to treat brain
function impairments,
memory loss, and dizziness.
Ginkgo extract is recognized to protect brain cells and to promote brain blood
flow. Additionally,
it participates in the pre-synaptic choline uptake and acetylcholine release,
while it upregulates the post-
synaptic acetylcholine muscarinic receptor. Although research on some small
partial effects of Ginkgo
exists, most investigations reported positive results. Similarly, although
several clinical studies are present,
all the referred papers consistently show that Ginkgo helps memory
improvement. In particular, a previous
clinical study indicated a significant memory improvement effect from low
doses of 3 administrations of
19.2 mg per day. Moreover, it is categorized as a medical product in Korea and
recognized as effective on
memory reduction and for attentional disorders. Ginkgo extract may be obtained
from Ginkgo biloba leaf,
purified, and standardized. In an embodiment of this invention, a material
that contains about 24% Ginkgo
flavonoids and 6% triterpene lactones, which is appropriate under the USP
pharmacopeia, is used.
In the present invention, Ginkgo biloba extract is preferably administered in
an amount of about
60-240 mg per day; more preferably, about 100-150 mg per day. Compositions of
the present invention
.. preferably include this dosage.
Active Vitamin Blend
This is a blend of water-soluble vitamins comprised of Folic acid, Vitamin
B12, Vitamin B6, and
Vitamin B1, which affects memory and neural protection both directly and
indirectly. Specifically, the
active form of each vitamin with a high absorption rate and support for brain
performance is desirable.
Furthermore, the blend above evenly blocks the homocysteine synthesis
mechanism, which has adverse
effects on many organs (including the brain) and efficiently blocks
homocysteine, which is a known cause
of neural brain damage, delays communication between cranial nerves, and brain
contraction. It can be an
essential prescription either for people with weak homeostenosis at stressful
situations or elderly people.
Finally, it can be used as part of a composition of the present invention in
combination with the Multipath
Blend above, or without.
23
Date Recue/Date Received 2022-04-14
Each of the ingredients discussed below ¨ Folic acid, Vitamin B12, Vitamin B6,
and Vitamin B1 ¨
may be added to a composition according to the present invention as a
preferred embodiment. However,
in another preferred embodiment, all of these ingredients would be
administered together as part of the
composition of the present invention, for instance in the same discrete dose
unit, or for instance in separate
dose units on the same day, in the preferred dosages provided below.
Active Folic acid (5-Methylfolate)
Folic acid, also referred to as vitamin B9, is a water-soluble vitamin that
participates in red blood
cell synthesis, nucleic acid synthesis, and fetal development. Deficiency can
occur due to malabsorption,
low consumption, and an increase in demand. Folic acid plays a role in
homocysteine methylation,
providing the methyl group that converts methionine into s-adenosyl methionine
during brain function and
reducing homocysteine. Moreover, a previous clinical study described folic
acid administration tends to
improve memory and attention. In contrast, subjects with low folic acid values
showed impairments in both
word and object recall tasks. Supplementation of vitamin B12, vitamin B6, and
folic acid had a positive
impact on partial memory capacity measures. In this composition, folic acid
can either be present in its
natural condition (i.e., polyglutamate folate), in its synthetic state (i.e.,
monoglutamate folate) or its active
form (i.e., 5-methyltetrahydrofolate). Preferably, 5-methyltetrahydrofolate is
used.
In the present invention, folic acid is preferably administered in an amount
of about 0.065-1 mg
per day; more preferably, about 0.4 mg per day. Compositions of the present
invention preferably include
this dosage. Also, the active form of folic acid (5-Methylfolate) is
preferably administered in an amount of
about 1-15 mg per day; more preferably 2-7.5 mg dosage per day.
Active Vitamin B12 (Cobalamin)
Vitamin B12, also referred to as cobalamin, is a type of water-soluble vitamin
that plays various
roles, including metabolic support, generation of neurons, DNA, RNA, and red
blood cells, prevention of
dementia, and mental health. Deficiency in vitamin B12 commonly occurs in
people with an unhealthy diet,
elderly people, and vegetarians. Vitamin B12 functions as an essential co-
factor of the one-carbon cycle for
the synthesis of neurotransmitters, such as acetylcholine. It activates
methionine synthase within the
methionine cycle and reduces the amount of homocysteine. A clinical study
revealed that low consumption
of vitamin B12, vitamin B6, and folic acid could increase the probability of
MCl/dementia. Additionally, a
low concentration of vitamin B12 is associated with low memory capacity. In
this composition, vitamin
B12 can be used in either its general forms (i.e., cyanocobalamin and
hydroxycobalamin) or active forms
24
Date Recue/Date Received 2022-04-14
(i.e., methylcobalamin and adenosylcobalamin). Methylcobalamin has the highest
biological activity and is
preferably used.
In the present invention, vitamin B12 in its active form, methylcobalamin, is
preferably
administered in an amount of about 0.5-6 mg per day; more preferably, about 1-
4 mg per day. Compositions
of the present invention preferably include this dosage.
Active Vitamin B6 (Pyridoxal 5'-phosphate)
Vitamin B6, also referred to as Pyridoxine, is a water-soluble vitamin that
participates in the
metabolism of nutrients, such as several amino acids, the synthesis of red
blood cells and neurotransmitters,
and in gene expression. Although rare, vitamin B6 deficiency can occur due to
hyperthyroidism, excessive
consumption of proteins, and abuse of antibiotics. Vitamin B6 is an essential
cofactor of the folate cycle
and activates cystathionine 0-synthase, which synthesizes the generated
homocysteine into attenuated
substances, such as cysteine. With regards to the clinical evidence, clinical
evaluation described vitamin
B6 supplements to improve information retention considerably. In fact, vitamin
B12, vitamin B6, and folic
acid supplements were shown to have a considerable positive influence on some
memory capacity
measures. Low consumption of vitamin B12, vitamin B6, and folic acid can
increase the probability of
MCl/dementia. In this composition, Vitamin B6 can be used as pyridoxine,
pyridoxal, pyridoxamine, or the
phosphorylated foun of each. Desirably, Pyridoxal 5'-phosphate, which has the
highest biological activity,
should be used.
In the present invention, vitamin B6 in its active forms (Pyridoxal 5'-
phosphate) is preferably
administered in an amount of about 20-100 mg per day; more preferably, about
30-80 mg per day.
Compositions of the present invention preferably include this dosage.
Active Vitamin B1 (Benfotiamine)
Vitamin B1, also referred to as thiamine, is a supportive enzyme that
participates in many stages
of body sugar metabolism by converting it into thiamine pyrophosphate.
Considering that all cells require
energy, thiamine deficiency can affect all organs. However, thiamine is not
produced by the body and does
not have a large reservoir. Therefore, its low consumption can result in
thiamine deficiency within a few
weeks to months. The role of thiamine in the brain in the generation of
pyruvate dehydrogenase, an essential
enzyme in the generation of acetylcholine, is of crucial importance. Wemicke
syndrome is a nervous system
disorder related to thiamine deficiency which results in the paralysis of the
eye muscles due to functional
impairments in the diencephalon and midbrain, decreased ability to walk, and
dysfunction in consciousness.
This condition progresses to the Korsakoff syndrome if not treated promptly,
which is a continuous memory
Date Recue/Date Received 2022-04-14
and learning disorder. In this composition, Vitamin B1 has the general form of
thiamine as well as the active
forms of benfotiamine and fiusultiarnine. Preferably, the active form is used.
In the present invention, vitamin B1 in its active form (Benfotiamine) is
preferably administered in
an amount of about 5-100 mg per day; more preferably, about 25-50 mg per day.
Compositions of the
.. present invention preferably include this dosage.
COMPOSITIONS
Compositions of the present invention are defined throughout the entire
application.
Compositions of the present invention are shown in the various Examples to
provide synergistic effects.
The lack of a specific assertion of synergy or synergistic effect this
application or lack of a notation of
statistical significance including in any given Example is not intended to
indicate a lack of synergy or
statistical significance, unless expressly indicated as such. Further
statistical analyses may be carried out
based on the data in the Examples as needed.
A composition according to the present invention comprises purified Bombyx
mori cocoon silk
peptide fiber and refined Buglossoides arvensis seed oil, and optionally also
comprises blueberry extract.
In one embodiment, the purified Bombyx mori cocoon silk peptide fiber is
Peptylin , the refined
Buglossoides arvensis seed oil is NeurXcel Seed Oil in the form of a micro-
encapsulated powder
providing a 50% NeurXcel oil payload in a modified starch matrix or is in the
foim of liquid oil. The
Blueberry extract is North American and/or wild Canadian blueberry extract, in
some embodiments of
this invention. Other pharmaceutically acceptable ingredients or additives may
also be included in the
present compositions, for instance as described throughout this application.
A composition of the present invention may include refined Buglossoides
arvensis seed oil to
purified Bombyx mori cocoon silk peptide fiber in a mass balance ratio of from
about 0.1: 1 to 10: 1. In
another embodiment, said ratio is from about 0.4: 1 to 5:1. In another
embodiment, said ratio is about 2.5
: 1 to 0.625:1. This factors in non-active micro-encapsulation carriers. In
another embodiment, said ratio
of active ingredients is about 0.1 : 1, 0.4 : 1, 0.625 : 1,2.5 : 1, or 5:1.
Ratios of blueberry powder w/w to the other capsule ingredients (e.g. B. mori
fiber and B.
arvensis oil) may be in a ratio of about 1:20 to about 1:6 (about 5.3% w/w to
about 16.7%) in BrainiLex
powder. In the Braini powder, typically the blueberry extract powder is about
4%. In general, in a
composition of this invention, a blueberry extract powder may be about 2% w/w
of the composition to
about 40%.
26
Date Recue/Date Received 2022-04-14
In an embodiment of the present invention, a composition for oral
administration comprises the
following: purified Bombyx mori cocoon silk peptide fiber (Peptylin ) and
refined Buglossoides arvensis
seed oil, optionally Blueberry Extract, as well as one or more of the
following: Bacopa monnieri extract,
Huperzine A extracted from Huperzia chinensis, Acetyl-L-carnitine, Panax
ginseng extract, Citicoline,
Ginkgo biloba extract, active Folic acid (5-Methylfolate), active Vitamin B12
(Cobalamin), and active
Vitamin B6 (Pyridoxine aka Pyridoxal 5'-phosphate); in another embodiment, all
of the above are included
in the composition.
In an embodiment of the present invention, a composition for oral
administration comprises the
following: purified Bombyx mori cocoon silk peptide fiber (Peptylin ) and
refined Buglossoides arvensis
seed oil, optionally, wild Canadian Blueberry (Vaccinium angustifolium)
extract, as well as one or more of
the following: rice starch, maltodextrin, vegetable cellulose (if the
composition is formulated in capsule
form), vegetable starch, corn syrup solids, natural flavor, mixed tocopherols,
ascorbyl palmitate; in another
preferred embodiment, all of the above are included in the composition. In
other embodiments, such as the
Braini formulation discussed in the in vivo Examples, other ingredients may
include non-GMO modified
food starch, non-GMO corn syrup solids, rice starch, vegetable cellulose
(capsule shell), rosemary extract
(anti-oxidant), natural tocopherols (anti-oxidant), ascorbyl palmitate (anti-
oxidant), natural flavors.
In an embodiment of the present invention, a composition for oral
administration to improve
cognitive performance among dyslexic and/or dyspraxic individuals comprises
the following: purified
Bombyx mori cocoon silk peptide fiber (Peptylin ) and refined Buglossoides
arvensis seed oil, wild
Canadian Bluberry extract or North American Blueberry (Vaccinium corymbosum)
extract, as well as one
or more of the following: Zinc (as zinc citrate, zinc glycinate, zinc
gluconate, or zinc picolonate); and
docosahexaenoic acid (DHA) derived from Schizochytrium spp algae.
In an embodiment of the present invention, a composition comprises 400mg
Peptylin , 500 mg
NeurXcel seed oil in powdered form, and 100 mg Blueberry extract. In an
embodiment, this composition
is administered, for instance in one or more discrete dose units, daily to a
subject.
These and other preferred components are more clearly defined below, and the
basis of the
pharmacological effects of each component on memory improvement is mentioned
based on clinical trials.
Table 1 discloses some embodiments of compositions of the present invention:
TABLE 1
Servings Scale
Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr.
2-4/day
(mg/capsule) 8 9 10 11 12 13 14 15 16 17
27
Date Recue/Date Received 2022-04-14
Compositions PEPTYLIN
100 200 100 100 100 200 200 200 100 100
of present NeurXcel seed oil 125 250 125 125
125 250 250 250 125 125
invention Blueberry
25 50 25 25 25 50 50 50 25 25
Multipath Acetyl-L-carnitine 125 125 125 125
125
Memory Bacopa 50 50 50 50
50 50
Blend Panax Ginseng 50 50 50 50 50
50
Citicoline 125 125 125 125
125
Ginkgo extract 30 30 30 30 30
30
Active 5-Methylfolate 1.25 2.5 1.25 2.5
1.25 1.25
Vitamin Methylcobalamin 0.75 1.5 0.75 1.5
0.75 0.75
Blend Pyridoxal 5'-phosphate 15 30 15 30
15 15
Benfotiamine 12.5 25 12.5 25
12.5 12.5
Excipients MCC
qs qs qs qs qs Qs qs qs qs qs
Silicon dioxide
Magnesium stearate
HPMC capsule
Others
The above preparations ("Pr.") represent compositions according to the present
invention. Preparations
described herein are not intended as limiting. Various other preparations are
possible according to the
present invention, including the full variety of dosing ranges indicated
above.
A composition according to the present invention is in one embodiment orally
administered. A
composition of the present invention may be formulated into nutraceutical or
pharmaceutical dosage
forms comprising for instance tablets, capsules, powders, liquids, chewables
such as gummies,
transdermals, injectables, dietary supplements, topical creams, lozenges,
pills, and so forth. In one
particular embodiment, a composition of the present invention is formulated
into a gummi blend. In
another embodiment, a composition is formulated into a dual capsule form,
comprising powdered active
ingredient(s) in an inner capsule, surrounded by liquid ingredient(s) in an
outer capsule. A composition of
the present invention may further comprise one or more excipients, additives,
and/or other substances.
In an embodiment, a composition of this invention is a synergistic
composition, Braini .
Braini is a bulk blended dietary supplement powder comprising Peptylin
powder (with 1 gram of
Braini powder providing about 400 mg Peptylin( ); NeurXcel oil
microencapsulated powder (with 1
gram of Braini powder providing about 500 mg NeurXcel oil encapsulated
powder); and Blueberry
extract powder (Vaccinium corymbosum) (with 1 gram of Braini powder providing
about 100 mg
Blueberry extract powder, BLUEd'Or, with a ratio of 75:1 frozen fresh
blueberries:powder). 500 mg
NeurXcel microencapsulated powder includes about 250 mg NeurXcel oil and
about 250mg of
microencapsulated powder composed primarily of modified food starch, corn
syrup solids, antioxidants,
28
Date Recue/Date Received 2022-04-14
and natural flavors. 1 gram of the Braini powder contains a complete daily
intake of plant-based omega-
3-6-9 fatty acids in a daily dose.
Compositions of the present invention provided synergistic results in vitro as
discussed for
instance throughout this application and Examples 1-13. Also, synergistic
effects of the present invention
are apparent for instance as compared with a Korean study which, evaluating
Peptylin (BF-7) as
compared with placebo, showed improved CTT (reaction time) scores in healthy
children. BF-7 achieved
a 23% improvement in reaction time compared with placebo (p<0.05). By
comparison, Example 19 of the
present application shows that in the administration of Braini to healthy
young adults, 11 of 13 subjects
receiving active product improved their SAT-RT (shifting attention test,
correct response reaction time)
by a group average of 84 milliseconds, compared to the placebo group in which
only 10 of 18 subjects
improved their SAT-RT for a group average of only 2.5 milliseconds. The
improvement in the active
Braini cohort was highly statistically significant (p<0.007). As a numeric
percentage improvement
compared with the placebo cohort, 84 milliseconds vs. 2.5 milliseconds is a
remarkable improvement in
reaction time, greater than 3300%. Although the CNS Vital Signs test suite is
different than the Color
Trails Making Test used in the BF-7 study, both test suites are well-
recognized standardized measures of
cognitive performance. Further, the BF-7 and Braini trails used the same
active dose of BF-7 (aka
Peptylin ) at 400 mg/day, however the BF-7 trial was carried out over 16
weeks, while the Braini trial
was carried out over only 4 weeks. This outcome shows that Braini
synergistically outperforms BF-7
alone in terms of standardized controlled clinical trial reaction time
outcomes compared with placebo.
As mentioned above, in an embodiment, Braini powder is enclosed in capsules
("Braini
capsules"). In an embodiment, the capsules are made of HPMC (hydroxypropyl
methylcellulose), and
hold about 500 mg of powder, so that an average daily dose for a human adult
(1 gram Braini powder)
may be administered with 2 capsules/day. In an embodiment, the Braini powder
is added to capsules in
smaller amounts, so that 3 or 4 capsules/day are needed to administer the
above daily amount, but also so
that smaller amounts may be administered for instance to children or others
that may be administered a
smaller dose. Braini capsules were orally administered to humans in all in
vivo Examples below,
providing daily amounts of 400 mg Peptylin , 500 mg NeurXcel
microencapsulated powder (including
250 mg NeurXcel seed oil), and 100 mg Vaccinium corymbosum Blueberry extract
powder (BLUEd'Or,
with a ratio of 75:1 frozen fresh blueberries:powder). As indicated above, a
daily dose of a composition
of this invention may be greater than 1 gram, for instance to accommodate
increased doses of required
ingredients (B. mori fiber (Peptylin and NeurXcel oil) and/or other
ingredients, excipients, and the
like. In an embodiment, Peptylin and Blueberry extract of this invention are
at least 95% pure,
preferably at least 97-99% pure, without additional ingredients.
29
Date Recue/Date Received 2022-04-14
In an embodiment, a composition of this invention is a synergistic
composition, "Braini Lex .
Braini Lex is a bulk blended dietary supplement powder comprising Peptylin
powder (about 400 mg
Peptylin(0 per recommended daily dose); NeurXcel microencapsulated powder
(about 500 mg
NeurXcel oil encapsulated powder per recommended daily dose); Blueberry
extract powder (Vaccinium
corymbosum) (about 100 mg Blueberry extract powder per recommended daily dose;
where the extract
powder is BLUEd'Or, with a ratio of 75:1 frozen fresh blueberries:powder); as
well as about 100 mg algal
docosahexaenoic acid (DHA) and about 25 mg zinc glycinate. 500 mg NeurXcel
microencapsulated
powder includes about 250 mg NeurXcel oil and about 250mg of
microencapsulated powder composed
primarily of modified food starch, corn syrup solids, antioxidants, and
natural flavors. The above amounts
represent a preferred daily dose of Braini Lex for a human adult. 500 mg
NeurXcel
microencapsulated powder includes 250 mg NeurXcel 1) oil and 250mg of
microencapsulated powder
composed primarily of modified food starch, corn syrup solids, antioxidants,
and natural flavors. The
Braini Lex powder contains a complete daily intake of plant-based omega-3-6-9
fatty acids in a daily
dose.
In an embodiment, Braini Lex powder is enclosed in capsules ("Braini
capsules"); for
instance HPMC capsules as discussed above, with total powder taken as a daily
dose adjusted to
accommodate the addition of algal DHA (e.g. Algarithm) and zinc glycinate
(e.g. Novotech
Nutraceuticals).
In an embodiment, a composition of the present invention (including but not
limited to Braini
powder, Braini capsules, Braini Lex powder, Braini Lex capsules) may be
administered in an
amount of about 10mg-600 mg total composition per kg body weight. In an
embodiment, bulk powders
are administered in amounts of about 50-600 mg/kg body weight, and in another
embodiment, capsules
are administered in amount of about 12.5 to 150 kg/body weight. The upper
limits to these ranges may
include for instance about 3 tablespoons of Braini or Braini Lex bulk powder
in a child's daily dose.
A child's daily dose may be the size of an adult dose or, for instance a
reduce amount, such as about 10%
to 90%, about 30%-70%, or about 50% of an adult dose.
In an embodiment, a composition of the present invention includes an
agglomerate of purified
Bombyx mori cocoon silk peptide fiber and refined Buglossoides arvensis seed
oil, and optionally a
Blueberry Extract. In an embodiment, the agglomerate is of purified Bombyx
rnori cocoon silk peptide
fiber in powdered form, refined Buglossoides arvensis seed oil in
microencapsulated powdered form, and
Blueberry Extract in powdered form, for instance as used in the Braini
compositions described above.
In an embodiment, the agglomerate is formed by mixing the above-mentioned
constituents together, such
that the agglomerate shows chemical and physical changes and different
properties from the constituents
Date Recue/Date Received 2022-04-14
alone. Agglomerates according to the present invention are shown for instance
by scanning electron
micrograph and HPLC mass spectrometry, discussed in Examples 22 and 23, below.
In an embodiment, a composition of the present invention has a shelf-life of
about 1-2 years. In
an embodiment, the shelf-life for Braini or Braini Lex powder or capsules is
about 18 months.
The present invention may be further understood in connection with the
following Examples and
embodiments. The following non-limiting Examples and embodiments described
throughout this
application are provided to illustrate the invention.
EXAMPLES
The aforementioned experimental ingredients were confirmed to result in memory
improvements
in clinical trials. The inventors have conducted in vitro and in vivo
experimental trials to understand
optimal interactions and synergies in various formulated compositional ratios,
as shown in the following
examples.
IN VITRO TESTS
Examples 1-5 disclose the first scientific finding of human SH-SY5Y neural
cell challenge
recovery improvements resulting from combining purified Bombyx mori cocoon
silk peptide fiber with
refined Buglossoides arvensis seed oil and other components. The use of human
SH-SY5Y cell cultures
as models for neurological systems and for instance neurodegenerative
disorders has been published. [71-
74]. Please also refer to references listed below.
The tests in Examples 1-5 were designed to mimic the effects of excessive
oxidation of human
neural cells, and assess neuroprotective or neurotoxic action by compositions
defined by Formulas A-E.
Formulas A-E are as shown in Table 2 for Examples 1-5. Formulas B-E describe
compositions according
to the present invention. The formulation is liquid. Peptylin , Aframomum
melegueta extract, and
Blueberry Extract are powders, and refined Buglossoides arvensis seed oil is
liquid. For example, for
Sample A, 50mg is prepared by dissolving in 1 ml DMSO. Dilute with Media to
set final test
concentration. The above method is common in in vitro tests using cell lines.
EXA11vIPLE 1
Cell viability test
SH-SY5Y (neuroblastoma) cells, from a human body-derived cell line obtained
from Korean Cell
Bank (Seoul, South Korea) were cultured in 100 ml 10% FBS/MEM (Gibco, US),
5x104 cells/well in 96-
well plates for 24 hours (37 C, 5% CO2). The 10% FBS/MEM was removed and 1%
FBS/MEM added to
the cells. Composition A, B, C, D, or E was administered. The concentration of
each component of
31
Date Recue/Date Received 2022-04-14
Compositions A-E is listed in Table 2 below. Normally, cells are grown for 1-2
days in 10% FBS/MEM.
Subsequently, it is replaced with 1% FBS/MEM and stabilized for one day before
processing samples.
Cell viability studies were conducted using MTT colorimeter analysis. Cells
were cultured for four
hours after the administration of the MTT solution (3-(4,5-dimethylthiazol-2-
y1)-2,5-diphenyltetrazolium
bromide) (Invitrogen/ThermoFisher, Carlsbad CA, USA). Because of an enzyme in
the mitochondria of a
surviving cell, tetrazolium unlinks and changes into formazan. The resulting
color significantly correlates
to the number of living cells, with a deeper/darker color indicating more
viable cells than a lighter color.
Color was quantified by measuring absorbance with a 570 nm
spectrophotometer/well-plate reader.
Amounts of sample treatment applied were lOul per well.
TABLE 2
Compositions
PEPTYLIN
(Purified Bombyx Aframomum
B. arvensis Blueberry Extract
Composition mori cocoon silk melegueta Extract
peptide fiber)
Seed Oil (m (pig)) 6-1g)
(-1,g)
Form. A 20 0 0 0
Form. B 20 50 2.5 0
Form.0 20 50 5 0
Form.D 20 50 0 2.5
Form.E 20 50 0 5
In Fig. 1, dilutions are shown in column bars from left to right: 1.00
(undiluted composition), 0.500,
0.250, 0.125, 0.0625. Dilutions were based on the above composite;
compositions re-generated by
concentration. As shown in Fig. 1, the results indicated no cytotoxicity was
present in any of the dilution
ratios (1 - 0.0625) of Compositions A-E.
EXAMPLE 2
Evaluation of improvement in SH-SY5Y Neuroblastoma cell viability in the
presence of
compositions of the present invention, in an oxidative environment (H202
exposure)
SH-SY5Y neuroblastoma cells were incubated with 100 .1 of 10% FBS/MEM in a
5x104 cells/well
96-well plate and cultured 24 hours, as in Example 1. The 10% FBS/MEM was
removed and 1% FBS/MEM
added to the cells. Composition A, B, C, D, or E was administered to the
wells. Normally, cells are grown
32
Date Recue/Date Received 2022-04-14
for 1-2 days in 10% FBS/MEM. Subequently, it is replaced with 1% FBS/MEM and
stabilized for one day
before processing samples. Cell room condition is 37 C, 5% CO2.
After 4 hours, H202 (a toxicity-inducing substance) was administered. In this
experiment, 0.25 uM
was treated and the MI __ -1 test solution used to evaluate cell viability, as
in Example 1. MU assay was
performed by adding MIT solution without removal.
In Fig. 2, dilutions are shown in column bars from left to right: 1.00
(undiluted composition), 0.5,
0.25. An "*" indicates statistical significance and synergistic effect when
compared with Formulation A;
p<0.05 by Student's t-test. As shown in Fig. 2, Formulas B-E showed improved
cell viability compared to
Formula A, with Formulas D and E showing statistically signifcant improvement
as compared with Folinula
A across 2 dilutions, and Formula B showing statistically significant
improvement across 1 dilution. An
increase in cell viability in an oxidative environment signifies that brain
cells can be protected through an
antioxidant mechanism.
EXAMPLE 3
Evaluation of improvement in SH-SY5Y Neuroblastoma cell viability in the
presence of
compositions of the present invention, in an oxidative environment (FeSO4
exposure)
SH-SY5Y neuroblastoma cells were incubated with 100 l of 10% FBS/MEM in a
5x104 cells/well
96-well plate and cultured 24 hours, as in Example 1. The 10% FBS/MEM was
removed and 1% FBS/MEM
added to the cells. Composition A, B, C, D, or E was administered to the
wells.
After 4 hours, FeSO4 (a toxicity-inducing substance) was administered. In this
experiment, 2.5 uM
was treated and the MTT test solution used to evaluate cell viability, as in
Example 1. MTT assay was
performed by adding MI __ -1 solution without removal.
In Fig. 3, dilutions are shown in column bars from left to right: 1.00
(undiluted composition), 0.5,
0.25. An "*" indicates statistical significance and synergistic effect when
compared with Formulation A;
p<0.05 by Student's t-test. As shown in Fig. 3, compositions B-E showed
improved cell viability compared
to composition A, similar to Example 2. Formula E shows statistically
significant improvement across all
3 dilutions shown, and Formulas B and C show statistically significant
improvement across the second
dilution. This confirmed the consistent tendency for concentration-dependent
cell protective effects in other
oxidated environments.
EXAMPLE 4
33
Date Recue/Date Received 2022-04-14
Evaluation of the inhibition of active oxygen production within SH-SY5Y
neuroblastoma cells by
compositions of the present invention (oxidative environment by 11202
administration)
Reactive Oxygen Species (ROS) within cells can be quantified by indexing using
the fluorescent
probe 2',7'-dichlorofluroescin diacetate (DCF-DA) (Sigma-Aldrich, USA). When
oxidized to the reactive
oxygen metabolite, the DCF-DA can be excited at 485 nm and releases
fluorescence at 530 nm.
SH-SY5Y neuroblastoma cells were incubated with 100 p.1 of 10% FBS/MEM in a
5x104 cells/well
96-well plate and cultured 24 hours, as in Example 1. The 10% FBS/MEM was
removed and 1% FBS/MEM
added to the cells. Composition A, B, C, D, or E was administered, and then
DCF-DA in keeping with the
DCF-DA assay described above.
After 4 hours, a 0.25 uM solution of H202 (a toxicity-inducing substance) was
administered.
Incubation time was for 2 hours before fluorescence test. Subsequently,
fluorescence was measured at 530
nm to assess the level of active oxygen production.
In Fig. 4, dilutions are shown in column bars from left to right: 1.00
(undiluted composition), 0.5,
0.25. An "*" indicates statistical significance and synergistic effect when
compared with Formula A;
p<0.05 by Student's t-test. As shown in Fig. 4, the results indicate that
compositions A-E inhibited the
production of ROS in a concentration-dependent manner, in concordance with
Examples 2 and 3. Formulas
C and E, compositions of the present invention, showed statistically
significant protection from ROS
generation when compared to Formula A. This tendency shows that in addition to
protecting cells, the
compositions can also causatively inhibit the generation of ROS, which can
cause additional oxidative
injury to brain cells.
EXAMPLE 5
Evaluation of the inhibition of active oxygen production within SH-SY5Y
neuroblastoma cells by
compositions of the present invention (oxidative environment by FeSO4
administration).
SH-SY5Y neuroblastoma cells were incubated with 1000 of 10% FBS/MEM in a 5x104
cells/well 96-well plate and cultured 24 hours, as in Examples 1 and 4. The
10% FBS/MEM was changed
to 1%, Composition A, B, C, D, or E administered as shown in Table 2 and then
the DCF-DA solution
administered in the correct concentration as discussed in Example 4.
After 4 hours, 2.5 uM FeSO4 (a toxicity-inducing substance) was administered.
Incubation time
was for 2 hours before fluorescence test. Subsequently, fluorescence was
measured at 530 nm to assess the
level of active oxygen production.
34
Date Recue/Date Received 2022-04-14
In Fig. 5, dilutions are shown in column bars from left to right: 1.00
(undiluted composition), 0.5,
0.25. An "*" indicates statistical significance and synergistic effect when
compared with Formulation A;
p<0.05 by Student's t-test. As shown in Fig. 5, the results indicate that
formulas A-E inhibited the
production of ROS in a concentration-dependent manner. Formula E, a
composition of the present
invention, showed significantly significant protection from ROS generation
when compared to Formula A.
The tendency for a consistent concentration-dependent inhibition of the
production of ROS in other
oxidatve environments was reconfirmed.
Through Examples 1-5 mentioned above, both an antioxidant function and an
active oxygen
inhibition within the brain cells is shown for all compositions tested, with
statistically significant
enhancement of results over Formula A for all tested compositions B-E of the
present invention: Formula
B, Examples 2 and 3; Formula C, Examples 3 and 4; Formula D, Example 2; and
Formula E, Examples 2,
3, 4, and 5.
EXAMPLE 6 AND EXAMPLE 7
Induced Cell Death Challenge
Compositions A-H include combinations of purified Bombyx mori cocoon silk
peptide fiber
(PEPTYLIN8), refined Buglossoides arvensis Seed Oil (NeurXcer), African ginger
extract (Aframomum
melegueta standardized to paradol), and Blueberry standardized extract, as set
out in Table 3 below:
TABLE 3
Compositions
Mg PEPTYLIN NeurXcel Paradol Blueberry NAC*
A 20 0 0 0 0
0 50 0 0 0
20 50 2.5 0 0
20 50 5 0 0
20 50 0 2.5 0
20 50 0 5 0
0 0 5 0 0
0 0 5 0
NAC 0 0 0 0 20
*NAC: N-Acetyl-L-cysteine
Compositions A and B include either purified Bombyx mori cocoon silk peptide
fiber or NeurXcel Seed
Oil, respectively, but not both; while Compositions G and H include neither.
Compositions C-F,
including both fibroin and NeurXcel Seed Oil, are compositions according to
the present invention.
Date Recue/Date Received 2022-04-14
SH-SY5Y cells were cultured as in Example 2. As shown in Fig. 6, after
challenge with H202,
Formula A (Peptylin alone, +77.5%) showed a recovery of +40.5% vs. no
treatment (+37%).
Accordingly Formula A inhibited induced cell death by +40.5%.
Formula F (+92.8%) performed the best of formulas of the present invention.
Formula F showed
a recovery of +55.8% vs. no treatment. Formula F synergistically inhibited
induced cell death +15.3%
better than Formula A (=92.8%-77.5%). This represents a 19.7% relative
improvement vs. Peptylin
alone (-92.8%/77.5%). Positive and Negative Controls validate the model, with
37% cell viability
remaining after exposure to H202 alone, and 100% cell viability after no
exposure to H202. The notation
(*) means statistically significant and synergistic increase in cell viability
as compared with Formula A.
The statistical analysis was performed by Student's t-tests. Each individual
in vitro assay was replicated 3
times to assure robustness of the data.
As shown in Fig. 7, after challenge with FeSO4, Formula A (Peptylin alone,
+76.9%) showed a
recovery of +27.8% vs. no treatment (+49.1%). Similar to the first experiment,
Formula F 1+94.9%)
performed the highest of the founulations. Specifically, formula F showed a
recovery of +45.8% vs. no
treatment. Therefore formula F inhibited induced cell death +18.0% better than
Formula A(= 94.9%-
76.9%). This represents a +23.4% relative improvement vs. Peptylin alone(=
94.9% /76.9%). The same
controls and replications were followed.
In both induced cell death challenge experiments, the best-performing
formulation significantly
out-performed Peptylin alone. The formulation achieved a 45-55% total
recovery of neural cells vs. no
treatment, an outright cell recovery of up to 95%, and it achieved a 20-23%
improvement in cell recovery
vs. Peptylin alone.
EXAMPLE 8 AND EXAMPLE 9
Reactive Oxygen Species Challenge
In Examples 8 and 9, SH-SY5Y cells were exposed in separate, parallel models
to H202 and
Fe SO4 to induce controlled cell death, but instead of analyzing cell
viability in the presence of various
active compounds, reactive oxygen species (ROS) generation was measured in the
presence of various
formulations, following peer-reviewed published methods. ROS is known
physiologically as a causative
factor in the formation of beta amyloid plaques and damage to key
neurotransmitter compounds such as
36
Date Recue/Date Received 2022-04-14
acetylcholine (ACh), and thus is implicated in progressive loss of cognitive
function for instance leading
to dementia and Alzheimer's disease.
In Figs. 8 and 9, greater inhibition of ROS is indicated by lower bars,
indicating lower ROS
generation. Formulations are shown in Table 3.
The results of Example 8 are shown in Fig. 8. Fig. 8 shows formula F achieved
the lowest 21.4%
ROS generation, compared to formula A (Peptylin alone) which achieved 29.4%
ROS generation.
Therefore formula F out-performed formula A by +8% (= 29.4% - 21.4%). This
represents a +27.2%
better, synergistic performance relative to Peptylin alone (= 8% / 29.4%).
Compared to the positive
control (+100% ROS generation), Peptylin alone showed a +70.6% (= 100% -
29.4%) ROS inhibition
effect and formula F showed a+ 78.6% ROS inhibition effect(= 100% -21.4%).
Similarly, the results of Example 9 (Fig. 9) show that after FeSO4 challenge,
formula F achieved
the lowest 36% ROS generation, compared to formula A (Peptylin alone) which
achieved 43.2% ROS
generation. Therefore formula F out-performed formula A by +7.2% (= 43.2%-
36%)(*). This represents
a +16.7% better, synergistic performance relative to Peptylin alone (= 7.2% /
43.2%). Compared to the
positive control (+100% ROS generation), Peptylin showed a +56.8% (= 100%-
43.2%) ROS inhibition
effect and formula F showed a +64% ROS inhibition effect(= 100% - 36%).
EXAMPLES 10-12
Examples 10-12 disclose similar results achieved with two different Blueberry
Extracts of the
present invention. The Wild Blueberry (Vaccinium angustifolium) powder tested
and disclosed for
instance at Fig. 10 is wild Canadian Blueberry extract (Blue d'Or) from Fruit
d'Or (labeled as
discussed above. The American Blueberry (Vaccinium corymbosum) extract
(VitaBlue ) is a powder
from Futureceuticals (labeled as "VB"), discussed above.
EXAMPLE 10
Cell viability tests
SH-SY5Y cells were plated on 96-well plates at a density of 5x104 cells/well
in 100 ul of 10%
FBS/MEM and incubated for 24 hours. The media was replaced with 90u1 of
1%FBS/MEM, and then the
compositions shown in the table were added to the cells. After the treatment,
Mug of MIT (344,5-
dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide)
(Invitrogen/ThermoFisher, Carlsbad CA, USA)
was aseptically added. The cells were incubated for about 2-4 hours and the
absorbance of the cells was
37
Date Recue/Date Received 2022-04-14
measured at a wavelength of 570 nm using an ELISA reader. Amounts of sample
treatments were 10u1
per well.
Results are displayed in Figs. 10 and 11 and Tables 4 (Fig. 10) and 5 (Fig.
11), with dilutions
ranging from 200-12.5 ug as applied shown from left to right in column bars
for each substance tested. In
Figs. 10 and 11, PP refers to Peptyling powder, GP to Grains of Paradise (15%
6-paradol powder), AFO
to NeurXcel oil, and WB to Wild blueberry powder, VB to American blueberry
powder (VitaBlue6).
Figs. 10 and 11 show no cytotoxicity was present in any of the dilution ratios
(1-0.0625) for any
of the substances tested.
TABLE 4
Conc.
BF GP AFO WB VB Control
(ug/mL)_____
200 1.115 1.019 1.103 1.033 1.065
1.280
100 1.126 1.048 1.163 1.053 1.099
1.223
, 50 1.178 1.014 1.143 1.129 1.152
1.215
25 1.146 . 1.081 1.122 , 1.105
1.083 1.257
3.0
12.5 1.135 1.048 1.092 1.114 1.166
1.224
-
TABLES
'
, - _______ -
Conc.
BF GB AF WB VB
F(Control)
(ug/mL)
50ug/m1 97% 83% 94% 93% 95% , 100%
_
25ug/m1 91% 86% 89% _ 88% 86% 100%
12.5n/1.n! 93% 86% 89% 91% 95% 100%
EXAMPLE 11
Compositions providing protection from 11202-induced cell death
SH-SY5Y cells were plated on 96-well plates at a density of 5x104 cells/well
in 100 ul of 10%
FBS/MEM and incubated for 24 hours. The media was replaced with 90u1 of
1%FBS/MEM.
38
Date Recue/Date Received 2022-04-14
Formulations shown in the table below were incubated with the cells for 4
hours (in triplicate, 3
dilutions). H202 was added to the cells and incubated with the cells for 24
hours. After the treatment,
lOug of MTT (3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide)
(Invitrogen/ThermoFisher,
Carlsbad CA, USA) was aseptically added. The cells were incubated for 3 to 4
hours and the absorbance
.. of the cells measured at 570nm wavelength using an ELISA reader.
TABLE 6
ug PEPTYLIN NeurXcel Paradol WB VB
H202
Formula A 20 0 0 0 0
Formula B 20 50 2.5 5 0
Formula C 20 50 2.5 0 5
Control (+)
Control (-)
Fig. 12 shows a statistically significant increase in cell viability for the
100% and 25% dilutions
of Formulas B and C as compared with Formula A, showing the synergistic effect
achieved with
compositions of the present invention with both blueberry extracts tested.
Results as shown in Fig. 12
show dilutions 100%, 50%, 25% from left to right for each Formula. Cell
viability tended to decrease as
the concentration decreased. In the comparison between formula A, B, and C,
the notation (*) means
statistically significant, synergistic increase in cell viability as compared
with Formula A. Statistical
analysis was performed by Student's t-tests.
TABLE 7
=
Formula A Formula B
Formula C Control (+) Control (-)
100% 91% 94% 94% 47% 100%
50% 89% 90% 90% = 47% 100%
25% 51% 58% ' 60% 47% 100%
Control (+) 47% - 47% 47%
Control (-) 100% 100% 100%
39
Date Recue/Date Received 2022-04-14
EXAMPLE 12
Compositions inhibiting H202-induced Reactive Oxygen Species generation
Reactive oxygen species generation induced by H202 was measured by incubation
with a
fluorescent probe 2',7'-dichlorofluorescin diacetate (DCF-DA). SH-SY5Y cells
were stained with 10AM
of DCF-DA. The cells were collected, and washed with PBS. Cells stained with
DCF-DA were incubated
then measured with excitation at 485 nm and emission at 530 nm by fluorometer.
Formulations used in this experiment were as follows:
TABLE 8
ug PEPTYLIN NeurXcel Paradol WB VB H202
Formula A 20 0 0 0 0
Formula B 20 50 2.5 5 0
Formula C 20 50 2.5 0 5
Control (+)
Control (-)
The positive control group, NAC, confirmed that SH-SY5Y cells were restored to
normal levels.
Fig. 13 shows a statistically significant inhibition of ROS generation for the
100% and 50%
dilutions of Formulas B and C as compared with Formula A, showing the
synergistic effect achieved with
both blueberry extracts tested. The results show that the higher the
concentration, the more the ROS
generation was suppressed. Results as shown in Fig. 13 show dilutions 100%,
50%, 25% in column bars
from left to right for each Formula. In the comparison between formula A, B,
and C, the notation (*)
means statistically significant increase in cell viability as compared with
Formula A. Statistical analysis
was performed by Student's t-tests.
IN VIVO TESTS
EXAMPLES 13-21
Individual subjects participated in voluntary open label empirical evidence
trials using a
synergistic composition of the subject invention, Braini . As discussed above,
Braini is a combination
of Peptylin powder (400 mg/day); NeurXcel microencapsulated powder (500 mg
powder/day); and
Blueberry extract powder (Vaccinium corymbosum) (100 mg/day). 500 mg NeurXcel
microencapsulated powder includes 250mg NeurXcel oil and 250mg of
microencapsulated powder
composed primarily of modified food starch, corn syrup solids, antioxidants,
and natural flavors. Braini
capsules in the above amounts were administered daily to human subjects of the
below Examples.
Administration to human subjects was for 28 days, unless indicated otherwise.
Date Recue/Date Received 2022-04-14
In the below Examples, subjects were tested before administration of Braini
capsules, and then
after administration of Brainie capsules. The following tests were conducted
according to CNS Vital
Signs (Morrisville, NC) testing protocols, which are reproduced or paraphrased
in part as follows: Subject
scores were used to assess their Verbal Memory, Psychomotor Speed, Reaction
Time, Cognitive
Flexibility, Processing Speed, Executive Function, and/or Motor Speed, as
described in the Examples
below. Scores help identify cognitive deficits and if present, level of
impairment, using an age-matched
normative comparison database. Standard scores (ss) for subjects of similar
age, and percentile ranks (Pr),
are auto-scored using an algorithm based on a normative data set of 1600+
subjects, ranging from ages 8-
90. In the age-matched normative sample, subjects were (1) in good health, (2)
had no past or present
psychiatric or neurological disorders, head injury, or learning disabilities,
and (3) the sample subjects
were free of any centrally-acting medications. CNS Vital Signs cognitive tests
are used by thousands of
researchers and physicians in over 50 counties, and for instance in the United
States by the US military
and the Veterans Administration.
The CNS Vital Signs normative data is presented in ten (10) age groups: less
than 10 years old,
.. 10-14 years old, 15-19 years old, 20-29 years old, 30-39 years old, 40-49
years old, 50-59 years old, 60-
69 years old, 70-79 years old, 80 years and older. Standard scores (ss) above
109 (>74 percentile) are
considered as Above Average, High Function and High Capacity; ss of 90-109 (25-
74 percentile) are
considered as Average, Normal Function and Normal Capacity; ss of 80-89 (9-24
percentile) are
considered as Low Average, Slight Deficit and Slight Impairment; ss of 70-79
(2-8 percentile) are
considered as Low, Moderate Deficit and Impairment Possible; ss of less than
70 (less than 2 percentile)
are considered as Very Low, Deficit and Impairment Likely. For CNS Vital Signs
results, the color Red
(R) indicates a "Very Low" score as compared with age-normed population
cohort; Orange (0) = "Low",
Yellow (Y) = "Low Average", Light Green (LG) = "Average", Dark Green (DG) =
"Above Average".
Reaction times are in milliseconds. Generally, higher scores indicate better
performance, with the
exception of Reaction Time, where a lower score ("*") indicates better
functioning. Patient Scores are
raw scores calculations generated from data values of individual subtests.
Percentile ranks are a
mathematical transformation of the standard score and an index of how the
subject scored compared with
other subjects of the same age on a scale of 1 to 99. Normal aging affects
performance on all CNS Vital
Signs tests. A subject's standard scores are based on data from normal
controls that are the same age.
Education and special skills may affect test performance, therefore concern
should be taken for subjects
that are very intelligent or well educated yet their scores are below average.
With any neuropsychological
tests, results can be affected by motivation or effort level; the Validity
Indicator will help identify those
41
Date Recue/Date Received 2022-04-14
patients. Further information is available on the CNS Vital Signs website,
www.cnsys.com; see for
instance the Brief Interpretation Guide.
The below tests are discussed in Examples below:
Verbal Memory (VBM) measures recognition memory for words and geometric
figures. Fifteen
words are presented, one by one, on the screen every two seconds. For
immediate recognition (learning
phase), the participant must identify those words nested among fifteen new
words. Then, after six more
tests, there is a delayed recognition memory trial. Subjects respond by using
the space bar on a keypad.
VBM is calculated as follows: VBM Correct Hits Immediate + VBM Correct Passes
Immediate + VBM
Correct Hits Delay + VBM Correct Passes Delay. VBM tests include Learning
Words, Memory for
Words, Word Recognition, Immediate and Delayed Recall. Real-life examples of
Verbal Memory
include remembering a scheduled test, recalling an appointment, taking
medications, and attending class.
VBM is considered a single test domain.
Psychomotor Speed measures how well a subject perceives, attends, responds to
visual-perceptual
information, and performs motor speed and fine motor coordination. Psychomotor
Speed is considered a
multiple test domain (combines tests for Motor Speed and Processing Speed). A
Finger Tapping Test and
a Symbol Digit Coding Test may be used to measure and calculate Psychomotor
Speed. Psychomotor
Speed is calculated as follows: Finger Tapping Test Right Taps Average +
Finger Tapping Test Left Taps
Average + Symbol Digit Coding Correct Responses. A Finger Tapping Test has
subjects press the space
bar on a keypad with their right index finger as many times as they can in 10
seconds. They do this once
for practice, and then there are three test trials. The test is repeated with
the left hand. A Finger Tapping
Test includes motor speed and fine motor control. A Symbol Digit Coding test
consists of serial
presentations of screens, each of which contains a bank of eight symbols above
and eight empty boxes
below. The participant types in the number on the number row that corresponds
to the symbol that is
highlighted. Only the digits from 2 through 9 are used; this is to avoid
confusion between "1" and "1" on
the keypad. The computer program does not allow a person to use a numerical
pad preventing a distinct
advantage for those who are skilled at using the numerical pad or for those
that are right- versus left-
handed. A Symbol Digit Coding test relates to complex information processing
accuracy, complex
attention, visual-perceptual speed, and information processing speed. Real-
life examples relating to
Psychomotor Speed include ability to perform simple motor skills and dexterity
through cognitive
functions such as used of precision instruments or tools, performing mental
and physical coordination
such as driving a car, playing a musical instrument.
42
Date Recue/Date Received 2022-04-14
Reaction Time measures how quickly a subject can react, in milliseconds, to a
simple and
increasingly complex direction set. Reaction time is considered a single test
domain. Stroop Test
Complex Reaction Time and Stroop Reaction Time may be used to measure and
calculate Reaction Time.
When considering Reaction Time results, a lower score indicates better
functioning (in contrast to other
tests described in this application). A Stroop Test has three parts. In the
first part, the words RED,
YELLOW, BLUE, and GREEN (printed in black) appear at random on the screen, and
the participant
presses the space bar as soon as the test subject sees the word. In the second
part, the words RED,
YELLOW, BLUE, and GREEN appear on the screen, printed in color. The
participant is asked to press
the space bar when the color of the word matches what the word says. In the
third part, the words RED,
YELLOW, BLUE, and GREEN appear on the screen, printed in color. The
participant is asked to press
the space bar when the color of the word does not match what the word says. A
Stroop Test relates to
simple reaction time, complex reaction time, Stroop reaction time,
inhibition/disinhibition, and frontal or
executive skills. Real-life examples relating to Reaction Time include driving
a car, attending to
conversation, tracking and responding to a set of simple instructions, taking
longer to decide what
response to make.
Cognitive Flexibility measures how well a subject is able to adapt to a
rapidly changing and
increasingly complex set of directions and/or to manipulate the information.
Cognitive Flexibility is
considered a multiple test domain (Executive Function and Stroop Test) and is
calculated as follows:
Shifting Attention Test Correct Responses ¨ Shifting Attention Test Errors ¨
Stroop Commission Errors.
The Shifting Attention Test is measure of ability to shift from one
instruction set to another quickly and
accurately. Participants are instructed to match geometric objects either by
shape or by color. Three
figures appear on the screen, one on top and two on the bottom. The top figure
is either a square or a
circle. The bottom figures are a square and a circle. The figures are either
red or blue (mixed randomly).
The participant is asked to match one of the bottom figures to the top figure.
The rules change at random
(i.e. match the figures by shape, for another, by color) and subject responds
by pressing the two shift
keys. The Shifting Attention Test relates to executive function, shifting
sets: rules, categories, and rapid
decision making; and reaction time. Real-life examples relating to Cognitive
Flexibility include
reasoning, switching tasks, decision-making, impulse control, strategy
formation, and attending to
conversation.
Processing Speed measures how well a subject recognizes and processes
information, that is,
perceiving, attending/responding to incoming information, motor speed, fine
motor coordination, and
visual-perceptual ability. Processing Speed is considered a single test
domain. Processing Speed is
43
Date Recue/Date Received 2022-04-14
calculated as follows: Symbol Digit Coding Correct Responses ¨ Symbol Digit
Coding Errors. Real-life
examples relating to Processing Speed include ability to recognize and
respond/react, that is, fitness-to-
drive, occupation issues, possible danger/risk signs or issues with accuracy
and detail.
Executive Function measures how well a subject recognizes rules, categories,
and manages or
navigates rapid decision making. Executive Function is considered a single
test domain. Executive
Function is calculated as follows: Shifting Attention Test Correct Responses ¨
Shifting Attention Test
Errors. Real-life examples relating to Executive Function may include the
ability to sequence tasks and
manage multiple tasks simultaneously as well as tracking and responding to a
set of instructions.
Executive Function is one of the most difficult neuro-cognitive performance
measures to influence,
especially without stimulants. Yet for instance in the context of the SAT-RT
test (Shifting Attention Test
¨ Reaction Time), healthy senior adult subjects experienced an improvement in
this parameter using
Braini capsules for 28 days, as compared with placebo, with an indicative p-
value = 0.05.
Motor Speed measures a subject's ability to perform movements to produce and
satisfy an
intention towards a manual action and goal. Motor Speed is considered a single
test domain. Motor Speed
is calculated as follows: Finger Tapping Test Right Taps Average + Finger
Tapping Test Left Taps
Average. Real-life examples relating to Motor Speed include the preparation
and production of simple
manual dexterity actions, such as manipulating and maneuvering objects.
EXAMPLE 13
Individual healthy seniors participated in a voluntary open label empirical
evidence trial using the
Braini composition identified above. Before administration of this
composition, individual (human)
subjects first took an online battery of standardized memory, cognitive
performance, and neuro-
physiological tests produced by CNS Vital Signs (Morrisville, NC). After 30
days of daily intake of the
composition, the subjects took a follow-up standardized CNS Vital Signs test
suite. Individual and
consolidated results for each subject are shown in Fig. 14. Significant,
synergistic improvements in 5 of 7
CNS Vital Signs memory, cognitive performance, and neuro-physiological scores
occurred over a cohort
of 8 healthy seniors with an average age of 70 years (range: 68-73 years).
Average cohort performance
improved significantly from +9.4% to +20.8% across the 5 assessment
parameters: Verbal Memory;
Processing Speed; Reaction Time; Psychomotor Speed; and Motor Speed. For the
other two assessment
parameters, only modest average cohort improvements occurred. Cognitive
Flexibility improved by
+0.9% and Executive Function improved by +2.8%.
EXAMPLE 14
44
Date Recue/Date Received 2022-04-14
Subjects diagnosed with neurodegenerative diseases such as Parkinson's disease
and Multiple
Sclerosis consumed a formulated embodiment of the subject invention (Braini
capsules, as described
above) for periods of up to 6 months under a physician's observational trial
model coupled to the
standardized CNS Vital Signs (Morrisville, NC) cognitive performance
assessment tool given at baseline
prior to taking the active composition and monthly thereafter. Such subjects
achieved superior and
significant, synergistic cognitive performance improvements over baseline
within 30-60 days of
continuous consumption of the referenced composition. Such subjects then
discontinued consuming the
formulation after an initial active product consumption phase of up to 4
months. Contrary to expectation,
the subjects' cognitive performance sustained at the significantly higher
levels over baseline for 30-60
days without significant changes to their dietary or lifestyle regimens, other
than the removal of the active
formulation, showing a synergistic, structural neuro-physiological effect
beyond what is expected from
available neuro-protective prescription drugs. After up to 60 days' non-
consumption of the embodiment,
the subjects' cognitive performance declined significantly, evidencing that
their neuro-physiological status
was starting to return to baseline conditions. Once the subjects resumed
consuming the active
formulation, in 30-60 days of resumed active supplementation, their cognitive
performance once again
improved significantly in at least 4 of 7 cognitive batteries (Verbal Memory,
Psychomotor Speed,
Reaction Time, Cognitive Flexibility, Processing Speed, Executive Function,
Motor Speed) under the
CNS Vital Signs (Morrisville, NC) assessment tool. See for example MS
Subject's performance
improvement in Fig. 15. The color Red (R) indicates a "Very Low" score as
compared with age-normed
population cohort; Orange (0) = "Low", Yellow (Y) = "Low Average", Light Green
(LG) = "Average",
Dark Green (DG) = "Above Average". The above references to color in CNS Vital
Signs data apply to
other Examples and data as well.
In addition to the above, evidence of remyelination and sustained Executive
Function and
Cognitive Flexibility in a subject with Multiple Sclerosis was seen with
Braini administration. A senior
adult (age 60) began oral administration of a composition of this invention,
Braini capsules as described
above, about 187 days after the first entry in the below Table "Days between
Tests 0". Administration
continued for about 4 months, and then ceased for about 6 weeks (beginning
just before "Days Between
Tests" below, period of 41 days)". At the end of the 6 weeks, the subject was
again administered the
Braini capsules, continuing to take Braini capsules for about 6 weeks
through the end of testing
indicated in the below table, and with continuous administration of Braini
capsules through the date of
filing this application (about 14 months). No adverse effects were reported by
the subject.
Date Recue/Date Received 2022-04-14
Table 9 row 1 shows that before administration of Braini capsules, the
subject tested in the
Very Low Category for Psychomotor Speed, Cognitive Flexibility, Processing
Speed, and Executive
Function. After four months of daily Braini administration, as shown by the
row marked as having 31
days between tests, the subject had steadily improved in each of these areas.
Upon resumption of
Braini administration, about 7 months after the initial administration,
Cognitive Flexibility, Executive
Function, and Processing Speed were dramatically improved in the subject, from
Very Low to Average
placement. Psychomotor Speed had also dramatically improved to indicate a high
functioning test
subject, still in the Low Average category. Other improvements may also be
seen in the below Table.
In addition, the spouse of the subject with Multiple Sclerosis, having known
the subject for 35
years, reported substantial improvements in the subject's abilities with
Braini administration. The
spouse commented on the subject's gradual deterioration in verbal memory with
the disease, and that
the subject's reaction time with Braini administration is significantly
improved, and post-Braini speech
is not as labored as pre-Braini speech.
Also, before Braini administration, the spouse reported the subject
experienced a noticeable
decline in motor function, speech, and reaction time. The spouse reported the
subject had always been
a "list maker" but that, as the disease progressed, the subject had stopped
planning and making lists.
However, Braini administration restored some of that function to the subject.
The spouse further
continued that after only a week to 10 days of Braini administration, the
subject with multiple sclerosis
began to improve noticeably. Also, the spouse commented that the subjects
local neurologist had noted
improvements in the subject's motor speed, in keeping with that observed with
data reported herein
from CNS Vital Signs testing. Also, the spouse commented that the subject's
specialist neurologist (Mass
General Hospital, Boston, MA) noted re-myelination in the subject's most
recent MRI images.
TABLE 9
Administration of Braini capsules to an adult senior with Multiple Sclerosis,
0-7 months
46
Date Recue/Date Received 2022-04-14
Verbs, Psychomotor Reaction Time
Cognitive Processing Executive Motor Speed
Memory Speed Flexibility Speed
Function
, ____________
Days between Tests 5 t d Scam.: )r c' soon s Std Scores Std
Stores Si c' `cores Sto S cot, s Std Scores
2161 77 92 ''.., 41e1 75 'Aiat4
, Alik A
________________ 17 96 I. 97 86 81 87 73
106 75 104 86 83 88 79
99 76 96 78 94 80 74
115 74 119 ,,9 87 88 74
115 " 75 99 71 83 72 79
112 71 106 79 85 81 73
' 112 84 92 75 92 78 86
= .
291 115 III 81 gi 100 99 94 100 g 80
Tables 10 and 11 show CNS Vital Signs testing results for the senior adult
with Multiple
Sclerosis about 18 months after beginning Braini administration, with
administration halted for about 6
weeks as mentioned above. The subject scored Average in Reaction Time,
Cognitive Flexibility,
Processing Speed, and Executive Function, and Low Average in Psychomotor Speed
and Motor Speed.
No Very Low or Low Average scores were assessed during this test, showing no
impairment or deficit in
the subject. These results show that chronic administration of Braini
capsules provided a continued
benefit to the subject, transforming the subject's profile from impairments in
several categories into a
subject with no apparent impairments or deficits. As of the date of the filing
of this application, the
subject continues to take Braini capsules with no adverse effects and with
continued improvement or
maintenance scores as compared with pre-Braini scores (21 months' since
beginning Braini capsule
administration). The data are consistent with providing neuroprotection and
remyelination in the subject.
In addition to the above data, the subject is described as dramatically
improved in speech, demeanor, and
cognitive function after long-term administration of Braini capsules.
TABLE 10
Improvement 21 Months After Beginning Administration of Braini Capsules
to an Adult Senior Subject With Multiple Sclerosis
Patient Percentile Range >74 25-74 9-24
2-8 <2
Profile
Standard Score Range >109 90-109 80-89
70-79 <70
Domain Patient
Standard Percentile Valid Above Average Low Low Very
Scores Score Score Score** Average
Low
Psychomotor
127 84 14 Yes X
Speed
47
Date Recue/Date Received 2022-04-14
Reaction
695 101 53 Yes X
Time*
Cognitive
35 99 47 Yes X
Flexibility
Processing
40 95 37 Yes X
Speed
Executive
38 100 50 Yes X
Function
Motor Speed 87 81 10 Yes X
TABLE 11A
Finger Tapping Test (FIT)
Tested Information Score Standard
Percentile
Right Taps Average 48 89 23
Left Taps Average 39 76 5
The FTT is a test of motor speed and fine motor control ability. There are
three rounds of tapping with
each hand. The F1-1 test measures the speed and the number of finger-taps with
each hand. Low scores
indicate motor slowing. Speed of manual motor activity varies with handedness.
Most people are faster
with their preferred hand but not always.
TABLE 11B
Symbol Digit Coding (SDC)
Tested Information Score Standard
Percentile
Correct Responses 40 93 32
Errors* 0 114 82
The SDC test measures speed of processing and draw upon several cognitive
processes simultaneously,
such as visual scanning, visual perception, visual memory, and motor
functions. Errors may be due to
impulsive responding, misperception, or confusion.
TABLE 11C
Stroop Test (ST)
Tested Information Score Standard
Percentile
Simple Reaction Time* 379 87 19
Complex Reaction Time 553 110 75
Correct*
48
Date Recue/Date Received 2022-04-14
Stroop Reaction Time 836 93 32
Correct*
Stoop Commission 3 92 30
Errors*
The ST measures simple and complex reaction time, inhibition / disinhibition,
mental flexibility or
directed attention. The ST helps assess how well a subject is able to adapt to
rapidly changing and
increasingly complex set of directions. Prolonged reaction times indicate
cognitive slowing / impairment.
Errors may be due to impulsive responding, misperception, or confusion.
TABLE 11D
Shifting Attention Test (SAT)
Tested Information Score Standard
Percentile
Correct Responses 42 94 34
Errors* 4 109 73
Correct Reaction Time* 1251 93 32
EXAMPLE 15
Subjects diagnosed with dyslexia consumed a formulated embodiment of the
subject invention for
at least 30 days under a physician's observational model coupled to the
standardized CNS Vital Signs
(Morrisville, NC) cognitive assessment tool given at baseline prior to taking
the active composition, and
again after 30 days of continuous consumption of the active composition. The
fact of the subjects'
dyslexia is captured readily in their relatively poor performance in the
Cognitive Flexibility and Executive
Function scores. See Fig. 16A where each subject scored normatively in the 30-
39 range ("Very Low") in
both assessments. Yet, unexpectedly, given that healthy non-dyslexic subjects
in Fig. 16A showed, on
average, relatively little improvement in Cognitive Flexibility and Executive
Function as a result of
consuming the active composition for at least 30 days, the dyslexic subjects
showed dramatic
improvements in Cognitive Flexibility and Executive Function scores (ranging
from +142% to +176%)
after 30 days, improving their normative results from "Very Low" to "Average"
in their respective
population age cohorts. This shows an unexpected, synergistic improvement in
functional cognition by
dyslexic subjects as compared to non-dyslexic subjects in the general
population.
Fig. 16B shows results from a physician's observational trial after Braini
administration for at
least 30 days in 6 dyslexic subjects (5 adults, 1 child). Data reported in
Fig. 16B includes data from the
subjects identified in Fig. 16A plus additional subjects, for a total of 6
dyslexic subjects studied. All
49
Date Recue/Date Received 2022-04-14
subjects studied experienced significant improvement in Cognitive Flexibility
and Executive Function
outcomes measures using the CNS Vital Signs standardized cognitive performance
assessment tool, after
taking Braini as directed for at least 30 days. A consistent pattern of
regular Braini use driving
improved Cognitive Flexibility and Executive Function outcomes is novel and
unique to dyslexic
subjects. As shown in Fig. 16B, after 30 days' administration of Braini ,
dyslexic subjects enjoyed from
16% to 187% improvement in Cognitive Flexibility and about 19% to 174% in
Executive Function.
Further, all 6 dyslexic subjects experienced notable improvements in their
Reaction Time
outcome measures. As it was discovered that participants in the university
controlled human clinical trials
experienced significant improvement in their SAT-RT scores, it is noteworthy
that dyslexic subjects'
overall Reaction Time scores consistently improved.
EXAMPLE 16
Individual healthy seniors participated in a voluntary open label empirical
evidence trial intended
to determine whether any significant changes in cognitive performance occur
after a subject consumed
Peptylin for 30 days and then switched to Braini (a composition of the
present invention) for an
additional 30 days. Before administration of the first composition (Peptylin ,
400 mg/day), individuals
first took an online battery of standardized memory, cognitive performance,
and neuro-physiological tests
produced by CNS Vital Signs (Morrisville, NC), noted as "SS I". After 30 days
of daily intake of the
Peptylin formulation, the subjects then took a follow-up standardized CNS
Vital Signs test suite.
Standardized scores were recorded, noted as "SS2". The subjects then switched
immediately to Braini
and consumed it as directed for an additional 30 days. The subjects took an
additional CNS Vital Signs
test suite, with recorded scores noted as "SS3". Individual and average
results for each subject are shown
in Fig. 17. After consuming Braini for 30 days, significant, synergistic
improvements vs. Peptylin alone
in Cognitive Flexibility and Executive Function (+21%, +23% respectively)
occurred. No other
significant changes in 5 other cognitive performance and neuro-physiological
test scores occurred, the
standard of significance being a 7% or greater change in scores. This shows
that there is a therapeutic
benefit in taking Braini over and above pure Peptylin , and that the observed
synergistic effect of in
vitro compositions of the present invention such as Braini vs. Peptylin in
Examples 1-12 (above)
correlates therapeutically to the significant improvements in Cognitive
Flexibility and Executive Function
in humans who consume Braini vs Peptylin .
EXAMPLE 17
Physician's Observational Data
Date Recue/Date Received 2022-04-14
Figs. 18-23 show the results of CNS Vital Signs testing on 43 human subjects
before and after
administration of Braini . Braini LLC has conducted open-label physician
observed studies comparing
healthy subjects' cognitive and neurophysiological performance using the CNS
Vital Signs online self-
test assay suite. These empirical studies have been carried out comparing a
subject's outcome scores on
Day 0 (prior to taking Braini ) and again after 30-60 days of taking Braini
as directed, using six of the
CNS Vital Signs standardized outcome measures. In some cases, subjects were re-
tested more than 60
days after their Day 0 test, because they delayed their initial start taking
Braini. This suite of tests is
notable for having been used in federally-backed neurological research,
including for the US military, by
over 10,000 physicians and 40 million test subjects. It is considered a
standard for assessing memory and
cognitive performance. General statements regarding CNS Vital Signs are
applicable to other Examples
using the CNS Vital Signs test suite.
Overall, 43 healthy subjects (age 11-80) completed the Braini Physician's
Observational
Trial. After taking Braini for at least 28 days, an average of 76% of subjects
experienced notable average
9-12% improvements in CNS Vital Signs (CNS VS) outcome measures, as follows:
TABLE 12
Summary of CNS Vital Signs outcome measures
CNS VS Outcome Measure Average Change
Psychomotor Speed +12.0%
Reaction Time +10.3%
Cognitive Flexibility +9.8%
Processing Speed +9.6%
Executive Function +9.8%
Motor Speed +9.0%
Compiled Average All +10.1%
Since Braini LLC started human subject testing in 2018, no subjects have
reported any serious adverse
events.
Subjects were tested for Psychomotor Speed, Reaction Time, Cognitive
Flexibility, Processing
Speed, Executive Function, and Motor Speed.
Psychomotor Speed
35 of the 43 subjects (about 81%) saw improvement (positive change) in
Psychomotor Speed
after Braini administration, ranging from about 1% to about 110% improvement
compared to testing
before Braini administration, as shown in Fig. 18. On average, Psychomotor
Speed improved about 12%
51
Date Recue/Date Received 2022-04-14
with Braini administration. As also noted in Fig. 18, 5 subjects did not
improve in the Psychomotor
Speed category with Braini administration, and 3 subjects experienced no
change.
Each Psychomotor Speed test result was noted as having a positive Validity
Indicator under the
test protocol (i.e. a reading of "Yes" to indicate valid test results). A
negative Validity Indicator would
.. suggest the test subject be evaluated to determine whether the subject
understood the test, put forth
their best effort, or has a clinical condition requiring further evaluation.
Reaction Time
35 of the 43 subjects (about 81%) saw improvement (positive change) in
Reaction Time after
Braini administration, ranging from about 1% to about 40% improvement
compared to testing before
Braini administration, as shown in Fig. 19. On average, Reaction Time improved
about 10% with Braini
administration. As also noted in Fig. 19, 6 subjects did not improve in the
Reaction Time category with
Braini administration, and 2 subjects experienced no change.
All but 2 test results (Subject ID #27, 36; test after Braini administration)
were noted as having
a positive Validity Indicator under the Reaction Time test protocol (i.e. a
reading of "Yes" to indicate
.. valid test results). A negative Validity Indicator would suggest the test
subject be evaluated to determine
whether the subject understood the test, put forth their best effort, or has a
clinical condition requiring
further evaluation.
Cognitive Flexibility
31 of the 43 subjects (about 72%) saw improvement (positive change) in
Cognitive Flexibility
after Braini administration, ranging from about 1% to about 51% improvement
compared to testing
before Braini administration, as shown in Fig. 20. On average, Cognitive
Flexibility improved about 10%
with Braini administration. As also noted in Fig. 20, 11 subjects did not
improve in the Cognitive
Flexibility category with Braini administration, and 1 subject experienced no
change.
All but 1 test result (Subject ID #17; test preceding Braini administration)
was noted as having a
positive Validity Indicator under the Cognitive Flexibility test protocol
(i.e. a reading of "Yes" to indicate
valid test results). A negative Validity Indicator would suggest the test
subject be evaluated to determine
whether the subject understood the test, put forth their best effort, or has a
clinical condition requiring
further evaluation.
Processing Speed
52
Date Recue/Date Received 2022-04-14
33 of the 43 subjects (about 77%) saw improvement (positive change) in
Processing Speed after
Braini administration, ranging from about 2% to about 60% improvement
compared to testing before
Braini administration, as shown in Fig. 21. On average, Processing Speed
increased about 10% with
Braini administration. As also noted in Fig. 21, 8 subjects did not improve
in the Processing Speed
category with Braini administration, and 2 subjects experienced no change.
Each Processing Speed test result was noted as having a positive Validity
Indicator under the test
protocol (i.e. a reading of "Yes" to indicate valid test results). A negative
Validity Indicator would suggest
the test subject be evaluated to determine whether the subject understood the
test, put forth their best
effort, or has a clinical condition requiring further evaluation.
Executive Function
31 of the 43 subjects (about 72%) saw improvement (positive change) in
Executive Function
after Braini administration, ranging from about 1% to about 50% improvement
compared to testing
before Braini administration, as shown in Fig. 22. On average, Executive
Function increased about 10%
with Braini administration. As also noted in Fig. 22, 10 subjects did not
improve in the Executive
Function category with Braini administration, and 2 subjects experienced no
change.
All but 2 test results (Subject ID #17, 32; test preceding Braini
administration) were noted as
having a positive Validity Indicator under the Reaction Time test protocol
(i.e. a reading of "Yes" to
indicate valid test results). A negative Validity Indicator would suggest the
test subject be evaluated to
determine whether the subject understood the test, put forth their best
effort, or has a clinical
.. condition requiring further evaluation.
Motor Speed
of the 43 subjects (about 70%) saw improvement (positive change) in Motor
Speed after
Braini administration, ranging from about 2% to about 97% improvement
compared to testing before
Braini administration, as shown in Fig. 23. On average, Motor Speed increased
about 9% with Braini
25 administration. As also noted in Fig. 23, 9 subjects did not improve in
the Motor Speed category with
Braini administration, and 4 subjects experienced no change.
Each Motor Speed test result was noted as having a positive Validity Indicator
under the test
protocol (i.e. a reading of "Yes" to indicate valid test results). A negative
Validity Indicator would suggest
53
Date Recue/Date Received 2022-04-14
the test subject be evaluated to determine whether the subject understood the
test, put forth their best
effort, or has a clinical condition requiring further evaluation.
Parkinson's Disease
Two subjects in the above study were diagnosed with Parkinson's Disease prior
to
administration with Braini capsules (Subject born 1952; 16-year diagnosis);
Subject born 1953, 10 year
diagnosis)). CNS Vital Signs Testing showed improvement in the subjects after
(post-Braini) about 30
consecutive days of Braini administration, as compared with the subjects'
test scores before Braini
administration (pre-Braini ), as shown in Tables 13 and 14.
TABLE 13
Improvements in Parkinson's Disease after 28 days' Braini Administration
CNS Vital Psychomotor Reaction Cognitive Processing Executive
Motor
Signs Speed Time Flexibility Speed Functioning Speed
Testing
Pre-
81 111 105 98 103 75
Braini
Post-
92 117 112 102 111 88
Braini
Score
+11 +6 +7 +4 +8 +13
Change
TABLE 14
Improvements in Parkinson's Disease after 28 days' Braini Administration
CNS Vital Psychomotor Reaction Cognitive Processing Executive
Motor
Signs Speed Time Flexibility Speed
Functioning Speed
Testing
Pre-
104 102 93 99 94 108
Braini
54
Date Recue/Date Received 2022-04-14
Post-
109 103 101 102 102 112
Braini
Score
+5 +1 +8 +3 +8 +4
Change
Dysgraphia and Anxiety Disorder
A young subject (11 years old) in the above study was diagnosed with
Dysgraphia and with
Anxiety Disorder prior to administration with Braini capsules. CNS Vital
Signs Testing showed
.. improvement in the subject after (post-Braini ) about 30 consecutive days
of Braini administration, as
compared with the subject's test scores before Braini administration (pre-
Braini ), as shown in Table
15.
TABLE 15
Improvements in Dysgraphia and in Anxiety Disorder after 28 days' Braini
Administration
CNS Vital Psychomotor Reaction Cognitive Processing Executive
Motor
Signs Speed Time Flexibility Speed Functioning Speed
Testing
Pre-
90 63 75 84 74 97
Braini
Post-
93 84 87 84 88 101
Braini
Score
+3 +21 +12 +0 +14 +4
Change
Dementia
A subject in the above study was diagnosed with Early Onset Dementia prior to
administration
with Braini capsules (Subject born 1962)). CNS Vital Signs Testing showed
improvement in the subject
after (post-Braini ) about 30 consecutive days of Braini administration, as
compared with the subject's
.. test scores before Braini administration (pre-Braini ), as shown in Table
16.
Date Recue/Date Received 2022-04-14
TABLE 16
Improvements in Early Onset Dementia after 30 days' Braini Administration
CNS Vital Psychomotor Reaction Cognitive Processing
Executive Motor
Signs Speed Time
Flexibility Speed Functioning Speed
Testing
Pre-
26 74 59 66 59 27
Braini
Post-
54 96 89 80 88 53
Braini
Score
+28 +22 +30 +14 +29 +27
Change
Attention-deficit disorders
Two subjects were diagnosed with ADHD (attention deficit-hyperactivity
disorder) prior to
administration with Braini capsules (Subjects born 1990, 1983). CNS Vital
Signs Testing showed
improvement in the subject after (post-Braini ) about 30 consecutive days of
Braini administration, as
compared with the subject's test scores before Braini administration (pre-
Braini ), as shown in Tables
17-18. Cognitive Flexibility and Executive Functioning in particular increased
with Braini administration
in each subject; Processing Speed or Reaction Time also improved. This data is
similar to that seen in
dyslexic subjects that were administered Braini capsules.
Without being bound by theory, these improvements may be due to subjects that
have some
difficulty with the uptake and/or processing of raw omega 3-6-9. For instance,
these subjects may lack
the ability to enzymatically digest raw omega. B. arvensis (Ahiflower ;
NeurXcel ) oil according to the
present invention includes omega SDA and GLA that have already been converted
and may be easier for
certain populations to absorb.
TABLE 17
Improvements in ADHD after 28 days' Braini Administration
Subject born 1990
56
Date Recue/Date Received 2022-04-14
CNS Vital Psychomotor Reaction Cognitive Processing Executive
Motor
Signs Speed Time Flexibility Speed Functioning Speed
Testing
Pre-
79 102 73 74 73 91
Braini
Post-
93 102 106 98 108 93
Braini
Score
+14 +0 +33 +24 +35 +2
Change
TABLE 18
Improvements in ADHD after 28 days' Braini Administration
Subject born 1983
CN5 Vital Psychomotor Reaction Cognitive Processing Executive
Motor
Signs Speed Time Flexibility Speed Functioning Speed
Testing
Pre-
103 73 108 10 107 102
Braini
Post-
106 101 118 10 122 104
Braini
Score
+3 +28 +10 -2 +15 +2
Change
EXAMPLE 18
Improvement in concentration in young adults with Braini administration
In a randomized double-blinded, placebo-controlled clinical trial with young
adults as subjects,
aged 18-30, the administration of Braini improved concentration by 85%, as
compared with placebo.
57
Date Recue/Date Received 2022-04-14
During the study, 23 active subjects were administered Braini for 28 days,
and 17 placebo
subjects took a placebo composition for the same number of days. The
DoubleTrouble concentration
test (Cambridge Brain Sciences, Toronto, Ontario, Canada) was taken by each
subject as a baseline
before beginning administration of Braini or placebo, and then taken again
upon completion of the
entire course. The Double Trouble test is based upon the Stroop task and
assesses response inhibition,
that is, the ability to concentrate on relevant information to make an
appropriate response, even when
distracting information or interference is present. Response inhibition is a
key component of
concentration. As mentioned above, the group that was administered Braini
experienced an 85%
improvement in the Double Trouble concentration test. See for instance Table
19 below, showing in
part an increase in the DoubleTrouble concentration scores of subjects
administered Braini , from 24.73
to about 45.74, showing 85% improvement; and see Fig. 24, where the upper line
reaching past an
average score of 45 in the DoubleTrouble entry shows Braini subjects'
improvement in concentration,
and the lower line plateauing between an average score of 40 and 42.5
represents average placebo
subject scores.
TABLE 19
time n mean sd time n mean sd
DigitSpan PairedAssoc
-
Placebo 1Baseline 24 6.29 1.12 Placebo Baseline 24 5.00 1.02
Placebo Post 18 6.83 1.29 Placebo Post 18
4.83 1.54
Braini I Baseline 26 6.58 1.10 Braini Baseline 26 4.69
0.79
Braini Post 23 6.65 1.19 Braini Post 23 4.87
0.69
DoubleTrouble SpatialSpan
Placebo Baseline 22 27.73 16.17 Placebo Baseline 24 6.17 0.87
Placebo Post 19 41.74 16.28 Placebo Post
19 6.16 1.12
1
Braini Baseline 26 24.73 15.89 Brain'
Baseline 26 5.85 1.01
Braini Post 23 45.74 14.79 Brain' Post
23 6.09 0.90
FeatureMatch
Placebo Baseline 24 118.58 34.53
Placebo Post 18 120.00 37.76 58
Braini Baseline 26 121.15 30.59
Brain l Post 23 123.13 37.58
Qatt_germai 9Red2QZQ4t4 ________
5
EXAMPLE 19
Improved Executive Function in Braini Clinical Trials for Young Adults and
Seniors
In two randomized double-blinded, placebo-controlled clinical trials, one in
healthy young adults
(ages 18-30) and one in healthy seniors (ages 50-80), subjects took either the
Braini supplement or
placebo as directed for 28 days.
The trials assessed subjects' overall cognitive performance at baseline prior
to starting
administration of Braini or placebo, and then again at 28 days, using the CNS
Vital Signs (Morrisville,
NC) computerized neuro-cognitive performance self-testing suite. After the
dietary study concluded, 10
outcome measures were assessed independently between the active Braini (A)
and placebo (P) cohorts
in each age group by the pharmaceutical statistical analysis firm
Pharmalnitiatives (Chapel Hill, NC).
YOUNG ADULT SUBJECTS
The university IRB-reviewed randomized double-blinded placebo-controlled study
of young adult
subjects included 30 participants with 14 active (administered Braini ) and 16
placebo subjects. Overall,
92% of all participants improved by at least 2.5% on the following 6 CNS Vital
Signs tests: Psychomotor
Speed, Reaction Time, Cognitive Flexibility, Processing Speed, Executive
Function, Motor Speed. 61%
of all subjects improved by at least 2.5% to 15.1% in Cognitive Flexibility.
= 77% of all subjects improved by at least 2.5% to 19.5% in Executive
Function.
= One subject improved 42.5% in Processing Speed; another subject improved
by 37.3%, and a third
improved by 29%.
Executive Function
Of the 6 CNS Vital Signs outcome measures scored for each subject in the above-
described trials,
scores related to Executive Function showed greatest improvement. As discussed
above, Executive
59
Date Recue/Date Received 2022-04-14
Function is one of the most difficult neuro-cognitive performance measures to
influence, especially
without stimulants. Executive Function measures how well a subject recognizes
rules, categories, and
manages or navigates rapid decision making, and relates to one's ability to
sequence tasks and manage
multiple tasks simultaneously as well as tracking and responding to a set of
instructions. This is a shifting
attention test.
Both studies had to be curtailed because of the coronavirus pandemic, which
limited subject
recruitment and made it harder to achieve statistical significance.
Nonetheless, after the clinical trial
concluded and the study was unblinded, the following key findings were
identified.
See for instance Fig. 25, a non-parametric model (Asymptotic Wilcoxon-Mann-
Whitney Test)
showing improvements in Executive Function (p=0.03; Z=2.152) in young adult
subjects administered
Braini (A) compositions of this invention, compared with a Placebo (P)
composition.
TABLE 20
Non-Parametric Model
Treatment Baseline (mean) Baseline (SD) Change
(mean) Change (SD)
A (Braini ) 101.7857 17.40989 6.166667
9.504385
P (Placebo) 110.6000 12.71303 -1.578947
9.645470
Some slides indicate that baseline scores were lower in those administered
Braini compositions,
showing that even if the placebo cohort performed better at Baseline on
average, the fact they were taking
a placebo still reverted to almost no overall change in this outcome measure.
If the results were biased
because this cohort was "smarter" they would be expected to achieve a higher
overall improvement after
28 days, but this didn't happen.
In Fig. 26, a parametric model shows that young adult subjects taking Braini e
(A, Active)
improved in Executive Function performance consistently with administration
Braini (A) regardless of
baseline, as compared with Placebo (P). Placebo results were mixed, but did
not show strong regression
to the mean.
TABLE 21
Parametric Model, Residuals and Coefficients
Residuals
Min 1Q Median 30 Max
-22.1667 -6.4211 -0.1667 7.5789
16.5789
Coefficients
Date Recue/Date Received 2022-04-14
Estimate Std. Error t value
Percentile
(Intercept) 6.167 2.769 2.227
0.0339*
Treatment (P) -7.746 3.537 -2.190
0.0367*
*Significance code 0.05
Residual standard error: 9.592 on 29 degrees of freedom (3 observations
deleted due to subject
absence). Multiple R-squared: 0.1419, Adjusted R-squared: 0.1123, F-
statistic:4.796 on 1 and 29 DF, p-
value: 0.03672
In Fig. 27, a multiple regression model incorporating baseline scores shows
improvement in
Executive Function in healthy young adult subjects with administration of
Braini (A) over Placebo (P)
(p=0.08).
Parallel lines with downward slopes support consistent effects across baseline
scores.
TABLE 22
Residuals
Min 10 Median 30 Max
-17.8865 -5.4223 -0.5217 5.8953
17.8757
Coefficients
Estimate Std. Error t value
Percentile
(Intercept) 26.5496 12.6713 2.095
0.0453*
Treatment (P) -6.3765 3.5363 -1.803
0.0821.
Baseline -0.1987 0.1207 -1.646
0.1109
*Significance code 0.05; =Significance code 0.1
Residual standard error: 9.321 on 28 degrees of freedom (3 observations
deleted due to subject
absence). Multiple R-squared: 0.2176, Adjusted R-squared: 0.1617, F-
statistic:3.894 on 2 and 28 DF, p-
value: 0.0322
Fig. 28 improves the above multiple regression model by analyzing absolute
deviation from the
scaled mean of 100 instead of baseline. The effect of very high values is
significant, showing clear and
beneficial improvement in young adult subjects administered Braini (A) as
compared with subjects
administered Placebo (P).
TABLE 23
Residuals
Min 10 Median 30 Max
-14.4238 -6.2144 0.5441 3.5068
18.6507
Coefficients
Estimate Std. Error t value
Percentile
(Intercept) 14.6370 3.2955 4.441
0.000127***
Treatment (P) -7.9453 2.9698 -2.675
0.012328*
61
Date Recue/Date Received 2022-04-14
Baseline -0.6236 0.1720 -3.626
0.001134**
***Significance code 0 '***', 0.001 '**', 0.01 '*'
Residual standard error: 8.053 on 28 degrees of freedom (3 observations
deleted due to subject
absence). Multiple R-squared: 0.4161, Adjusted R-squared: 0.3744, F-
statistic:9.976 on 2 and 28 DF, p-
value: 0.0005356
DISCUSSION
Executive Function in Healthy Young Adults
In healthy young adults aged 18-30, subjects taking Braini for 28 days
experienced significant
improvement over placebo in Executive Function (p=.0215). The Braini subjects
improved by an average
of 7.8% over the placebo subjects, with changes from baseline ranging from -
12.7% to +19.6% in the
Braini cohort compared with a range of -17.2% to +15.6% in the placebo cohort.
Also, the researchers
found that subjects taking Braini experienced significantly improved CNS
correct response shifting
attention reaction time (SAT-RT, important in Executive Function) compared
with placebo by an average
1/10th of a second (p=.007). Researchers consider a p-value of less than .05
to be significant.
SENIOR ADULT SUBJECTS
A university IRB-reviewed randomized double-blinded placebo-controlled study
of senior adult
subjects (ages 50-80) included 30 participants with 14 active (administered
Braini ) and 16 placebo
subjects. The trial was suspended early due to the coronavins pandemic, so
statistical power in the
subject pools was difficult to achieve.
As a cohort, the baseline scores of the active Braini subject group was
extremely high, with the
vast majority of the participants scoring in the "high function" category
(>110) in I or more CNS
outcome measures.
92.8% of active (Braini ) subjects had 2.5% or better improvement on at least
1 of 6 cognitive
performance measures.
Executive Function in Healthy Seniors
Healthy high-functioning seniors aged 55-80, after oral administration of
Braini for 28 days,
experienced significant improvement in Executive Function including SAT-RT vs
placebo (p value of
.05) achieving an average of 66.8 millisecond improvement in this parameter.
Reaction Time in Healthy Young Adult Subjects and in Healthy Seniors
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Date Recue/Date Received 2022-04-14
In both age groups, subjects experiencing a slower baseline Reaction Time
before Braini
administration experienced greater improvement in Reaction Times after 28 days
of Braini
administration, compared to subjects taking placebo.
EXAMPLE 20
CONTINUING EVIDENCE OF DRAMATIC IMPROVEMENT IN EXECUTIVE FUNCTION AND
COGNITIVE FLEXIBILTY OUTCOME MEASURES IN SUBJECTS WITH
DYSLEXIA AND/OR SELF-REPORTED LEARNING DIFFERENCES
A young adult (age 18) and a senior adult (age 62) were orally administered
Braini capsules of
this invention, as described above, for 28 days. In these and other dyslexic
subjects, the lowest test scores
are typically in Executive Function and Cognitive Flexibility. All tests and
scores shown in Table 24
(young adult) below had a positive Validity Indicator. In Table 25, a negative
Validity Indicator is
indicated by "1".
As shown in the below Tables, Cognitive Flexibility and Executive Function
dramatically
increased in the young adult and senior adult tested, from Low Average (young
adult) or Very Low
Average (senior adult) to Average functioning in these areas for both age
groups. Other improvements in
this area may also be seen in the below Tables and other data provided.
Without being bound by theory, these improvements may be due to subjects, such
as those
presenting with dyslexia and/or ADHD, that have difficulty with the uptake
and/or processing of raw
omega 3-6-9. For instance, these subjects may lack the ability to
enzymatically digest raw omega. B.
arvensis (Ahiflower ; NeurXcel ) oil according to the present invention
includes omega SDA and GLA
that have already been converted and may be easier for certain populations to
absorb.
In the young adult subject with dyslexia discussed in this Example, evidence
of dyslexia in CNS
Vital Signs scores was accompanied by a diagnosis of calcification of the
heart, possibly representing
that the subject's body cannot break down omegas and possibly, rather,
accumulates them. Fats were
severely restricted from his diet, including healthy fats such as from nuts
and avocados, because people
with his condition cannot digest them. The subject responded very well to 30
days' administration of
Braini capsules. Without being bound by theory, Braini capsules may have
improved absorption
and/or processing of SDA and GLA and facilitated SDA and GLA entry with B.
mori fiber of this invention
into the brain. B. mar! fiber (Peptylin ; BF-7; of the present invention) has
been shown to reduce
ischemic stroke in animal models.
63
Date Recue/Date Received 2022-04-14
TABLE 24
Improved Cognitive Flexibility and Executive Function in Young Adult having
Dyslexia
Patient Profile
Pre-Administration of Braini Post-Administration of
Braini
(Young Adult)
Patient Standard Patient Standard
Domain/Scores Percentile Status
Percentile Status
Score Score Score Score
Verbal Above
58 121 92 55 109 73 Ave.
Memory Ave.
Psychomotor
175 99 47 Ave. 160 90 25 Ave.
Speed
,
Reaction Very
Very
841 59 1 790 69 2
Time* Low
Low
Cognitive Low
33 81 10 49 104 61 Ave.
Flexibility Ave.
Processing
59 98 45 Ave. 54 92 30 Ave.
Speed
Executive Low
36 84 14 51 105 63 Ave.
Function Ave.
Low
Motor Speed 115 100 50 Ave. 101 89 23
Ave.
*Lower scores are better. (If no *, higher scores are better/an improvement).
Test Results ¨ Pre-Braini administration (Young Adult)
TABLE 24A
Verbal Memory Test (VBM)
Tested information Score Standard Percentile
Correct Hits - Immediate 15 118 88
Correct Passes - Immediate 13 84 14
Correct Hits - Delay 15 123 94
64
Date Recue/Date Received 2022-04-14
Correct Passes - Delay 15 113 81
Verbal Memory test: Subjects have to remember 15 words and recognize them in a
field of 15 distractors.
The test is repeated at the end of the battery. The VBM test measures how well
a subject can recognize,
remember, and retrieve words e.g. exploit or attend literal representations or
attribute. "Correct Hits"
refers to the number of target words recognized. Low scores indicate verbal
memory impairment.
TABLE 24B
Finger Tapping Test (FTT)
Tested Information Score Standard
Percentile
Right Taps Average 61 102 55
Left Taps Average 54 97 42
The FTT is a test of motor speed and fine motor control ability. There are
three rounds of tapping with
each hand. The F!! test measures the speed and the number of finger-taps with
each hand. Low scores
indicate motor slowing. Speed of manual motor activity varies with handedness.
Most people are faster
with their preferred hand but not always.
TABLE 24C
Symbol Digit Coding (SDC)
Tested Information Score Standard
Percentile
Correct Responses 60 98 45
Errors* 1 103 58
The SDC test measures speed of processing and draw upon several cognitive
processes simultaneously,
such as visual scanning, visual perception, visual memory, and motor
functions. Errors may be due to
impulsive responding, misperception, or confusion.
TABLE 24D
Stroop Test (ST)
Tested Information Score Standard Percentile
Simple Reaction Time* 411 54 1
Complex Reaction Time 787 61 1
Correct*
Stroop Reaction Time 895 65 1
Correct*
Stoop Commission 3 83 13
Errors*
The ST measures simple and complex reaction time, inhibition / disinhibition,
mental flexibility or
directed attention. The ST helps assess how well a subject is able to adapt to
rapidly changing and
increasingly complex set of directions. Prolonged reaction times indicate
cognitive slowing / impairment.
Errors may be due to impulsive responding, misperception, or confusion.
Date Recue/Date Received 2022-04-14
Test Results ¨ Post-Braini@ administration (Young Adult)
TABLE 24E
Verbal Memory Test (VBM)
Sub-tests Score Standard
Percentile
Correct Hits - Immediate 14 110 75
Correct Passes - Immediate 15 110 75
Correct Hits - Delay 12 104 61
Correct Passes - Delay 14 100 50
TABLE 24F
Finger Tapping Test (FTT)
Sub-tests Score Standard Percentile
Right Taps Average 56 95 37
Left Taps Average 45 83 13
TABLE 24G
Symbol Digit Coding (SDC)
Tested Information Score Standard Percentile
Correct Responses 59 97 42
Errors* 5 62 1
TABLE 24H
Stroop Test (ST)
Tested Information Score Standard Percentile
Simple Reaction Time* 396 59 1
Complex Reaction Time 692 79 8
Correct*
Stroop Reaction Time 887 67 1
Correct*
Stroop Commission Errors* 2 93 32
TABLE 25
Improved Cognitive Flexibility and Executive Function in Senior Adult Having
Dyslexia
Patient Profile Pre-Administration of Brainie
Post-Administration of Braini
66
Date Recue/Date Received 2022-04-14
(Senior Adult)
Patient Standard Patient Standard
Domain/Scores Percentile Status
Percentile Status
Score Score Score Score
Verbal Low
46 87 19 48 94 34
Ave.
Memory Ave.
Psychomotor
Above
163 107 68 Ave. 174 114 82
Speed
Ave.
Reaction Low
841 84 14 740 96 40
Ave.
Time* Ave.
Cognitive Very
_331 38 1 42 105 63
Ave.
Flexibility Low
Processing
Above
47 105 63 Ave. 55 115 84
Speed
Ave.
Executive Very
-321 37 1 43 104 61
Ave.
Function Low
Above
Motor Speed 116 108 70 Ave. 118 110 75
Ave.
*Lower scores are better. (If no *, higher scores are better/an improvement).
'Indicates a negative Validity Indicator.
Test Results ¨ Pre-Braini administration (Senior Adult)
TABLE 25A
Verbal Memory Test (VBM)
Tested Information Score Standard Percentile
Correct Hits - Immediate 12 102 55
Correct Passes - Immediate 13 86 18
Comet Hits - Delay 11 104 61
Correct Passes - Delay 10 64 1
TABLE 25B
Finger Tapping Test (F f1)
67
Date Recue/Date Received 2022-04-14
Tested Information Score Standard Percentile
Right Taps Average 62 113 81
Left Taps Average 54 102 55
TABLE 25C
Symbol Digit Coding (SDC)
Tested Information Score Standard Percentile
Correct Responses 47 103 58
Errors* 0 114 82
TABLE 25D
Stroop Test (ST)
Tested Information Score Standard
Percentile
Simple Reaction Time* 313 98 45
Complex Reaction Time 726 91 27
Correct*
Stroop Reaction Time 956 81 10
Correct*
Stoop Commission 1 103 58
Errors*
Test Results ¨ Post-Braini administration
TABLE 25E
Verbal Memory Test (VBM)
Tested Information Score Standard Percentile
Correct Hits - Immediate 12 102 55
Correct Passes - Immediate 13 86 18
Correct Hits - Delay 11 104 61
Correct Passes - Delay 12 82 12
TABLE 25F
Finger Tapping Test (FTT)
Tested Information Score Standard Percentile
Right Taps Average 63 114 82
Left Taps Average 55 104 61
TABLE 25G
68
Date Recue/Date Received 2022-04-14
Symbol Digit Coding (SDC)
Tested Information Score Standard
Percentile
Correct Responses 56 115 84
Errors* 1 103 58
TABLE 25H
Stroop Test (ST)
Tested Information Score Standard
Percentile
Simple Reaction 293 101 53
Time*
Complex Reaction 653 99 47
Time Correct*
Stroop Reaction Time 827 94 34
Correct*
Stroop Commission 1 103 58
Errors*
In dyslexic subjects, the lowest test scores are typically in Executive
Function and Cognitive Flexibility.
Dyslexic subjects administered a composition according to the present
invention (Braini capsules, as
above), for about 28 days, or more, showed very good, even strong improvement
in CNS Vital Signs test
outcome measures.
Executive Function is tightly linked to ADHD and other attention deficit
disorders. Four of the
CNS Vital Signs tests used in the Braini clinical trials described in this
application are used to measure
ADHD. They are Executive Function, Reaction Time, Cognitive Flexibility and
Processing Speed. CNS
Vital Signs is used throughout the world as a clinical tool to evaluate and
manage ADHD. Executive
Functioning, sometimes called executive control system, is generally
considered a frontal lobe
neurocognitive system that controls and manages other cognitive processes. It
is considered a higher-
order brain function, which includes attention, behavioral planning and
response inhibition, and the
manipulation of information in problem-solving tasks. Executive Function is
sometimes referred to as the
"command and control" or the "conductor" of many cognitive skills (Iverson et
al., J. Affect Disord.
132(3):360-367 (2011)). Likewise, Cognitive Flexibility is related to Mood
Disorders according to
Iverson et al. and CNS Vital Signs reports.
EXAMPLE 21
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Date Recue/Date Received 2022-04-14
A senior adult healthy woman self-administered Braini capsules as described
above, daily for
about 23 months. CNS Vital Signs testing was conducted and all scores improved
over time.
Specifically, the subject's Psychomotor Speed improved from 111 to 138 (24%
improvement),
and the subject's Motor Speed improved from 120 to 141 (18% improvement); test
scores are capped at
.. 130 in terms of percentile ranking and the like.
The subject's greatest improvement was seen in Processing Speed, improving
from 88 to 121
(38% improvement). Other improvements include in Reaction Time (from 96 to
102; 6% improvement),
Cognitive Flexibility (from 93 to 107; 15% improvement), and Executive
Function (from 94 to 109; 16%
improvement).
These results show that improvements from Braini show benefit over time,
including providing
neuroprotection with consistent administration.
EXAMPLE 22
In this Example, Scanning Electron Microscope (SEM) images of the Braini
composition
described above, and its individual constituents Peptylin powder, NeurXcel
powder, and wild blueberry
powder, show that the Braini composition contains bonding between the
ingredients and interactions to
indicate the formation of a new physical structure.
As shown below, each of the three initial components (Peptyline powder,
NeurXcel powder, and
wild blueberry powder) has a distinctive morphology that allows it to be
easily identified by shape in the
Braini compositions tested. The Braini compositions all comprise and in an
embodiment consist of
agglomerates where the individual particles are made up of two or more of the
initial components.
In the unencapsulated powders, there is merging between the primary particles
making up the
Braini agglomerates, more than would be seen for mere electrostatic
attraction.
In the encapsulated formulations, the degree of merging and interaction
between the ingredients is
even more evident than in the unencapsulated materials. There is a clear
change in morphology and a skin
of some material not seen in the micrographs of the initial ingredients
appears to be covering the
agglomerates and bonding them together.
Materials:
Braini Capsules
Braini Lex Capsules
Braini Powder
Braini Lex Powder
Date Recue/Date Received 2022-04-14
NeurXcel lot # 19100359c
Peptylin (BF-7) lot # 71908002
Wild dried blueberry powder lot # 18355
Methods:
Capsules were split with a scalpel and the powder inside analyzed. For each
sample, a small
amount of powder was dispersed onto a Leit tab (sticky pad made of carbon) and
mounted on an
aluminum stub. Each was coated with a 20 nm thick layer of gold using a
Polaron T sputter coater fitted
with a FT7690 film thickness monitor.
Images were obtained with a Carl Zeiss Evo-60 microscope fitted with a LaB6
emitter running at
1.96 A, the second emission current. The working distance was between 7-8 mm,
the EHT 20 kV, 100 pA
probe current, chamber vacuum of 5 x 10-6 mBar. Images were collected using
the Thornley-Everhart
secondary electron detector. Beam alignment was checked and corrected hourly.
Results:
NeurXcel powder is shown as discrete spheres in Fig. 29, ranging from 10-100
um in diameter.
The surface of the spheres is pitted and has a covering of smaller particles
of the order of 0.5 to 1 urn in
diameter. (Scale top: 100 urn; bottom: 10 urn).
Peptylin powder is shown in the form of a dispersion of angular crystal-like
material in Fig. 30.
Particle size varies from the millimeter to micron range. The surface of the
larger particles are covered
with smaller crystalline particles in the sub-micron size range. (Scale top:
100 um; bottom: 10um).
Wild blueberry powder is shown as relatively amorphous particles in Fig. 31
ranging from 10-50
um across. (Scale top: 100 urn; bottom: 10um).
Braini powder is analyzed in Fig. 32. The powder is easily dispersed, in the
form of discrete
particles. As shown in Fig. 32, each particle is an agglomerate of two or
three of the initial ingredients.
The magnified image clearly shows all three materials in one particle. (Scale
top: 100 um; bottom: 20um).
Figs. 33-35 further show Braini powder in the form of particles made of
agglomerates of
Peptylin powder, NeurXcel powder, and wild blueberry powder. Highlighted
areas provide evidence of
constituent ingredients merging and bonding together more than would be
expected for particles merely
held together by electrostatic attraction. (Scale: 20um).
71
Date Recue/Date Received 2022-04-14
In addition, in several areas (highlighted in Fig. 35), evidence can be seen
of a 'neck' of material
between adjoining particles, adhering them together. This merging of particles
is not seen in particles held
together by electrostatic forces (where the particles would remain unchanged)
and indicates interaction
between the materials to form a new physical structure. (Scale: 20um).
Braini Lex powder was analyzed and found to have similar characteristics to
the Braini
powder. As shown in Fig. 36, the Braini Lex particles are agglomerations of
the three ingredients
(Peptyline powder, NeurXcel powder, and wild blueberry powder), and there is
evidence of merging of
the ingredients in the formation of the particles. (Scale top: 100 um; bottom:
10 um).
Fig. 37 shows the encapsulated material from Braini Capsules is, on the
surface, different from
the Braini powder. The encapsulated material is composed of discrete
particles, recognizably containing
each of the ingredients. There is a very high degree of what appears to be a
coating over the entire surface
of the particles. This is possibly an excess of the material seen in small
necks in the Braini powder that
was holding the particles together. The processing of the three ingredients
has resulted in a clear change,
showing that this is not a simple mixture of the three components. (Scale top:
100 um; bottom: 10 urn).
Fig. 38 also shows that the encapsulated material from Braini Capsules
includes a high degree of
merging of the initial ingredients. (Scale top, middle, bottom: 100um).
As shown in Fig. 39, the encapsulated material from Braini Lex capsules also
includes a high
degree of merging of the initial ingredients, such that all three ingredients
have merged together to form
discrete particles. (Scale top: 100 urn; bottom: 10 um).
Finally, Fig. 40 shows further images of the Braini Lex capsule material,
showing a high degree
of merging of the Peptylin powder, NeurXcel powder, and wild blueberry
powder. (Scale top, middle,
bottom: 100um).
Further evidence of merging and transformation of particles as seen under the
Scanning Electron
Microscope may be seen by the substantial amount of empty space around the
particles throughout these
photomicrographs. Also, without being bound by theory, it is noted that the
encapsulated material tested
was prepared in an earlier batch than the powder provided for this experiment,
and so may have had
additional time to agglomerate.
EXAMPLE 23
HPLC mass spectrometry analysis of Braini ingredients and formulation
72
Date Recue/Date Received 2022-04-14
HPLC mass spectrometry was carried out on diluted ethanol extracts of Peptylin
, NeurXcel
(NeurXcek.) seed oil encapsulated powder, BLUEd'OR Wild Dried Blueberry powder
(60 mesh),
Braini bulk powder, Braini capsules, BrainiLexe bulk powder, and BrainiLexe
capsules.
Some striking differences are evident between the Peptylin, NeurXcel, and
Blueberry starting
compounds and the Braini and BrainiLexiD products shown in Fig. 41. For
instance, peak #17 in the
Peptylin spectra (middle) is the dominant peak in the starting material, but
is completely absent in the
products (either the powder or the capsules; top 2 spectra). Peak #16 in the
Peptylin spectra is not present
in the Braini product spectra. Also, Peak #6 is not present in the Braini
product spectra.
In addition, Peak #10 in the Peptylin spectra appears to have shifted to a
lower retention time
(Peak #11) in the Braini dietary supplement spectra. Peak #10 in the Peptylin
spectra is part of a group of
5 peaks. The retention times of Peaks #7 and #8 in the Peptylin spectra have
not altered in the Braini
dietary supplement spectra, but Peak #10 has shifted to a lower retention time
(Peak #11) in the Braini
product spectra. The disappearance of Peak #17 and Peak #16 from the Peptylin
spectra in particular
indicates that a component of the Peptylin starting material underwent a
transformation including some
form of molecular change when combined the other ingredients to form a Braini
product.
* * *
The present invention is directed to decreasing inflammation and/or oxidative
stress, and
providing neuroprotection, in a subject with compositions of this invention.
The below Examples study
cell viability and TNF-alpha production in cultured cells exposed to Peptylin
, NeurXcel , and other
substances. For the purposes of the present invention, NeurXcel oil (eg. FFA)
and NeurXcel EE oil may
be used interchangeably.
The Braini active complex of the present invention, comprising Peptylin +
NeurXcel and
optionally Blueberry Extract, exhibits a statistically significant (p<0.001)
anti-neuroinflammatory effect in
microglial RAW264.7 cells vs. NeurXcel alone (+7.8% better) and vs. Peptylin
alone (which by itself
exhibited no such effect). See e.g. Examples 24 and 25.
For instance, Peptylin(r) alone only slightly reduces the presence of
interleukin-6 (IL-6), while
NeurXcel alone has a higher effect in reducing the presence of IL-6 (in the
range of 24%-55% as shown
Example 25 below). In combination, Peptylin and NeurXcel together provide a
greater effect on IL-6
reduction (in a range of 52-68% reduction as shown Example 25 below).
In the same cell challenge model, the Braini active complex of Peptylin +
NeurXcel
significantly and unexpectedly outperforms omega-3 DHA by as much as 38%. DHA
is globally
73
Date Recue/Date Received 2022-04-14
recognized as part of the standard of care for preventing or ameliorating the
effects of age-related
dementia and for treating traumatic brain injuries, concussions, and other pro-
inflammatory insults to
the brain.
Overall, the Braini formulation comprising Peptylin and NeurXcel has shown a
significant anti-
inflammatory response, which is inclusive of all physiologic systems, examples
of which are
inflammatory diseases, auto-immune diseases, cardiovascular diseases, many
types of cancer,
inflammatory neurological diseases, inflammatory gastrointestinal diseases,
and other diseases or
conditions in which inflammation is inherent. In an embodiment, a method of
the present invention
includes a method of treating and/or preventing an inflammatory disease, an
auto-immune disease, a
cardiovascular disease, and a type of cancer. In an embodiment, such
inflammatory disease is an
inflammatory neurological disease or an inflammatory gastrointestinal disease.
In an embodiment, the present invention is a method of treating or preventing
inflammation
and/or oxidative stress. In an embodiment, the present invention is a method
of treating brain trauma,
or brain injury, decreasing neuroinflammation, treating or preventing ALS
(Amyotrophic Lateral
Sclerosis), treating or preventing inflammatory diseases including systemic,
infectious diseases, treating
auto-immune diseases such as Multiple Sclerosis, treating or preventing an
auto-immune disease;
treating and/or preventing arthritis including rheumatoid arthritis, lupus,
inflammatory bowel disease
(IBD), Crohn's disease, Addison's disease, Grave's disease, myasthenia gravis,
Hashimoto's disease,
celiac disease, ulcerative colitis, gastritis; treating and/or preventing
cardiovascular disease and/or
aiding recovery from a cardiovascular incident, a stroke, calcification of the
heart, myocardial infarction;
treating a type of cancer or epilepsy; treating and/or preventing an
inflammatory neurological disease;
treating and/or preventing meningoencephalitis, inflammatory mechanisms in
neural conditions (such
as inflammatory mechanisms in Alzheimer's, Parkinson's, Huntington's disease,
ALS, stroke, traumatic
brain injury). See for instance Degan et al. (2018). Without being bound by
theory, the present
invention activates glial cells and complement-mediated pathways, synthesis of
inflammation
mediators, and/or recruits leukocytes.
In an embodiment, the present invention decreases inflammation in the body. In
an
embodiment, the present invention decreases oxidative stress in the body. In
an embodiment, the
present invention promotes neurogenesis in the body. In an embodiment, a
method of the present
invention treats and/or prevents, and/or decreases inflammation and oxidative
stress, in leaky brain and
related neurological and psychiatric disorders, and in major depressive
disorder. See for instance Morris
74
Date Recue/Date Received 2022-04-14
et al. (2018). In an embodiment, a method of the present invention may
decrease inflammation and/or
oxidative stress and thereby treat, prevent, or aid in treatment/prevention of
Alzheimer's disease,
chronic pain, neurodegenerative disorders, brain disease. See for instance
Salter (2017) and Kwon et al.
(2020).
In an embodiment, a method of treatment and/or prevention according to this
invention
comprises the steps of providing a composition having an effective amount of
Buglossoides arvensis
seed oil and purified Bombyx mori cocoon silk peptide fiber, and optionally
blueberry extract, and
administering an effective amount of the composition to a subject to treat
and/or prevent as specified.
In an embodiment, a method of decreasing inflammation and/or decreasing
oxidative stress according
to this invention comprises the steps of providing a composition having an
effective amount of
Buglossoides arvensis seed oil and purified Bombyx mori cocoon silk peptide
fiber, and optionally
blueberry extract, and administering an effective amount of the composition to
a subject to treat and/or
prevent as specified. In an embodiment, an "effective amount" is as defined
throughout this
application, and in particular with regard to a method of treating and/or
preventing a disease, condition,
or disorder, or decreasing inflammation and/or oxidative stress, a mass
balance ratio of oil:peptide fiber
of about 0.15-0.5:1, for instance about 0.18-about 0.43:1, and about 0.25-
0.35:1, and about 0.3:1. In an
embodiment, a daily dose of amounts of refined B. arvensis oil and B. mori
peptide fiber according to
this invention include ranges of amounts as defined throughout this
application and in keeping with the
above ratios. In an embodiment, an effective amount according to this
invention includes as a
composition and/or a daily dose a combination of about 100-5500 mg Peptylin
(for instance 1093-5464
mg or 100-1000mg or 200-1000nng or 400-600mg) and of about 25-3000mg NeurXcel
(for instance
about 25-750mg or 250-750mg). Timeframes for administration of the present
compositions in a
method of the present invention include those described throughout this
application, and for instance 1
day, 2-6 days, 1 week, 2 weeks, 1 day-1 month, 1 month-2months or more, and so
on.
In an embodiment, the present invention includes a method of a sequential
treatment or
administration with refined Buglossoides arvensis oil (NeurXcel ) of the
present invention. Said method
includes the steps of providing a composition comprising an effective amount
of refined Buglossoides
arvensis seed oil (e.g. NeurXcel ) as defined above, and then administering
the composition to a subject
already taking a daily or regular dose of Peptylin . In an embodiment, such a
composition, and/or a
daily dose of said refined Buglossoides arvensis seed oil, is up to 3000 mg,
including for instance 25-
Date Recue/Date Received 2022-04-14
3000mg, 50-1000 mg, 100-800 mg, 200-750mg, 250-750mg, 25-400 mg, or other
amount appropriate to
the subject. In an embodiment, the subject is human.
The present invention uses the synergistic effects of different natural-based
products (silk
fibroin peptide (Peptylin ), NeurXcel (including plant-based omega), and
optionally blueberry extract)
to promote health in all aspects of the nervous system ¨ central, peripheral,
and autonomic ¨to
decrease inflammation, decrease oxidative stress, and promote neurogenesis.
Compositions and
methods of the present invention may protect against oxidative stress. These
activities, collectively,
both promote health and help to decrease the burden of inflammatory conditions
in the nervous
system, including but not limited to cognitive decline, multiple sclerosis,
Parkinson's Disease,
Alzheimer's disease, dementia, cognitive decline, a seizure disorder,
depression, and anxiety. In an
embodiment, the method of the present invention may treat Alzheimer's disease,
dementia, cognitive
decline, and other conditions arising from elevated blood sugars (see e.g. the
below Examples and anti-
inflammatory responses from elevated saccharide LPS (lipopolysaccharide).
Generally, no effect on cell viability in WST-8 assays was found after
exposure of the cells for
instance to EPA EE (eicosapentaenoic acid, ethyl ester), Peptylin , Peptylin
in combination with
NeurXcel , Peptylin in combination with EPA EE, NeurXcel Oil EE, or NeurXcel
Oil FFA (as defined in
above). In one study, Peptylin alone was found to increase cell viability by
10-25%.
In cell cultures challenged with LPS to stimulate TNF-alpha production,
compared with
lipopolysaccharide (LPS) controls, EPA EE alone was found to reduce TNF-alpha
production in BV-2 cells
.. by 25% in one study, 18% in another study, and 27-33% in a third study. In
RAW264.7 cells, EPA EE
reduced TNF-alpha production by 32%. Peptylin reduced TNF-alpha production by
31-39% in BV-2
cells. Lower concentrations of 5Ougiml negated Peptylie's pro-inflammatory
effect. Peptylin
combined with EPA EE reduced TNF-alpha production by 35-42% in BV-2 cells.
Peptylin in combination
with NeurXcel in one study achieved a 30-56% reduction in TNF-alpha
production in RAW264.7 cells.
NeurXcel Oil EE alone reduced TNF-alpha production in RAW264.7 cells by 31-
58%, and then again, by
37-52%. NeurXcel Oil EE was not cytoxic at lower concentrations, but became
cytotoxic at higher
concentrations (>300ug/m1). NeurXcel Oil FFA did not affect TNF-alpha
production in some preliminary
studies.
Summary of Studies on Peptylin and NeurXcel on LPS-induced TNF-alpha
production in BV-2 and
RAW264.7 cells
76
Date Recue/Date Received 2023-09-14
In cell viability studies where EPA EE had no effect, Peptylin alone
increased cell viability by 10-
25%. NeurXcel Oil EE and NeurXcel Oil FFA (considered physiologically
equivalent for the purposes of
this invention as discussed above) also had no effect on cell viability. Where
a 25% reduction in LPS-
induced TNF-a production was seen by EPA EE exposure to BV-2 cells, a 31-39%
reduction in TNF-a
production was found with Peptylin alone, and a 35-42% reduction in TNF-a
production found with
Peptylie+EPA EE. NeurXcel Oil alone (EE or FFA) and Peptylin+NeurXcel had no
effect on LPS-induced
TNF-a production in BV-2 cells. NeurXcel Oil EE reduced viability of RAW264.7
cells by 14-19%. Also in
that study, NeurXcel Oil EE reduced LPS-induced TNF-a production by 31-58%,
compared with
reductions of 21-42% (DHA EE), 32% (EPA EE), and 35-71% (toxic amount of SDA
EE) (see above
discussion of omega-3 and omega-6 studies).
In a study using RAW264.7 cells, where Peptylin , Peptylin + NeurXcel , and
NeurXcel Oil EE
had no effect on cell viability, the effect of Peptylin alone caused an
increase of 3-71% in LPS-induced
TNF-a production, causing an antagonist effect. Peptylin at a 50ug/m1
exposure increased TNF-a
production by 3%; 100 ug/ml increased TNF-a production by 38%; 150 ug Peptylin
/ml increased TNF-a
production by 41%; and 200 ug Peptylin /m1 increased TNF-a production by 71%.
In contrast, NeurXcel
Oil EE reduced TNF-a production by 35-45% (35% with a 30 uM concentration of
NeurXcel and 45%
with a concentration of 50uM NeurXcel ). Taken together, 50 ug/m1Peptylie+
50uM NeurXcel
increased TNF-a production by 10%; 100 ug/m1Peptylie+ 50uM NeurXcel increased
TNF-a production
by 17%; 150 ug/ml Peptylin + 50uM NeurXcel increased TNF-a production by 13%;
and 200 ug/ml
Peptylin + 50uM NeurXcel increased TNF-a production by 21%.
In a study challenging RAW264.7 cells in 3 batches (discussed above), where
SDA EE reduced
LPS-induced TNF-a production by 16-24% and DGLA EE reduced TNF-a production by
11-18%, NeurXcel
Oil EE reduced TNF-a production by 55-79%. In another study, Peptylin ,
Peptylie+NeurXcelt and
NeurXcel Oil EE had no effect on RAW264.7 cell viability. 50ug/m1Peptylin
did not increase or
decrease LPS-induced TNF-a production, however, in combination with 50uM
NeurXcel , Peptylin and
NeurXcel reduced TNF-a production by 30%; and 50 ug/ml Peptylin and 100uM
NeurXcel reduced
TNF-a production by 56%. Taken alone, NeurXcel 50uM reduced TNF-a production
by 37% and 100uM
NeurXcel reduced TNF-a production by 52%. Overall, Peptylie+NeurXcel taken
together reduced
LPS-induced TNF-a production by a statistically significant 30-56%, whereas
Peptylin alone had no
effect on TNF-a production, and NeurXcel Oil EE (50,100uM) reduced TNF-a
production by 37-52%. In a
further study, Peptylie+NeurXcel did not diminish RAW264.7 cell viability.
Peptylin (60 ug/ml) alone
77
Date Recue/Date Received 2023-09-14
did not affect LPS-induced TNF-a production, however, Peptylin (60 ug/ml) in
combination with 50uM
and 100 uM NeurXcel reduced TNF-a production by 44 and 57% respectively.
Taken alone, NeurXcel
reduced TNF-a production 39 and 54% respectively. Overall, PeptylinN-NeurXcel
reduced TNF-a
production 44-57%, whereas Peptylin alone had no effect and NeurXcel alone
reduced TNF-a
production by 39-54%.
In a further study, Peptylin and Peptylie+NeurXcel did not diminish RAW264.7
cell viability,
but where Peptylin had no effect on LPS-induced TNF-a production and NeurXcel
reduced TNF-a
production by 42-50%, Peptylie+NeurXcel reduced TNF-a by statistically
significant 49-58% (p<0.001).
Peptylin was used in an amount of 60 ug/ml in this study; NeurXcel was used
at 50 uM and 100 uM
(43% and 50% reduction in TNF-a, respectively), and Peptylin
(60ug/m1)+NeurXcel (50uM and 100uM)
reduced TNF-a by 49% and 58%, respectively.
It is noted that lower concentrations of Peptylin , such as 50ug-60ug/ml,
negate pro-
inflammatory effects, and that optimal TNF-a suppression occurs at
6Oug/m1Peptylie+100uM
NeurXcel . Suppression of TNF-a production is improved by 7-8% with
Peptylie+NeurXcer, compared
with NeurXcel alone (p<0.001). Peptylie+NeurXcel provide a synergistic
effect on TNF-a production.
NeurXcel is not cytotoxic at lower concentrations, but is cytotoxic at higher
concentrations
(>300ug/m1). Optimal TNF-a suppression with NeurXcer+Peptylin or NeurXcel
alone is at 100 ug/ml
NeurXcel .
In an additional study, LPS-induced production of IL-6 was measured in
RAW264.7 cells.
Exposure of Peptylin alone to the LPS-treated cells resulted in a decrease in
IL-6 production of 17.20%.
NeurXcel at 30uM, 50uM, and 70uM concentrations reduced LPS-induced IL-6
production by 24.16%,
36.11%, and 54.57%, respectively. NeurXce16(30uM, 50uM,
70uM)+PeptylinN50ug/m1) reduced LPS-
induced IL-6 production by 51.67%, 67.69%, and 66.69%, respectively.
Overall, Peptylin alone reduced the presence of IL-6 by a relatively small
amount (about 17%)
compared with the upper range achieve by NeurXcel alone (24-55%). However,
Peptylie+NeurXcel
combined provided a greater effect on IL-6 reduction (52-68%). The combined
reduction of IL-6
Peptylin NeurXcel was overall greater than Peptylin alone and NeurXcel
alone, and the reduction
of IL-6 by NeurXcel alone was greater than by Peptylin alone.
EXAMPLE 24
Effect of Peptylin and NeurXcel on LPS-induced Microglial Activation in
RAW264.7 Cells
1. EXECUTIVE SUMMARY
78
Date Recue/Date Received 2023-09-14
1.1 Aim
The aim of the present study is to assay the effect of NeurXcel and Peptylin
, used in combination or
alone, in the LPS-induced TNF-a production in the mouse macrophage RAW264.7
cell line.
1.2 Methods
RAW264.7 cells were seeded in 96 well plates at a density of 20,000
cells/well. On day 2, the cells were
serum starved for 24h in DMEM. Then, the cells were pre-treated with 60m/m1 of
Peptylin alone or in
combination with 50 p,M or 100 jaM NeurXcel EE diluted in DMEM. Appropriate
vehicle controls have
been included, i.e. 0.5% Et0H + 1.2% H20. On day 4 the cells were LPS-induced
(500 ng/ml) for 1.5h and
the supernatants were collected. A WST-8 assay was performed to measure cell
viability and the
supernatants were used for an ELISA assay to measure TNF-a levels. All the
experiments were carried
out with 9 replicates (for control and LPS treated cells) or 24 replicates
(for cells treated with NX and/or
PT). A t-test was used to compare the media of TNF-a levels between the
treatment of NX alone and in
combination with PT.
1.3 Results
According to the WST-8 assay none of the treatments affected cell viability.
The PT alone did not reduce
TNF-a levels. The treatment with 50 M NX induced a reduction of 42% in TNF-a
and the cotreatment
with PT further reduced these levels 49%. The TNF-a was reduced 50% when cells
were treated with 100
j.tM NX while the co-treatment with PT induced a reduction of 58%. in both
cases there was a significant
difference between treating the cells with NX alone or in combination with PT
(p<0.001).
1.4 Conclusion
The NeurXcel reduces the TNF-a levels in RAW264.7 cells induced with LPS. The
co-treatment of cells
with Peptylin significantly improves the protective effect of NeurXcel .
1. INTRODUCTION
Microglia are the primary antigen-presenting cells in the central nervous
system. These immune-like
cells can be activated in response to injury, disease, or inflammation,
leading to the secretion of a
variety of factors such as proinflammatory cytokines, prostaglandins, and
reactive oxygen/nitrogen
species, each of which can cause neuronal damage. LPS triggers an array of
microglial response by
interacting with the membrane receptor Toll-like receptor 4 (TLR4), leading to
the production of
proinflammatory mediates (e.g., cytokines and interleukins, TNFa, and IFNy)
and the self-activation of
the nuclear factor NF-KB system. In this assay, the effect of Peptylin and
NeurXcel , alone or in
combination, in the production of INFa was measured in LPS-induced RAW264.7
cells.
79
Date Recue/Date Received 2023-09-14
3. MATERIALS AND METHODS
3.1 Reagents and Equipment
Reagents and equipment in this Example are the same as disclosed in Example 1.
3.2 Test Compounds
TABLE 42
Test Compound: Peptylin
Test Item Peptylin (powdered), a Purified
Bombyx mori Cocoon
Silk Peptide Fiber of this invention
Manufacturer Famenity Co., Uiwang-si, S. Korea
Storage Conditions The compound was protected from
light and stored at
room temperature. After reconstitution of the
powder, samples were kept at 4 C.
Dilution protocol Peptylin powder was dissolved in
water to a
concentration of 5 mg/ml. From this stock, the final 60
p.g/m1concentration was prepared in DMEM (final
H20 concentration of 1.2%).
TABLE 43
Test Compound NeurXcel EE
Test Item NeurXcel , ethyl ester (NeurXcel
EE), a Refined
Buglossoides arvensis Seed Oil of this invention
Manufacturer Nature's Crops International Ltd.,
Kensington, PE,
Canada
Storage Conditions The compound was protected from
light and stored at
-20 C.
Molecular Weight 305
Dilution protocol NeurXcel EE oil was provided as a
100% active
compound. First, 20mM and 10 mM dilutions were
prepared in 100% ethanol, from which the 100 and 50
uM working concentrations were prepared in DMEM
medium (final ethanol concentration of 0.5%).
3.3 Cell Culture
The mouse RAW264.7 cells were cultured in DMEM medium supplemented with 10%
FBS in T75 flasks
for 3 days. Cultures were maintained at 37 C in a humidified atmosphere with
5% CO2.
3.4 Experimental procedure RAW264.7 cells were seeded in 96-well plates at a
density of 20,000
cells/well in DMEM + 10% FBS. The next day, the cells were washed once with
DPBS and serum starved
for 24h in 150 iL of DMEM. Then, the cells were treated with 150 liL of 60
p.g/m1 of Peptylin and 100 or
Date Recite/Date Received 2023-09-14
50 p.M NeurXcel EE that were used either alone or in combination. Appropriate
vehicle controls were
also included in the assay, i.e. 0.5% ethanol + 1.2% H20. After 24h, the cells
were incubated for 1.5h
with 10 'IL of LPS (7500 ng/ml) to reach a final concentration of 500 ng/ml.
After the incubation, the
supernatants were collected and a WST-8 assay was carried out. CCK-8 reagent
(WST-8) was diluted 1:10
in DMEM media and 100 IA were added to each well and the plate was incubated
at 372C. After 15
minutes the absorbance was measured at 450 nm using the Synergy II microplate
reader. WST-8 is
bioreduced by cellular dehydrogenases to an orange formazan product that is
soluble in tissue culture
medium. The amount of formazan produced is directly proportional to the number
of living cells. The
ELISA assay was performed following the manufacturer's instructions. The TNF-a
levels produced under
each condition were normalized to the corresponding vehicle control (with or
without Et0H). All the
experiments were carried out with 9 replicates (for control and LPS treated
cells) or 24 replicates (for
cells treated with NX and/or PT). To determine if the levels of TNF-a between
the treatment of NX alone
and in combination with PT were significantly different, a t-test was carried
out. Using Microsoft Excel
the following formula was used to calculate the p-value:
=T.TEST(array1,array2,tails,type), where array1
corresponds to the TNF-a values for one treatment, array2 corresponds to TNF-a
values for the co-
treatment, one-tail test has been considered and type is 2 (two sample equal
variance t-test). A p-value
of 0.05 was used as the cutoff for significance.
4 RESULTS
4.1 Cell viability
RAW264.7 cells were serum starved for 24 hours, pre-treated for 24h with 60
pig/m1 of Peptylin and
100 or 50 I.LM NeurXcel , alone or in combination. Then, the cells were
induced for 1.5h with 500 ng/ml
LPS. After the treatments, the supernatants were collected and the cells were
incubated with the CCK-8
reagent for 15 minutes. The absorbance was measured at 450 nm using the
Synergy II rnicroplate reader
(Fig. 42).
.. Cell viability was determined by WST-8 analyzing the amount of formazan
product produced by the cells,
which is proportional to the number of living cells. Control: cells maintained
in DMEM; LPS: LPS-induced
cells in DMEM + 0.5% EtOH + 1.2% H20; PT: cells pre-treated with 60 p.g/mlof
Peptylin for 24h; 50 NX
and 100 NX: cells treated with 50 or 1001.1M NX for 24h; 50NX+PT and 100NX+PT:
cells pre-treated with
50 or 100 I.LM NX and 60 p.g/m1 PT for 24h. Then the cells were induced with
500 ng/ml LPS for 1.5h. The
data has been normalized to the corresponding LPS control which is considered
to be 100% viability. The
81
Date Recue/Date Received 2023-09-14
data represent the mean SD of an experiment performed in nine replicates
(for Cntr and LPS) or 24
replicates (for PT and NX treatments).
According to the WST-8 assay, none of the treatments affected cell viability.
4.2 INF-a production
After the treatments, the supernatants were collected and an ELISA assay was
performed. First, the
standard curve was generated (Fig. 43). Using these data, the pg/ml of TNF-a
produced by the cells was
calculated (Fig. 44).
TABLE 44
ELISA Assay (Standardized Curve)
Correlation 0.999311167
Slope 0.921410268
Intercept -6.449858739
Blank 0.0575
Bmax 4.22914434
[1/2Bmax] 1096.61752
Iterations 236
.. The TNF-a standard was measured in 8 serial 1:2 dilutions ranging from 0-
4000 pg/ml.
TNF-a production was measured by ELISA. Control: cells maintained in DMEM;
LPS: LPS-induced cells in
DMEM + 0.5% Et0H + 1.2% H20; PT: cells pre-treated with 60 pg/mlof Peptylin
for 24h; 50 NX and 100
NX: cells treated with 50 or 100 Al NX for 24h; 50NX+PT and 100NX+PT: cells
pre-treated with 50 or
100 M NX and 60 dm' PT for 24h. Then the cells were induced with 500 ng/ml
LPS for 1.5h. The data
represent the mean SD of an experiment performed in nine replicates (for
Cntr and LPS) or 24
replicates (for PT and NX treatments). NS: non-significant; *** p<0.001.
Peptylin (60 g/ml) did not affect the TNF-a levels. The treatment of cells
with 50 p.M NeurXcel
reduced 43% TNF-a levels while the co-treatment with PT reduced TNF-a 49%.
According to the ttest
analysis, there is a significant difference between treating the cells with 50
p.M NX alone or in
combination with PT (p=5.6X10-4). Besides, the TNF-a levels were reduced 50%
when cells were treated
with 100 M NX and 58% when cells were co-treated with NX and PT. There is
also a significant
difference between treating the cells with 100 p.M NX alone or in combination
with PT (p=1.2X10-6).
5 CONCLUSIONS
According to the WST-8 assay, none of the treatments affect the cell viability
in RAW264.7 cells. The
treatment of cells with Peptylin (60 pg/ml) did not affect TNF-a levels. The
treatment of cells with 50 or
82
Date Recite/Date Received 2023-09-14
100 p.M NX reduced TNF-a levels 43 and 50%, respectively, while the co-
treatment with PT induced a
reduction of 49 and 58%, respectively. According to the t-test analysis there
is a significant difference in
the TNF-a levels between treating the cells with NeurXcel alone or in
combination with Peptylin.
EXAMPLE 25
LPS-Induced Microglial Activation Assay: Neuroinflammation-related in vitro
model
1 EXECUTIVE SUMMARY
1.1 Aim The aim of the present study is to assay the effect of NeurXcel and
Peptylin, used in
combination or alone, in the LPS-induced 1L-6 production in the mouse
macrophage RAW264.7 cell line.
1.2 Methods Cells were seeded in 96 well plates. On day 2, cells were serum
starved for 24h.
Then, cells were pretreated with 70, 50 or 30 iiM NeurXcel alone or combined
with 50 pernIPeptylin or
Peptylin alone diluted in DMEM. Appropriate vehicle control was included, i.e.
0.5% ethanol. On day 4,
cells were LPS-induced (500 ng/m1) for 6h, supernatants were collected and an
IL-6 ELISA was carried
out. Cell viability was tested with a WST-8 assay.
1.3 Results According to the WST-8 assay Peptylin had not an effect on cell
viability while
pretreatment with NX very slightly increased the number of viable cells at the
end of the experiment.
Pretreatment with NX had a protective effect on IL-6 release in response to
LPS-mediated inflammation
in a dose-dependent manner. Pretreatment with Peptylin produced a decrease in
the amount of IL6
secreted and, in combination with NX, it enhances the protection of NX against
LPS-induced
inflammation in RAW264.7 cells.
1.4 Conclusion Taking into account the data from the previous experiments
(Reports 21 and 22),
we can conclude that NX have a protective effect on IL-6 secretion in LPS-
induced inflammation in
RAW264.7 cells. Furthermore, the combination of NX and Peptylin appears to
increase the protective
effect of NX.
2 INTRODUCTION Microglia are the primary antigen-presenting cells in the
central nervous
system. These immune-like cells can be activated in response to injury,
disease, or inflammation, leading
to the secretion of a variety of factors such as proinflammatory cytokines,
prostaglandins, and reactive
oxygen/nitrogen species, each of which can cause neuronal damage. LPS triggers
an array of microglial
response by interacting with the membrane receptor Toll-like receptor 4
(TLR4), leading to the
production of proinflammatory mediates (e.g., cytokines and interleukins,
TNFa, and IFNy) and the self-
activation of the nuclear factor NF-KB system. In this assay, the effect of
different concentrations of
83
Date Recue/Date Received 2023-09-14
NeurXcel, both alone and in combination with Peptylin will be measured in LPS-
induced RAW264.7 cells
to assess the production of IL-6.
3 MATERIALS AND METHODS
3.1 Reagents and Equipment
TABLE 45
Reagent / Equipment and Catalogue and Lot Numbers Supplier
RAW264.7 (# Cat. ATCC TIB-71) ATCC
Dulbecco's Modified Eagle's Medium ¨high glucose- (# Sigma-Aldrich
Cat. D6429)
FBS (# Cat. F7524) Sigma-Aldrich
Penicillin/ Streptomycin (# Cat. 15240) Gibco
Trypsin-EDTA 0.5% (w/v) (# Cat. 25300) Gibco
PBS (# Cat. D8537) Sigma-Aldrich
Mouse IL-6 DuoSet ELISA (# Cat. DY406) - R&D Systems
DuoSet ELISA Ancillary Reagent Kit 2 (# Cat. DY008) R&D Systems
Lipopolysaccharide, LPS (It Cat. L4391) Sigma-Aldrich
Incubator (# Model 381 S/N 314342) Thermo Scientific
Synergy II microplate reader BioTek Instruments
3.2. Test compounds
TABLE 46
Test Compound: Peptylie
Test Item Peptylin (powdered), a Purified
Bombyx mori Cocoon
Silk Peptide Fiber of this invention
Manufacturer Famenity Co., Uiwang-si, S. Korea
Storage Conditions The compound was protected from
light and stored at
room temperature. After reconstitution of the
powder, samples were kept at 4 C.
84
Date Recite/Date Received 2023-09-14
Dilution protocol Peptylin powder was dissolved in
water to a
concentration of 5 mg/ml. From this stock, the final 50
u.g/mIconcentration was prepared in DMEM.
TABLE 47
Test Compound NeurXcel EE
Test Item NeurXcel , ethyl ester (NeurXcel
EE), a Refined
Buglossoides arvensis Seed Oil of this invention
Manufacturer Nature's Crops International Ltd.,
Kensington, PE,
Canada
Storage Conditions The compound was protected from
light and stored at
-20 C.
Molecular Weight 305
Dilution protocol NeurXcel EE oil was provided as a
100% active
compound. First, 10mM and 6mM dilutions were
prepared in 100% ethanol, from which the 70, 50, and
30 uM working concentrations were prepared in
DMEM medium (final ethanol concentration of 0.5%).
3.3 Cell culture
RAW264.7 cells were cultured in DMEM medium supplemented with 10% FBS in T75
flasks for 3
days. Cultures were maintained at 37gC in a humidified atmosphere with 5% CO2.
3.4 Experimental procedure
Cells were seeded in 96 well plates at a density of 30,000 cells/well in DMEM
+ 10% FBS. On day
2 the cells were washed once with DPBS and serum starved for 24h in DMEM.
Then, cells were
pretreated with 70, 50 or 30 p.M NeurXcel alone or combined with 50
iig/mIPeptylin or Peptylin alone
diluted in DMEM. Appropriate vehicle control was included, i.e. 0.5% ethanol.
On day 4 cells were LPS-
induced (500 ng/ml) for 6 hours. After the incubation, the supernatants were
collected and a WST-8
assay was carried out. CCK-8 reagent (WST-8) was diluted 1:10 in DMEM media
and 100 'IL were added
to each well and the plate was incubated at 372C. After 15 minutes the
absorbance was measured at
450 nm using the Synergy II microplate reader. WST-8 is bioreduced by cellular
dehydrogenases to an
orange formazan product that is soluble in tissue culture medium. The amount
of formazan produced is
directly proportional to the number of living cells. The ELISA assay was
performed following the
manufacturer's instructions. All the experiments were carried out in
triplicate.
4 RESULTS
Date Recite/Date Received 2023-09-14
4.1 Cell viability
RAW264.7 cells were serum starved for 24 hours, pre-treated for 24h with 70,
50 or 30 iiM
NeurXcel alone or combined with 50 p.g/m1 Peptylin or Peptylin alone diluted
in DMEM and induced for
6h with 500 ng/ml LPS. After the treatments, the supernatants were collected
and the cells were
.. incubated with the CCK-8 reagent for 15 minutes. The absorbance was
measured at 450 nm using the
Synergy II microplate reader. Peptylin had no effect on cell viability while
pretreatment with NX very
slightly increased the number of viable cells (Figure 54). In Fig. 45, data
were normalized to the
corresponding vehicle control (LPS-induced cells in DMEM + 0.5% Ethanol) that
is considered as 100%
viability. The data represent mean SD of an experiment performed in
triplicate.
4.2 IL-6 production
After the treatments, the supernatants were collected and an ELISA assay was
performed. First,
the standard curve was generated (Figure 55, Table 48).
TABLE 48
Standard Curve for ELISA Assay
Correlation 0.99857428
Slope 0.9966385
Intercept -6.45637192
Blank 0.077
Bmax 4.51697613
[1/2Bmax] 650.764773
Iterations 246
Standards were measured in 8 serial 1:2 dilutions ranging from 0 to 2000
pg/ml.
Using these data, the pg/ml of IL-6 produced by the cells were calculated
(Figure 56).
Pretreatment with NX had a protective effect on IL-6 release in response to
LPS-mediated inflammation
in a dose-dependent manner (% of reduction 24, 36 and 54% when pretreating
with 30, 50 and 70 M
NeurXcel respectively). Pretreatment with Peptylin produced a 17% decrease
in the amount of IL-6
secreted and, in combination with NX, it appears to very slightly enhance the
protection of NX against
LPS-induced inflammation (% of reduction 51, 67 and 66% when pretreating with
30, 50 and 70 iiM
86
Date Recite/Date Received 2023-09-14
NeurXcel combined with 50 pg/mIPeptylin respectively). In Fig. 47, IL-6
production was measured by
ELISA, and data represent the mean SD of an experiment performed in
triplicate.
TABLE 49
WST-8 Assay, Raw Data
Abs 450 nm Average SD Norm SD
..õ.,
Control , 0,565 0,476 0,505
0,51533333 0,04539089 93,3011467 _8,21802562
LPS 0,542 , 0,556 0,559 0,55233333 _0,00907377 _ 100
1,64280719
Pep 0,469 0,534 0,493
0,49866667 I 0,03286842 90,2836451 5,95083124
INX30 . 0,614 0,613 0,64
0,62233333 0,01530795 112,673506 2,77150573
-
INX50 0,624 0,594 0,598
0,60533333 0,01628906 109,595655 2,949135
NX70 , 0,554 0,565 0,576 0,565
0,011 102,293301 1,991551
INX3O+Pep 0,63 0,59 0,565
0,595 0,03278719 107,724804 5,93612419
INX5O+Pep 0,541 0,562 0,541 _ ....
0,5481 0,01212436 99,2154496 2,19511569
IN X7O+Pe p 0,537 0,594 0,529
0,55333333 0,03544479 100,18105 6,41728294
Table 49 shows raw data for each condition. Average in the Table refers to
average of six
replicates. SD refers to Standard Deviation, Norm refers to normalization of
all data to corresponding
LPS control which is considered to be 100% of cell viability.
Table 50 shows data for the IL-6 ELISA Standard curve shown in Fig. 46.
TABLE 50
IL-6 ELISA Standard Curve
87
Date Recite/Date Received 2023-09-14
II pdmIIL-6 Abs 450 nm
______________________________________ 0 0,077
15,625 0,195
________________________________________ 31025 0,276
________________________________________ 620,5 0,436
1111011011
125 0,756
________________________________________ 250 1,255
500 2,154
õ
________________________________________ 1000 2,833
2000 3,38
TABLE 51
IL-6 ELISA Assay
__________________________________ 1 ____ pg/ml IL-6 <,,.1._ Average
SD %of Reduction'
Control 0,077 0,083 0,077 <.c 0,861 0 0
100
IPS 1,545 1,001 1,694 321 412
372J368,333333 45,6106713 0
_ _
Pep 1,493 1,538 1,454 304 318 _____________________ 22
304,66666/ 13,0128142 17,28506787
NX30 1,172 1,577 1,466 212. 331 295
279,3333331 61,0273163 24,16289593
_ _
INX50 1,376 1,203 1,196 268 220 218
235,333333 28,3078317 36,10859729
INX70 1,019 0,984 0,965 174 166 162
167,333333 6,11010093 54,57013575
NX30-Pep 0,947 1,179 0,965 15' 214i 162J ,
178 31,24099871 51,67420814
.. . _ _
INX5O+Pep 0,695 0,695 0,897 105' 105 147j 119
24,2487113 67,69230769
INX7O+Pep 0,786 0,777 0,793 1231 12.1
1241 122,666667 1,52752523 66,69683258
Table 51 shows raw data for each condition. Using data obtained from the
standard curve the
amount of IL-6 in pg/ml was measured, as shown in Fig. 47. Average in the
Table refers to average of the
replicates, SD refers to Standard Deviation, % of Reduction represents the
decrease in IL-6 (%) compared
with LPS control.
5 CONCLUSIONS
According to the WST-8 assay, treatment with Peptylin does not affect cell
viability in
RAW264.7 cells while NX very slightly increased the number of viable cells.
NeurXcel has a protective
effect on IL-6 secretion in LPS-induced inflammation in RAW264.7 cells. The
treatment of cells with
88
Date Recite/Date Received 2023-09-14
Peptylin (50 g/ml) alone produced a very slight decrease in the amount of IL-
6 secreted and the
combination of NX and Peptylin increased the protective effect of NX.
The use of the terms "a," "an," "the," and similar referents in the context of
describing the
present invention (especially in the context of the claims) are to be
construed to cover both the singular
and the plural, unless otherwise indicated herein or clearly contradicted by
context. Recitation of
ranges of values herein are merely intended to serve as a shorthand method of
referring individually to
each separate value falling within the range, unless otherwise indicated
herein, and each separate value
is incorporated into the specification as if it were individually recited
herein. Use of the term "about" is
intended to describe values either above or below the stated value in a range
of approximately 20%; in
other embodiments, the values may range in value above or below the stated
value in a range of
approximately 5%; in other embodiments, the values may range in value above
or below the stated
value in a range of approximately 2%; in other embodiments, the values may
range in value above or
below the stated value in a range of approximately 1%. The preceding ranges
are intended to be made
clear by context, and no further limitation is implied. Reference to "about"
an amount in a range applies
to the entire range, for instance about 1-10 is intended to mean about 1 to
about 10. All method steps
described herein can be performed in any suitable order unless otherwise
indicated herein or otherwise
clearly contradicted by context. The use of any and all examples, or exemplary
language (e.g., "such as")
provided herein, is intended merely to better illuminate the invention and
does not pose a limitation on
the scope of the invention unless otherwise stated. No language in the
specification should be
construed as indicating any non-claimed element as essential to the practice
of the invention.
While in the foregoing specification this invention has been described in
relation to certain
embodiments thereof, and many details have been put forth for the purpose of
illustration, it will be
apparent to those skilled in the art that the invention is susceptible to
additional embodiments and that
certain of the details described herein can be varied considerably without
departing from the basic
principles of the invention.
The present invention may be embodied in other specific forms without
departing from the
spirit or essential attributes thereof and, accordingly, reference should be
made to the appended claims,
rather than to the foregoing specification, as indicating the scope of the
invention.
References to trademarked source materials are intended to be exemplary but
not limiting
throughout this application.
89
Date Recue/Date Received 2023-09-14
apparent to those skilled in the art that the invention is susceptible to
additional embodiments and that
certain of the details described herein can be varied considerably without
departing from the basic
principles of the invention.
The present invention may be embodied in other specific forms without
departing from the
spirit or essential attributes thereof and, accordingly, reference should be
made to the appended claims,
rather than to the foregoing specification, as indicating the scope of the
invention.
References to trademarked source materials are intended to be exemplary but
not limiting
throughout this application.
Date Recue/Date Received 2023-09-14
