Note: Descriptions are shown in the official language in which they were submitted.
COMPOSITIONS AND METHODS FOR TREATING THE EYE
CROSS REFERENCE TO RELATED APPLICATION
The present application claims the benefit of the earlier filing date of
United States
provisional patent applications 62/937,458 and 62/937,473, each, filed
November 19, 2019
and United States patent application 17/099,185 filed November 16, 2020 the
entirety of each
such application is hereby incorporated by reference herein as if fully set
forth herein.
FIELD OF THE INVENTION
The present invention relates to compositions comprising one or more compounds
and/or extracts which induce, promote and/or improve
production/release/delivery/excretion
of mucin from and/or in the cornea, and methods of using the compositions to
treat the eye.
BACKGROUND OF THE INVENTION
"Dry eye is a multifactorial disease of the ocular surface characterized by a
loss of
homeostasis of the tear film, and accompanied by ocular symptoms, in which
tear film
instability and hyperosmolarity, ocular surface inflammation and damage, and
neurosensory
abnormalities play etiological roles." Craig, J.P. et al. TFOS DEWS II
definition and
classification report. Ocul Surf 2017; 15: 276-283. Dry eye can result from
abnormal or
inadequate tear formation, and deficiency in mucin secretion (i.e.,
keratoconjunctivitis sicca).
Dry eye symptoms can be manifest as a result of various underlying disorders
such as
autoimmune disorders that damage lacrimal (i.e., tear-producing) glands, such
as rheumatoid
arthritis, Sjogren's syndrome, systemic lupus erythrematosus, and systemic
sclerosis and
sarcoidosis. Dry eye can also be induced following eye surgery, such as Lasik0
surgery.
Dry eye is estimated to affect more than 13 million individuals in the United
States.
Regardless of the underlying pathology, dry eye commonly involves the rapid
breakdown of the pre-ocular tear film, resulting in dehydration of the exposed
outer surface.
Normal tear formation is required to keep the cornea and conjunctiva moist,
and this in turn
helps to prevent ulceration of both, as well as to maintain corneal
transparency. In addition,
tears facilitate movement of the eyelid over the eye surface (e.g., blinking)
and removal of
foreign substances from the eye. Tears also normally contain lysozyme which is
useful in
preventing infection in the eye. Dry eye can be associated with mild
discomfort to severe
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Date Recue/Date Received 2020-11-19
pain in the eye. When it occurs for prolonged periods of time, it can cause
blurred vision,
grittiness and/or burning sensation, and itchiness. If the condition is
allowed to persist
without treatment, it can further lead to corneal ulcers and/or scarring.
Dry eye symptoms include eye pain or fatigue, increased blinking, and
bloodshot
eyes. Further, bacteria may enter through a scratch and cause infection, and
if the scratch is
deep enough it can even affect the vision of the person. In addition to
eyestrain, causes of dry
eye include Sjogren's syndrome, Stevens-Johnson syndrome, burns and injury to
the eye, and
side effects of hypotensive drugs, tranquilizers, eyedrops for treating
glaucoma, and other
such drugs.
Eyedrops are an effective way to treat dry eye. Such eyedrops typically
include dry-
eye treatment actives - a common active in such eyedrops is hyaluronic acid.
Hyaluronic acid
is a biologically derived macromolecular substance, has extremely high water
retention and
characteristic properties such as high viscoelasticity, good thickening
property, and good
thread-forming ability, and has been used as a humectant in topical agents for
treating various
kinds of skin problems and so forth. In the case of dry eye caused by
Sjogren's syndrome, in
which dryness is seen over' the entire body, the application of eyedrops
containing hyaluronic
acid is effective. However, when instilled as an eyedrop, hyaluronic acid has
a relatively
short residence time on the cornea, so the effect of hyaluronic acid eyedrops
lasts only about
2 or 3 hours, which means that the patient must apply the drops more
frequently (such as 5 to
.. 10 times a day).
Hyaluronic acid (HA) is produced by corneal epithelial cells in the eye.
Notably,
significantly higher hyaluronic acid concentrations have been found in the
corneas of younger
human population than in the older. (See Pacella, E., Pascella, F., De Paolis,
G., et al.
Glycosaminoglycans in the human cornea: age-related changes. Ophthalmol. Eye
Dis. 7:1-5,
2015).
In addition, the tear film is the body's natural defense against dry eye. The
tear film
contains ocular mucins and is essential for maintaining the homeostasis of the
wet ocular
surface. Mucins are produced, among other places, by corneal epithelial cells
in the eye.
Mucins are glycoproteins expressed by epithelial tissues of mucosal surfaces.
They protect
tissues by functioning as antioxidants and providing lubrication. Mucin genes
associated
with the tear film include MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16.
Mucin is also useful as an antimicrobial and both Mucin and hyaluronic acid
are
useful for general wound healing, and is essential for overall eye health
maintenance.
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Date Recue/Date Received 2020-11-19
There is therefore a need for an ophthalmic pharmaceutical composition that
would
promote and/or improve the production and/or release of hyaluronic acid and/or
mucin from
and/or in the cornea.
The present inventors have discovered extracts, or sources of extracts, of the
genus
Pichia which can induce, promote and/or improve
production/release/delivery/excretion of
hyaluronic acid and/or mucin from and/or in the cornea.
Accordingly an aspect of the present invention relates to microemulsion
compositions
comprising a safe and effective amount of one or more extracts, or sources of
extracts, of the
genus Pichia to induce, promote and/or improve
production/release/delivery/excretion of
hyaluronic acid and/or mucin from and/or in the cornea.
Another aspect of the present invention relates to methods for inducing,
promoting
and/or improving production/release/delivery/excretion of hyaluronic acid
and/or mucin
from and/or in the cornea comprising the step of administering ophthalmic
microemulsion
compositions comprising a safe and effective amount of one or more extracts,
or sources of
extracts, of the genus Pichia
Another aspect of the present invention relates to methods for treating (e.g.,
reducing)
and/or preventing dry eye, or the symptoms associated with dry eye, comprising
the step of
administering ophthalmic microemulsion compositions comprising one or more
extracts, or
sources of extracts, of the genus Pichia.
A further aspect of the present invention relates to methods for treating
(e.g.,
reducing) and/or preventing dry eye, or the symptoms associated with dry eye,
comprising
the step of administering ophthalmic microemulsion compositions comprising one
or more
extracts, or sources of extracts, of the genus Pichia.
Another aspect of the present invention relates to methods of preventing
and/or
treating (e.g., reducing) eye symptoms associated with dry eye and/or
resulting from
decreased or low-level production/release/delivery/excretion of hyaluronic
acid and/or mucin
from and/or in the cornea by administering compositions comprising a safe and
effective
amount of one or more extracts, or sources of extracts, of the genus Pichia.
One aspect of the present invention relates to compositions comprising a safe
and
effective amount of safe and effective amount of one or more extracts, or
sources of extracts,
of the genus Pichia to induce, promote and/or improve
production/release/delivery/excretion
of hyaluronic acid and/or mucin from and/or in the cornea, which compositions
can be
administered to patients having a MUC5AC concentration in tears lower than 6
(or about 6),
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Date Recue/Date Received 2020-11-19
optionally 8 (or about 8), nanograms per milligram of proteins, such that the
concentration of
MUC5AC in the tears is raised to (or, is made to be) equal to or greater than
8 (or about 8)
nanograms to 15 (or about 15) nanograms, optionally from 9 (or about 9)
nanograms to 12 (or
about 12) nanograms, per milligram of proteins.
In certain embodiments, the above-described concentration of MUC5AC in the
tears
(i.e., equal to or greater than 8 (or about 8) nanograms to 15 (or about 15)
nanograms,
optionally from 9 (or about 9) nanograms to 12 (or about 12) nanograms, per
milligram of
proteins), resulting from the compounds and/or extracts which induce, promote
and/or
improve production/release/delivery/excretion of hyaluronic acid and/or mucin
from and/or
in the cornea is maintained for a period of up to, at least, about 2 hours,
optionally about 4
hours, optionally about 6 hours, optionally about 8 hours, optionally about 10
hours,
optionally about 12 hours, or optionally from about 12 to about 24 hours.
Concentrations of MUC5AC in tears detailed above are determined using the
Uchino
Method (described below in the definitions).
A similar aspect of the present invention relates to methods for inducing,
promoting
and/or improving production/release/delivery/excretion of hyaluronic acid from
and/or in the
cornea comprising the step of administering ophthalmic compositions comprising
safe and
effective amount of one or more extracts, or sources of extracts, of the genus
Pichia to
induce, promote and/or improve production/release/delivery/excretion of
hyaluronic acid
and/or mucin from and/or in the cornea, which compositions can be administered
to patients
having a hyaluronic acid concentration, in their tears, lower than 10 (or
about 10) nanograms,
optionally lower than 15 (or about 15) nanograms, optionally lower than 20 (or
about 20)
nanograms, or optionally lower than 25 (or about 25) nanograms, per milligram
of proteins,
such that the concentration of hyaluronic acid in their tears is raised to
(or, is made to be)
equal to or greater than 10 (or about 10), optionally equal to or greater than
15 (or about 15),
optionally equal to or greater than 20 (or about 20), optionally equal to or
greater than 25 (or
about 25) nanograms, optionally equal to or greater than 30 (or about 30),
optionally equal to
or greater than 35 (or about 35), optionally equal to or greater than 40 (or
about 40), or
optionally equal to or greater than 45 (or about 45) nanograms per milligram
of proteins to
100 (or about 100), optionally 90 (or about 90), optionally 80 (or about 80),
optionally 70 (or
about 70), or optionally 60 (or about 60) nanograms per milligram of proteins.
In certain embodiments, the above-described concentration of hyaluronic acid,
in the
patients' tears, resulting from the compounds and/or extracts of the present
invention is
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Date Recue/Date Received 2020-11-19
maintained for a period of up to, at least, about 2 hours, optionally about 4
hours, optionally
about 6 hours, optionally about 8 hours, optionally about 10 hours, optionally
about 12 hours,
or optionally from about 12 to about 24 hours.
Concentrations of hyaluronic acid detailed above are determined using the
Dreyfuss
Method (described below in the definitions).
A further aspect of the present invention relates to methods of promoting
healing or
increasing the rate of healing of wounds in and/or on the eye (e.g., non-dry
eye associated,
eye trauma, postoperative surgical or nonspecific wounds) of a patient by
administering
compositions comprising a safe and effective amount of one or more extracts,
or sources of
extracts, of the genus Pichia which induce, promote and/or improve
production/release/delivery/excretion of hyaluronic acid or mucin from and/or
in the cornea
(namely, to increase production/release/delivery/excretion of hyaluronic acid
or mucin from
and/or in the cornea), in certain embodiments, beyond the respective
concentration levels of
hyaluronic acid or mucin ordinarily produced by such patient without (or
absent)
administration of the compositions comprising a safe and effective amount of
the one or more
extracts, or sources of extracts, of the genus Pichia).
A still further aspect of the present invention relates to methods for
improving the
antimicrobial properties in tears (or, the tear film of the eye) of a patient
by administering
compositions comprising a safe and effective amount of one or more extracts,
or sources of
extracts, of the genus Pichia which induce, promote and/or improve
production/release/delivery/excretion of hyaluronic acid and/or mucin from
and/or in the
cornea (namely, to increase production/release/delivery/excretion of mucin
from and/or in the
cornea, in certain embodiments, beyond the concentration level of mucin
ordinarily produced
by such patient without (or absent) the administration of the compositions
comprising a safe
and effective amount of such one or more extracts, or sources of extracts, of
the genus
Pichia).
SUMMARY OF THE INVENTION
The present invention relates to a microemulsion composition for treating the
eye
comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia; and
ii) an ophthalmologically acceptable carrier
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Date Recue/Date Received 2020-11-19
wherein the microemulsion droplets or particles have a maximum dimension of
less than
1,500 A.
The present invention relates to methods for
producing/releasing/delivering/excreting
mucin from and/or in the cornea comprising the step of administering
(optionally, in a patient
in need of such production/release/deliverance/excretion of mucin) a
composition, in certain
embodiments an ophthalmic microemulsion composition, comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia;
ii) optionally, a safe and effective amount of a permeation enhancer; and
iii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less than
1,500 A.
The present invention relates to methods for preventing or treating the
symptoms
associated with dry eye comprising the step of topically administering
(optionally, in a patient
need of such prevention or treatment) a microemulsion composition comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia;
ii) one or more demulcents or soothing agents;
iii) optionally, a safe and effective amount of a permeation enhancer; and
iv) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less than
1,500 A.
The present invention relates to methods for maintaining the concentration of
MU5AC in tears within the range of equal to or greater than 8 nanograms to 15
nanograms
per milligrams of protein, comprising the step of administering (optionally,
in a patient in
need of such maintenance) a microemulsion composition comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia;
ii) optionally, a safe and effective amount of a permeation enhancer; and
iii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less than
1,500 A.
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Date Recue/Date Received 2020-11-19
The present invention relates to methods for treating a patient having
decreased or
low-level production/release/delivery/excretion of mucin from and/or in the
cornea
comprising the step of topically administering to the eye the patient a
microemulsion
composition comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia;
ii) optionally, a safe and effective amount of a permeation enhancer; and
iii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less than
1,500 A.
The present invention relates to methods for preventing or treating the
symptoms
associated with dry eye comprising the step of topically administering to a
patient (optionally,
in a patient need of such prevention micromulsion or reduction in dry eye
symptoms) a
microemulsion composition comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia;
ii) optionally, a safe and effective amount of a permeation enhancer; and
iii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less than
1,500 A.
The present invention relates to methods for promoting healing or increasing
the rate
of healing of wounds in and/or on the eye (optionally, of a patient in need of
such eye wound
healing), comprising the step of administering a microemulsion compositions
(i.e., which
increase production/release/delivery/excretion of mucin and/or hyaluronic acid
from and/or
in the cornea, in certain embodiments, beyond the concentration level of mucin
produced by
such patient without (or absent) administration of the microemulsion
compositions
comprising a safe and effective amount of one or more extracts, or sources of
extracts, of the
genus Pichia) comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia; and
ii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less than
1,500 A.
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Date Recue/Date Received 2020-11-19
The present invention relates to methods for improving the antimicrobial
properties in
tears (or, the tear film of the eye) of a patent (optionally, in a patient
need of such
antimicrobial properties), comprising the step of administering a
microemulsion compositions
(i.e., which increase production/release/delivery/excretion of mucin and/or
hyaluronic acid
from and/or in the cornea, in certain embodiments, beyond the concentration
level of mucin
produced by such patient without (or absent) administration of the
microemulsion
compositions comprising a safe and effective amount of one or more extracts,
or sources of
extracts, of the genus Pichia) comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia;
ii) optionally, a safe and effective amount of a permeation enhancer; and
iii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less than
1,500 A.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 depicts bar graphs showing increase in MUC4 expression in corneal
epithelial cells after adding Pichia anomala extract to growth medium
containing corneal
epithelial cells..
Figure 2 depicts bar graphs showing increase in mucin-1 secretion in corneal
epithelial cells after adding Pichia anomala extract to growth medium
containing corneal
epithelial cells..
Figure 3 depicts bar graphs showing increased Mucin-1 production in corneal
tissue
cells after topical applications of Pichia anomala extract.
Figure 4 depicts bar graphs showing increased HA production in corneal
epithelial
cells after topical applications of Pichia anomala extract at 24 and 48 hours.
Figure 5 depicts bar graphs showing statistically significant increase in HA
production in corneal epithelial cells at 48 hours after placing corneal
epithelial cells in
growth medium containing Pichia anomala extract.
DETAILED DESCRIPTION OF INVENTION
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Date Recue/Date Received 2020-11-19
It is believed that one skilled in the art can, based upon the description
herein, utilize
this invention to its fullest extent. The following specific embodiments can
be construed as
merely illustrative, and not limitative of the remainder of the disclosure in
any way
whatsoever.
The compositions of the present invention can comprise, consist of, or consist
essentially of the elements, steps and limitations of the invention described
herein, as well
any of the additional or optional ingredients, components, or limitations
described herein.
The term "comprising" (and its grammatical variations) as used herein is used
in the
inclusive sense of "having" or "including" and not in the exclusive sense of
"consisting only
of" The terms "a" and "the" as used herein are understood to encompass the
plural as well as
the singular.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
the invention
belongs. Also, all publications, patent applications, patents, and other
references mentioned
herein are incorporated by reference in their entirety to the extent that they
are not
inconsistent with this specification. As used herein, all percentages are by
weight of the total
composition unless otherwise specified
As used herein, the terms "cornea" or "corneal" is, includes and/or relates
to, the
transparent front part of the eye that covers the iris, pupil, and anterior
chamber, the layers of
which transparent front part include the corneal epithelium layer (comprising
corneal
epithelial cells), Bowman's layer (also known as the anterior limiting
membrane), Corneal
stroma (also substantia propria), Descemet's membrane (also posterior limiting
membrane),
and Corneal endothelium (simple squamous or low cuboidal monolayer,
approximately 5 pin
thick, of mitochondria-rich cells).
The functions of the various layers are as follows:
Epithelium provides:
= provides barrier to chemicals and water;
= provides barrier to microbes;
= provides smooth optical surface as an internal part of the tear film-
cornea
interface, contributing to refractive poser of the eye; and
= houses Langerhans cells which perform important immunological functions.
Bowman's layer:
= aides in maintaining the corneal shape.
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Date Recue/Date Received 2020-11-19
Corneal stroma:
= provides mechanical strength to cornea;
= provides transparency of cornea; and
= acts as refracting lens.
Descernet's membrane:
= acts as resting layer for endothelial cells.
Corneal endothelium:
= maintains corneal clarity by removing water from the corneal stroma.
The corneal epithelium layer is composed fairly uniformly of 5-7 layers of
cells. The
corneal epithelium is about 50 p. in thickness. The epithelium is uniform to
provide a smooth
regular surface and is made up of nonkeratinized stratified squamous
epithelium. Without
being limited by theory, it is believed that, in the case of hyaluronic acid
production, the
corneal epithelium layer further contains tight junction structures (i.e.,
those structures [e.g.,
membrane or film barriers] typically inhibiting chemical permeation into
tissues) which may
obstruct, or act as a barrier, to diffusion of the one or more extracts, or
sources of extracts, of
the genus Pichia, reducing diffusion of the one or more extracts, or sources
of extracts, of the
genus Pichia across the corneal epithelium and into the corneal tissues,
thereby, reducing the
concentration levels of the extract that can accumulate within the corneal
tissues and contact
the cellular portions of such tissues responsible for hyaluronic acid
production.
As used herein, the phrase "decreased or low-level
production/release/delivery/excretion of hyaluronic acid from and/or in the
cornea" means a
concentration of hyaluronic acid which is less than the concentration of
hyaluronic acid in the
tears of a normal (i.e., non-diseased) person, or, in certain embodiments,
less than 25 (or
about 25) nanograms per milligram of proteins, as determined using the method
described in
Dreyfuss JL, Regatieri CV, Coelho B, et al. Altered hyaluronic acid content in
tear fluid of
patients with adenoviral conjunctivitis. An Acad Bras Cienc. 2015;87(1):455-
462. That
method (the Dreyfuss Method) is reproduced below:
= Sample Collection
For collecting the tears, Schirmer strips were placed in the temporal side of
each eye
under the eyelid, during 5 minutes, without any use of topical anesthetics.
The strips
were dried at room temperature and stored at ¨20 C until analysis.
= Tear Sample Preparation
Date Recue/Date Received 2020-11-19
Tear compounds were eluted from the Schirmer strips using 100 pL of distilled
water,
and hyaluronic acid and protein content analyses were performed.
= Hyaluronic Acid Measurement
Hyaluronic acid content in tear fluids was assayed by a non-isotopic
fluoroassay (See
Martins Jr, Passerotti CC, Maciel RM, Sampaio Lo, Dietrich CP and Nader HB.
2003.) Practical determination of hyaluronan by a new noncompetitive
fluorescence-
based assay on serum of normal and cirrhotic patients. Anal Biochem 319: 65-
72.)
Eluted tear fluids and standard concentrations of hyaluronic acid (Sigma, St.
Louis,
MO) were added to 96 multiwell plates (FluoroNUNC Maxisorp-microtiterplates,
Roskilde, Denmark) previously coated with hyaluronic acid -binding protein.
The
plates were then sequentially incubated with biotinylated hyaluronic acid -
binding
protein and europiumlabeled streptavidin (Amershan, Piscataway, NJ).
Afterwards,
the europium remaining in the solid phase was released by an enhancement
solution
and the fluorescence was measured using a time-resolved flurometer (Perkin-
Elmer
Life Sciences-Wallac Oy, Turku, Finland). The data (counts/s) were processed
automatically using the MultiCalc software program (Perkin-Elmer Life Sciences-
Wallac Oy) and values are expressed as ng/mg protein.
= Protein Analysis
Total tear protein concentration was determined using a colorimetric assay kit
according to the manufacturer's instructions (Protein Assay Kit from Bio-Rad,
Hercules, CA). The protein profile was analyzed through sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (See
Laemmli UK. 1970.) Cleavage of structural proteins during the assembly of the
head
of bacteriophage T4. Nature 227: 680-685.). Briefly, 10 Kg of protein from the
tear
samples were applied to a 3-20% linear gradient polyacrylamide gel under
reducing
conditions. After electrophoresis, the gels were stained by comassie blue (Bio-
Rad,
Hercules, CA). Each protein band was quantified by densitometry using the
software
ImageJ Version 10.2 for Mac (U.S. National Institutes of Health, Bethesda,
Maryland,
USA). The results are expressed by arbitrary densitometric units (ADU).
As used herein, the phrase "decreased or low-level
production/release/delivery/excretion of mucin from and/or in the cornea"
means a
concentration of MUC5AC which is less than the concentration of MUC5AC in the
tears of a
normal (i.e., non-diseased) person, or, in certain embodiments, less than 6,
optionally less
than 8, nanograms per milligram of proteins, as determined using the method
described in
Uchino Y, Uchino M, Yokoi N, et al. Alteration of Tear Mucin SAC in Office
Workers Using
Visual Display Terminals: The Osaka Study. JAMA Ophthalmol. n2014;132(8):985-
992.
That method (the Uchino Method) is reproduced below:
= MUC5AC Concentration in Tears
The concentration of the secreted mucin MUC5AC in the tear samples was
quantified by enzyme-linked immunoassay (E90756Hu; USCN Life Science). (See
Maker AV, Katabi N, Gonen M, et al. Pancreatic cyst fluid and serum mucin
levels
predict dysplasia in intraductal papillary mucinous neoplasms of the pancreas.
Ann
Surg Oncol. 2011;18(1):199-206.) All samples were analyzed according to the
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Date Recue/Date Received 2020-11-19
manufacturer's guidelines. Absorbance was measured at 450 nm, and the standard
solutions in the kit were recombinant human MUC5AC. A protein assay reagent
kit
(BCA Protein Assay Kit; Pierce) was used to determine the protein
concentration in
the tear samples. The MUC5AC concentration was normalized to the tear protein
content and expressed as MUC5AC protein (nanograms) per tear total protein
(milligrams).
As used herein, a composition that is "essentially free" of an ingredient
means the
composition that has about 2% or less of that ingredient by weight based on
the total weight
of the composition. Preferably, a composition that is essentially free of an
ingredient has
about 1% or less, more preferably about 0.5% or less, more preferably about
0.1% or less,
more preferably about 0.05 or less, more preferably about 0.01% or less by
weight based on
the total weight of composition of the ingredient. In certain more preferred
embodiments, a
composition that is essentially free of an ingredient is free of the
ingredient, i.e. has none of
that ingredient in the composition.
As used herein, "microemulsion" means emulsions possessing one or more of the
following characteristics: i) they are formed spontaneously or substantially
spontaneously
when their components are brought into contact, that is without substantial
energy supply,
e.g. in the absence of heating or the use of high shear equipment or other
substantial
agitation; ii) they exhibit thermodynamic stability; iii) they are monophasic;
iv) they are
substantially non-opaque, i.e. are transparent or opalescent when viewed by
optical
microscopic means; and/or v) in their undisturbed state, they are optically
isotropic, though
an anisotropic structure may be observable using e.g. x-ray technique. The
particles of a
microemulsion may be spherical, though other structures are feasible, e.g.
liquid crystals with
lamellar, hexagonal or isotropic symmetries. Generally, microemulsions
comprise droplets or
particles having a maximum dimension (e.g. diameter) of less than 1,500 A,
preferably less
than 1000 A, preferably less than 500 A, but greater than 100 A. Included
within this
definition of microemulsion are self-emulsifying drug delivery systems
(SEDDS). SEDDS
are isotropic mixtures of oil, surfactant (with or without co-surfactant) and
co-solvent which
spontaneously emulsify when exposed to an aqueous medium with gentle
agitation. SEDDS
may be used to improve bioavailability of poorly water soluble drugs via oral
administration.
The addition of a co-solvent facilitate the formation of a self-emulsifying
system as it
significantly reduces the interfacial tension. In so doing, it creates a fluid
interfacial film
with sufficient flexibility to take up different curvatures required to form
microemulsion over
a wide range of compositions. Further detail regarding SEDDS can be found in
US Pat.
12
Date Recue/Date Received 2020-11-19
Publication US2018/0036233A1 to Shabaik et al., herein incorporated by
reference in its
entirety.
As used herein, "ophthalmologically acceptable" means that the ingredients
which the
term describes are suitable for use in contact with tissues (e.g., the soft
tissues of the eye or
periorbital skin tissues) without undue toxicity, incompatibility,
instability, irritation, allergic
response, and the like. As will be recognized by one of skill in the art,
cosmetically/
dermatologically acceptable salts are acidic/anionic or basic/cationic salts.
As used herein, the term "safe and effective amount" means an amount of
disclosed
the extract, compound or of the composition sufficient to induce, promote
and/or improve the
production/release/delivery/excretion of hyaluronic acid and/or mucin from
and/or in one or
more layer of the cornea, but low enough to avoid serious side effects. The
safe and effective
amount of the compound, extract, or composition will vary with e.g. the age,
health and
environmental exposure of the end user, the duration and nature of the
treatment, the specific
extract, ingredient, or composition employed, the particular
ophthalmologically-acceptable
carrier utilized, and like factors.
In certain embodiments, the present invention as disclosed herein may be
practiced in
the absence of any compound or element (or group of compounds or elements)
which is not
specifically disclosed herein.
In general, IUPAC nomenclature rules are used herein and according to the
following
term definitions.
The term "C1-8 alkyl," whether used alone or as part of a substituent group,
refers to a
saturated aliphatic branched or straight-chain monovalent hydrocarbon radical
having from 1-
8 carbon atoms. For example, "C1-8a1ky1" specifically includes the radicals
methyl, ethyl, 1-
propyl, 2-propyl, 1-butyl, 2-butyl, tert-butyl, 1-butyl, 1-pentyl, 2-pentyl, 3-
pentyl, 1-hexyl, 2-
hexyl, 3- hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 1-octyl, 2-octyl, 3-octyl and
the like. Said term
may also refer to the corresponding alkyldiyl radical. Alkyl and alkyldiyl
radicals may be
attached to a core molecule via a terminal carbon atom or via a carbon atom
within the chain.
Similarly, any number of substituent variables may be attached to an alkyl or
alkyldiyl radical
when allowed by available valences.
The term "C1-4a1ky1," whether used alone or as part of a substituent group,
refers to a
saturated aliphatic branched or straight-chain monovalent hydrocarbon radical
or alkyldiyl
linking group having a specified number of carbon atoms, wherein the radical
is derived by
the removal of one hydrogen atom from a carbon atom and the alkyldiyl linking
group is
13
Date Recue/Date Received 2020-11-19
derived by the removal of one hydrogen atom from each of two carbon atoms in
the chain.
The term "C1-4a1ky1" refers to a radical having from 1-4 carbon atoms in a
linear or branched
arrangement. For example, "C1-4a1ky1" specifically includes the radicals
methyl, ethyl, 1-
propyl, 2-propyl, 1-butyl, 2-butyl, tert- butyl, 1-butyl, and the like. Alkyl
and alkyldiyl
radicals may be attached to a core molecule via a terminal carbon atom or via
a carbon atom
within the chain. Similarly, any number of substituent variables may be
attached to an alkyl
or alkyldiyl radical when allowed by available valences.
The term "C2-4a1keny1" refers to an alkenyl radical having from 2-4 carbon
atoms.
For example, specifically includes the radicals ethenyl, propenyl, allyl (2-
propenyl), butenyl
and the like. As described above, an alkenyl radical may be similarly attached
to a core
molecule and further substituted where indicated.
The term "halo" as such or in combination with other terms means halogen atom,
such as fluoro, chloro, bromo or iodo.
The term "substituted," refers to a core molecule in which one or more
hydrogen
atoms have been replaced with that amount of substituents allowed by available
valences.
Substitution is not limited to the core molecule, but may also occur on a
substituent radical,
whereby the radical becomes a linking group.
The term "independently selected" refers to two or more substituents that may
be
selected from a substituent variable group, wherein the selected substituents
may be the same
or different.
The term "dependently selected" refers to one or more substituent variables
that are
specified in an indicated combination for substitution in a core molecule
(e.g. variables that
refer to groups of substituents appearing in a tabular list of compounds).
Acceptable salts from inorganic bases include, for example, sodium or
potassium
salts, and the like. Acceptable salts from organic bases include, for example,
salts formed
with primary, secondary, or tertiary amines, and the like.
Compounds and/or Extracts which Induce, Promote and/or Improve
Production/Release/Delivery/Excretion of Hyaluronic Acid and/or Mucin in the
Cornea.
The present invention comprises one or more compounds and/or extracts which
induce, promote and/or improve production/release/delivery/excretion of
hyaluronic acid
and/or mucin from and/or in the cornea.
14
Date Recue/Date Received 2020-11-19
In certain embodiments, the compounds and/or extracts which induce, promote
and/or
improve production/release/delivery/excretion of hyaluronic acid and/or mucin
from and/or
in the cornea are, or comprise, extracts, or sources of extracts, of the genus
Pichia.
Pichia is a genus of yeasts in the family Saccharomycetaceae. More than 100
species
of this genus are known. Suitable species for use in the compositions of the
present invention
include (selected from or selected from the group consisting of) Pichia
anomala, Pichia
Pichia norvegensis, and Pichia ohmeri. Pichia anomala (formerly named
Hansenula anomala) can be found in raw milk and cheese. The extracts of yeasts
of the
genus Pichia are rich in mannans, polysaccharides composed of mannose
monomers.
Extracts or sources of extracts of the genus Pichia may be isolated from the
fruit or other
aerial parts of a plant. Any ophthalmologically acceptable extract of the
genus of Pichia may
be used. Mixtures of extracts, or sources of extract, of the above species
from the genus of
Pichia may also be used.
In certain embodiments, the extracts, or sources of extracts, from genus of
Pichia used in the
present invention are extracts of Pichia anomala. Extracts of Pichia anomala
can be isolated
from the fruit or other aerial parts of a plant. In certain embodiments, a
suitable extract of
Pichia anomala is produced from a strain of Pichia anomala present on fruit or
leaves of a
kiwi plant. In another embodiment, extract of Pichia anomala is commercially
available
from Silab-France as PRO-LIPISKIN or UNFLAMAGYLO, where the extract is
produced
from a strain of Pichia anomala present on sugar cane. Preferred for use
herein is an extract
of Pichia anomala characterized as having:
- a solids content of between 26 and 40 g/l,
- a pH between 6 and 7,
- a protein content between 2 and 170 g/l, and
- a sugars content (mannose curve) ranging between 18 and 30 g/1
(within which characterization falls PRO-LIPISKIN and Hyalurodine (Silab-
France).
Hyalurodine is the Pichia anomala extract used in the Examples (below). In one
embodiment, the Pichia anomala extract is obtained in accordance with the
following process
as described in FR2897266 and FR2938768, both of which are incorporated by
herein by
reference.
In certain embodiments, the extraction process described below eliminates much
of
the protein from a Pichia extract and concentrates the active in terms of
mannan. The
Date Recue/Date Received 2020-11-19
process comprises at least one enzymatic hydrolysis step of the proteins, to
obtain peptides
and small proteins and, in certain embodiments, a further step of removing
these small
peptides and proteins by filtration based on the selection of the size of such
molecules.
In certain embodiments, the extracts of Pichia genus, including Pichia anomala
extract, are obtained by an extraction process involving one or more
hydrolysis enzyme (s) to
hydrolyze proteins in Pichia genus, either successively or simultaneously.
In certain embodiments, enzymatic hydrolysis is used to break-down proteins in
the
extract of the Pichia genus into protein fractions having weight average
molecular weights of
less than 5000Da. Suitable hydrolysis enzymes include, but is not limited to,
at least one
peptidase, in certain embodiments, chosen from papain, trypsin, chymotrypsin,
subtilisin,
pepsin, thermolysin, pronase, flavastacine, enterokinase, factor Xa protease,
Turin,
bromelain, proteinase K, genenase I, thermitase, carboxypeptidase A,
carboxypeptidase B,
collagenase or mixtures thereof
In certain embodiments, the enzymes used to obtain the Pichia genus extract
are
inactivated prior to separation of the resulting soluble and insoluble phases.
In one embodiment, the Pichia anomala extract is characterized as having:
- a solids content of between 5 and 300 g/l,
- a pH between 4 and 9,
- a protein content between 2 and 10, preferably 3-9, g/l, and
- a sugars content (mannose curve) ranging between 1 and 100 g/1
In certain embodiments, extract of Pichia genus comprises a mannan content of
greater than or equal to 30% of the total weight of dried Pichia genus
extract, or optionally at
a mannan content of at least 50% by weight of the total weight of the dried
Pichia genus
extract.
In certain embodiments, the phase of Pichia genus extract is dried (and the
solids
content is measured) by passing the Pichia genus extract containing phase
through oven heat
of 105 C (or about 105 C) in the presence of sand until a constant weight is
obtained/observed.
In certain embodiments, the solids (or dry matter) content of the dried
extract of
Pichia genus is between 10 and 200 g/l, or optionally between 26 and 40 g/l.
16
Date Recue/Date Received 2020-11-19
In certain embodiments, the pH measured by the potentiometric method at room
temperature (25 C) leads to values of between 4.5 and 8.5, optionally between
6.0 and 7Ø
Determination of total sugar content, the DUBOIS method can be used. In the
presence of concentrated sulphuric acid and phenol, reducing sugars give an
orange yellow
compound. From a standard range (preferably using a mannose assay curve), the
total sugar
content of a sample can be determined. In certain embodiments, the total sugar
content in the
dried extract of Pichia genus is between 7 and 145 g/l, or optionally between
18 and 29 g/l.
In certain embodiments, the dried extract of Pichia genus contains at least
30% of total sugars
by weight of dried extract of Pichia genus compared to the total solids by
weight of dried
extract of Pichia genus, optionally, at least 50% by weight of dried extract
of Pichia genus.
In certain embodiments, the carbohydrate fraction of the dried extract of
Pichia genus
is composed of mannose and glucose in the form of (or essentially in the form
of)
oligosaccharides and polysaccharides having a weight average molecular weight
of from
about 180 to about 800,000 Da, optionally, from about 5000 to about 515,000
Da, optionally
from about 6000 to about 270,000 Da. In certain embodiments, at least 70% (or
about 70%),
optionally at least 75% (or about 75%), at least 80% (or about 80%),
optionally at least 85%
(or about 85%), at least 90% (or about 90%), optionally at least 95% (or about
95%), or
100% (or about 100%) of the oligosaccharides and polysaccharides in the dried
extract of
Pichia genus fall within the aforementioned weight average molecular weight
ranges.
Determination of the protein content protein is obtained by the Kjedhal
method. In
certain embodiments, the dried extract of Pichia genus has a protein content
of between 4 and
90 g/l, or optionally between 12 and 18 g/l. The dried extract of Pichia genus
contains less
than 45% of proteins, or optionally less than 30%, of the total solids in the
dried extract of
Pichia genus.
In certain embodiments, the dried extract of Pichia genus comprises mannans,
mannoses polymerized in the form of oligosaccharides and polysaccharides,
whose weight
average molecular weight is from about 180 to about 800,000 Da, optionally,
from about
5000 to about 515,000 Da, optionally from about 6000 to about 270,000 Da.
dried extract of
Pichia genus. In certain embodiments, at least 70% (or about 70%), optionally
at least 75%
(or about 75%), at least 80% (or about 80%), optionally at least 85% (or about
85%), at least
90% (or about 90%), optionally at least 95% (or about 95%), or 100% (or about
100%) of the
17
Date Recue/Date Received 2020-11-19
oligosaccharides and polysaccharides in the dried extract of Pichia genus fall
within the
aforementioned weight average molecular weight ranges.
In certain embodiments, the extraction process includes a step, after the step
of
enzymatic hydrolysis of proteins, of removing (e.g., through filtration)
proteins having a
weight average molecular weight of less than 5000Da. Accordingly, the extracts
of the
Pichia genus are free of or substantially free of proteins and/or peptides
having a weight
average molecular weight of less than 5000Da.
In certain embodiments, the above described extraction process may include a
step of
deodorizing, bleaching and / or stabilizing the extract of Pichia genus before
the filtrations.
The filtrations can be the following: - Press filtration, and - sterilizing
filtration.
In certain embodiments, the extracts used belong to the variety Pichia
anomala. A
particular non-limiting example of a production process is described below.
- Extracts (or yeasts) of Pichia anomala are cultured in a culture medium
adapted to their development, then centrifuged to recover the biomass,
- The biomass is then milled in a ball mill. Then the ground material is
resuspended in water at a concentration of 50 grams per liter before
enzymatic hydrolysis in basic medium at 30 C for 6 hours,
- After hydrolysis, the product is centrifuged and filtered before
sterilization,
- By successive filtrations on filters of different sizes, a hydrolyzate
containing at least 30% of mannans relative to the total weight of the
solids is obtained and/or proteins of specific weight average molecular
weight are obtained. (The hydrolyzate obtained is in the form of a clear
liquid aqueous solution of light yellow color.)
In certain embodiments, the Pichia anomala extract is obtained in accordance
with
the following manufacturing examples:
1. Pichia anomala extract A:
I. Extraction of the Pichia anomala extract A:
18
Date Recue/Date Received 2020-11-19
Preparation of Pichia anomala extract A includes the following steps:
- culture of yeast Pichia anomala in an environment adapted to their
development,
- centrifugation to retrieve the biomass,
- solubilization of biomass,
- enzymatic hydrolysis at basic pH,
- separation of the soluble and insoluble phases,
- heat treatment,
- filtration, and
- sterile filtration.
Characterization of the Pichia anomala extract A:
The Pichia anomala extract A obtain above is characterized by:
- - a solids content of between 48 and 84 g/l,
- - a pH between 4 and 9,
- - a protein content between 19 and 48 g/l, and
- - a total sugar content between 10 and 42 g/l.
2. Pichia anomala extract B
I. Extraction of the Pichia anomala extract B:
Preparation of Pichia anomala extract B includes the following steps:
- culture of yeast Pichia anomala in an environment adapted to their
development,
- centrifugation to retrieve the biomass,
- solubilization of biomass,
- enzymatic hydrolysis at acid pH,
- separation of the soluble and insoluble phases,
- heat treatment,
- filtration, and
- sterile filtration.
Characterization of the Pichia anomala extract B:
The Pichia anomala extract B obtain above is characterized by:
- - a solids content of between 58 and 95 g/l,
- - a pH between 4 and 9,
- - a protein content between 23 and 54 g/l, and
- - a total sugar content between 12 and 32 g/l.
3. Pichia anomala extract C
I. Extraction of the Pichia anomala extract C:
Preparation of Pichia anomala extract C includes the following steps:
19
Date Recue/Date Received 2020-11-19
- culture of yeast Pichia anomala in an environment adapted to their
development,
- centrifugation to retrieve the biomass,
- solubilization of biomass,
- successive enzymatic hydrolyses in basic medium,
- separation of the soluble and insoluble phases,
- heat treatment,
- filtration, and
- sterile filtration.
Characterization of the Pichia anomala extract C:
The Pichia anomala extract C obtain above is characterized by:
- - a solids content of between 91 and 195 g/l,
- - a pH between 4 and 9,
- - a protein content between 36 and 111 g/l, and
- - a total sugar content between 18 and 65 g/l.
4. Pichia anomala extract D
I. Extraction of the Pichia anomala extract D:
Preparation of Pichia anomala extract D includes the following steps:
- culture of yeast Pichia anomala in an environment adapted to their
development,
- centrifugation to retrieve the biomass,
- solubilization of biomass,
- hydrolyzed simultaneously with at least two enzymes at acid pH,
- separation of the soluble and insoluble phases,
- heat treatment,
- filtration, and
- sterile filtration.
Characterization of the Pichia anomala extract D:
The Pichia anomala extract D obtain above is characterized by:
- - a solids content of between 5 and 53 g/l,
- - a pH between 4 and 9,
- - a protein content between 2 and 30 g/l, and
- - a total sugar content between 1 and 18 g/l.
5. Pichia anomala extract E
I. Extraction of the Pichia anomala extract E:
Preparation of Pichia anomala extract E includes the following steps:
Date Recue/Date Received 2020-11-19
- culture of yeast Pichia anomala in an environment adapted to their
development,
- centrifugation to retrieve the biomass,
- solubilization of biomass in a hydroglycolique environment,
- hydrolyzed simultaneously with at least two enzymes at acid pH,
- separation of the soluble and insoluble phases,
- heat treatment,
- filtration, and
- sterile filtration.
Characterization of the Pichia anomala extract E:
The Pichia anomala extract E obtain above is characterized by:
- - a solids content of between 172 and 300 g/l,
- - a pH between 4 and 9,
- - a protein content between 69 and 170 g/l, and
- - a total sugar content between 34 and 100 g/l.
Preferably, the Pichia anomala extract is obtained using the process for
obtaining Pichia
anomala extract D as described above.
In certain embodiments, the extract, or source of extracts, of the genus
Pichia
comprises oligosaccharides and polysaccharides having an average degree of
polymerization
of from DP 1 to DP 4444, optionally from DP 30 to DP 2860, optionally from DP
35 to DP
1500. In certain embodiments, at least 70% (or about 70%), optionally at least
75% (or about
75%), at least 80% (or about 80%), optionally at least 85% (or about 85%), at
least 90% (or
about 90%), optionally at least 95% (or about 95%), or 100% (or about 100%) of
the
oligosaccharides and polysaccharides in the dried extract of Pichia genus fall
within the
aforementioned average degree of polymerization ranges.
In certain embodiments, the extract, or source of extracts, of the genus
Pichia are
present in the compositions of the present invention so as to provide a Pichia
extract
concentration in, or contacting, corneal tissue cells in the user (i.e.,
internal corneal fluid
levels), after topical application, of at least 0.3mg/m1 (or about 0.3 mg/ml),
optionally, at least
0.5 mg/ml (or about 0.5 mg/ml), optionally, at least 1 mg/ml (or about 1
mg/ml), optionally,
at least 1.5 mg/ml (or about 1.5mg/m1), optionally, at least 2 mg/ml (or about
2 mg/ml),
optionally, at least 2.5 mg/ml (or about 2.5mg/m1), optionally, at least
3mg/m1 (or about
3mg/m1), optionally, at least 3.5 mg/ml (or about 3.5mg/m1), optionally, at
least 4 mg/ml (or
21
Date Recue/Date Received 2020-11-19
about 4 mg/ml), optionally, at least 4.5 mg/ml (or about 4.5 mg/ml),
optionally, at least 5
mg/ml (or about 5 mg/ml), or at least 5.5 mg/ml (or about 5.5 mg/ml),
optionally, at least 6
mg/ml (or about 6 mg/ml), or optionally, at least 6.5 mg/ml (or about 6.5
mg/ml), optionally,
at least 7 mg/ml (or about 7 mg/ml), optionally, at least 7.5 mg/ml (or about
7.5 mg/ml),
optionally, at least 8 mg/ml (or about 8 mg/ml), optionally, at least 8.5
mg/ml (or about 8.5
mg/ml), optionally, at least 9 mg/ml (or about 9 mg/ml), optionally, at least
8.5 mg/ml (or
about 8.5 mg/ml), optionally, at least 9 mg/ml (or about 9 mg/ml), optionally,
at least 9.5
mg/ml (or about 9.5 mg/ml), or optionally, at least 10 mg/ml (or about
10mg/m1) to 100
mg/ml (or about 100 mg/ml), optionally, to 95 mg/ml (or about 95 mg/ml),
optionally, to 90
mg/ml (or about 90 mg/ml), optionally, to 85 mg/ml (or about 85mg/m1),
optionally, to 80
mg/ml (or about 80 mg/ml), optionally, to 75 mg/ml (or about 75 mg/ml),
optionally, to 70
mg/ml (or about 70 mg/ml), optionally, to 65 mg/ml (or about 65 mg/ml),
optionally, to 60
mg/ml (or about 60 mg/ml), optionally, to 55 mg/ml (or about 55 mg/ml),
optionally, to 50
mg/ml (or about 50 mg/ml), optionally, to 45 mg/ml (or about 45 mg/ml),
optionally, to 40
mg/ml (or about 40 mg/ml), optionally, to 35 mg/ml (or about 35 mg/ml),
optionally, to 30
mg/ml (or about 30 mg/ml), optionally, to 25 mg/ml (or about 25 mg/ml),
optionally, to 20
mg/ml (or about 20 mg/ml), or optionally, to 15 mg/ml (or about 15 mg/ml).
In certain embodiments, the extract, or source of extracts, of the genus
Pichia are
present in the compositions of the present invention at a concentration of
from 0.01% (or
about 0.01%), optionally, from 0.05% (or about 0.05%), optionally, from 0.1%
(or about
0.1%), optionally, from 0.5% (or about 0.5%), optionally, from 1% (or about
1%), optionally,
from 1.5% (or about 1.5%), optionally, from 2% (or about 2%), optionally, from
2.5% (or
about 2.5%), optionally, from 3% (or about 3%), optionally, from 3.5%, (or
about 3.5%),
optionally, from 4% (or about 4%), optionally, from 4.5% (or about 4.5%),
optionally, from
5% (or about 5%), optionally, from 5.5% (or about 5.5%), optionally, from 6%
(or about
6%), optionally, from 6.5% (or about 6.5%), optionally, from 7% (or about 7%),
optionally,
from 7.5% (or about 7.5%), optionally, from 8% (or about 8%), optionally, from
8.5% (or
about 8.5%), optionally, from 9% (or about 9%), optionally, from 9.5% (or
about 9.5%),
optionally, from 10% (or about 10%), optionally, from 10.5% (or about 10.5%),
optionally,
from 11% (or about 11%), optionally, from 11.5% (or about 11.5%), optionally,
from 12%
(or about 12%), optionally, from 12.5% (or about 12.5%), optionally, from 13%
(or about
13%), optionally, from 13.5% (or about 13.5%), optionally, from 14% (or about
14%),
optionally, from 14.5% (or about 14.5%), optionally, from 15% (or about 15%),
optionally,
22
Date Recue/Date Received 2020-11-19
from 15.5% (or about 15.5%), optionally, from 16% (or about 16%), optionally,
from 16.5%
(or about 16.5%), optionally, from 17% (or about 17%), optionally, from 17.5%
(or about
17.5%), optionally, from 18% (or about 18%), optionally, from 18.5% (or about
18.5%),
optionally, from 19% (or about 19%), optionally, from 19.5% (or about 19.5%),
optionally,
from 20% (or about 20%), optionally, from 20.5% (or about 20.5%) to 30% (or
about 30%),
optionally, to 35% (or about 35%), optionally, to 40% (or about 40%),
optionally, to 45% (or
about 45%), optionally, to 50% (or about 50%), optionally, to 55% (or about
55%),
optionally, to 60% (or about 60%), optionally, to 65% (or about 65%),
optionally, to 70% (or
about 70%), optionally, to 75% (or about 75%), optionally, to 80% (or about
80%),
optionally, to 85% (or about 85%), optionally, to 90% (or about 90%),
optionally, to 95% (or
about 95%), or optionally, to 100% (or about 100%), by weight, of the total
composition.
Permeation Enhancer
In certain embodiments, the compositions of the present invention optionally
comprise a permeation enhancer.
Suitable permeation enhancers include (selected from or selected from the
group
consisting of) either alone or in combination, surfactants such as saponins,
polyoxyethylene,
polyoxyethylene ethers of fatty acids such as polyoxyethylene 4-, 9-, 10-, and
23-lauryl ether,
polyoxyethylene 10-and 20-cetyl ether, polyoxyethylene 10-and 20-stearyl
ether, sorbitan
monooleate, sorbitan monolaurate, polyoxyethylene monolaurate, polyoxyethylene
sorbitans
such as polyoxyethylene sorbitan monolaurate, decamethonium, decamethonium
bromide,
and dodecyltrimethylammonium bromide; chelators such natural polyacids (e.g.,
citric acid),
phosphate salts (e.g., disodium pyrophosphate), phosphonates, bisphosphonates
(e.g.,
etridronic acid), aminocarboxylic acids (e.g., ethylenediaminetetraacetic acid
(EDTA) and
disodium EDTA) and ethylenediamine-N,N'-disuccinic acid (EDDS)); bile salts
and acids
such as cholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholic
acid, taurocholic
acid, taurodeoxycholic acid, sodium cholate, sodium glycocholate,
glycocholate, sodium
deoxycholate, sodium taurocholate, sodium glycodeoxycholate, sodium
taurodeoxycholate,
chenodeoxycholic acid, and urosdeoxycholic acid; fusidic acid derivatives,
glycyrrhizic acid,
and ammonium glycyrrhizide, with saponin EDTA, fusidic acid, polyoxyethylene 9-
lauryl
ether, polyoxyethylene 20-stearylether, glycocholate, or mixtures of any of
the above.
23
Date Recue/Date Received 2020-11-19
The concentration of permeation enhancer administered should be the minimum
amount needed to sufficiently increase absorption of the compound and/or
extract through the
mucous or other barrier membranes of the eye. Generally, concentrations
ranging from
0.01% (or about 0.01%), optionally, from 0.05% (or about 0.05%), optionally,
from 0.1% (or
about 0.1%), optionally, from 0.15% (or about 0.15%), optionally, from 0.2%
(or about
0.2%), optionally, from 0.25% (or about 0.25%) to 2% (or about 2%),
optionally, to 2.5% (or
about 2.5%), optionally, to 3% (or about 3%), optionally, to 3.5%, (or about
3.5%),
optionally, to 4% (or about 4%), optionally, to 4.5% (or about 4.5%),
optionally, to 5% (or
about 5%), optionally, to 5.5% (or about 5.5%), optionally, to 6% (or about
6%), optionally,
to 6.5% (or about 6.5%), optionally, to 7% (or about 7%), optionally, to 7.5%
(or about
7.5%), optionally, to 8% (or about 8%), optionally, to 8.5% (or about 8.5%),
optionally, to
9% (or about 9%), optionally, to 9.5% (or about 9.5%), optionally, to 10% (or
about 10%),
optionally, to 10.5% (or about 10.5%), optionally, to 11% (or about 11%),
optionally, to
11.5% (or about 11.5%), optionally, to 12% (or about 12%), optionally, to
12.5% (or about
12.5%), optionally, to 13% (or about 13%), optionally, to 13.5% (or about
13.5%),
optionally, to 14% (or about 14%), optionally, to 14.5% (or about 14.5%),
optionally, to 15%
(or about 15%), optionally, to 15.5% (or about 15.5%), optionally, to 16% (or
about 16%),
optionally, to 16.5% (or about 16.5%), optionally, to 17% (or about 17%),
optionally, to
17.5% (or about 17.5%), optionally, to 18% (or about 18%), optionally, to
18.5% (or about
18.5%), optionally, to 19% (or about 19%), optionally, to 19.5% (or about
19.5%),
optionally, to 20% (or about 20%), of the total composition (w/v), are useful
in the
compositions of the present invention.
Ophthalmologically Acceptable Carrier
The compositions of the present invention also comprise an aqueous, oil-in-
water
emulsion, water-in-oil emulsion carrier, oil-in-water microemulsion, or water-
in-oil
microemulsion carrier. The carrier is ophthalmologically acceptable. Useful
oil-in-water and
water-oil-carriers can be found in US Patent Publication 20030165545A1 and US
Patents
9480645, 8828412 and 8496976, each of which patent documents are herein
incorporated by
reference in its entirety.
In certain embodiments, the compositions of the present invention include
emulsions,
optionally, self-emulsifying emulsions, including an oily component, such as
one or more
24
Date Recue/Date Received 2020-11-19
oils, for example, and without limitation, mineral oil and/or one or more
other conventional
well known and/or commercially available oils suitable for use in the present
invention; a
surfactant component which includes three or more surfactants; and an aqueous
component
which includes an aqueous phase. In addition, a number of additional
components may be
included in the present compositions. The compositions of the present
invention are
substantially non-toxic and/or non-irritating and/or non-damaging to the eye
and can provide
a protective function for ocular cells and tissues.
One or more oils or oily substances are used to form the present compositions.
Any
suitable oil or oily substance or combinations of oils or oily substances may
be employed
provided such oils and/or oily substances are effective in the present
compositions, and do
not cause any substantial or significant detrimental effect to the human or
animal to whom
the composition is administered, or to the contact lens being treated, or the
wearing of the
treated contact lens, or to the wearer of the treated contact lens. The oily
component may, for
example, and without limitation, be a higher fatty acid glyceride, for
example, castor oil, corn
oil, sunflower oil and the like and mixtures thereof The oily component may
include one or
more non-polar oils such as mineral oil, silicone oil and the like and
mixtures thereof
The ophthalmologically acceptable carrier (or, compositions of the present
invention)
may optionally comprise one or more additional excipients and/or one or more
additional
active ingredients. Examples of such optional components are described below.
Excipients commonly used in ophthalmic compositions include, but are not
limited to,
demulcents, tonicity agents, preservatives, chelating agents, buffering agents
(other than and
in addition to the organic acids of the present invention), and surfactants.
Other excipients
comprise solubilizing agents, stabilizing agents, comfort-enhancing agents,
polymers,
emollients, pH-adjusting agents (other than and in addition to the organic
acids of the present
invention), and/or lubricants. Any of a variety of excipients may be used in
the compositions
of the present invention including water, mixtures of water and water-miscible
solvents, such
as vegetable oils or mineral oils comprising from 0.5% to 5% non-toxic water-
soluble
polymers, natural products, such as agar and acacia, starch derivatives, such
as starch acetate
and hydroxypropyl starch, and also other synthetic products such as polyvinyl
alcohol,
polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, and
preferably cross-
linked polyacrylic acid and mixtures thereof
Date Recue/Date Received 2020-11-19
Demulcents or soothing agents used with embodiments of the present invention
include, but are not limited to, cellulose derivatives (such hydroxyethyl
cellulose, methyl
cellulose, hypromellose or mixtures thereof), hyaluronic acid or salt thereof
(such as sodium
hyaluronate), tamarind seed extract, glycerin, polyvinyl pyrrolidone,
polyethylene oxide,
polyethylene glycol, propylene glycol and polyacrylic acid and mixtures
thereof In certain
embodiments, one or more of hyaluronic acid, propylene glycol, tamarind seed
extract,
glycerin and/or polyethylene glycol 400 are the demulcents or soothing agents.
In certain
embodiments, the demulcent or soothing agent is selected from hyaluronic acid,
tamarind
seed extract or mixtures thereof
Compositions of the present invention are ophthalmologically suitable for
application
to a subject's eyes. The term "aqueous" typically denotes an aqueous
formulation wherein
the excipient is > about 50%, more preferably > about 75% and in particular >
about 90% by
weight water. In certain embodiments, the compositions of the present
invention are
essentially free of compounds which irritate the eye. In certain embodiments,
the
compositions of the present invention are essentially free of free fatty acids
and Ci to C4
alcohols. In certain embodiments, the compositions of the present invention
are comprise
less than 40% (or about 40%), optionally, less than 35% (or about 35%),
optionally, less than
30% (or about 30%), optionally less than 25% (or about 25%), optionally, less
than 20% (or
about 20%), optionally, less than 15% (or about 15%), optionally less than 10%
(or about
10%), or optionally, less than 5% (or about 5%), by weight of the total
composition, of a non-
alcohol, organic excipient or solvent. These drops may be delivered from a
single dose
ampoule which may preferably be sterile and thus render bacteriostatic
components of the
formulation unnecessary. Alternatively, the drops may be delivered from a
multi-dose bottle
which may preferably comprise a device which extracts any preservative from
the
composition as it is delivered, such devices being known in the art.
In certain embodiments, the compositions of the present invention are
isotonic, or
slightly hypotonic in order to combat any hypertonicity of tears caused by
evaporation and/or
disease. This may require a tonicity agent to bring the osmolality of the
formulation to a
level at or near 210-320 milliosmoles per kilogram (mOsm/kg). The compositions
of the
present invention generally have an osmolality in the range of 220-320
mOsm/kg, or,
optionally, have an osmolality in the range of 235-300 mOsm/kg. The ophthalmic
compositions will generally be formulated as sterile aqueous solutions.
26
Date Recue/Date Received 2020-11-19
The osmolality of the compositions of the present invention may be adjusted
with
tonicity agents to a value which is compatible with the intended use of the
compositions. For
example, the osmolality of the composition may be adjusted to approximate the
osmotic
pressure of normal tear fluid, which is equivalent to about 0.9 w/v % of
sodium chloride in
.. water. Examples of suitable tonicity adjusting agents include, without
limitation, sodium,
potassium, calcium and magnesium chloride; dextrose; glycerin; propylene
glycol; mannitol;
sorbitol and the like and mixtures thereof In one embodiment, a combination of
sodium
chloride and potassium chloride are used to adjust the tonicity of the
composition.
The compositions of the present invention can also be used to administer
pharmaceutically active compounds. Such compounds include, but are not limited
to,
glaucoma therapeutics, pain relievers, anti-inflammatory and anti-allergy
medications, and
anti-microbials. More specific examples of pharmaceutically active compounds
include
betaxolol, timolol, pilocarpine, carbonic anhydrase inhibitors and
prostglandins;
dopaminergic antagonists; post-surgical antihypertensive agents, such as para-
amino
clonidine (apraclonidine); anti-infectives such as ciprofloxacin,
moxifloxacin, and
tobramycin; non-steroidal and steroidal anti-inflammatories, such as naproxen,
diclofenac,
nepafenac, suprofen, ketorolac, tetrahydrocortisol and dexamethasone; dry eye
therapeutics
such as PDE4 inhibitors; and anti-allergy medications such as H1/H4
inhibitors, H4
inhibitors, olopatadine or mixtures thereof
It is also contemplated that the concentrations of the ingredients comprising
the
formulations of the present invention can vary. A person of ordinary skill in
the art would
understand that the concentrations can vary depending on the addition,
substitution, and/or
subtraction of ingredients in a given formulation.
In certain embodiments, the compositions of the present invention may have a
pH
which is compatible with the intended use, and is often in the range of 4 (or
about 4) to 10 (or
about 10), optionally between 6 (or about 6) to 8 (to about 8), optionally
between 6.5 (or
about 6.5) to 7.5 (or about 7.5), or optionally between 6.8 (or about 6.8) to
7.2 (or about 7.2).
In certain embodiments, a variety of conventional buffers may be employed,
such as
phosphate, borate, citrate, acetate, histidine, tris, bis-tris and the like
and mixtures thereof
Borate buffers include boric acid and its salts, such as sodium or potassium
borate.
Potassium tetraborate or potassium metaborate, which produce boric acid or a
salt of boric
27
Date Recue/Date Received 2020-11-19
acid in solution, may also be employed. Hydrated salts such as sodium borate
decahydrate
can also be used. Phosphate buffers include phosphoric acid and its salts; for
example,
M 2HPO4 and MH2PO4, wherein M is an alkali metal such as sodium and potassium.
Hydrated salts can also be used. In one embodiment of the present invention,
Na2HPO4.7H20 and NaH2P02.H20 are used as buffers. The term phosphate also
includes
compounds that produce phosphoric acid or a salt of phosphoric acid in
solution.
Additionally, organic counter-ions for the above buffers may also be employed.
The
concentration of buffer generally varies from about 0.01 to 2.5 w/v % and more
preferably
varies from about 0.05 to about 0.5 w/v %.
In certain embodiments, the viscosity of the compositions of the present
invention
range from about 1 to about 500 cps, optionally from about 10 to about 200
cps, or optionally
from about 10 to about 100 cps, when measured using a TA Instrument AR 2000
rheometer.
The TA Instrument AR 2000 rheometer should be used with the AR2000 flow test
method of
the TA Rheological Advantage software with a 40 mm steel plate geometry; the
viscosity
ranges should be obtained by measuring steady state flow controlling shear
rate from 0 sec
to 200 sec
In certain embodiments, the compositions of the present invention are useful
as, and
in the form of, eye-drop solution, eye wash solution, contact lens lubricating
and/or rewetting
solution, spray, mist or any other manner of administering a composition to
the eye.
The compositions of the present invention may also be useful as, and in the
form of,
packing solutions for contact lenses. In certain embodiments, as packing
solutions, the
compositions of the present invention may be sealed in blister packaging and,
also, suitable
for undergoing sterilization.
Examples of blister packages and sterilization techniques are disclosed in the
following references which are hereby incorporated by reference in their
entirety, U.S. Pat.
Nos. D435,966; 4,691,820; 5,467,868; 5,704,468; 5,823,327; 6,050,398,
5,696,686;
6,018,931; 5,577,367; and 5,488,815. This portion of the manufacturing process
presents
another method of treating the ophthalmic devices with anti-allergic agent,
namely adding
anti-allergic agents to a solution prior to sealing the package, and
subsequently sterilizing the
package. This is the preferred method of treating ophthalmic devices with anti-
allergic
agents.
28
Date Recue/Date Received 2020-11-19
Sterilization can take place at different temperatures and periods of time.
The
preferred sterilization conditions range from about 100 C for about 8 hours to
about 150 C
for about 0.5 minute. More preferred sterilization conditions range from about
115 C for
about 2.5 hours to about 130 C for about 5.0 minutes. The most preferred
sterilization
conditions are about 124 C for about 18 minutes.
When used as packing solutions, the compositions of the present invention may
be
water-based solutions. Typical packing solutions include, without limitation,
saline solutions,
other buffered solutions, and deionized water. In certain embodiments, the
packing solution
is an aqueous solution of deioinized water or saline solution containing salts
including,
without limitation, sodium chloride, sodium borate, sodium phosphate, sodium
hydrogenphosphate, sodium dihydrogenphosphate, or the corresponding potassium
salts of
the same. These ingredients are generally combined to form buffered solutions
that include
an acid and its conjugate base, so that addition of acids and bases cause only
a relatively
small change in pH. In certain embodiments, the pH of the packing solution is
as described
above. The buffered solutions may additionally include 2-(N-
morpholino)ethanesulfonic acid
(MES), sodium hydroxide, 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol,
n-tris(hydroxymethyl)methy1-2-aminoethanesulfonic acid, citric acid, sodium
citrate, sodium
carbonate, sodium bicarbonate, acetic acid, sodium acetate, ethylenediamine
tetraacetic acid
and the like and combinations thereof Preferably, the solution is a borate
buffered or
phosphate buffered saline solution or deionized water. The particularly
preferred solution
contains about 500 ppm to about 18,500 ppm sodium borate, most particularly
preferred
about 1000 ppm of sodium borate.
If any ingredients incorporated into the packing solutions are subject to
oxidative
degradation, agents that stabilize packing solutions containing such
ingredients may be
added. Such "oxidative stabilization agents" include but are not limited to
chelants such as
EDTA, Dequest, Desferal, silica, chitin derivatives such as chitosan,
cellulose and its
derivatives, and N,N,N',N',N", N"-hexa(2-pyridy1)-1,3,5-
tris(aminomethyObenzene, and
certain macrocyclic ligands such as crown ethers, ligand containing knots and
catenands.
See, David A. Leigh et al Angew. Chem Int. Ed., 2001, 40, No. 8, pgs. 1538-
1542 and Jean-
Claude Chambron et al. Pure & Appl. Chem., 1990, Vol. 62, No. 6, pgs. 1027-
1034.
Oxidative stabilization agents may include other compounds that inhibit
oxidations such as
those selected from the group consisting of 2,2',2",6,6',6"-Hexa-(1,1-
dimethylethy1)4,4',4"-
29
Date Recue/Date Received 2020-11-19
[(2,4,6-trimethy1-1,3,5-benzenetriy1)-trismethylenel-triphenol (Irganox 1330),
1,3,5tris[3,5-
di(1,1-dimethylethy1)4-hydroxybenzy11-1H,3H,5H-1,3,5-triazine-2,4,6-trione,
pentaerythrityl
tetrakis[3-[3,5-di(1,1-dimethylethyl)-4-hydroxyphenyll-propionate], octadecy1-
3-[3,5-di(1,1-
dimethylethyl)-4-hydroxyphenyll-propionate, tris[2,4-di(1,1-dimethylethyl)-
phenyll-
phosphite, 2,2'-di(octadecyloxy)-5,5'-spirobi(1,3,2-dioxaphosphorinane),
dioctadecyl
disulphide, didodecy1-3,3'-thiodipropionate, dioctadecy1-3,3'-
thiodipropionate,
butylhydroxytoluene, ethylene bis[3,3-di[3-(1,1-dimethylethyl)-4-
hydroxyphenyllbutyratel
and mixtures thereof The preferred oxidative stabilization agents are
diethylenetriaminepentaacetic acid ("DTPA"), or salts of DTPA such as
CaNa3DTPA,
ZnNa3DTPA, and Ca2DTPA. See, U.S. App. Pat. No. 60/783,557 filed on, March 17,
2006,
entitled "Methods for Stabilizing Oxidatively Unstable Pharmaceutical
Compositions" and its
corresponding non-provisional filing which are hereby incorporated by
reference in their
entirety. In certain embodiments, the concentration of oxidative stabilization
agents in the
solution be from about 2.5 nmoles/liter to about, 5000 nmoles/liter,
optionally, from about
20 nmoles/liter to about 1000 nmoles/liter, optionally from about 100
nmoles/liter to about
1000 nmoles/liter, or optionally from about 100 nmoles/liter to about 500
nmoles/liter.
In particular embodiments, the compositions of the present invention are
formulated
for administration at any frequency of administration, including once a week,
once every five
days, once every three days, once every two days, twice a day, three times a
day, four times a
day, five times a day, six times a day, eight times a day, every hour, or
greater frequency.
Such dosing frequency is also maintained for a varying duration of time
depending on the
therapeutic needs of the user. The duration of a particular therapeutic
regimen may vary from
one-time dosing to a regimen that extends for months or years. One of ordinary
skill in the
art would be familiar with determining a therapeutic regimen for a specific
indication.
The composition and products containing such compositions of this invention
may be
prepared using methodology that is well known by an artisan of ordinary skill.
EXAMPLES
Any compositions of the present invention as described in following examples
illustrate specific embodiments of compositions of the present invention, but
are not intended
Date Recue/Date Received 2020-11-19
to be limiting thereof Other modifications can be undertaken by the skilled
artisan without
departing from the spirit and scope of this invention.
The following test methods were used in the Examples:
Example 1
To reduce obstruction potentially caused by tight junction structures (i.e.,
those
surface structures [e.g., membrane or film barriers] typically inhibiting
chemical permeation
into tissues) , induction of mucin secretion in human epicorneal 3D tissues
was observed by
contacting the corneal tissues with growth media containing Pichia anomala
ferment extracts
(Hyalurodine) (i.e., "treated" media) such that the bottom cell layer of the
corneal tissues
(i.e., cell layer which don't have tight junction structures inhibiting
chemical permeation into
tissues) were immersed in the growth media. By contacting the bottom cell
layer of the
corneal tissues with the growth media containing the Pichia anomala ferment
extracts, the
extract can directly contact and move upward into the inner corneal tissues
without having to
first pass through any tight junction structures or other surface barrier,
providing improved
bioavailability of Pichia anomala into corneal tissue cells.
Human epicorneal 3D tissues in growth media treated to have 1.56 mg/ml and
1.95
mg/ml concentrations of Pichia anomala ferment extracts (Hyalurodine) showed
an increase
of MUC4 gene expressions in the human epicorneal 3D tissues.
EpiCorneal 3D human tissues were purchased from MatTek Company (Ashland, MA,
USA). Upon receiving the epicorneal 3D human tissues, they were incubated in
MatTek
assay medium overnight following the manufacturer's instruction. The
epicorneal 3D human
tissues were divided into three treatment groups with three tissues per group.
Pichia anomala
ferment extracts (Hyalurodine) were added, respectively, into the culture
medium containing
the human epicorneal tissues of two of the treatment groups to produce media
concentrations
of 1.56 mg/ml and 1.95 mg/ml, respectively. The epicorneal tissues in all
treatment groups
were allowed to incubate for two days. The Pichia anomala ferment extract was
supplied by
Silab, St. Viance, France. After the two days, the human epicorneal 3D tissues
were cut in
halves and half of the tissues were lysed in 350 pL lysis buffer, consisting
of 100 parts RLT
buffer (RNeasy Mini kit, Qiagen, Valencia, CA), to one part 2-mercaptoethanol.
RNA was
31
Date Recue/Date Received 2020-11-19
extracted from the solutions using the RNeasy Mini Kit (Qiagen, Valencia, CA)
according to
manufacturer's instructions and RNA was eluted in 25 pL RNase-free water.
Reverse transcription (RT) was performed using the Applied Biosystems High
Capacity Reverse Transcription Kit (ThermoFisher Scientific, Bridgewater, NJ).
Gene
expression assays sold under the tradename TAQMAN for mucin-4 (MUC4),
polymerase
(RNA) II polypeptide A (POLR2A), and Master Mix were purchased from
ThermoFisher
Scientific (Bridgewater, NJ). qPCR analysis was performed using TaqMan Master
Mix
(ThermoFisher Scientific, Bridgewater, NJ), and run on a real time PCR system
sold under
the tradename QUANTSTUDIO 7 Flex System (ThermoFisher Scientific, Bridgewater,
NJ).
The expression of the MUC4 gene was normalized against the expression of the
human
POLR2A housekeeping gene. The fold changes were calculated in comparison to
the
untreated control (UT) and two-tailed two-sample Student t-tests (Microsoft
Office Excel
2007; Microsoft, Redmond, WA, USA) were performed. Results were shown in
Figure 1,
indicating increased fold changes of MUC4 in corneal tissue cells with
increased
concentration of Pichia anomala ferment extract in media. The "untreated"
sample was
prepared the same as described this example except no Pichia anomala is added
to media
Example 2
Human epicorneal 3D tissues in growth media treated to contain 0.39 mg/ml,
0.78
mg/ml, 1.56 mg/ml and 1.95 mg/ml concentrations of Pichia anomala ferment
extracts
(Hyalurodine) showed an increase of MUC4 gene expressions in human epicorneal
3D
tissues. *, p < 0.05.
EpiCorneal 3D human tissues were purchased from MatTek Company (Ashland, MA,
USA). Upon receiving the epicorneal 3D human tissues, theywere incubated in
MatTek
assay medium overnight following the manufacturer's instruction. The
epicorneal 3D human
tissues were divided into five treatment groups with three tissues per group.
Pichia anomala
ferment extracts (Hyalurodine) were added, respectively, into the culture
medium of four of
the treatment groups to produce media concentrations of 0.39 mg/ml, 0.78
mg/ml, 1.56
mg/ml and 1.95 mg/ml, respectively. The epicorneal tissues of all five of the
treatment
groups allowed to incubate for two days. The Pichia anomala ferment extract
was supplied
by Silab, St. Viance, France. After the two days, culture media were collected
for measuring
32
Date Recue/Date Received 2020-11-19
mucin 1 secretion using human mucin-1 (CA15-3) enzyme-linked immunosorbent
assay
(ELISA) kit (EHMUC1, ThermoFisher Scientific, Bridgewater, NJ) following the
manufacturer's protocol. To assess activity, the colorimetric change was
measured using a
microplate reader (SpectraMax M2E, Molecular Devices, Sunnyvale, CA, USA).
This assay
employs the standard enzyme-linked immunosorbent assay technique, so there is
a linear
correlation between Mucin-1 concentration in the sample and the colorimetric
change. A
standard curve was generated, with the Mucin-1 concentration on the x-axis and
absorbance
on the y-axis to indicate corresponding Mucin-1 concentration. Results were
shown in Figure
2, indicating increased Mucin-1 production in corneal tissue cells with
increased
environmental concentration of Pichia anomala ferment extract in media.
Example 3
Compositions having the 1.95 mg/ml, 7.8 mg/ml, 19.5 mg/ml and 39 mg/ml
concentrations of Pichia anomala extracts in PBS showed an increase of mucin-1
secretion in
human epicorneal 3D tissues when applied topically to corneal tissue cells. *,
p < 0.05.
EpiCorneal 3D human tissues were purchased from MatTek Company (Ashland, MA,
USA). Upon receiving the epicorneal 3D human tissues, they were incubated in
MatTek
assay medium overnight following the manufacturer's instruction. The
epicorneal 3D human
tissues were divided into three treatment groups with three tissues per group.
Four
.. concentrations of Pichia anomala ferment extracts (Hyalurodine) 1.95 mg/ml,
7.8 mg/ml,
19.5 mg/ml and 39 mg/ml were suspended in aqueous phosphate buffered solution
(PBS)
vehicles and were topically applied, respectively, to the corneal epithelium
surface of four of
the treatment groups. The epicorneal tissues of all five treatment groups were
allowed to
incubate for two days. The Pichia anomala ferment extract was supplied by
Silab, St.
Viance, France. After the two days, culture media were collected for measuring
mucin 1
secretion using human mucin-1 (CA15-3) enzyme-linked immunosorbent assay
(ELISA) kit
(EHMUC1, ThermoFisher Scientific, Bridgewater, NJ) following the
manufacturer's
protocol. To assess activity, the colorimetric change was measured using a
microplate reader
(SpectraMax M2E, Molecular Devices, Sunnyvale, CA, USA). This assay employs
the
standard enzyme-linked immunosorbent assay technique, so there is a linear
correlation
between Mucin-1 concentration in the sample and the colorimetric change. A
standard curve
was generated, with the Mucin-1 concentration on the x-axis and absorbance on
the y-axis to
33
Date Recue/Date Received 2020-11-19
indicate corresponding Mucin-1 concentration. Results were shown in Figure 3.
(Kiwi is
used in Figure 2 to represent Pichia anomala ferment extract).
The results show that increased Mucin-1 production in corneal tissue cells was
observed after topical application of compositions having the 1.95 mg/ml, 7.8
mg/ml, 19.5
mg/ml and 39 mg/ml concentrations of Pichia anomala extracts (in PBS).
Example 4
Pichia anomala ferment extracts (Hyalurodine) induced hyaluronic acid
secretion in
human epicorneal 3D tissues when applied topically to corneal tissue cells.
Without being
.. limited by theory, it is believed that such topical application requires
diffusion of the Pichia
anomala ferment extracts through tight junction structures of the human
epicorneal tissue
(i.e., those structures [e.g., membrane or film barriers] typically inhibiting
chemical
permeation into tissues) to reach the inside of the epicorneal tissue.
EpiCorneal 3D human tissues were purchased from MatTek Company (Ashland, MA,
USA). Upon receiving the epicorneal 3D human tissues, they were incubated in
MatTek
assay medium overnight following the manufacturer's instruction. The
epicorneal 3D human
tissues were divided into five treatment groups with three tissues per group.
Four
concentrations of Pichia anomala ferment extracts (Hyalurodine) 1.95 mg/ml,
7.8 mg/ml,
19.5 mg/ml and 39 mg/mlin aqueous phosphate buffered solution (PBS) vehicle
were
topically applied, respectively, to the corneal epithelium surface of four of
the treatment
groups (such that the Pichia anomala ferment extracts were delivered through
the above-
mentioned tight junction structures). The epicorneal tissues in all five
treatment groups were
allowed to incubate for two days. The Pichia anomala ferment extract was
supplied by Silab,
St. Viance, France. After the two days, the culture media were collected for
measuring
hyaluronic acid (HA) secretion using HA enzyme-linked immunosorbent assay
(ELISA) kit
(K-1200, Echelon, Salt Lake City, UT, USA) following the manufacturer's
protocol. To
assess activity, the colorimetric change was measured using a microplate
reader (SpectraMax
M2E, Molecular Devices, Sunnyvale, CA, USA). This assay employs the
competitive
enzyme-linked immunosorbent assay technique, so there is an inverse
correlation between
HA concentration in the sample and the colorimetric change. A standard curve
was
generated, with the HA concentration on the x-axis and absorbance on the y-
axis to indicate
corresponding HA concentration. Results were shown in Figure 4.
34
Date Recue/Date Received 2020-11-19
While the results suggest that topical application at the tested concentration
directionally indicate induction of HA production in the corneal cells,
factors such as tight
junction structures (or, other surface membrane or film layer barriers) may
reduce the
concentration of Pichia anomala reaching the internal corneal tissues. In such
situations,
embodiments of the present invention incorporating penetration enhancers may
be useful.
Example 5
To reduce obstruction potentially caused by the above tight junction
structures (or,
membrane or film barriers), induction of hyaluronic acid secretion in human
epicorneal 3D
tissues was observed by contacting the corneal tissues with growth media
containing Pichia
anomala ferment extracts (Hyalurodine) (i.e., "treated" media) such that the
bottom cell
layer of the corneal tissues (i.e., cell layers which don't have tight
junction structures
inhibiting chemical permeation into tissues) were immersed in the growth
media. By
contacting the bottom cell layer of the corneal tissues with the growth media
containing the
Pichia anomala ferment extracts, the extract can directly contact and move
upward into the
inner corneal tissues without having to first pass through any tight junction
structures or other
surface barrier, providing improved bioavailability of Pichia anomala into
corneal tissue
cells.
EpiCorneal 3D human tissues were purchased from MatTek Company (Ashland, MA,
USA). Upon receiving the epicorneal 3D human tissues, these tissues were
incubated in
MatTek assay medium overnight following the manufacturer's instruction. The
epicorneal
3D human tissues were divided into five treatment groups with three tissues
per group.
Pichia anomala ferment extracts (Hyalurodine) were added into the culture
medium of four
of the treatment groups to produce media concentrations of 0.39 mg/ml, 0.78
mg/ml, 1.56
mg/ml and 1.95 mg/ml, respectively, to contact the bottom cell layer of the
human epicorneal
tissues. The epicorneal tissues of all five of the treatment groups were
allowed to incubate for
two days. The Pichia anomala ferment extract was supplied by Silab, St.
Viance, France.
After the two days, the culture media were collected for measuring hyaluronic
acid (HA)
secretion using HA enzyme-linked immunosorbent assay (ELISA) kit (K-1200,
Echelon, Salt
Lake City, UT, USA) following the manufacturer's protocol. To assess activity,
the
colorimetric change was measured using a microplate reader (SpectraMax M2E,
Molecular
Date Recue/Date Received 2020-11-19
Devices, Sunnyvale, CA, USA). This assay employs the competitive enzyme-linked
immunosorbent assay technique, so there is an inverse correlation between HA
concentration
in the sample and the colorimetric change. A standard curve was generated,
with the HA
concentration on the x-axis and absorbance on the y-axis to indicate
corresponding HA
concentration. Results were shown in Figure 5.
The results show that a statistically significant increase in HA production in
corneal
tissue cells was observed at such tissue environmental concentrations of
Pichia anomala of at
least 0.3 mg/ml (i.e., concentrations of Pichia anomala extract contacting
such internal
corneal tissue cells [e.g., corneal fluid level concentrations]).
Example 6
Table 1 illustrates the components of such formulations (as shown in
formulations 6A
and 6B), which components can be incorporated as described below using
conventional
mixing technology.
36
Date Recue/Date Received 2020-11-19
Table 1
6A 6B
Useful for Relief of Useful for Relief
of
Dry Eye Irritation Dry Eye Irritation
for Contact Lenses
INGREDIENT %w/w amount %w/w amount
per batch per
(gms) batch
(gms)
Sodium 0.20 2.0 0.15 1.5
Hyaluronate
Pichia anomala 2.0 20.0 5.0 50.0
extract
Polysorbate 80 1.0 10.0 2.0 20.0
Polysorbate 20 5.0 50.0 10.0 100.0
Polyethylene 0.25 2.5 0 0
Glycol 400
Boric Acid 0.60 6.0 0.60 6.0
Sodium Borate 0.05 0.50 0.05 0.50
Sodium
Chloride*
Potassium 0.10 1.0 0.10 1.0
Chloride
Calcium 0.006 0.06 0.006 0.06
Chloride
Dihydrate
Magnesium 0.006 0.06 0.006 0.06
Chloride
Sodium 0.014 0.14 0.014 0.14
Chlorite
Dihydrate
37
Date Recue/Date Received 2020-11-19
Polyquatemium 0.0015 0.015 0.0015 0.015
42
(33%aqueous)
Sodium 0.014 0.14 0.014 0.14
Chlorite
Dihydrate
1N Sodium
Hydroxide
solution**
1N
Hydrochloric
Acid solution**
Purified
Water***
total 100.00 1000.0 g 100.00 1000.00
* adjust to tonicity of 280-290 mOsm/Kg
** adjust to pH 7.2
*** q.s to 100%w/w
For Examples 6A-6B: The Sodium Hyaluronate can be supplied by CONTIPRO A.S.
(DOLNI, DOBROUC, CZECH REPUBLIC)
For Examples 6A and 6B: The Pichia anomala extract supplied by SILAB (SAINT
VIANCE, FRANCE).
For Examples 6A-6B: The Polysorbate 20 can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 6A-6B: The Polysorbate 80 can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 6A: The Polyethylene Glycol 400 can be supplied by Clariant
Produkte
(BURGKIRCHEN, GERMANY).
For Examples 6A-6B: The Boric Acid can be supplied by Merck KGaA (DARMSTADT,
GERMANY).
38
Date Recue/Date Received 2020-11-19
For Examples 6A-6B: The Sodium Borate can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 6A-6B: The Sodium Chloride can be supplied by Caldic (DUSSELDORF,
GERMANY).
For Examples 6A-6B: The Potassium Chloride can be supplied by Merck KGaA
(DARMSTADT, GERMANY).
For Examples 6A-6B: The Calcium Chloride Dihydrate can be supplied by Merck
KGaA
(DARMSTADT, GERMANY).
For Examples 6A-6B: The Magnesium Chloride can be supplied by KGaA (DARMSTADT,
GERMANY).
For Examples 6A-6B: The Polyquaternium-42 (33% aqueous) can be supplied by DSM
BIOMEDICAL (BERKELEY, CA, USA).
For Examples 6A-6B: The Sodium Chlorite Dihydrate can be supplied by Oxychem
(WICHITA, KS, USA)
For Examples 6A-6B: The 1N Sodium Hydroxide can be supplied by VWR (RADNER,
PA,
USA).
For Examples 6A-6B: The 1N Hydrochloric acid can be supplied by VWR (RADNER,
PA,
USA).
Solution 6A can be prepared as follows:
1. To a 1500 ml beaker is added 800 grams of Purified Water USP.
2. To the above is added 10 g of Polysorbate 80 and 50 g of Polysorbate 20.
The
solution is mixed until both are fully mixed and dissolved.
3. To the above is added 20.0 g of Pichia anomala extract. The solution is
mixed until
the Pichia anomala extract is dissolved.
4. The solution is filtered through a 0.45micron filter and returned to a
1500 ml beaker.
5. To the solution of Step 4 is added 2.0 grams of Sodium Hyaluronate. The
solution is
mixed to fully dissolve the Sodium Hyaluronate.
6. The following ingredients are next added sequentially, allowing for each
to dissolve
before adding the next: 2.5 grams Polyethylene Glycol 400, 6.0 grams Boric
acid,
0.05gram Sodium Borate, 1.0 gram Potassium Chloride, 0.06 gram Calcium
Chloride
Dihydrate, 0.06 gram Magnesium Chloride, and 0.0015 grams Polyquaternium-42
(aqueous).
39
Date Recue/Date Received 2020-11-19
7. While continuing to mix, 0.14gram Sodium Chlorite Dihydrate is added and
mixed to
dissolve.
8. The tonicity of the formula is determined and adjusted to 280 mOsm/Kg
with Sodium
Chloride.
9. The pH of the formula is adjusted to pH of 7.2 using the 1N Sodium
Hydroxide
and/or 1N Hydrochloric acid.
10. The solution is brought to 1000.0 grams using Purified Water USP and
mixed for 10
minutes to be fully uniform.
11. The solution is filtered using a 0.22 micron filter.
Solution 6B can be prepared as follows:
1. To a 1500 ml beaker is added 800 grams of Purified Water USP.
2. To the above is added 20 g of Polysorbate 10 and 100 g of Polysorbate
20. The
solution is mixed until both are fully mixed and dissolved.
3. To the above is added 50 g of Pichia anomala extract. The solution is
mixed until the
Pichia anomala extract is dissolved.
4. The solution is filtered through a 0.45micron filter and returned to a
1500 ml beaker.
5. To the solution of Step 4 is added 1.5 grams of Sodium Hyaluronate. The
solution is
mixed to fully dissolve the Sodium Hyaluronate.
6. The following ingredients are next added sequentially, allowing for each
to dissolve
before adding the next: 6.0 grams Boric acid, 0.05 gram Sodium Borate, 1.0
gram
Potassium Chloride, 0.06 gram Calcium Chloride Dihydrate, 0.06 gram Magnesium
Chloride and 0.0015 grams Polyquaternium-42 (aqueous).
7. While continuing to mix, 0.14 gram Sodium Chlorite Dihydrate is added
and mixed to
dissolve.
8. The tonicity of the formula is determined and adjusted to 280 mOsm/Kg
with Sodium
Chloride.
9. The pH of the formula is adjusted to pH of 7.2 using the 1N Sodium
Hydroxide
and/or 1N Hydrochloric acid.
10. The solution is brought to 1000.0 grams using Purified Water USP and
mixed for 10
minutes to be fully uniform.
11. The solution is filtered using a 0.22 micron filter.
Date Recue/Date Received 2020-11-19
Example 7
Table 2 illustrates the components of formulations of the present invention
(as shown
in formulations 7A and 7B), which components can be incorporated as described
below using
conventional mixing technology.
Table 2
7A 7B
Useful for Relief Useful for Relief
of Dry Eye of Dry Eye
Irritation Irritation for
Contact Lenses
INGREDIENT %w/w amount %w/w amount
per per
batch batch
(gms) (gms)
Pichia anomala 1.0 10.0 2.0 20.0
extract
Polysorbate 80 1.0 10.0 1.0 10.0
Polysorbate 20 3.0 30.0 5.0 50.0
Glycerin 0.25 2.5 0.25 2.5
Hypromellose 0.198 1.98 0.198 1.98
E3 2910
Boric Acid 0.40 4.0 0.40 4.0
Sodium Borate 0.022 0.22 0.022 0.22
Disodium 0.027 0.27 0.027 0.27
Phosphate
Sodium Citrate 0.40 4.0 0.40 4.0
Dihydrate
Sodium
Chloride*
Potassium 0.10 1.0 0.10 1.0
Chloride
41
Date Recue/Date Received 2020-11-19
50% Aqueous 0.057 0.57 0.057 0.57
Solution of
Sodium Lactate
Magnesium 0.013 0.13 0.013 0.13
Chloride
Glucose 0.0036 0.036 0.0036 0.036
Glycine 0.0000 0.0002 0.0000 0.0002
2 2
Ascorbic Acid 0.0000 0.0001 0.0000 0.0001
1 1
Disodium 0.01 0.1 0.05 0.5
Edetate
Polyquaternium 0.0030 0.030 0.0015 0.015
42
(33%aqueous)
Sodium 0.014 0.14 0.014 0.14
Chlorite
Dihydrate
1N Sodium
Hydroxide
solution**
1N
Hydrochloric
Acid solution**
Purified
Water***
total 100.00 1000.00 100.00 1000.00
* adjust to tonicity of 280-290 mOsm/Kg
** adjust to pH 7.2
*** q.s to 100.00% volume
42
Date Recue/Date Received 2020-11-19
For Examples 7A and 7B: The Pichia anomala extract can be supplied by SILAB
(SAINT
VIANCE, FRANCE).
For Examples 7A-7B: The Polysorbate 20 can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 7A-7B: The Polysorbate 80 can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 7A-7B The Boric Acid can be supplied by Merck KGaA (DARMSTADT,
GERMANY).
For Examples 7A-7B: The Sodium Borate can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 7A-7B: The Sodium Chloride can be supplied by Caldic (DUSSELDORF,
GERMANY).
For Examples 7A-7B: The Potassium Chloride can be supplied by Merck KGaA
(DARMSTADT, GERMANY).
For Examples 7A-7B: The Hypromellose E3 2910 can be supplied by DOW CHEMICAL
(PLAQUEMINE, LOUISIANA, USA).
For Examples 7A-7B: The Glycerin can be supplied by Emery Oleochemicals GmbH
(DUSSELDORF, GERMANY).
For Examples 7A-7B: The Disodium Phosphate can be supplied by Merck KGaA
(DARMSTADT, GERMANY).
For Examples 7A-7B: The Sodium Citrate can be supplied by Merck KGaA
(DARMSTADT, GERMANY).
For Examples 7A-7B: The Sodium Lactate can be supplied as Sodium Lactate (50%
aqueous) by Merck KGaA (DARMSTADT, GERMANY).
For Examples 7A-7B: The Glucose can be supplied by Roquette Freres (LASTREM,
FRANCE).
For Examples 7A-7B: The Glycine can be supplied by Merck KGaA (DARMSTADT,
GERMANY).
For Examples 7A-7B: The Ascorbic Acid can be supplied by DSM NUTRITIONAL
Products (DRAKEMYRE, SCOTLAND, UK).
For Examples 7A-7B: The Polyquatemium 42 to be supplied as Polyquatemium 42
(33%
aqueous) by DSM BIOMEDICAL, (BERKELEY, CA).
43
Date Recue/Date Received 2020-11-19
For examples 7A-7B: The Disodium Edetate can be supplied by Merck NV/SA
(OVERUSE,
BELGIUM).
For Examples 7A-7B: The 1N Sodium Hydroxide can be supplied by VWR (RADNER,
PA,
USA).
For Examples 7A-7B: The 1N Hydrochloric acid can be supplied by VWR (RADNER,
PA,
USA).
For Examples 7A-7B: The Sodium Chlorite Dihydrate to be supplied by Oxychem
(WICHITA, KS, USA)
Solution 7A can be prepared as follows:
1. To a 1500 ml beaker is added 800 grams of Purified Water USP.
2. To the above is added 10 g of Polysorbate 80 and 30 g of Polysorbate 20.
The solution
is mixed until both are fully mixed and dissolved.
3. To the above is added 10.0 g of Pichia anomala extract. The solution is
mixed until
the Pichia anomala extract is dissolved.
4. The solution is filtered through a 0.45micron filter and returned to a
1500 ml beaker.
5. To the above is added 1.98 g of Hypromellose E3 Premium. The solution is
mixed
until the Hypromellose E3 Premium dissolved.
6. The following ingredients are next added sequentially, allowing each to
dissolve
before adding the next: 2.50 grams Glycerin, 4.0 grams Boric acid, 0.22 gram
Sodium Borate, 0.27gram Disodium Phosphate, 4.00 grams Sodium Citrate
Dihydrate, 1 gram Potassium Chloride, 0.57 gram Sodium Lactate (50% aqueous),
0.13 gram Magnesium Chloride, 0.036 gram Glucose, 0.0002 gram Glycine, 0.0001
gram Ascorbic acid, 0.10 gram Disodium Edetate, 0.030 gram Polyquaternium-42
(33%aqueous), and 0.14 gram Sodium Chlorite.
7. The tonicity of the solution is determined and adjusted to 280 mOsm with
Sodium
Chloride.
8. The pH of the solution is measured and adjusted to 7.2 with 1N Sodium
Hydroxide
and/or 1N Hydrochloric acid.
9. The solution is brought to a volume of 1,000.00 grams with Purified
Water and mixed
for 10 minutes.
10. The solution is filtered using a 0.22 micron filter.
44
Date Recue/Date Received 2020-11-19
Solution 7B can be prepared as follows:
1. To a 1500 ml beaker is added 800 grams of Purified Water USP.
2. To the above is added 10 g of Polysorbate 80 and 50 g of Polysorbate 20.
The solution
is mixed until both are fully mixed and dissolved.
3. To the above is added 20.0 g of Pichia anomala extract. The solution is
mixed until
the Pichia anomala extract is dissolved.
4. The solution is filtered through a 0.45micron filter and returned to a
1500 ml beaker.
5. To the above is added 1.98 g of Hypromellose E3 Premium. The solution is
mixed
until the Hypromellose E3 Premium dissolved.
6. The following ingredients are next added sequentially, allowing each to
dissolve
before adding the next: 2.50 grams Glycerin, 4.0 grams Boric acid, 0.22 gram
Sodium Borate, 0.27gram Disodium Phosphate, 4.00 grams Sodium Citrate
Dihydrate, 1 gram Potassium Chloride, 0.57 gram Sodium Lactate (50% aqueous),
0.13 gram Magnesium Chloride, 0.036 gram Glucose, 0.0002 gram Glycine, 0.0001
gram Ascorbic acid, 0.05 gram Disodium Edetate, 0.015 gram Polyquaternium-42
(33%aqueous), and 0.14 gram Sodium Chlorite.
7. The tonicity of the solution is determined and adjusted to 280 mOsm with
Sodium
Chloride.
8. The pH of the solution is measured and adjusted to 7.2 with 1N Sodium
Hydroxide
and/or 1N Hydrochloric acid.
9. The solution is brought to a volume of 1,000.00 grams with Purified
Water and mixed
for 10 minutes.
10. The solution is filtered using a 0.22 micron filter.
45
Date Recue/Date Received 2020-11-19
Example 8
Table 3 illustrates the components of such formulations (as shown in
formulations 8A
and 8B), which components can be incorporated as described below using
conventional
mixing technology.
Table 3
8A 8B
Useful for Relief of Useful for Relief
of
Dry Eye Irritation Dry Eye Irritation
for Contact Lenses
INGREDIENT %w/w amount %w/w amount
per batch per
(gms) batch
(gms)
Sodium 0.20 2.0 0.15 1.5
Hyaluronate
Kiwi Extract 2.0 20.0 5.0 50.0
Polysorbate 80 1.0 10.0 2.0 20.0
Polysorbate 20 5.0 50.0 10.0 100.0
Polyethylene 0.25 2.5 0 0
Glycol 400
Boric Acid 0.60 6.0 0.60 6.0
Sodium Borate 0.05 0.50 0.05 0.50
Sodium
Chloride*
Potassium 0.10 1.0 0.10 1.0
Chloride
Calcium 0.006 0.06 0.006 0.06
Chloride
Dihydrate
Magnesium 0.006 0.06 0.006 0.06
Chloride
46
Date Recue/Date Received 2020-11-19
Sodium 0.014 0.14 0.014 0.14
Chlorite
Dihydrate
Polyquatemium 0.0015 0.015 0.0015 0.015
42
(33%aqueous)
Sodium 0.014 0.14 0.014 0.14
Chlorite
Dihydrate
1N Sodium
Hydroxide
solution**
1N
Hydrochloric
Acid solution**
Purified
Water***
total 100.00 1000.0 g 100.00 1000.00
* adjust to tonicity of 280-290 mOsm/Kg
** adjust to pH 7.2
*** q.s to 100%w/w
For Examples 8A-8B: The Sodium Hyaluronate can be supplied by CONTIPRO A.S.
(DOLNI, DOBROUC, CZECH REPUBLIC)
For Examples 8A and 8B: The Kiwi Extract (Pichia anomala yeast extract) can be
supplied
by SILAB (SAINT VIANCE, FRANCE).
For Examples 8A-8B: The Polysorbate 20 can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 8A-8B: The Polysorbate 80 can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 8A: The Polyethylene Glycol 400 can be supplied by Clariant
Produkte
(BURGKIRCHEN, GERMANY).
47
Date Recue/Date Received 2020-11-19
For Examples 8A-8B: The Boric Acid can be supplied by Merck KGaA (DARMSTADT,
GERMANY).
For Examples 8A-8B: The Sodium Borate can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 8A-8B: The Sodium Chloride can be supplied by Caldic (DUSSELDORF,
GERMANY).
For Examples 8A-8B: The Potassium Chloride can be supplied by Merck KGaA
(DARMSTADT, GERMANY).
For Examples 8A-8B: The Calcium Chloride Dihydrate can be supplied by Merck
KGaA
(DARMSTADT, GERMANY).
For Examples 8A-8B: The Magnesium Chloride can be supplied by KGaA (DARMSTADT,
GERMANY).
For Examples 8A-8B: The Polyquaternium-42 (33% aqueous) can be supplied by DSM
BIOMEDICAL (BERKELEY, CA, USA).
For Examples 8A-8B: The Sodium Chlorite Dihydrate can be supplied by Oxychem
(WICHITA, KS, USA)
For Examples 8A-8B: The 1N Sodium Hydroxide can be supplied by VWR (RADNER,
PA,
USA).
For Examples 8A-8B: The 1N Hydrochloric acid can be supplied by VWR (RADNER,
PA,
USA).
Solution 8A can be prepared as follows:
1. To a 1500 ml beaker is added 800 grams of Purified Water USP.
2. To the above is added 10 g of Polysorbate 80 and 50 g of Polysorbate 20.
The
solution is mixed until both are fully mixed and dissolved.
3. To the above is added 20.0 g of Kiwi Extract. The solution is mixed
until the Kiwi
Extract is dissolved.
4. The solution is filtered through a 0.45micron filter and returned to a
1500 ml beaker.
5. To the solution of Step 4 is added 2.0 grams of Sodium Hyaluronate. The
solution is
mixed to fully dissolve the Sodium Hyaluronate.
6. The following ingredients are next added sequentially, allowing for each
to dissolve
before adding the next: 2.5 grams Polyethylene Glycol 400, 6.0 grams Boric
acid,
0.05gram Sodium Borate, 1.0 gram Potassium Chloride, 0.06 gram Calcium
Chloride
48
Date Recue/Date Received 2020-11-19
Dihydrate, 0.06 gram Magnesium Chloride, and 0.0015 grams Polyquatemium-42
(aqueous).
7. While continuing to mix, 0.14gram Sodium Chlorite Dihydrate is added and
mixed to
dissolve.
8. The tonicity of the formula is determined and adjusted to 280 mOsm/Kg
with Sodium
Chloride.
9. The pH of the formula is adjusted to pH of 7.2 using the 1N Sodium
Hydroxide
and/or 1N Hydrochloric acid.
10. The solution is brought to 1000.0 grams using Purified Water USP and
mixed for 10
minutes to be fully uniform.
11. The solution is filtered using a 0.22 micron filter.
49
Date Recue/Date Received 2020-11-19
Solution 8B can be prepared as follows:
1. To a 1500 ml beaker is added 800 grams of Purified Water USP.
2. To the above is added 20 g of Polysorbate 10 and 100 g of Polysorbate
20. The
solution is mixed until both are fully mixed and dissolved.
3. To the above is added 50 g of Kiwi Extract. The solution is mixed until
the Kiwi
Extract is dissolved.
4. The solution is filtered through a 0.45micron filter and returned to a
1500 ml beaker.
5. To the solution of Step 4 is added 1.5 grams of Sodium Hyaluronate. The
solution is
mixed to fully dissolve the Sodium Hyaluronate.
6. The following ingredients are next added sequentially, allowing for each
to dissolve
before adding the next: 6.0 grams Boric acid, 0.05 gram Sodium Borate, 1.0
gram
Potassium Chloride, 0.06 gram Calcium Chloride Dihydrate, 0.06 gram Magnesium
Chloride and 0.0015 grams Polyquaternium-42 (aqueous).
7. While continuing to mix, 0.14 gram Sodium Chlorite Dihydrate is added
and mixed to
dissolve.
8. The tonicity of the formula is determined and adjusted to 280 mOsm/Kg
with Sodium
Chloride.
9. The pH of the formula is adjusted to pH of 7.2 using the 1N Sodium
Hydroxide
and/or 1N Hydrochloric acid.
10. The solution is brought to 1000.0 grams using Purified Water USP and
mixed for 10
minutes to be fully uniform.
11. The solution is filtered using a 0.22 micron filter.
Example 9
Table 4 illustrates the components of formulations of the present invention
(as shown
in formulations 9A and 9B), which components can be incorporated as described
below using
conventional mixing technology.
Table 4
9A 9B
Useful for
Relief of Dry
Date Recue/Date Received 2020-11-19
Useful for Eye Irritation
Relief of Dry for Contact
Eye Irritation Lenses
INGREDIENT %w/w amoun %w/w amoun
t per t per
batch batch
(gms) (gms)
Kiwi Extract 1.0 10.0 2.0 20.0
Polysorbate 80 1.0 10.0 1.0 10.0
Polysorbate 20 3.0 30.0 5.0 50.0
Glycerin 0.25 2.5 0.25 2.5
Hypromellose 0.198 1.98 0.198 1.98
E3 2910
Boric Acid 0.40 4.0 0.40 4.0
Sodium Borate 0.022 0.22 0.022 0.22
Disodium 0.027 0.27 0.027 0.27
Phosphate
Sodium Citrate 0.40 4.0 0.40 4.0
Dihydrate
Sodium
Chloride*
Potassium 0.10 1.0 0.10 1.0
Chloride
50% Aqueous 0.057 0.57 0.057 0.57
Solution of
Sodium Lactate
Magnesium 0.013 0.13 0.013 0.13
Chloride
Glucose 0.0036 0.036 0.0036 0.036
Glycine 0.0000 0.0002 0.0000 0.0002
2 2
51
Date Recue/Date Received 2020-11-19
Ascorbic Acid 0.0000 0.0001 0.0000 0.0001
1 1
Disodium 0.01 0.1 0.05 0.5
Edetate
Polyquaternium 0.0030 0.030 0.0015 0.015
42
(33%aqueous)
Sodium 0.014 0.14 0.014 0.14
Chlorite
Dihydrate
1N Sodium
Hydroxide
solution**
1N
Hydrochloric
Acid solution**
Purified
Water***
total 100.00 1000.0 100.00 1000.0
0 g
* adjust to tonicity of 280-290 mOsm/Kg
** adjust to pH 7.2
*** q.s to 100.00% volume
For Examples 9A and 9B: The Kiwi Extract (Pichia anomala yeast extract) can be
supplied
by SILAB (SAINT VIANCE, FRANCE).
For Examples 9A-9B: The Polysorbate 20 can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 9A-9B: The Polysorbate 80 can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 9A-9B The Boric Acid can be supplied by Merck KGaA (DARMSTADT,
GERMANY).
52
Date Recue/Date Received 2020-11-19
For Examples 9A-9B: The Sodium Borate can be supplied by Merck KGaA
(DARMSTADT,
GERMANY).
For Examples 9A-9B: The Sodium Chloride can be supplied by Caldic (DUSSELDORF,
GERMANY).
For Examples 9A-9B: The Potassium Chloride can be supplied by Merck KGaA
(DARMSTADT, GERMANY).
For Examples 9A-9B: The Hypromellose E3 2910 can be supplied by DOW CHEMICAL
(PLAQUEMINE, LOUISIANA, USA).
For Examples 9A-9B: The Glycerin can be supplied by Emery Oleochemicals GmbH
(DUSSELDORF, GERMANY).
For Examples 9A-9B: The Disodium Phosphate can be supplied by Merck KGaA
(DARMSTADT, GERMANY).
For Examples 9A-9B: The Sodium Citrate can be supplied by Merck KGaA
(DARMSTADT, GERMANY).
For Examples 9A-9B: The Sodium Lactate can be supplied as Sodium Lactate (50%
aqueous) by Merck KGaA (DARMSTADT, GERMANY).
For Examples 9A-9B: The Glucose can be supplied by Roquette Freres (LASTREM,
FRANCE).
For Examples 9A-9B: The Glycine can be supplied by Merck KGaA (DARMSTADT,
GERMANY).
For Examples 9A-9B: The Ascorbic Acid can be supplied by DSM NUTRITIONAL
Products (DRAKEMYRE, SCOTLAND, UK).
For Examples 9A-9B: The Polyquaternium 42 to be supplied as Polyquaternium 42
(33%
aqueous) by DSM BIOMEDICAL, (BERKELEY, CA).
For examples 9A-9B: The Disodium Edetate can be supplied by Merck NV/SA
(OVERUSE,
BELGIUM).
For Examples 9A-9B: The 1N Sodium Hydroxide can be supplied by VWR (RADNER,
PA,
USA).
For Examples 9A-9B: The 1N Hydrochloric acid can be supplied by VWR (RADNER,
PA,
USA).
For Examples 9A-9B: The Sodium Chlorite Dihydrate to be supplied by Oxychem
(WICHITA, KS, USA)
53
Date Recue/Date Received 2020-11-19
Solution 9A can be prepared as follows:
1. To a 1500 ml beaker is added 800 grams of Purified Water USP.
2. To the above is added 10 g of Polysorbate 80 and 30 g of Polysorbate 20.
The solution
is mixed until both are fully mixed and dissolved.
3. To the above is added 10.0 g of Kiwi Extract. The solution is mixed
until the Kiwi
Extract is dissolved.
4. The solution is filtered through a 0.45micron filter and returned to a
1500 ml beaker.
5. To the above is added 1.98 g of Hypromellose E3 Premium. The solution is
mixed
until the Hypromellose E3 Premium dissolved.
6. The following ingredients are next added sequentially, allowing each to
dissolve
before adding the next: 2.50 grams Glycerin, 4.0 grams Boric acid, 0.22 gram
Sodium Borate, 0.27gram Disodium Phosphate, 4.00 grams Sodium Citrate
Dihydrate, 1 gram Potassium Chloride, 0.57 gram Sodium Lactate (50% aqueous),
0.13 gram Magnesium Chloride, 0.036 gram Glucose, 0.0002 gram Glycine, 0.0001
gram Ascorbic acid, 0.10 gram Disodium Edetate, 0.030 gram Polyquaternium-42
(33%aqueous), and 0.14 gram Sodium Chlorite.
7. The tonicity of the solution is determined and adjusted to 280 mOsm with
Sodium
Chloride.
8. The pH of the solution is measured and adjusted to 7.2 with 1N Sodium
Hydroxide
and/or 1N Hydrochloric acid.
9. The solution is brought to a volume of 1,000.00 grams with Purified
Water and mixed
for 10 minutes.
10. The solution is filtered using a 0.22 micron filter.
Solution 9B can be prepared as follows:
1. To a 1500 ml beaker is added 800 grams of Purified Water USP.
2. To the above is added 10 g of Polysorbate 80 and 50 g of Polysorbate 20.
The solution
is mixed until both are fully mixed and dissolved.
3. To the above is added 20.0 g of Kiwi Extract. The solution is mixed
until the Kiwi
Extract is dissolved.
4. The solution is filtered through a 0.45micron filter and returned to a
1500 ml beaker.
54
Date Recue/Date Received 2020-11-19
5. To the above is added 1.98 g of Hypromellose E3 Premium. The solution is
mixed
until the Hypromellose E3 Premium dissolved.
6. The following ingredients are next added sequentially, allowing each to
dissolve
before adding the next: 2.50 grams Glycerin, 4.0 grams Boric acid, 0.22 gram
Sodium Borate, 0.27gram Disodium Phosphate, 4.00 grams Sodium Citrate
Dihydrate, 1 gram Potassium Chloride, 0.57 gram Sodium Lactate (50% aqueous),
0.13 gram Magnesium Chloride, 0.036 gram Glucose, 0.0002 gram Glycine, 0.0001
gram Ascorbic acid, 0.05 gram Disodium Edetate, 0.015 gram Polyquaternium-42
(33%aqueous), and 0.14 gram Sodium Chlorite.
7. The tonicity of the solution is determined and adjusted to 280 mOsm with
Sodium
Chloride.
8. The pH of the solution is measured and adjusted to 7.2 with 1N Sodium
Hydroxide
and/or 1N Hydrochloric acid.
9. The solution is brought to a volume of 1,000.00 grams with Purified
Water and mixed
for 10 minutes.
10. The solution is filtered using a 0.22 micron filter.
Date Recue/Date Received 2020-11-19
Embodiments of the Present Invention
1. A microemulsion composition or microemulsion for treating the eye
comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia; and
ii) an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less
than 1,500 A.
2. The composition of embodiment 1 (or, any of the following embodiments),
wherein
the genus of Pichia is selected from Pichia anomala, Pichia guilhermondil,
Pichia
norvegensis, Pichia ohmeri and mixtures thereof
3. The composition of embodiments land/or 2 (or, any of the following
embodiments),
wherein the genus of Pichia is Pichia anomala.
4. The composition of any one of or combination of embodiments 1 to 3 (or,
any of the
following embodiments), where in the one or more extracts, or sources of
extracts, of
the genus Pichia are present at a concentration of from about 0.01% to about
100% by
weight of the total composition.
5. The composition of any one of or combination of embodiments 1 to 4 (or,
any of the
following embodiments), where in the one or more extracts, or sources of
extracts, of
the genus Pichia are present at a concentration of from about 0.05% to about
95% by
weight of the total composition.
6. The composition of any one of or combination of embodiments 1 to 5 (or,
any of the
following embodiments), where in the one or more extracts, or sources of
extracts, of
the genus Pichia are present at a concentration of from about 0.1% to about
90% by
weight of the total composition.
7. The composition of any one of or combination of embodiments 1 to 6 (or,
any of the
following embodiments), comprising a permeation enhancer.
56
Date Recue/Date Received 2020-11-19
8. The composition of any one of or combination of embodiments 1 to 7 (or,
any of the
following embodiments), wherein the permeation enhancer is present at a
concentration of from about 0.01% to about 20% (w/v) of the total composition.
9. The composition of any one of or combination of embodiments 1 to 8 (or,
any of the
following embodiments), wherein the permeation enhancer is present at a
concentration of from about 0.1% to 10% (w/v) of the total composition.
10. The composition of any one of or combination of embodiments 1 to 9 (or,
any of the
following embodiments), wherein the permeation enhancer is present at a
concentration of from about 0.25% to 5% (w/v) of the total composition,
11. The composition of any one of or combination of embodiments 1 to 10 (or,
any of the
following embodiments), wherein the permeation enhancer is selected from
polyoxyethylene, polyoxyethylene ethers of fatty acids, sorbitan monooleate,
sorbitan
monolaurate, polyoxyethylene monolaurate, polyoxyethylene sorbitan
monolaurate,
fusidic acid and derivatives thereof, EDTA, disodium EDTA, cholic acid,
deoxycholic
acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid,
taurodeoxycholic
acid, sodium cholate, sodium glycocholate, glycocholate, sodium deoxycholate,
sodium taurocholate, sodium glycodeoxycholate, sodium taurodeoxycholate,
chenodeoxycholic acid, urosdeoxycholic acid, saponins, glycyrrhizic acid,
ammonium
glycyrrhizide, decamethonium, decamethonium bromide, and
dodecyltrimethylammonium bromide or mixtures of any of the above.
12. The composition of any one of or combination of embodiments 1 to 11 (or,
any of the
following embodiments), wherein the Pichia genus extract comprises
oligosaccharides and polysaccharide having a weight average molecular weight
of
from about 180 to about 800,000 Da.
13. The composition of any one of or combination of embodiments 1 to 12 (or,
any of the
following embodiments), wherein the Pichia genus extract comprises
57
Date Recue/Date Received 2020-11-19
oligosaccharides and polysaccharide having an average degree of polymerization
of
from DP 1 to DP 4444.
14. A method for producing/releasing/delivering/excreting mucin from and/or in
the
cornea comprising the step of administering a microemulsion composition
comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia; and
ii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less
than 1,500 A.
15. The method according to embodiment 14 (or, any of the following
embodiments),
wherein the genus of Pichia is selected from Pichia anomala, Pichia
guilhermondil,
Pichia norvegensis, Pichia ohmeri and mixtures thereof
16. The method according to embodiments 14 and/or 15 (or, any of the following
embodiments), wherein the genus of Pichia is Pichia anomala.
17. A method for maintaining the concentration of MU5AC in tears within the
range of
equal to or greater than 8 nanograms to 15 nanograms per milligrams of
protein,
comprising the step of administering a microemulsion composition comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia; and
ii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less than
1,500 A.
18. A method for treating a patient having decreased or low-level
production/release/delivery/excretion of mucin from and/or in the cornea
comprising
the step of topically administering to the eye the patient a microemultion
composition
comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia; and
ii) optionally, an ophthalmologically acceptable carrier
58
Date Recue/Date Received 2020-11-19
wherein the microemulsion droplets or particles have a maximum dimension of
less
than 1,500 A.
19. A method for promoting healing or increasing the rate of healing of wounds
in and/or
on the eye of a patient, comprising the step of administering a microemulsion
composition comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia; and
ii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less
than 1,500 A.
20. A method for improving the antimicrobial properties in tears of a patient,
comprising
the step of administering a microemulsion composition comprising:
i) one or more extracts, or sources of extracts, of the genus Pichia; and
ii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less
than 1,500 A.
21. A method for treating a patient having decreased or low-level
production/release/delivery/excretion of hyaluronic acid from and/or in the
cornea
comprising the step of topically administering to the eye of the patient a
microemulaion composition comprising:
i) a safe and effective amount of one or more extracts, or sources of
extracts, of
the genus Pichia to achieve a Pichia genus extract concentration in the
corneal
fluid in the corneal tissues of the eye of at least about 0.3mg/m1;
ii) optionally, a safe and effective amount of a permeation enhancer; and
iii) optionally, an ophthalmologically acceptable carrier
wherein the microemulsion droplets or particles have a maximum dimension of
less
than 1,500 A.
22. The method of embodiment 21 (or, any of the following embodiments),
wherein the
Pichia genus extract in the composition comprises oligosaccharides and
59
Date Recue/Date Received 2020-11-19
polysaccharide having a weight average molecular weight of from about 180 to
about
800,000 Da.
23. The method of embodiment 21 and/or 22 (or, any of the following
embodiments),
wherein the Pichia genus extract in the composition comprises oligosaccharides
and
polysaccharide having an average degree of polymerization of from DP 1 to DP
4444.
24. The method of any one of or combination of embodiments 21 to 23 (or, any
of the
following embodiments), wherein the composition comprises a permeation
enhancer.
25. The method of any one of or combination of embodiments 21 to24 (or, any of
the
following embodiments), wherein the permeation enhancer is present at a
concentration of from about 0.01% to about 20% (w/v) of the total composition.
26. The method of any one of or combination of embodiments 21 to 25 (or, any
of the
following embodiments), wherein the permeation enhancer is present at a
concentration of from about 0.1% to 10% (w/v) of the total composition.
27. The method of any one of or combination of embodiments 21 to 26 (or, any
of the
following embodiments), wherein the permeation enhancer is present at a
concentration of from about 0.25% to 5% (w/v) of the total composition,
28. The method of any one of or combination of embodiments 21 to 27 (or, any
of the
following embodiments), wherein the permeation enhancer is selected from
polyoxyethylene, polyoxyethylene ethers of fatty acids, sorbitan monooleate,
sorbitan monolaurate, polyoxyethylene monolaurate, polyoxyethylene sorbitan
monolaurate, fusidic acid and derivatives thereof, EDTA, disodium EDTA, cholic
acid, deoxycholic acid, glycocholic acid, glycodeoxycholic acid, taurocholic
acid,
taurodeoxycholic acid, sodium cholate, sodium glycocholate, glycocholate,
sodium
deoxycholate, sodium taurocholate, sodium glycodeoxycholate, sodium
taurodeoxycholate, chenodeoxycholic acid, urosdeoxycholic acid, saponins,
Date Recue/Date Received 2020-11-19
glycyrrhizic acid, ammonium glycyrrhizide, decamethonium, decamethonium
bromide, and dodecyltrimethylammonium bromide or mixtures of any of the above.
29. The method of any one of or combination of embodiments 21 to 28 (or, any
of the
following embodiments), wherein the composition is administered when the
hyaluronic acid concentration, in the patient's tears, is lower than about 10
nanograms per milligram of proteins.
30. The method of any one of or combination of embodiments 21 to 29 (or, any
of the
following embodiments), wherein the composition is administered when the
hyaluronic acid concentration, in the patient's tears, is lower than about 15
nanograms per milligram of proteins.
31. The method of any one of or combination of embodiments 21 to 30 (or, any
of the
following embodiments), wherein the composition is administered when the
hyaluronic acid concentration, in the patient's tears, is lower than about 20
nanograms per milligram of proteins.
32. The method of any one of or combination of embodiments 21 to 31 (or, any
of the
following embodiments), wherein the composition is administered when the
hyaluronic acid concentration, in the patient's tears, is lower than about 25
nanograms per milligram of proteins.
33. The method of any one of or combination of embodiments 21 to 32 (or, any
of the
following embodiments), wherein the composition is administered to the
patient's
eye to raise the concentration of hyaluronic acid in the patient's tears to be
equal to
or greater than about 10 nanograms per milligram of proteins.
34. The method of any one of or combination of embodiments 21 to 33 (or, any
of the
following embodiments), wherein the composition is administered to the
patient's
eye to raise the concentration of hyaluronic acid in the patient's tears to be
equal to
or greater than about 15 nanograms per milligram of proteins.
61
Date Recue/Date Received 2020-11-19
35. The method of any one of or combination of embodiments 21 to 34 (or, any
of the
following embodiments), wherein the composition is administered to the
patient's
eye to raise the concentration of hyaluronic acid in the patient's tears to be
equal to
or greater than about 20 nanograms per milligram of proteins.
36. The method of any one of or combination of embodiments 21 to 35 (or, any
of the
following embodiments), wherein the composition is administered to the
patient's
eye to raise the concentration of hyaluronic acid in the patient's tears to be
equal to
or greater than about 25 nanograms per milligram of proteins.
62
Date Recue/Date Received 2020-11-19
