U.S. patent application number 16/636470 was filed with the patent office on 2021-10-21 for treating dry eye disorders.
The applicant listed for this patent is YUNNAN BAIAOTAIKE BIOTECHNOLOGY CO, LTD. Invention is credited to Dan Yang.
Application Number | 20210322506 16/636470 |
Document ID | / |
Family ID | 1000005722668 |
Filed Date | 2021-10-21 |
United States Patent
Application |
20210322506 |
Kind Code |
A1 |
Yang; Dan |
October 21, 2021 |
TREATING DRY EYE DISORDERS
Abstract
This disclosure is related to compositions and methods for
treating dry dye disorders.
Inventors: |
Yang; Dan; (Kunming,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YUNNAN BAIAOTAIKE BIOTECHNOLOGY CO, LTD |
Kunming |
|
CN |
|
|
Family ID: |
1000005722668 |
Appl. No.: |
16/636470 |
Filed: |
January 8, 2020 |
PCT Filed: |
January 8, 2020 |
PCT NO: |
PCT/CN2020/070798 |
371 Date: |
February 4, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/481 20130101;
A61K 38/13 20130101; A61K 31/045 20130101; A61K 2236/50 20130101;
A61K 36/815 20130101; A61K 36/287 20130101; A61K 36/482 20130101;
A61P 27/02 20180101; A61K 36/889 20130101 |
International
Class: |
A61K 36/889 20060101
A61K036/889; A61K 36/815 20060101 A61K036/815; A61K 36/481 20060101
A61K036/481; A61K 36/287 20060101 A61K036/287; A61K 36/482 20060101
A61K036/482; A61K 31/045 20060101 A61K031/045; A61K 38/13 20060101
A61K038/13; A61P 27/02 20060101 A61P027/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 8, 2019 |
CN |
PCT/CN2019/070856 |
Claims
1. A method of treating a dry eye disorder or alleviating symptoms
of a dry eye disorder, comprising administering to a subject in
need thereof an effective amount of a pharmaceutical composition
comprising deacidified coconut oil.
2. The method of claim 1, wherein the dry eye disorder is dry eye
syndrome.
3. The method of claim 1 or 2, wherein the composition is
administered to the patient's eye as an eye drop.
4. The method of any one of claims 1-3, wherein the composition
consists essentially of deacidified coconut oil.
5. The method of any one of claims 1-4, wherein the deacidified
coconut oil administered to the patient is made by a process
comprising the following steps: providing coconut oil; mixing the
coconut oil with a basic solution (pH>8), thereby obtaining a
mixture comprising a water phase and an oil phase; separating the
water phase and the oil phase from the mixture; and collecting
deacidified coconut oil from the oil phase.
6. The method of claim 5, wherein the process further comprises:
mixing coconut meat or coconut powder with a base, thereby
obtaining a coconut meat mixture; heating and pressing the coconut
meat mixture, thereby obtaining an extract; and collecting coconut
oil from the extract.
7. The method of any one of claims 1-6, wherein prior to
administering the pharmaceutical composition to the subject,
artificial tear eye drops are administered to the subject.
8. The method of any one of claims 1-7, wherein the method further
comprises orally administering to the subject an effective amount
of a composition comprising one, two, or all of the following
ingredients: (1) wolfberries (Lycium barbarum or Lycium chinense)
or a wolfberry extract; (2) Astragalus root or an Astragalus root
extract; (3) chrysanthemum or a chrysanthemum extract to the
subject.
9. The method of claim 8, wherein the method comprises orally
administering an effective amount of wolfberries (Lycium barbarum
or Lycium chinense) or a wolfberry extract to the subject.
10. The method of claim 8, wherein the method further comprises
orally administering an effective amount of Astragalus root or an
Astragalus root extract to the subject.
11. The method of claim 8, wherein the method further comprises
orally administering an effective amount of chrysanthemum or a
chrysanthemum extract to the subject.
12. The method of any one of claims 1-11, wherein the method
further comprises administering a heat therapy to the eye of the
subject.
13. The method of claim 12, wherein the heat therapy comprises
applying a thermal pad comprising a herb composition on the eye of
the subject.
14. The method of claim 12 or 13, wherein the herb composition
comprises Cassiae semen and borneol.
15. The method of claim 14, wherein the weight percentage of
Cassiae semen in the herb composition is from 50% to 90%.
16. The method of claim 14, wherein the weight percentage of
borneol in the herb composition is from 10% to 50%.
17. A pharmaceutical composition comprising deacidified coconut
oil.
18. The composition of claim 17, wherein the pharmaceutical
composition consists of deacidified coconut oil.
19. The method of claim 17 or 18, wherein the deacidified coconut
oil is made by a process comprising the following steps: providing
coconut oil; mixing the coconut oil with a basic solution
(pH>8), thereby obtaining a mixture comprising a water phase and
an oil phase; separating the water phase and the oil phase from the
mixture; and collecting deacidified coconut oil from the oil
phase.
20. The method of claim 19, wherein the process further comprises:
mixing coconut meat or coconut powder with a base, thereby
obtaining a coconut meat mixture; heating and pressing the coconut
meat mixture, thereby obtaining an extract; and collecting coconut
oil from the extract.
21. A method of improving the efficacy of an artificial tear eye
drops in a subject, the method comprising administering an
artificial tear eye drop to the subject; and administering to the
subject an effective amount of a composition comprising deacidified
coconut oil after the artificial tear eye drop is administered to
the subject.
22. The method of claim 21, wherein the composition consists
essentially of deacidified coconut oil.
23. The method of claim 21 or 22, wherein the composition
comprising deacidified coconut oil is administered within 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 minutes after the artificial tear eye drop
is administered to the subject.
24. A herb composition comprising: (1) wolfberries (Lycium barbarum
or Lycium chinense) or a wolfberry extract; (2) Astragalus root or
an Astragalus root extract; and (3) chrysanthemum or a
chrysanthemum extract.
25. The herb composition of claim 24, wherein the ratio of (1),
(2), and (3) is about 1:1:1.
26. A thermal pad comprising a herb composition, wherein the herb
composition comprises Cassiae semen and borneol.
27. The thermal pad of claim 26, wherein the weight percentage of
Cassiae semen in the herb composition is from 50% to 90%.
28. The thermal pad of claim 26, wherein the weight percentage of
borneol in the herb composition is from 10% to 50%.
29. A method of making a composition comprising deacidified coconut
oil, the method comprising: mixing coconut meat or coconut powder
with a base or a basic solution, thereby obtaining a coconut meat
mixture; heating and pressing the coconut meat mixture, thereby
obtaining an extract; and collecting the coconut oil from the
extract.
30. The method of claim 29, wherein the method further comprises:
after collecting coconut oil from the extract, mixing the coconut
oil with a basic solution (pH>7), thereby obtaining a mixture
comprising a water phase and an oil phase; separating the water
phase and the oil phase from the mixture; and collecting
deacidified coconut oil from the oil phase.
31. The method of claim 31, wherein the method further comprises:
filtering the deacidified coconut oil through membrane filtration,
thereby obtaining a filtered deacidified coconut oil; and
sterilizing the filtered deacidified coconut oil.
32. A method of making a composition comprising deacidified coconut
oil, the method comprising: mixing coconut oil with a base or a
basic solution, thereby obtaining a mixture; and collecting
deacidified coconut oil.
33. The method of claim 32, wherein the method comprises mixing
coconut oil with a basic solution; separating the water phase and
the oil phase from the mixture; and collecting deacidified coconut
oil.
34. The method of claim 33, wherein the method further comprises:
filtering the deacidified coconut oil through membrane filtration,
thereby obtaining a filtered deacidified coconut oil; and
sterilizing the filtered deacidified coconut oil.
35. A composition comprising deacidified coconut oil and
cyclosporine.
36. The composition of claim 35, wherein cyclosporine has a
concentration of 0.05%.
37. A method of treating a dry eye disorder or alleviating symptoms
of a dry eye disorder, comprising administering to a subject in
need thereof an effective amount of a pharmaceutical composition
comprising deacidified coconut oil and cyclosporine.
38. The method of anyone of claims 1-14, further comprising
administering an effective amount of cyclosporine to the subject.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit of PCT/CN2019/070856,
filed on Jan. 8, 2019. The entire contents of the foregoing are
incorporated herein by reference.
TECHNICAL FIELD
[0002] This disclosure is related to compositions and methods for
treating dry dye disorders.
BACKGROUND
[0003] Dry eye disorder (e.g., dry eye syndrome) is a common eye
disease. It is the condition of having dry eyes. Some common
symptoms of dry eye disorders include e.g., irritation, redness,
discharge, inflammation, easily fatigued eyes, and blurred vision.
In some severe cases, scarring of the cornea may occur. Dry eye
disorder affects about 5-34% of the population worldwide. In China,
it affects at least 17% of people.
[0004] Treatments for dry eye disorder usually involves artificial
tear eye drops. The application of artificial tear eye drops
usually can provide temporary relief of dry eye disorder. In most
cases, artificial tear eye drops need to be periodically reapplied.
This can be inconvenient, particularly if the need to reapply eye
drops occurs too frequently. In fact, it is quite common for a
patient to have to apply artificial tear solution ten to twenty
times over the course of the day. Such dosing is cumbersome and
time-consuming, and increases the exposure of the eye to
preservative agents present in many artificial tears. In addition,
for patients with severe dry eye disorders or chronic dry eye
disorders, the application of artificial tear eye drops is usually
ineffective. There remains a need for safe and effective therapies
to treat dry eye disorders.
SUMMARY
[0005] This disclosure is related to compositions and methods for
treating dry dye disorders.
[0006] In one aspect, the present disclosure provides methods of
treating a dry eye disorder or alleviating symptoms of a dry eye
disorder. The methods involve administering to a subject in need
thereof an effective amount of a pharmaceutical composition
comprising deacidified coconut oil.
[0007] In some embodiments, the dry eye disorder is dry eye
syndrome. In some embodiments, the composition is administered to
the patient's eye as an eye drop.
[0008] In some embodiments, the composition consists essentially of
deacidified coconut oil.
[0009] In some embodiments, the deacidified coconut oil
administered to the patient is made by a process comprising one or
more the following steps: providing coconut oil; mixing the coconut
oil with a basic solution (pH>8), thereby obtaining a mixture
comprising a water phase and an oil phase; separating the water
phase and the oil phase from the mixture; and collecting
deacidified coconut oil from the oil phase.
[0010] In some embodiments, the process further includes one or
more of the following steps: mixing coconut meat or coconut powder
with a base, thereby obtaining a coconut meat mixture; heating and
pressing the coconut meat mixture, thereby obtaining an extract;
and collecting coconut oil from the extract.
[0011] In some embodiments, prior to administering the
pharmaceutical composition to the subject, artificial tear eye
drops are administered to the subject.
[0012] In some embodiments, the methods further include one or more
of the following steps: orally administering to the subject an
effective amount of a composition comprising one, two, or all of
the following ingredients: wolfberries (Lycium barbarum or Lycium
chinense) or a wolfberry extract; Astragalus root or an Astragalus
root extract; chrysanthemum or a chrysanthemum extract to the
subject.
[0013] In some embodiments, the methods involve orally
administering an effective amount of wolfberries (Lycium barbarum
or Lycium chinense) or a wolfberry extract to the subject.
[0014] In some embodiments, the methods involve orally
administering an effective amount of Astragalus root or an
Astragalus root extract to the subject.
[0015] In some embodiments, the methods involve orally
administering an effective amount of chrysanthemum or a
chrysanthemum extract to the subject.
[0016] In some embodiments, the methods further involve
administering a heat therapy to the eye of the subject.
[0017] In some embodiments, the heat therapy comprises applying a
thermal pad comprising a herb composition on the eye of the
subject.
[0018] In some embodiments, the herb composition contains Cassiae
semen and borneol. In some embodiments, the weight percentage of
Cassiae semen in the herb composition is from 50% to 90%.
[0019] In some embodiments, the weight percentage of borneol in the
herb composition is from 10% to 50%.
[0020] In one aspect, the disclosure provides a pharmaceutical
composition comprising deacidified coconut oil. In some
embodiments, the pharmaceutical composition consists of deacidified
coconut oil.
[0021] In some embodiments, the deacidified coconut oil is made by
a process comprising one or more of the following steps: providing
coconut oil; mixing the coconut oil with a basic solution
(pH>8), thereby obtaining a mixture comprising a water phase and
an oil phase; separating the water phase and the oil phase from the
mixture; and collecting deacidified coconut oil from the oil
phase.
[0022] In some embodiments, the process further comprises: mixing
coconut meat or coconut powder with a base, thereby obtaining a
coconut meat mixture; heating and pressing the coconut meat
mixture, thereby obtaining an extract; and collecting coconut oil
from the extract.
[0023] In one aspect, the present disclosure provides methods of
improving the efficacy of an artificial tear eye drops in a
subject. The methods involve administering an artificial tear eye
drop to the subject; and administering to the subject an effective
amount of a composition comprising deacidified coconut oil after
the artificial tear eye drop is administered to the subject.
[0024] In some embodiments, the composition consists essentially of
deacidified coconut oil.
[0025] In some embodiments, the composition comprising deacidified
coconut oil is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
minutes after the artificial tear eye drop is administered to the
subject.
[0026] In one aspect, the present disclosure provides a herb
composition comprising: [0027] (1) wolfberries (Lycium barbarum or
Lycium chinense) or a wolfberry extract; [0028] (2) Astragalus root
or an Astragalus root extract; and [0029] (3) chrysanthemum or a
chrysanthemum extract.
[0030] In some embodiments, the ratio of (1), (2), and (3) is about
1:1:1.
[0031] In one aspect, the present disclosure provides a thermal pad
comprising a herb composition. In some embodiments, the herb
composition comprises Cassiae semen and borneol.
[0032] In some embodiments, the weight percentage of Cassiae semen
in the herb composition is from 50% to 90%.
[0033] In some embodiments, the weight percentage of borneol in the
herb composition is from 10% to 50%.
[0034] In one aspect, the present disclosure provides methods of
making a composition comprising deacidified coconut oil. The
methods involve mixing coconut meat or coconut powder with a base
or a basic solution, thereby obtaining a coconut meat mixture;
heating and pressing the coconut meat mixture, thereby obtaining an
extract; and collecting the coconut oil from the extract.
[0035] In some embodiments, the methods further involve after
collecting coconut oil from the extract, mixing the coconut oil
with a basic solution (pH>7), thereby obtaining a mixture
comprising a water phase and an oil phase; separating the water
phase and the oil phase from the mixture; and collecting
deacidified coconut oil from the oil phase.
[0036] In some embodiments, the methods further involve filtering
the deacidified coconut oil through membrane filtration, thereby
obtaining a filtered deacidified coconut oil; and sterilizing the
filtered deacidified coconut oil.
[0037] In one aspect, the present disclosure provides methods of
making a composition comprising deacidified coconut oil. The
methods involve mixing coconut oil with a base or a basic solution,
thereby obtaining a mixture; and collecting deacidified coconut
oil.
[0038] In some embodiments, the methods involve mixing coconut oil
with a basic solution; separating the water phase and the oil phase
from the mixture; and collecting deacidified coconut oil.
[0039] In some embodiments, the methods further involve filtering
the deacidified coconut oil through membrane filtration, thereby
obtaining a filtered deacidified coconut oil; and sterilizing the
filtered deacidified coconut oil.
[0040] In one aspect, the disclosure also relates to a composition
comprising deacidified coconut oil and cyclosporine.
[0041] In one aspect, the disclosure provides a method of treating
a dry eye disorder or alleviating symptoms of a dry eye disorder,
comprising administering to a subject in need thereof an effective
amount of a pharmaceutical composition comprising deacidified
coconut oil and cyclosporine.
[0042] In some embodiments, the method further comprises
administering an effective amount of cyclosporine to the
subject.
[0043] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0044] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0045] FIG. 1 is a schematic diagram showing 5 regions of the
cornea of eye. Number 1-5 are exemplary scores indicating the
percentage of colored area within each region.
[0046] FIG. 2 is a bar histogram showing average body weight of
mice in each group. "c" indicates that compared with Negative
control group, "the differences were statistically significant
(p<0.05). AT indicates artificial tears TA indicates test
composition.
[0047] FIG. 3 is a bar histogram showing average tear secretion of
mice in each group. "a" indicates that compared with Age-matched
control group, "c" indicates that compared with Negative control
group, "*" indicates that compared with that before modeling, the
differences were statistically significant (p<0.05). AT
indicates artificial tears TA indicates test composition.
[0048] FIG. 4 is a bar histogram showing average corneal sodium
fluorescein staining score of mice in each group. "a" indicates
that compared with Age-matched control group, "b" indicates that
compared with Model control group, "c" indicates that compared with
Negative control group, "*" indicates that compared with that
before modelling, the differences were statistically significant
(p<0.05). AT indicates artificial tears TA indicates test
composition.
[0049] FIG. 5 is a bar histogram showing ratio of corneal
fluorescein sodium staining score of mice in each group. "b"
indicates that compared with Model control group, "c" indicates
that compared with Negative control group, the differences were
statistically significant (p<0.05). AT indicates artificial
tears TA indicates test composition.
[0050] FIG. 6 is a bar histogram showing average body weight of
mice in each group.
[0051] FIG. 7 shows the total ion chromatogram of QC (ESL+).
[0052] FIG. 8 shows the total ion chromatogram of QC (ESL-).
[0053] FIG. 9 shows an exemplary total ion chromatogram of a sample
of the QC group (ECL+).
[0054] FIG. 10 shows an exemplary total ion chromatogram of a
sample of the QC group (ECL-).
[0055] FIG. 11 shows an exemplary total ion chromatogram of a
sample of the deacidified coconut oil (Prco) group (ECL+).
[0056] FIG. 12 shows an exemplary total ion chromatogram of a
sample of the deacidified coconut oil group (ECL-).
[0057] FIG. 13 shows an exemplary total ion chromatogram of a
sample of the original coconut oil (Orco) group (ECL+).
[0058] FIG. 14 shows an exemplary total ion chromatogram of a
sample of the original coconut oil group (ECL-).
[0059] FIG. 15 shows the PCA scores plot of all samples (ESI+).
[0060] FIG. 16 shows the PCA scores plot of all samples (ESI-).
[0061] FIG. 17 shows the PCA scores plot of both the Prco and Orco
groups (ESI+).
[0062] FIG. 18 shows the PCA scores plot of both the Prco and Orco
groups (ESI-).
[0063] FIG. 19A shows the PLS-DA scores plot of both the Prco and
Orco group (ESI+).
[0064] FIG. 19B shows the fitted model and calculated parameters of
both the Prco and Orco group (ESI+).
[0065] FIG. 20A shows the PLS-DA scores plot of both the Prco and
Orco group (ESI-).
[0066] FIG. 20B shows the fitted model and calculated parameters of
both the Prco and Orco group (ESI-).
[0067] FIG. 21 shows the OPLS-DA scores plot of both the Prco and
Orco group (ESI+).
[0068] FIG. 22 shows the OPLS-DA scores plot of both the Prco and
Orco group (ESI-).
[0069] FIG. 23 shows the total ion chromatogram of QC (ESL+).
[0070] FIG. 24 shows the total ion chromatogram of QC (ESL-).
[0071] FIG. 25 shows an exemplary total ion chromatogram of the QC
group (ECL+).
[0072] FIG. 26 shows an exemplary total ion chromatogram of the QC
group (ECL-).
[0073] FIG. 27 shows an exemplary total ion chromatogram of the
product group (ECL+).
[0074] FIG. 28 shows an exemplary total ion chromatogram of the
product group (ECL-).
[0075] FIG. 29 shows an exemplary total ion chromatogram of the
stock solution group (ECL+).
[0076] FIG. 30 shows an exemplary total ion chromatogram of the
stock solution group (ECL-).
[0077] FIG. 31 shows the PCA score plot of all samples (ESI+). Orco
is the stock solution group; Prco is the product group; and QC is
the QC group.
[0078] FIG. 32 shows the PCA score plot of all samples (ESI-). Orco
is the stock solution group; Prco is the product group; and QC is
the QC group.
[0079] FIG. 33 shows the PCA score plot of both the Prco and Orco
groups (ESI+). Orco is the stock solution group; Prco is the
product group.
[0080] FIG. 34 shows the PCA score plot of both the Prco and Orco
groups (ESI-). Orco is the stock solution group; Prco is the
product group.
[0081] FIG. 35A shows the PLS-DA score plot of both the Prco and
Orco group (ESI+). Orco is the stock solution group; Prco is the
product group.
[0082] FIG. 35B shows the fitted model and calculated parameters of
both the Prco and Orco group (ESI+).
[0083] FIG. 36A shows the PLS-DA score plot of both the Prco and
Orco group (ESI-). Orco is the stock solution group; Prco is the
product group.
[0084] FIG. 36B shows the fitted model and calculated parameters of
both the Prco and Orco group (ESI-).
[0085] FIG. 37 shows the OPLS-DA score plot of both the Prco and
Orco group (ESI+). Orco is the stock solution group; Prco is the
product group.
[0086] FIG. 38 shows the OPLS-DA score plot of both the Prco and
Orco group (ESI-). Orco is the stock solution group; Prco is the
product group.
[0087] FIG. 39 is a pie graph showing the percentage of lipid
classes (ESI+) in the stock solution group. Orco is the stock
solution group.
[0088] FIG. 40 is a pie graph showing the percentage of lipid
classes (ESI+) in the product group. Pcro is the product group.
[0089] FIG. 41 is a pie graph showing the percentage of lipid
classes (ESI-) in the product group. Pcro is the product group.
[0090] FIG. 42 is a pie graph showing the percentage of lipid
classes (ESI-) in the stock solution group. Orco is the stock
solution group.
[0091] FIG. 43 summarizes the differences between the deacidified
coconut oil (Prco) group and the original coconut oil (Orco) group
detected under ECL+ model (metabolite analysis), showing retention
time (RT), molecular weight (detected), name, molecular weight
(predicated), A ppm, VIP, P value for T-test, fold change
(log2(Prco/Orco)), average of Orco, and average of Prco.
[0092] FIG. 44 summarizes the differences between the deacidified
coconut oil (Prco) group and the original coconut oil (Orco) group
detected under ECL- model (metabolite analysis), showing retention
time (RT), molecular weight (detected), name, molecular weight
(predicated), .DELTA.ppm, VIP, P value for T-test, fold change
(log2(Prco/Orco)), average of Orco, and average of Prco.
[0093] FIG. 45 summarizes the differences between the deacidified
coconut oil (Prco) group and the original coconut oil (Orco) group
detected under ECL+ model (lipidomics analysis), showing VIP, lipid
ion, lipid group, class, fatty acid, FA1, FA2, FA3, calculated m/z,
ion formula, retention time (RT), average of Prco, average of Orco,
Prco/Orco, fold change (log2 (Prco/Orco)), P value for T-test.
[0094] FIG. 46 summarizes the differences between the deacidified
coconut oil (Prco) group and the original coconut oil (Orco) group
detected under ECL- model (lipidomics analysis), showing VIP, lipid
ion, lipid group, class, fatty acid, FA1, FA2, FA3, FA4, calculated
m/z, ion formula, retention time (RT), average of Prco, average of
Orco, Prco/Orco, fold change (log2(Prco/Orco)), P value for
T-test.
[0095] FIG. 47 shows an eye ball structure. The lipid is located on
top of the tear.
[0096] FIG. 48 shows a closed airtight structure comprising eyelid
and the tear film.
[0097] FIG. 49 shows an eye ball structure with dry eye disease.
Lack of tears is shown in black and while.
[0098] FIG. 50A shows a glass tube containing cyclosporine
dissolved in deacidified coconut oil at a concentration of
0.25%.
[0099] FIG. 50B shows a glass tube containing cyclosporine
dissolved in DMSO at a concentration of 2.5%.
[0100] FIG. 50C shows a glass tube containing cyclosporine
dissolved in olive oil at a concentration of 2.5%.
[0101] FIG. 51A shows a glass tube containing cyclosporine-DMSO
solution (2.5%) mixed with deacidified coconut oil with a volume
ratio of 1:9.
[0102] FIG. 51B shows a glass tube containing cyclosporine-olive
oil solution (2.5%) mixed with deacidified coconut oil with a
volume ratio of 1:9.
[0103] FIG. 52A shows a glass tube containing cyclosporine-DMSO
solution (5%) mixed with deacidified coconut oil with a volume
ratio of 1:19.
[0104] FIG. 52B shows a glass tube containing cyclosporine-olive
oil solution (5%) mixed with deacidified coconut oil with a volume
ratio of 1:19.
[0105] FIG. 53A shows a centrifuge tube containing cyclosporine
dissolved in deacidified coconut oil.
[0106] FIG. 53B shows a centrifuge tube containing cyclosporine
dissolved in deacidified coconut oil.
[0107] FIG. 53C shows a centrifuge tube containing cyclosporine
dissolved in DMSO/deacidified coconut oil mixed solution.
[0108] FIG. 53D shows a centrifuge tube containing cyclosporine
dissolved in DMSO/deacidified coconut oil mixed solution.
DETAILED DESCRIPTION
[0109] This disclosure relates to the diagnosis, alleviation, cure,
and treatment of dry eye disorders. Particularly, the disclosure
provides an ophthalmic composition for treating dry eye disorders.
The ophthalmic composition can comprise, consist essentially of, or
consist of deacidified coconut oil. As used herein, the term
"deacidified coconut oil" refers to coconut oil (which can be
prepared as described herein or commercially obtained) that has
been at least partially deacidified by treating the coconut oil
with a base. The deacidification process causes a change in the
chemical compositions of the coconut oil so that the deacidified
coconut oil causes less irritation to the eye when it is
administered to the eye of a subject. As used herein, the term
"coconut oil" refers to an oil composition that is collected or
derived from coconut, or a composition that has ingredients that
are essentially the same as the oil derived from coconut.
[0110] The ophthalmic composition comprising coconut oil described
herein can provide relief, e.g., long-term relief, for dry eye
disorders. Regular coconut oil (e.g., virgin coconut oil) has
irritants and is unsuitable for use as eye drops. The deacidified
coconut oil can be administered to the subject alone or in
combination with artificial tear eye drops. When it is used in
combination with artificial tears, the deacidified coconut oil eye
drop can also greatly enhance the efficacy of the artificial
tears.
[0111] The present disclosure also provides herb tea compositions
and heat therapies. The herb tea and heat therapies can be use
alone or in combination with deacidified coconut oil eye drops. The
herb tea and heat therapies can improve the therapeutic effects of
deacidified coconut oil eye drops.
[0112] Together, the methods described herein can provide rapid
relief of symptoms of dry eye disorders and improve the effects of
artificial tear eye drops. Particularly, the methods described
herein can provide a relatively long-term effect, obviating the
need of administering artificial tear eye drops 10-20 times per
day.
[0113] Dry Eye Disorders
[0114] The tear film is a consistent layer of tears on the surface
of the eye. It is essential to keep the eyes healthy, comfortable
and seeing well. Tears bathe the eye's surface to keep it moist and
wash away dust, debris and microorganisms. The normal tear film
typically has three important components: a lipid component, a
watery component, and a mucous-like (mucin) component. Each tear
component is produced by different glands on or near the eye. The
lipid component is produced by meibomian glands in the eyelids. The
watery component is produced by lacrimal glands located behind the
outer aspect of the upper eyelids. The mucin component is produced
by goblet cells in the conjunctiva that covers the white of the eye
(sclera).
[0115] In order to remain transparent, cornea has no blood vessels.
The oxygen and nutrients needed by its surface cells are
transported by tears, as are its metabolic wastes. Tears can clean
up normal shed epithelial debris, metabolized carbon dioxide and
water. It delivers nutrients from the limbus blood vessels and
oxygen exchanged from the air to the cornea. Therefore, the tear is
equivalent to the blood of the cornea and it is the necessary
foundation of a healthy cornea.
[0116] As shown in FIG. 47, the lipid component provides a physical
barrier such that tears do not drip out of the eye. In addition,
the physical barrier can keep the tears from evaporation. Because
oxygen is fat-soluble, oxygen can freely pass through the lipid
layer into tears to supply the cornea for metabolism. In addition,
the tear film and eyelid closed together can form a closed airtight
structure (FIG. 48), to protect the eyes from the invasion of
pathogens in the atmosphere. Therefore, if the tear is equivalent
to the blood of the cornea, the lipid layer is equivalent to
transparent blood vessels.
[0117] A problem with any of these sources of tear film components
can result in tear instability and dry eyes (FIG. 49). For example,
if the meibomian glands cannot produce or secrete enough oil
(meibum), the tear film may evaporate too quickly--a condition
called "evaporative dry eye" or "meibomian gland dysfunction."
[0118] Dry eye disorder refers to a lack of sufficient lubrication
and moisture on the surface of the eye, or dysfunction of the tear
film. Dry eye disorder can be temporary, acute, or chronic. Chronic
dry eye disorders include e.g., dry eye syndrome, keratitis sicca,
keratoconjunctivitis sicca, dysfunctional tear syndrome, lacrimal
keratoconjunctivitis, evaporative tear deficiency, aqueous tear
deficiency, meibomian gland dysfunction, and LASIK-induced
neurotrophic epitheliopathy (LNE). Dry eye syndrome is caused by a
chronic a chronic lack of sufficient lubrication and moisture on
the surface of the eye. Keratitis sicca refers to dryness and
inflammation of the cornea.
[0119] Keratoconjunctivitis sicca refers to dry eye that affects
both the cornea and the conjunctiva. Dysfunctional tear syndrome
generally refers to inadequate quality of tears.
[0120] Consequences of dry eye disorders range from subtle but
constant eye irritation to significant inflammation and even
scarring of the front surface of the eye. Symptoms of dry eye
disorders (e.g., dry eye syndrome) include, e.g., burning
sensation, itchiness, aching sensations, heavy eyes, fatigued eyes,
sore eyes, dryness sensation, red eyes, photophobia (sensitivity to
light), blurred vision, inflammation, and a foreign body sensation.
The foreign body sensation is a common symptom. It refers to the
feeling that grit or some other object or material is "in" the eye.
In some cases, watery eyes can also be a symptom of dry eye
syndrome. This is because dryness on the eye's surface sometimes
will over-stimulate production of the watery component of tears as
a protective mechanism. However, this "reflex tearing" cannot stay
on the eye long enough to correct the underlying dry eye condition.
In some cases, dry eye disorders can affect the outcomes of LASIK
(laser-assisted in situ keratomileusis) and cataract surgery. In
severe cases, vision can be substantially impaired.
[0121] Dry eye occurs when the eye does not produce enough tears or
when the tears evaporate too quickly. There can be many causes for
dry eye disorders, including e.g., contact lens use, meibomian
gland dysfunction, allergies, pregnancy, Sjogren's syndrome,
vitamin A deficiency, LASIK surgery, and certain medications such
as antihistamines, some blood pressure medication, hormone
replacement therapy, and antidepressants.
[0122] Skilled practitioners will appreciate that diagnosis can be
mostly based on the symptoms. In some cases, tests can be performed
to determine whether the quantity and the quality of the tears are
sufficient. A slit lamp examination is often used to diagnose dry
eyes and to document any damage to the eye. The Schirmer's test can
measure the amount of moisture bathing the eye. This test is useful
for determining the severity of the condition. A tear breakup time
(TBUT) test measures the time it takes for tears to break up in the
eye. The tear breakup time can be determined after placing a drop
of fluorescein in the cul-de-sac. Thus, in some aspects, this
disclosure also provides methods of identifying a subject having
dry eye disorders and dye eye symptoms (e.g., from mild to severe
dry eye symptoms).
[0123] Many factors can increase the risk of dry eye disorders.
These factors include, e.g., computer use, contact lens wear,
aging, menopause, air conditioning, air heating, arid climates, dry
or windy weather conditions, smoking, diabetes, thyroid-associated
diseases, lupus, rheumatoid arthritis, Sjogren's syndrome,
medications (e.g., antihistamines, antidepressants, blood pressure
medications and birth control pills), eyelid problems (e.g.,
lagophthalmos), LASIK, and corneal refractive surgery, etc.
[0124] The present disclosure provides methods and compositions of
treating various dry eye disorders as described herein.
[0125] Deacidified Coconut Oil
[0126] Coconut oil is an edible, non-toxic oil extracted from the
kernel or meat of mature coconuts harvested from the coconut palm
(Cocos nucifera). It is characterized by high content of saturated
fat. It is slow to be oxidized and, thus, resistant to
rancidification. It can last up to six months at 24.degree. C.
without spoiling.
[0127] Coconut oil is 99% fat (or lipid), composed mainly of
saturated fats (82% of total). In some cases, coconut oil includes
one or more of the following fatty acids: octanoic acid (e.g.,
caprylic acid) (C8:0), citric acid, dodecanoic acid (e.g., lauric
acid) (C12:0), tetradecanoic acid (e.g., myristic acid) (C14:0),
hexadecanoic acid (e.g., palmitic acid) (C16:0), octadecyl acid
(C18:0), and/or octadecenoic acid (e.g., oleic acid) (C18:1). In
some cases, about half of the saturated fat content of coconut oil
is lauric acid (e.g., about 41.8 grams per 100 grams), while other
significant saturated fats are myristic acid (e.g., about 16.7
grams per 100 grams), palmitic acid (e.g., about 8.6 grams per 100
grams), and caprylic acid (e.g., about 6.8 grams per 100 grams).
Monounsaturated fats can be, e.g., about 5% to 10% (e.g., 6%) of
total composition, and polyunsaturated fats can be, e.g., about 1%
to 5% (e.g., 2%). Coconut oil also may also include
phytosterols.
[0128] Coconut oil can be extracted through dry or wet processing.
Dry processing requires that the meat or kernel be extracted from
the shell and dried using fire, sunlight, or kilns to create copra.
The copra is pressed or dissolved with solvents, producing the
coconut oil and a high-protein, high-fiber mash.
[0129] The all-wet process uses raw coconut rather than dried
copra, and the protein in the coconut creates an emulsion of oil
and water. The more challenging step is breaking up the emulsion to
recover the oil. This used to be done by prolonged boiling, but
this produces a discolored oil and is not economical. Modern
techniques use centrifuges and pre-treatments with heat. Despite
numerous variations and technologies, wet processing may be less
viable than dry processing due to a 10-15% lower yield, even taking
into account the losses due to spoilage and pests with dry
processing.
[0130] Virgin coconut oil refers to coconut oil extracted from
coconuts without subjecting to substantial chemical or physical
changes. The virgin coconut oil is closer to its natural form
(Jayasekara et al., "Processing technologies for virgin coconut oil
and coconut based confectionaries and beverages." Proceedings of
International Coconut Summit (2007): 7-11). Virgin coconut oil is
typically extracted by cold compression or cold milling of copra
with a moisture content of around six percent. After using
processes such as fermentation, churning (centrifugal separation),
and refrigeration, the oil is separated from the water or moisture.
In some cases, virgin coconut oil can be extracted directly by cold
compression of fresh dried coconut meat. This process is called
micro expelling.
[0131] It has been determined that that unmodified coconut oil and
virgin coconut oil has irritants and are not suitable for being
used in an ophthalmic composition, such as eye drops. However, when
modified as described herein, e.g., via deacidification, coconut
oil can be applied to the eye. The present disclosure provides
deacidified coconut oil compositions, which are different from the
unmodified coconut oil and virgin coconut oil in the composition,
and are safe to be used in ophthalmic compositions. The deacidified
coconut oil can be made by the methods described herein. In some
embodiments, the deacidified coconut oil is made by a process
comprising mixing the coconut oil with a basic solution (pH>7);
separating the water phase and the oil phase from the mixture; and
collecting deacidified coconut oil from the oil phase.
[0132] While not intending to be bound by any theory, it is
believed that the deacidification process removes soluble
ingredients (e.g., free glycerol) and soluble fatty acids in the
coconut oil. Thus, it is likely that the deacidified fatty acids
contain a higher percentage of lipids that are resistant to
deacidification (or resistant to saponification) and/or a higher
percentage of insoluble fatty acids (fatty acids with a relatively
long hydrocarbon carbon chain, e.g., with C10 or higher). The
deacidified coconut oil is more stable and is less likely to cause
irritation when it is administered to eyes.
[0133] The pH of the ophthalmic solution is preferably 6.0 to 8.5,
more preferably 7.0 to 8.0. A pH of lower than 6.0 tends to cause
eye irritation, while a pH of higher than 8.5 is out of the
physiological pH range. In some embodiments, the pH of the
ophthalmic solution is about 7.
[0134] In some embodiments, the ophthalmic solution does not
contain any preservatives, such as antimicrobial preservatives
(e.g., antibiotics, sorbic acid, sodium sorbate and sorbates,
benzoic acid, sodium benzoate and benzoates, hydroxybenzoate and
derivatives, sulfur dioxide and sulfites, nitrite, nitrate, lactic
acid, propionic acid and/or sodium propionate) or antioxidants
(e.g., ascorbic acid, sodium ascorbate, butylated hydroxytoluene,
butylated hydroxyanisole, gallic acid and sodium gallate, sulfur
dioxide and sulfites, and/or tocopherols).
[0135] In some embodiments, the deacidified coconut oil has an
enriched level of Stigmastentriol as compared to original coconut
oil (e.g., the log2 of the ratio between the deacidified coconut
oil and the original coconut oil is at least or about 5, 6, 7, 8,
9, 10, or 11). In some embodiments, the deacidified coconut oil has
an enriched level of Campest-4-en-3-one as compared to original
coconut oil (e.g., the log2 of the ratio between the deacidified
coconut oil and the original coconut oil is at least or about 4, 5,
6, 7, 8, 9, or 10). In some embodiments, the deacidified coconut
oil has an enriched level of Stigmasterol as compared to original
coconut oil (e.g., the log2 of the ratio between the deacidified
coconut oil and the original coconut oil is at least or about 3, 4,
5, 6, 7, 8, or 9). In some embodiments, the deacidified coconut oil
has an enriched level of Stigmast-22-ene-3,6-dione as compared to
original coconut oil (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is at least or
about 3, 4, 5, 6, 7, 8, or 9). In some embodiments, the deacidified
coconut oil has an enriched level of ubiquinone-4 as compared to
original coconut oil (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is at least or
about 3, 4, 5, 6, 7, 8, or 9). In some embodiments, the deacidified
coconut oil has an enriched level of Vitamin D3 as compared to
original coconut oil (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is at least or
about 3, 4, 5, 6, 7, 8, or 9).
[0136] In some embodiments, the deacidified coconut oil has a
decreased level of 3-hexenoic acid as compared to original coconut
oil (e.g., the log2 of the ratio is less than or about -7, -8, -9,
-10, -11, -12, or -13). In some embodiments, the deacidified
coconut oil has a decreased level of 5, 8-tetradecadienoic acid as
compared to original coconut oil (e.g., the log2 of the ratio is
less than or about -4, -5, -6, -7, -8, -9, or -10). In some
embodiments, the deacidified coconut oil has a decreased level of
Indole as compared to original coconut oil (e.g., the log2 of the
ratio is less than or about -4, -5, -6, -7, -8, -9, or -10). In
some embodiments, the deacidified coconut oil has a decreased level
of isolecucine as compared to original coconut oil (e.g., the log2
of the ratio is less than or about 0, -1, or -2). In some
embodiments, the deacidified coconut oil has a decreased level of
valine as compared to original coconut oil (e.g., the log2 of the
ratio is less than or about 0, -1, -2, -3, or -4). In some
embodiments, the deacidified coconut oil has a decreased level of
glutamate as compared to original coconut oil (e.g., the log2 of
the ratio is less than or about 0, -1, -2, -3, -4, -5, or -6). In
some embodiments, the deacidified coconut oil has a decreased level
of beta-alanine as compared to original coconut oil (e.g., the log2
of the ratio is less than or about 0, -1, -2, -3, -4, -5, or
-6).
[0137] In some embodiments, the deacidified coconut oil has an
enriched level of Piperochromenoic acid as compared to original
coconut oil (e.g., the log2 of the ratio between the deacidified
coconut oil and the original coconut oil is at least or about 3, 4,
5, 6, 7, 8, or 9). In some embodiments, the deacidified coconut oil
has an enriched level of LysoPA(a-25:0/0:0) as compared to original
coconut oil (e.g., the log2 of the ratio between the deacidified
coconut oil and the original coconut oil is at least or about 2, 3,
4, 5, 6, 7, 8, or 9). In some embodiments, the deacidified coconut
oil has an enriched level of LysoPA(24:0/0:0) as compared to
original coconut oil (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is at least or
about 3, 4, 5, 6, 7, 8, 9, or 10).
[0138] In some embodiments, the deacidified coconut oil has a
decreased level of sucrose as compared to original coconut oil
(e.g., the log2 of the ratio is less than or about -5, -6, -7, -8,
-9, -10, or -11). In some embodiments, the deacidified coconut oil
has a decreased level of citric acid as compared to original
coconut oil (e.g., the log2 of the ratio is less than or about -4,
-5, -6, -7, -8, -9, -10, or -11). In some embodiments, the
deacidified coconut oil has a decreased level of mannitol as
compared to original coconut oil (e.g., the log2 of the ratio is
less than or about -3, -4, -5, -6, -7, -8, -9, -10, or -11). In
some embodiments, the deacidified coconut oil has a decreased level
of glucose or glucose-6-phosphate as compared to original coconut
oil (e.g., the log2 of the ratio is less than or about -1, -2, -3,
-4, -5, -6, or -7).
[0139] In some embodiments, the deacidified coconut oil comprises
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, or all of Stigmastentriol, Campest-4-en-3-one,
DG(8:0/8:0/0:0), MG(0:0/15:0/0:0), Stigmast-22-ene-3,6-dione,
Stigmasterol, Hexadecanedioic acid, Delta 8,14-Sterol, PA(8:0/8:0),
Vitamin D3, Ubiquinone-4, (R)-2-Hydroxysterculic acid, Betulin,
PA(8:0/14:0), PA(8:0/12:0), MG(17:0/0:0/0:0), LysoPA(18:0e/0:0),
Ergosterol, Stigmastane-3,6-dione, Vitamin K1, TG(10:0/8:0/8:0),
MG(0:0/14:0/0:0), DG(8:0/10:0/0:0), Nervonic acid, Methyl
cinnamate, Cinnamic acid, Pantothenic Acid, TG(13:0/13:0/8:0),
Ganodosterone, MG(18:0/0:0/0:0), TG(12:0/12:0/8:0), Tridecanoic
acid, Linoleic acid, MG(16:0/0:0/0:0), TG(8:0/8:0/14:0), Pangamic
acid, Camelledionol, DG(8:0/0:0/14:0), m-Hydroxyphenylpyruvic acid,
DG(18:1n9/0:0/20:4n3), DG(12:0/12:0/0:0), Phosphocholine,
Stearamide, LysoPA(24:0/0:0), Piperochromenoic acid,
LysoPA(a-25:0/0:0), DG(8:0/0:0/15:0), LysoPA(i-20:0/0:0),
PA(8:0/16:0), 2-Stearoylglycerophosphoglycerol, LysoPA(18:0e/0:0),
LysoPA(22:0/0:0), Cerebronic acid, PA(8:0/14:0),
DG(20:4n3/0:0/20:4n3), LysoPA(18:0/0:0), DG(10:0/0:0/19:0),
DG(12:0/15:0/0:0), PA(22:0/8:0), PA(8:0/20:0), and PA(22:0/13:0).
In some embodiments, these chemicals have a higher concentration as
compared to original coconut oil.
[0140] In some embodiments, the deacidified coconut oil has an
enriched level of DG as compared to original coconut oil (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is at least or about 0, 1, 2, 3, 4, 5, 6, 7,
8, or 9). In some embodiments, the deacidified coconut oil has an
enriched level of ChE as compared to original coconut oil (e.g.,
the log2 of the ratio between the deacidified coconut oil and the
original coconut oil is at least or about 0, 1, 2, or 3). In some
embodiments, the deacidified coconut oil has an enriched level of
ZyE or StE as compared to original coconut oil (e.g., the log2 of
the ratio between the deacidified coconut oil and the original
coconut oil is at least or about 0, 1, 2, or 3).
[0141] In some embodiments, the deacidified coconut oil has a
decreased level of LPE as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -2, -3, -4, -5, -6, -7,
or -8). In some embodiments, the deacidified coconut oil has a
decreased level of PE as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -2, -3, -4, -5, -6, -7,
or -8). In some embodiments, the deacidified coconut oil has a
decreased level of Co as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -1, -2, -3, or -4). In
some embodiments, the deacidified coconut oil has a decreased level
of LPC as compared to original coconut oil (e.g., the log2 of the
ratio is less than or about -1, -2,-3, -4, -5, -6, -7, or -8). In
some embodiments, the deacidified coconut oil has a decreased level
of CerG1 as compared to original coconut oil (e.g., the log2 of the
ratio is less than or about -1, -2,-3, -4, or -5).
[0142] In some embodiments, the deacidified coconut oil has an
enriched level of LdMePE as compared to original coconut oil (e.g.,
the log2 of the ratio between the deacidified coconut oil and the
original coconut oil is at least or about 1, 2, 3, 4, 5, 6, or 7).
In some embodiments, the deacidified coconut oil has an enriched
level of PAF as compared to original coconut oil (e.g., the log2 of
the ratio between the deacidified coconut oil and the original
coconut oil is at least or about 1, 2, 3, 4, or 5). In some
embodiments, the deacidified coconut oil has an enriched level of
DGMG as compared to original coconut oil (e.g., the log2 of the
ratio between the deacidified coconut oil and the original coconut
oil is at least or about 1, 2, 3, 4, or 5). In some embodiments,
the deacidified coconut oil has an enriched level of MGMG as
compared to original coconut oil (e.g., the log2 of the ratio
between the deacidified coconut oil and the original coconut oil is
at least or about 1, 2, 3, 4, or 5). In some embodiments, the
deacidified coconut oil has an enriched level of LPMe as compared
to original coconut oil (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is at least or
about 1, 2, 3, 4, 5, 6, or 7).
[0143] In some embodiments, the deacidified coconut oil has a
decreased level of DGDG as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -3, -4, -5, -6, -7, -8,
or -9). In some embodiments, the deacidified coconut oil has a
decreased level of cPA as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -3, -4, -5, -6, -7, -8,
or -9). In some embodiments, the deacidified coconut oil has a
decreased level of LPI as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -2, -3, -4, -5, -6, -7,
or -8). In some embodiments, the deacidified coconut oil has a
decreased level of LPE as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -1, -2, -3, -4, -5, -6,
or -7). In some embodiments, the deacidified coconut oil has a
decreased level of PC as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -1, -2, -3, -4, -5, -6,
or -7). In some embodiments, the deacidified coconut oil has a
decreased level of dMePE as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -1, -2, -3, -4, -5, -6,
or -7). In some embodiments, the deacidified coconut oil has a
decreased level of MGDG as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -1, -2, -3, -4, -5, -6,
or -7). In some embodiments, the deacidified coconut oil has a
decreased level of PI as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -1, -2, -3, -4, -5, -6,
or -7). In some embodiments, the deacidified coconut oil has a
decreased level of PE as compared to original coconut oil (e.g.,
the log2 of the ratio is less than or about -1, -2, -3, -4, or -5).
In some embodiments, the deacidified coconut oil has a decreased
level of PG as compared to original coconut oil (e.g., the log2 of
the ratio is less than or about -1, -2, -3, -4, or -5). In some
embodiments, the deacidified coconut oil has a decreased level of
PMe as compared to original coconut oil (e.g., the log2 of the
ratio is less than or about 0, -1, or -2). In some embodiments, the
deacidified coconut oil has a decreased level of LPG as compared to
original coconut oil (e.g., the log2 of the ratio is less than or
about 0, -1, or -2). In some embodiments, the deacidified coconut
oil has a decreased level of PEt as compared to original coconut
oil (e.g., the log2 of the ratio is less than or about 0, -1, or
-2).
[0144] In some embodiments, the deacidified coconut oil comprises
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of DG, ChE, ZyE, StE,
LdMePE, PAF, DGMG, MGMG, LPMe and CL.
[0145] In some embodiments, these chemicals have a higher
concentration as compared to original coconut oil.
[0146] In some embodiments, the deacidified coconut oil does not
comprise (e.g., a detectable amount of) cardiolipin (CL)
(18:1/18:1/18:1/20:0) or digalactosyldiacylglycerol (DGDG)
(16:0/18:1). In some embodiments, the methods described herein
(e.g., lipidomics analysis) cannot detect CL(18:1/18:1/18:1/20:0)-H
or DGDG (16:0/18:1)+HCOO from the deacidified coconut oil.
[0147] Cyclosporine Cyclosporine ophthalmic solution (or emulsion)
is a prescription eye drop that helps patients increase their eyes'
natural ability to produce tears. It is used to treat a type of
chronic dry eye condition caused by inflammation. Cyclosporine is
also available in oral and injectable formulations that are used to
treat various conditions including treatment or prevention of
rejection of transplanted organs, psoriasis, and rheumatoid
arthritis. Following oral administration or injection, cyclosporine
is absorbed in the blood stream and works systemically to suppress
the body's immune system. However, cyclosporine eye drop emulsion
is thought to work differently. Cyclosporine is believed to work
locally in the eye as a partial modulator of the immune system.
Tear production is thought to be decreased when lymphocytes, a type
of white blood cell of the immune system, die and accumulate in the
tear glands. Cyclosporine reverses this condition, increasing tear
production. Cyclosporine does not produce its effect immediately.
An increase in tear production may not be noticed until 3 to 6
months after starting treatment.
[0148] Because cyclosporine is not soluble in water, the
composition described herein (e.g., deacidified coconut oil) can be
used as a solvent for cyclosporine. In some embodiments,
cyclosporine can be used for dry eye treatment at a concentration
of about or at least 0.01 mg/ml, e.g., about or at least 0.05
mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6
mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2
mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, or 10 mg/ml. In some embodiments,
the concentration is about or at least 0.5 mg/ml.
[0149] In some embodiments, the concentration for cyclosporine is
about or at least 0.01%, e.g., about or at least 0.02%, 0.03%,
0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (weight percentage or volume
percentage). In some embodiments, the concentration is less than
0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%,
0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (weight
percentage or volume percentage). In some embodiments, the
concentration is from 0.01% to 0.1% (weight percentage or volume
percentage), e.g., 0.05% or 0.09%.
[0150] In some embodiments, the solution can additionally comprise
DMSO and/or olive oil.
[0151] In some embodiments, cyclosporine can be used for dry eye
treatment in combination with one or more artificial tear (e.g.,
carboxymethylcellulose, dextran, glycerin, hypromellose,
polyethylene glycol 400 (PEG 400), polysorbate, polyvinyl alcohol,
povidone, or propylene glycol), lubricant, unprocessed coconut oil
samples as described herein, deacidified coconut oil samples as
described herein, or any dry eye treatment compounds known in the
art.
[0152] Herb Tea
[0153] The present disclosure provides herb tea compositions for
treating dry eye disorders. In some embodiments, the herb tea
composition comprises one or more of the following ingredients:
[0154] (1) wolfberries (Lycium barbarum or Lycium chinense) or a
wolfberry extract;
[0155] (2) Astragalus root or an Astragalus root extract;
[0156] (3) chrysanthemum or a chrysanthemum extract to the
subject.
[0157] Wolfberries (also known as "goji" in Chinese) are the fruit
of either Lycium barbarum or Lycium chinense. The fruit has been
used as an ingredient in traditional Chinese medicine.
[0158] Astragalus root (also known as "huangqi" in Chinese) is the
root of Astragalus propinquus. It is commonly used in traditional
Chinese medicine. Chemical constituents of the roots include
polysaccharides and triterpenoids (such as astragalosides), as well
as isoflavones (including e.g., kumatakenin, calycosin, and
formononetin) and their glycosides and malonates.
[0159] Chrysanthemum (also known as "juhua" in Chinese) are
flowering plants of the genus Chrysanthemum in the family
Asteraceae. Chrysanthemum can be used in the tea and is also widely
used in traditional Chinese medicine.
[0160] In some embodiments, the herb tea composition (e.g., for one
serving) can comprise about or at least 1 gram, e.g., about or at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 30, 40, or 50 grams of wolfberries (e.g., 10 g). In some
embodiments, the herb tea composition (e.g., for one serving) can
comprise about or at least 1 gram, e.g., about or at least 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,
or 50 grams of Astragalus root (e.g., 10 g). In some embodiments,
the herb tea composition (e.g., for one serving) can comprise about
or at least 1 gram, e.g., about or at least 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 grams of
Chrysanthemum (or Chrysanthemum flowers) (e.g., 10 g).
[0161] In some embodiments, the weight percentage of wolfberries in
the herb tea composition is at least or about 10%, e.g., at least
or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some
embodiments, the herb tea composition comprises wolfberries, and
the weight percentage of wolfberries in the herb tea composition is
less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
[0162] In some embodiments, the weight percentage of Astragalus
root in the herb tea composition is about or at least 10%, e.g.,
about or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In
some embodiments, the weight percentage of Astragalus root in the
herb tea composition is less than 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or 90%.
[0163] In some embodiments, the weight percentage of Chrysanthemum
(or Chrysanthemum flowers) in the herb tea composition is about or
at least 10%, e.g., about or at least 20%, 30%, 40%, 50%, 60%, 70%,
80%, or 90%. In some embodiments, the weight percentage of
Chrysanthemum (or Chrysanthemum flowers) in the herb tea
composition is less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or
90%.
[0164] In some embodiments, the ratio of three ingredients (1),
(2), and (3) is about 1:1:1, 2:1:1, 1:2:1, 1:1:2, 1:2:2, 2:1:2, or
2:2:1.
[0165] The herb tea compositions can be administered to the subject
as needed. The subject can drink herb tea several times per day,
e.g., about or at least 1 time (e.g., about or at least 2, 3, 4, 5,
6, 7, 8, 9, or 10 times) per day. In some embodiments, the subject
can drink the herb tea periodically for an extended period of time,
e.g., about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or
about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
In some embodiments, the subject can drink herb tea prior to or
after the administration of deacidified coconut oil eye drops.
[0166] In some embodiments, for convenience, the herb tea
composition can be placed in herb tea pods. These herb tea pods can
be used as single-serve containers (e.g., tea cup, tea pods, K-cup,
tea capsule). The powder of the herb tea composition can be stored
in the single-serve containers. In some embodiments, the containers
are filled with nitrogen to increase storage time. The nitrogen can
prevent wolfberries powder from forming an aggregate. The
containers can be further sealed. In some embodiments, the herb tea
pods can be used in an appropriate coffee machine or tea maker.
[0167] In some embodiments, the extracts of wolfberries, Astragalus
roots, or chrysanthemum can be used. In some embodiments, the
extracts can be made in the form of pills, tablets or capsules. The
pills, tablets or capsules can be administered on a regimen of 1 to
4 times per day, preferably once or twice per day. This dosing
regimen can be adjusted to provide the optimal therapeutic
response.
[0168] Heat Therapies
[0169] Also provided herein are heat therapies. The heat therapies
can be used alone or in combination with other methods described
herein (e.g., deacidified coconut oil eye drops and/or herb tea) to
treat dry eye disorders. In some embodiments, a thermal pad
comprising an herb composition is used. As used herein, the term
"thermal pad" or "heat pad" refers to a pad used for warming of
parts of the body in order to provide therapeutic effects. The
thermal pad can have a shape of pre-formed square or rectangle.
Thermal pads can be filled with medical compositions (e.g., herb
compositions).
[0170] In some embodiments, the herb composition comprises Cassiae
semen and/or borneol.
[0171] Cassiae semen (also known as "Juemingzi" in Chinese) is the
dry and mature seed of Cassia obtusifolia or Cassia tora, which
belong to the Cassia genus of Leguminosae. It is cultivated in
Korea, Japan and China, and is commonly consumed as a roasted tea.
In traditional Chinese medicine, it has been used in treatments for
hyperlipemia, diabetes mellitus, Alzheimer's disease, acute liver
injury, inflammation, photophobia, headache, dizziness and
hypertension. A detailed description of Cassiae semen can be found
e.g., in Dong et al. "Cassiae semen: A review of its phytochemistry
and pharmacology." Molecular medicine reports 16.3 (2017):
2331-2346, which is incorporated herein by reference in its
entirety.
[0172] Borneol is a bicyclic organic compound and a terpene
derivative. It can be found in several species of Heterotheca,
Artemisia, Callicarpa, Dipterocarpaceae, Blumea balsamifera and
Kaempferia galanga. In some cases, the borneol resin refers to the
resin obtained from Dryobalanops aromatica or from Blumea
balsamifera. The typical form of borneol is in thin, semi-opaque,
whitish angular pieces or crystals. Borneol (as a crude resin) is
used internally and externally in the practice of Chinese
medicine.
[0173] The disclosure provides a thermal pad comprising Cassiae
semen and/or borneol that can be used to treat dry eye disorders or
alleviate symptoms of dry eye disorders.
[0174] In some embodiments, the thermal pad comprises about or at
least 50 g, e.g., about or at least 100 g, 150 g, 200 g, 250 g, 300
g, 350 g, 400 g, 450 g, or 500 g of Cassiae semen (e.g., 200 g). In
some embodiments, the thermal pad comprises about or at least 50 g,
e.g., about or at least 60 g, 70, 80 g, 90 g, 100 g, 150 g, 200 g,
250 g, 300 g, 350 g, 400 g, 450 g, or 500 g of borneol (e.g., 80
g).
[0175] In some embodiments, the herb composition in the thermal pad
is about or at least 50 g, e.g., about or at least 100 g, 150 g,
200 g, 250 g, 300 g, 350 g, 400 g, 450 g, or 500 g. In some
embodiments, the weight percentage of Cassiae semen in the herb
composition is about or at least 10%, e.g., about or at least 20%,
30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the
weight percentage of borneol in the herb composition is about or at
least 10%, e.g., about or at least 20%, 30%, 40%, 50%, 60%, 70%,
80%, or 90%. In some embodiments, the weight percentage of Cassiae
semen in the herb composition is less than 90%, e.g., less than
10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. In some embodiments, the
weight percentage of borneol in the herb composition is less than
90%, e.g., less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%.
[0176] In some embodiments, the weight ratio of Cassiae semen to
Borneol is about 5:1, 5:2, 5:3, 5:4, 1:1, 4:5, 3:5, 2:5, or 1:5. In
some embodiments, the weight ratio of Cassiae semen to Borneol is
about 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, or 1:4.
[0177] In some embodiments, the thermal pad has a size or a volume
of at least or about 100 mm.sup.3, e.g., at least or about 200
mm.sup.3, 300 mm.sup.3, 400 mm.sup.3, 500 mm.sup.3, 600 mm.sup.3,
700 mm.sup.3, 800 mm.sup.3, 900 mm.sup.3, or 1000 mm.sup.3.
[0178] In some embodiments, the thermal pad is heated (e.g., in a
steamer) for a sufficient period of time (e.g., about or at least 5
minutes, 10 minutes, or 15 minutes). When the temperature on the
surface of thermal pad reaches an appropriate temperature (e.g.,
from 40 to 70.degree. C., from 40 to 65.degree. C., from 40 to
60.degree. C., from 45 to 70.degree. C., from 45 to 65.degree. C.,
from 45 to 60.degree. C., from 50 to 70.degree. C., from 50 to
65.degree. C., or from 50 to 60.degree. C., e.g., about 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
or 60.degree. C.), the thermal pad can be placed on the eyes of the
subject for a sufficient period of time (e.g., about or at least 5,
10, 15, 20, 25, or 30 minutes). In some embodiments, the thermal
pad is big enough to cover the eye affected by dry eye syndrome, or
both eyes of the subject.
[0179] The heat therapies can be administered to the subject once
per day, twice per day, or three times per day. In some
embodiments, the heat therapies are administered to the patient in
the morning, or in the evening, or both. For patients with severe
symptoms, the heat therapies can be additionally administered to
the patient around noon. In some embodiments, the heat therapies
are administered to the subject several times a week, e.g., once
per week, twice per week, three times per week, four times per
week, five times per week, six times per week, or seven times per
week, or more.
[0180] In some embodiments, the heat therapies are administered to
the patients prior to or after the administration of deacidified
coconut oil eye drops. In some embodiments, the thermal pad can be
in the form a flexible eye cover. In some embodiments, the size of
thermal pad is adjustable, and/or is sized to fit the head of the
subject.
[0181] In some embodiments, eye massage are performed before or
after the heat therapy. The heat therapies and/or eye massage can
increase the secretion of lipids from meibomian glands, increase
the flow in the meibomian gland ducts, and resolve the clogging of
meibomian gland ducts.
[0182] Methods of Treating Dry Eye Disorders
[0183] The methods described herein include methods for the
treatment of disorders associated with dry eye disorders (e.g., dry
eye syndrome, keratitis sicca, keratoconjunctivitis sicca,
dysfunctional tear syndrome, lacrimal keratoconjunctivitis,
evaporative tear deficiency, or aqueous tear deficiency). In some
embodiments, the disorder is chronic. In some embodiments, the
disorder is dry eye syndrome. Generally, the methods include
administering a therapeutically effective amount of the composition
as described herein (e.g., deacidified coconut oil), to a subject
who is in need of, or who has been determined to be in need of,
such treatment.
[0184] As used in this context, to "treat" means to ameliorate at
least one symptom of the dry eye disorder. Often, the treatment can
result in a reduction of irritation, dryness sensation, burning
sensation, itchiness, or pain. In some aspects, the methods
described herein can also be used to increase tear production,
improve the quantity and/or quality of tear, reduce ocular
discomfort, improve ocular surface health, protect the ocular
surface during environmentally challenging conditions (e.g., dry or
windy conditions), increase the amount or concentration of one or
more lacrimal proteins on the ocular surface (e.g., epithelial
growth factor, lactoferin, lacritin, prolactin,
adrenocorticotropic, leucine enkephalin, ALS2CL, ARHGEF19,
KIAA1109, PLXNAL POLG, WIPI1, ZMIZ2 or other proteins of the tear
proteome), or enhance tear clearance.
[0185] The terms "subject" and "patient" are used interchangeably
throughout the specification and describe an animal, human or
non-human, to whom treatment according to the methods of the
present invention is provided. Veterinary and non-veterinary
applications are contemplated by the present invention. Human
patients can be adult humans or juvenile humans (e.g., humans below
the age of 18 years old). In addition to humans, patients include
but are not limited to mice, rats, hamsters, guinea-pigs, rabbits,
ferrets, cats, dogs, and primates. Included are, for example,
non-human primates (e.g., monkey, chimpanzee, gorilla, and the
like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets,
rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine,
canine, feline, bovine, and other domestic, farm, and zoo
animals.
[0186] In some embodiments, the subject is a human. The human
subject can be a male or a female (e.g., a post-menopausal woman).
In some embodiments, the human patient is at least or about 30
years old, e.g., at least or about 35, 40, 45, 50, 55, 60, 65, 70,
75, or 80 years old. In some embodiments, the patient can have the
dry eye disorder for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or
more, years.
[0187] In some embodiments, the deacidified coconut oil eye drops
are administered after the subject is treated by artificial tear
eye drops. While not intending to be bound by any theory, it is
believed that the deacidified coconut oil can form a lipid layer,
and the lipid layer can keep the tear film from evaporating too
quickly and increase lubrication.
[0188] Various artificial tear eye drops are known in the art.
Examples of artificial tear can include, but are not limited to,
water and/or buffered, isotonic saline solutions. In some
embodiments, the aqueous solutions can contain water-soluble
polymers that render the solutions more viscous and thus less
easily shed by the eye. In some embodiments, artificial tear
compositions can include one or more of the following ingredients:
carboxymethyl cellulose, polyvinyl alcohol, hydroxypropyl
methylcellulose (a.k.a. HPMC or hypromellose), hydroxypropyl
cellulose and hyaluronic acid (a.k.a. hyaluronan, HA). In some
embodiments, artificial tear compositions can include one or more
of the following ingredients: carboxymethylcellulose, dextran,
glycerin, hypromellose, polyethylene glycol 400 (PEG 400),
polysorbate, polyvinyl alcohol, povidone, and propylene glycol. In
some embodiments, artificial tear compositions can include
polyvinyl alcohol (e.g., 0.1% to 1%, e.g., about 0.5% by volume
percentage) and/or povidone (e.g., 0.1% to 1%, e.g., about 0.6% by
volume percentage).
[0189] In some embodiments, the deacidified coconut oil eye drop is
administered before, during, or after the artificial tear eye drop
is administered to the subject.
[0190] In some embodiments, the deacidified coconut oil eye drop is
administered within a period of time after the artificial tear eye
drop is administered to the subject, e.g., within 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 minutes. In some embodiments, the period of time is
from 1 to 30 minutes, from 1 to 20 minutes, from 1 to 15 minutes,
from 1 to 10 minutes, from 1 to 5 minutes, from 5 to 20 minutes,
from 5 to 15 minutes, from 5 to 10 minutes, or from 10 to 20
minutes.
[0191] In some embodiments, the deacidified coconut oil eye drop is
administered before, during, or after the cyclosporine is
administered to the subject. In some embodiments, the deacidified
coconut oil eye drop is administered within a period of time after
the cyclosporine is administered to the subject, e.g., within 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the period
of time is from 1 to 30 minutes, from 1 to 20 minutes, from 1 to 15
minutes, from 1 to 10 minutes, from 1 to 5 minutes, from 5 to 20
minutes, from 5 to 15 minutes, from 5 to 10 minutes, or from 10 to
20 minutes.
[0192] In some embodiments, a composition comprising deacidified
coconut oil and cyclosporine is administered to the subject.
[0193] The deacidified coconut oil and the compositions described
herein can be used in combination with some other methods (e.g.,
medical procedures), compositions, or mediations to treat dry eye
disorders. For example, artificial tear solutions, saline, topical
steroids, topical immunosuppressants (e.g., ciclospori),
diquafosol, lifitegrast, or ciclosporin can be administered to the
subjects along with the deacidified coconut oil. In some
embodiments, an anti-inflammation medication (e.g., cyclosporine or
corticosteroids) is administered to the subject in order to reduce
eyelid inflammation and/or to control cornea inflammation (e.g.,
cyclosporine). In some embodiments, antibiotics can be administered
to the subject. In some embodiments, the hydroxypropyl cellulose
(Lacrisert) insert can be administered to a subject between the
lower eyelid and the eyeball. The insert can dissolve slowly,
releasing a substance to lubricate the eye. In some embodiments,
tear-stimulating drugs (e.g., cholinergics, pilocarpine, or
cevimeline) can be administered to a subject to help increase tear
production. These drugs can be available as pills, gel or
eyedrops.
[0194] In some embodiments, procedures can be performed to close
the tear ducts to reduce tear loss. This procedure can be done by
partially or completely closing the tear ducts, which are designed
to drain tears away. In some embodiments, tear ducts can be plugged
with tiny silicone plugs (punctal plugs). These are removable. In
some embodiments, tear ducts can be plugged with a procedure that
uses heat (e.g., thermal cautery).
[0195] In some embodiments, a subject can wear special contact
lenses (e.g., scleral lenses or bandage lenses). The special
contact lens can protect the surface of the eyes and trap moisture.
In some embodiments, these contact lenses are covered by
deacidified coconut oil described herein.
[0196] In some embodiments, procedures can be performed to unblock
oil glands. In some embodiments, the procedure is LipiFlow thermal
pulsation. During the treatment, a device that is similar to an
eyecup is placed over the affected eye. It can deliver a gentle,
warm massage to the lower eyelid.
[0197] In some embodiments, light therapy or eyelid massage can be
performed. In some embodiments, a technique called intense-pulsed
light therapy followed by massage of the eyelids can be used to
treat severe dry eyes.
[0198] In some embodiments, tarsorrhaphy can be performed in
addition to the administration of the pharmaceutical compositions
described herein. The tarsorrhaphy procedure can reduce the
palpebral fissure (eyelid separation), leading to a reduction in
tear evaporation.
[0199] In some embodiments, the methods described herein does not
cause irritation to the subject.
[0200] In some embodiments, the methods described herein can
improve Schirmer's test score by at least 10%, e.g., at least 20%,
30%, 40%, 50%, 60%, 70%, 80%, or 90%. For example, some patients
can have 15 mm wetting after 5 minutes in a Schirmer's test.
[0201] In some embodiments, the methods described herein can
increase tear secretion by at least 10%, e.g., at least 20%, 30%,
40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the methods
described herein can reduce corneal fluorescein sodium staining
score by at least 10%, e.g., at least 20%, 30%, 40%, or 50%.
[0202] Methods of Making Deacidified Coconut Oil
[0203] The present disclosure provides methods of making a
composition comprising deacidified coconut oil. The methods can
involve one or more the following steps: mixing coconut meat,
coconut copra or coconut powder with a base; heating and pressing
the coconut meat mixture, thereby obtaining an extract; collecting
the coconut oil from the extract; mixing the coconut oil with a
basic solution (pH>7), thereby obtaining a mixture comprising a
water phase and an oil phase; separating the water phase and the
oil phase from the mixture; and/or collecting deacidified coconut
oil from the oil phase.
[0204] The coconut meat, coconut copra or coconut powder are
readily available and can be obtained commercially. In some
embodiments, the coconut meat, coconut copra or coconut powder are
dried coconut meat, coconut copra or coconut powder. In some
embodiments, the methods also involve producing coconut meat,
coconut copra or coconut powder from coconuts. Methods of producing
coconut meat, coconut copra or coconut powder are known in the art.
For example, the methods can involve removing the shell of
coconuts, breaking the shell up, and/or drying. Copra can be made
by smoke drying, sun drying, or kiln drying. During sun drying,
halved nuts are drained of water, and left with the meat facing the
sky. They can then be washed to remove mold-creating contaminants.
After a few days, the meat can be removed from the shell with ease,
and the drying process is complete after three to five more days
(up to seven in total). Sun drying is often combined with kiln
drying, eight hours of exposure to sunlight means the time spent in
a kiln can be reduced by a day and the hot air the shells are
exposed to in the kiln is more easily able to remove the remaining
moisture. This process can also be reversed, partially drying the
copra in the kiln and finishing the process with sunlight. In some
embodiments, the methods can involve filtering the deacidified
coconut oil through membrane filtration, thereby obtaining a
filtered deacidified coconut oil. In some instances, the filtered
deacidified coconut oil may be sterilized, e.g., using heat
sterilization methods known in the art.
[0205] In some embodiments of making deacidified coconut oil, the
coconut meat or coconut powder is dried in a suitable dryer (e.g.,
a solar dyer, a forced drought tray-type dryer or a vacuum
dryer).
[0206] In some embodiments, the coconut meat or coconut powder is
collected, and is mixed with a base (e.g., sodium carbonate
powder). In some embodiments, the base is a dried powder. In some
embodiments, the base is an aqueous solution. The base can be NaOH,
KOH, Mg(OH).sub.2, Ca(OH).sub.2, Na.sub.2CO.sub.3, NaHCO.sub.3,
Na.sub.2CO.sub.3, K.sub.2CO.sub.3, or KHCO.sub.3. In some
embodiments, the coconut meat or coconut powder can be mixed with a
chemical compound, wherein if the compound is mixed with water, it
will generate a base aqueous solution. In some embodiments, the
chemical compound is CaO.
[0207] The mixture is then heated to an appropriate temperature
(e.g., about 60 to 90, 70 to 80, or 70 to 75.degree. C.) in an
appropriate apparatus. In some embodiments, the temperature is
about or at least 60, e.g., about or at least 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95.degree. C. In
some embodiments, the temperature is below 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, or 95.degree. C. Skilled practitioners
will appreciate that any suitable apparatus may be used, e.g., a
heating apparatus such as a steam jacketed kettle.
[0208] The heated coconut meat or coconut powder is then pressed,
by e.g., an expeller, a hand-pressing machine, a screw type oil
press, hydraulic pressing, or a hydraulic jack type oil press.
[0209] The extract is then cooled (e.g., to room temperature, or to
a temperature that is about or below 20.degree. C., e.g., about or
below 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,
2, or 1.degree. C.). In some embodiments, the extract is cooled to
a temperature that is about or at least 10.degree. C., e.g., about
or at least 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.degree. C. During the
cooling process, the water phase and the oil phase are separated.
Skilled practitioners will appreciate that cooling can be performed
using any active or passive cooling method, e.g., refrigeration or
allowing the mixture to cool to ambient temperature. The coconut
oil can be collected from the oil phase. In some embodiments, the
temperature is cooled to below the melting point of the coconut oil
(e.g., around 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10.degree.
C.). The coconut oil is then solidified, facilitating the
collection of the coconut oil from the extract. In some
embodiments, the cooling process can be operated in a refrigerator,
in a cooled rom, or in a cooling apparatus.
[0210] In some embodiments, the coconut oil is treated with a base
solution. The base aqueous solution can be e.g., the aqueous
solution of NaOH, KOH, Mg(OH).sub.2, Ca(OH).sub.2,
Na.sub.2CO.sub.3, NaHCO.sub.3, Na.sub.2CO.sub.3, K.sub.2CO.sub.3,
or KHCO.sub.3, or any combination thereof. In some embodiments, the
pH of the solution can be, e.g., from 7.5 to 11, from 7.5 to 10,
from 7.5 to 9, from 8 to 11, from 8 to 10, from 8 to 9, or from 9
to 11. In some embodiments, the aqueous solution is a saturated
solution (e.g., at the room temperature, at the standard condition,
or at 20 or 25.degree. C.). In some embodiments, the solution is a
saturated solution of sodium bicarbonate. The amount of the base
solution should be sufficient to react with all acids that are free
to react with the base. In some embodiments, the weight ratio of
the extract to the sodium bicarbonate solution is equal to or less
than about 1:1, e.g., equal to or less than about 1:1.1, 1:1.2,
1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, or
1:5.
[0211] In some embodiments, the starting material is regular
coconut oil (e.g., coconut oil that can be obtained commercially,
virgin coconut oil, untreated coconut oil). The regular coconut oil
can be treated with a base (e.g., NaOH, KOH, Mg(OH).sub.2,
Ca(OH).sub.2, Na.sub.2CO.sub.3, NaHCO.sub.3, Na.sub.2CO.sub.3,
K.sub.2CO.sub.3, or KHCO.sub.3) or a basic solution as described
herein (e.g., the aqueous solution of NaOH, KOH, Mg(OH).sub.2,
Ca(OH).sub.2, Na.sub.2CO.sub.3, NaHCO.sub.3, Na.sub.2CO.sub.3,
K.sub.2CO.sub.3, or KHCO.sub.3, or any combination thereof) and
processed as described above and/or as described elsewhere
herein.
[0212] The mixture is then cooled (e.g., to room temperature), or
to a temperature that is below 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.degree. C. In some
embodiments, the extract is cooled to a temperature that is above
10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.degree. C. In some embodiments,
the temperature is cooled to below the melting point of the
deacidified coconut oil (e.g., around 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10.degree. C.). In some embodiments, the coconut oil is
solidified, facilitating the collection. In some embodiments, the
cooling process can be operated in a refrigerator, in a cooled rom,
or in a cooling apparatus.
[0213] In some embodiments, the coconut oil can be then collected
and filtered, e.g., through a membrane under a pressure. In some
embodiments, the filter size is at least or about 5, e.g., at least
or about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
nm. In some embodiments, the filter size is less than 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm. In some
embodiments, the filter size is from 10 to 15 nm, from 5 to 20 nm,
or from 10 to 20 nm. In some embodiments, the pressure is greater
than 1 kg/cm.sup.2 (1 kg/cm.sup.2=98.0665 kPa). In some
embodiments, the pressure is less than 2 kg/cm.sup.2, less than 3
kg/cm.sup.2, or 4 kg/cm.sup.2.
[0214] The filtered coconut oil may be sterilized (e.g., at a
temperature above 100, 110, 120, 130, 140, or 150.degree. C., or at
about 134.degree. C.). In some embodiments, the temperature is less
than 100, 110, 120, 130, 140, or 150.degree. C. Skilled
practitioners will appreciate that any art-known method of
sterilization may be utilized.
[0215] In some embodiments, the methods increase the amount of
Stigmastentriol (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is at least or
about 5, 6, 7, 8, 9, 10, or 11). In some embodiments, the methods
increase the amount of Campest-4-en-3-one (e.g., the log2 of the
ratio between the deacidified coconut oil and the original coconut
oil is at least or about 4, 5, 6, 7, 8, 9, or 10). In some
embodiments, the methods increase the amount of Stigmasterol (e.g.,
the log2 of the ratio between the deacidified coconut oil and the
original coconut oil is at least or about 3, 4, 5, 6, 7, 8, or 9).
In some embodiments, the methods increase the amount of
Stigmast-22-ene-3,6-dione (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is at least or
about 3, 4, 5, 6, 7, 8, or 9). In some embodiments, the methods
increase the amount of ubiquinone-4 (e.g., the log2 of the ratio
between the deacidified coconut oil and the original coconut oil is
at least or about 3, 4, 5, 6, 7, 8, or 9). In some embodiments, the
methods increase the amount of Vitamin D3 (e.g., the log2 of the
ratio between the deacidified coconut oil and the original coconut
oil is at least or about 3, 4, 5, 6, 7, 8, or 9).
[0216] In some embodiments, the methods decrease 3-hexenoic acid
(e.g., the log2 of the ratio between the deacidified coconut oil
and the original coconut oil is less than or about -7, -8, -9, -10,
-11, -12, or -13). In some embodiments, the methods decrease 5,
8-tetradecadienoic acid (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is less than
or about -4, -5, -6, -7, -8, -9, or -10). In some embodiments, the
methods decrease Indole (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is less than
or about -4, -5, -6, -7, -8, -9, or -10). In some embodiments, the
methods decrease isolecucine (e.g., the log2 of the ratio between
the deacidified coconut oil and the original coconut oil is less
than or about 0, -1, or -2). In some embodiments, the methods
decrease valine (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is less than
or about 0, -1, -2, -3, or -4). In some embodiments, the methods
decrease glutamate (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is less than
or about 0, -1, -2, -3, -4, -5, or -6). In some embodiments, the
methods decrease beta-alanine (e.g., the log2 of the ratio between
the deacidified coconut oil and the original coconut oil is less
than or about 0, -1, -2, -3, -4, -5, or -6).
[0217] In some embodiments, the methods increase the amount of
Piperochromenoic acid (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is at least or
about 3, 4, 5, 6, 7, 8, or 9). In some embodiments, the methods
increase the amount of LysoPA(a-25:0/0:0) (e.g., the log2 of the
ratio between the deacidified coconut oil and the original coconut
oil is at least or about 2, 3, 4, 5, 6, 7, 8, or 9). In some
embodiments, the methods increase the amount of LysoPA(24:0/0:0)
(e.g., the log2 of the ratio between the deacidified coconut oil
and the original coconut oil is at least or about, 4, 5, 6, 7, 8,
9, or 10).
[0218] In some embodiments, the methods decrease sucrose (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about 5, -6, -7, -8, -9, -10,
or -11). In some embodiments, the methods decrease citric acid
(e.g., the log2 of the ratio between the deacidified coconut oil
and the original coconut oil is less than or about -4, -5, -6, -7,
-8, -9, -10, or -11). In some embodiments, the methods decrease
mannitol (e.g., the log2 of the ratio between the deacidified
coconut oil and the original coconut oil is less than or about -3,
-4, -5, -6, -7, -8, -9, -10, or -11). In some embodiments, the
methods decrease glucose (e.g., the log2 of the ratio between the
deacidified coconut oil and the original coconut oil is less than
or about -1, -2, -3, -4, -5, -6, or -7).
[0219] In some embodiments, the methods increase the amount of DG
(e.g., the log2 of the ratio between the deacidified coconut oil
and the original coconut oil is at least or about 0, 1, 2, 3, 4, 5,
6, 7, 8, or 9). In some embodiments, the methods increase the
amount of ChE (e.g., the log2 of the ratio between the deacidified
coconut oil and the original coconut oil is at least or about 0, 1,
2, or 3). In some embodiments, the methods increase the amount of
ZyE (e.g., the log2 of the ratio between the deacidified coconut
oil and the original coconut oil is at least or about 0, 1, 2, or
3).
[0220] In some embodiments, the methods decrease LPE (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -2, -3, -4, -5, -6, -7,
or -8). In some embodiments, the methods decrease PE (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -2, -3, -4, -5, -6, -7,
or -8). In some embodiments, the methods decrease Co (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -1, -2, -3, or -4). In
some embodiments, the methods decrease LPC (e.g., the log2 of the
ratio between the deacidified coconut oil and the original coconut
oil is less than or about -1, -2,-3, -4, -5, -6, -7, or -8). In
some embodiments, the methods decrease CerG1 (e.g., the log2 of the
ratio between the deacidified coconut oil and the original coconut
oil is less than or about -1, -2,-3, -4, or -5).
[0221] In some embodiments, the methods increase the amount of
LdMePE (e.g., the log2 of the ratio between the deacidified coconut
oil and the original coconut oil is at least or about 1, 2, 3, 4,
5, 6, or 7). In some embodiments, the methods increase the amount
of PAF (e.g., the log2 of the ratio between the deacidified coconut
oil and the original coconut oil is at least or about 1, 2, 3, 4,
or 5). In some embodiments, the methods increase the amount of DGMG
(e.g., the log2 of the ratio between the deacidified coconut oil
and the original coconut oil is at least or about 1, 2, 3, 4, or
5). In some embodiments, the methods increase the amount of MGMG
(e.g., the log2 of the ratio between the deacidified coconut oil
and the original coconut oil is at least or about 1, 2, 3, 4, or
5). In some embodiments, the methods increase the amount of LPMe
(e.g., the log2 of the ratio between the deacidified coconut oil
and the original coconut oil is at least or about 1, 2, 3, 4, 5, 6,
or 7).
[0222] In some embodiments, the methods decrease DGDG (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -3, -4, -5, -6, -7, -8,
or -9). In some embodiments, the methods decrease cPA (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -3, -4, -5, -6, -7, -8,
or -9). In some embodiments, the methods decrease LPI (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -2, -3, -4, -5, -6, -7,
or -8). In some embodiments, the methods decrease LPE (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -1, -2, -3, -4, -5, -6,
or -7). In some embodiments, the methods decrease PC (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -1, -2, -3, -4, -5, -6,
or -7). In some embodiments, the methods decrease dMePE (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -1, -2, -3, -4, -5, -6,
or -7). In some embodiments, the methods decrease MGDG (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -1,-2, -3, -4, -5, -6,
or -7). In some embodiments, the methods decrease PI (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -1, -2, -3, -4, -5, -6,
or -7). In some embodiments, the methods decrease PE (e.g., the
log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about -1, -2, -3, -4, or -5).
In some embodiments, the methods decrease PG (e.g., the log2 of the
ratio between the deacidified coconut oil and the original coconut
oil is less than or about -1, -2, -3, -4, or -5). In some
embodiments, the methods decrease PMe (e.g., the log2 of the ratio
between the deacidified coconut oil and the original coconut oil is
less than or about 0, -1, or -2). In some embodiments, the methods
decrease LPG (e.g., the log2 of the ratio between the deacidified
coconut oil and the original coconut oil is less than or about 0,
-1, or -2). In some embodiments, the methods decrease PEt (e.g.,
the log2 of the ratio between the deacidified coconut oil and the
original coconut oil is less than or about 0, -1, or -2).
[0223] Dosage
[0224] An "effective amount" is an amount sufficient to effect
beneficial or desired results. For example, a therapeutic amount is
one that achieves the desired therapeutic effect. This amount can
be the same or different from a prophylactically effective amount,
which is an amount necessary to prevent onset of disease or disease
symptoms. An effective amount can be administered in one or more
administrations, applications or dosages. The compositions (e.g.,
deacidified coconut oil or herb tea) can be administered one from
one or more times per day to one or more times per week; including
once every other day. The skilled artisan will appreciate that
certain factors may influence the dosage and timing required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of the therapeutic compounds described herein can
include a single treatment or a series of treatments.
[0225] Dosage, toxicity and therapeutic efficacy of the therapeutic
compositions can be determined by standard pharmaceutical
procedures in experimental animals, e.g., for determining the LD50
(the dose lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50.
[0226] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration
utilized.
[0227] Skilled practitioners will appreciate, however, that the
specific dose level for any particular patient will depend upon a
variety of factors. Such factors include the age, body weight,
general health, sex, and diet of the patient. Other factors include
the time and route of administration, rate of excretion, drug
combination, and the type and severity of the particular disease
undergoing therapy.
[0228] In the treatment of the disorders as described herein, an
appropriate dosage level of deacidified coconut oil can be about 1
to 10 drops, 1 to 9 drops, 1 to 8 drops, 1 to 7 drops, 1 to 6
drops, 1 to 5 drops, 1 to 4 drops, 1 to 3 drops, 1 to 2 drops, 2 to
5 drops, 3 to 5 drops, or 2 to 3 drops per administration. For
example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (e.g., 1 or 2) drops of the
deacidified coconut oil can be administered to the subject. Each
drop can be, e.g., 0.1 to 1 ml, 0.1 to 0.9 ml, 0.1 to 0.8 ml, 0.1
to 0.7 ml, 0.1 to 0.6 ml, 0.1 to 0.5 ml, 0.1 to 0.4 ml, 0.1 to 0.3
ml, 0.1 to 0.2 ml, 0.2 to 1 ml, 0.2 to 0.8 ml, 0.2 to 0.7 ml, 0.3
to 1 ml, 0.3 to 0.8 ml, 0.3 to 0.7 ml, 0.4 to 1 ml, 0.4 to 0.9 ml,
0.4 to 0.8 ml, 0.4 to 0.7 ml, 0.4 to 0.6 ml, 0.1 to 0.5 ml, 0.5 to
1 ml, or 0.1 to 1 ml. In some embodiments, each drop is about 0.05
ml. The deacidified coconut oil can be administered to the subject
about or at least 1 times per day, e.g., at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, or 12 times per day, or about or at least 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, or 12 times every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 hours. In some embodiments, deacidified coconut oil
can be administered to the subject less than 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 times per day. In some embodiments, deacidified
coconut oil can be administered to the subject 1 to 10 times, 1 to
9 times, 1 to 8 times, 1 to 7 times, 1 to 6 times, 1 to 5 times, 1
to 4 times, 1 to 3 times, 1 to 2 times, 2 to 5 times, 3 to 5 times,
or 2 to 3 times per day. In some embodiments, the deacidified
coconut oil eye drop is administered to the subject every 4, 5, 6,
7, 8, 9 or 10 hours. In some embodiments, the therapeutic effects
(e.g., relief of symptoms of dry eye disorders) of deacidified
coconut oil eye drop can last e.g., 1 to 12 hours, 1 to 11 hours, 1
to 10 hours, 1 to 9 hours, 1 to 8 hours, 1 to 7 hours, 1 to 6
hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours, 2 to
12 hours, 2 to 11 hours, 2 to 10 hours, 2 to 9 hours, 2 to 8 hours,
2 to 7 hours, 2 to 6 hours, 2 to 5 hours, 3 to 12 hours, 3 to 10
hours, 3 to 8 hours, 3 to 6 hours, or 4 to 6 hours. In some
embodiments, the therapeutic effects can last about or about or at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, 1 day, 2 days, 3
days, 4 days, or 5 days (e.g., about or at least 6 hours, 12 hours,
or 1 day).
[0229] In some embodiments, the length of the treatment period is
between 2 days and 1 year, including e.g., about or at least 2
days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15
days, 20 days, 28 days, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.
[0230] In some embodiments, the composition described herein is
administered to the subject twice a day. In some embodiments, about
or at least 10 uL is administered to one eye for each
administration.
[0231] Pharmaceutical Compositions and Methods of
Administration
[0232] The methods described herein include the use of an
ophthalmic compositions comprising deacidified coconut oil as an
active ingredient or a composition comprising the active agents of
the herb tea described herein.
[0233] The compositions can include a pharmaceutically acceptable
carrier. As used herein, the language "pharmaceutically acceptable
carrier" includes saline, solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration.
[0234] Oral compositions (e.g., compositions comprising active
agents of the herb tea described herein) generally include an inert
diluent or an edible carrier. For the purpose of oral therapeutic
administration, the active agents can be incorporated with
excipients and used in the form of tablets, troches, or capsules,
e.g., gelatin capsules. Oral compositions can also be prepared
using a fluid carrier for use as a mouthwash. Pharmaceutically
compatible binding agents, and/or adjuvant materials can be
included as part of the composition. The tablets, pills, capsules,
troches and the like can contain any of the following ingredients,
or compounds of a similar nature: a binder such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch
or lactose, a disintegrating agent such as alginic acid, Primogel,
or corn starch; a lubricant such as magnesium stearate or Sterotes;
a glidant such as colloidal silicon dioxide; a sweetening agent
such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl salicylate, or orange flavoring.
[0235] In some embodiments, the active agents are prepared with
carriers that will protect the therapeutic compounds against rapid
elimination from the body. Biodegradable, biocompatible polymers
can be used, such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Such formulations can be prepared using standard techniques, or
obtained commercially.
[0236] In some embodiments, the active agents are prepared in a
nebulized form. Thus, the deacidified coconut oil can be
administered to a subject as sprays. In some embodiments, the
deacidified coconut oil can be added to contacts. Thus, the
deacidfied coconut oil is administered to a subject, when the
subject wears the contacts.
[0237] In some embodiments, the pharmaceutical composition consists
of or consists essentially of deacidified coconut oil. In some
embodiments, the pharmaceutical composition can optionally have
various ingredients other than deacidified coconut oil, such as
sugars, electrolytes, amino acids, vitamins, lipids, and medicinal
additives. Examples of these ingredients include sugars such as
glucose, maltose, etc., oligosaccharides, mannitol, and sugar
alcohols such as sorbitol; electrolytes such as sodium chloride,
sodium hydrogenphosphate, potassium chloride, magnesium sulfate,
and calcium chloride; amino acids such as glycine and alanine;
vitamins such as thiamin hydrochloride, sodium riboflavin
phosphate, pyridoxine hydrochloride, nicotinic acid amide, folic
acid, biotin, vitamin A, L-ascorbic acid, and a-glycosyl ascorbic
acid; and derivatives of these. These ingredients may be compounded
in suitable combinations as needed.
[0238] In some embodiments, the pharmaceutical composition can
include preservatives such as methyl parahydroxybenzoate, sodium
dehydroacetate, and benzalkonium chloride; stabilizers such as
sodium edetate and sodium hydrogensulfite; buffers such as borax,
boric acid, and sodium hydrogencarbonate; thickeners such as methyl
cellulose, carboxymethyl cellulose, chondroitin sulfuric acid,
polyvinyl alcohol, and pullulan; and dissolution improvers such as
Polysorbate 80.
[0239] In some embodiments, the compositions can additionally
include one or more of the following ingredients: carboxymethyl
cellulose, polyvinyl alcohol, hydroxypropyl methylcellulose (a.k.a.
HPMC or hypromellose), hydroxypropyl cellulose and hyaluronic acid
(a.k.a. hyaluronan, HA). In some embodiments, the compositions can
additionally include one or more of the following ingredients:
carboxymethylcellulose, dextran, glycerin, hypromellose,
polyethylene glycol 400 (PEG 400), polysorbate, polyvinyl alcohol,
povidone, and propylene glycol.
[0240] Skilled practitioners will appreciate that other
compositions that can be used to treat dry eye can be included in
the pharmaceutical compositions or the treatments described herein.
For example, artificial tear solutions, saline, steroids,
immunosuppressants (e.g., ciclospori), diquafosol, lifitegrast, or
ciclosporin can be included in the pharmaceutical compositions or
can be administered to the subjects with the pharmaceutical
compositions comprising deacidified coconut oil.
[0241] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration. In some embodiments, the kit also include
artificial tears.
EXAMPLES
[0242] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
[0243] Coconut Oil Extraction and Deacidification
[0244] The coconut meat or coconut powder was collected, and was
mixed with sodium carbonate powder. The mixture was then heated to
about 70.degree. C. and then was screw pressed. The extract was
then cooled to about 5.degree. C. The water phase and the oil phase
were separated. The coconut oil was collected. The coconut oil was
then mixed with a saturated solution of sodium bicarbonate (the
weight ratio of the extract to the sodium bicarbonate solution is
about 1:1.1). The mixture was then cooled to about 5.degree. C. The
coconut oil was then collected and was then filtered through 13 nm
membrane filtration under a pressure not greater than 4 kg/cm.sup.2
(about 392.266 kPa). The filtered coconut oil was then sterilized
at about 134.degree. C. and stored for future use.
[0245] Both the sodium carbonate powder and the saturated solution
of sodium bicarbonate were used to deacidify the coconut oil. Some
coconut oil was also prepared without the deacidification process
for comparison purpose.
Example 2
[0246] Testing deacidified coconut oil eye drops in human
subjects
[0247] Safety
[0248] The deacidified coconut oil eye drops were tested in ten
subjects after their consent. Among them, four were healthy
subjects, and six had dry eye syndromes. None of the subjects,
including the four healthy subjects, experienced any irritation or
discomfort. Only in some of these cases, immediately after the
deacidified coconut oil was applied to the eye, some patients
experienced mild blurred visions for a short period of time (e.g.,
seeing colored stripes around strong light). The patterns of
stripes was similar to those on soap bubbles, which are caused by
the thin film interference. The mild blurred vision was typically
resolved within hours or just a few days. Among the subjects with
dry eye syndromes, the deacidified coconut oil eye drops were
administered to the subjects for an extended period of time. No
adverse effects were observed. The results at least suggest the
deacidified coconut oil was generally safe and not toxic to
human.
[0249] For comparison purposes, coconut oil without deacidification
was also tested in at least two human subjects. The coconut oil
immediately caused irritation, redness, and blisters. The results
suggest that coconut oil without deacidification is not suitable
for being used as eye drops.
[0250] Furthermore, coconut oil (cold-pressed) was purchased from
the company Haizhiye (Hainan Province, China). The coconut oil was
extracted without being heated and the decalcification process. The
coconut oil was also known as virgin coconut oil. The coconut oil
(cold-pressed) was tested in one human subject. The coconut oil
(cold-pressed) immediately caused strong irritation in the eyes.
The results indicate that coconut oil (cold-pressed) cannot be used
as eye drops.
[0251] The results above indicate that the regular coconut oil has
irritants for eyes and is not suitable for being used as eye drops,
and the deacidification process can effectively remove the
irritants.
[0252] Efficacy
[0253] Six patients with dry eye syndromes were treated with
deacidified coconut oil eye drops after their consent. The symptoms
were alleviated in all of the six cases. These cases are described
in more detail below:
[0254] Case 1: A 52 year-old male patient received a surgery in
2007 to prevent trichiasis. After the operation, the patient
started to have the sensation of dryness in both eyes. In 2012, it
was determined that the tarsal glands had shrunk by two thirds of
its size, and the patient was diagnosed as having dry eye syndrome.
The tarsal glands may be damaged during the surgery in 2007. The
patient experienced the symptoms of dry eye syndrome several times
a year. The patient had the sensation of dryness, pain, and severe
itching, and could not perform normal daily functions, including
e.g., using the cellphone or the computer. Other symptoms included
headache, insomnia, sensitivity to light and wind, and difficulties
to open eyes. The patient were prescribed with artificial tear eye
drops. However, the treatments were not effective. The quality of
the patient's life was severely affected.
[0255] In 2017, the patient was treated with deacidified coconut
oil eye drop. The deacidified coconut oil eye drop was administered
along with the artificial tear eye drops. The artificial tear eye
drops were administered to the patient first, and then the
deacidified coconut oil eye drops were administered. After one
year's treatment, the patient's right eye was fully recovered. The
symptoms for the left eye was not yet fully recovered, but
significant improvements were observed. Particularly, immediately
after the treatment, certain symptoms, including e.g., extreme
itchiness, headache, insomnia, sensitivity to light and wind were
resolved. In the following check-up in the Department of
Ophthalmology at Yunnan No. 2 People's Hospital, it was determined
that the patient's right eye was found to be normal, and the
patient's left eye was diagnosed as having only mild dry eye
syndrome.
[0256] Case 2: The female patient was 47 years old. She had dry eye
syndrome for at least 5 years before being treated with deacidified
coconut oil eye drop. The dry eye syndrome caused great discomfort,
and the disease severely affected her work performance. The patient
was treated by various treatments (including artificial tear eye
drops), but there was no improvement in her symptoms. In early
2018, she was treated with deacidified coconut oil eye drop (one
administration per day, one eye drop for one administration). The
deacidified coconut oil eye drop was administered along with
artificial tear eye drops. The treatment immediately alleviated her
pain. She continued to use the deacidified coconut oil eye drops.
The sensation of dryness and pain were resolved during the
treatment period.
[0257] Case 3: The patient was a 56 years old female. She had dry
eye syndrome for at least 6 years before being treated with
deacidified coconut oil eye drops. She was also diagnosed of
chronic keratoconjunctivitis. Her symptoms included dryness and
redness in her eyes. In early 2018, she was treated with
deacidified coconut oil eye drops. The deacidified coconut oil eye
drops were administered along with artificial tear eye drops. The
treatment alleviated the symptoms of the dry eye syndrome. The
redness in the eyes was resolved.
[0258] Case 4: The patient was a 52-year-old female. She had
moderate dry eye syndrome for years. After being treated with
deacidified coconut oil eye drops along with artificial tear eye
drops, the symptoms for dry every syndrome were resolved.
[0259] Case 5: The patient was a woman, who was more than 70 years
old. She had severe dry eye syndrome for several years. After being
treated with deacidified coconut oil eye drops along with
artificial tear eye drops, her symptoms were resolved.
[0260] Case 6: The patient was a man, who was more than 60 year
old. He had dry eye syndrome for a few years. After being treated
with deacidified coconut oil eye drops along with artificial tear
eye drops, his symptoms were resolved as well.
[0261] As all of these cases had chronic dry eye syndrome for
years, the results before and after the treatment with deacidified
coconut oil drops suggest that deacidified coconut oil drops are
effective for treating dry eye disorders.
Example 3
[0262] Methods of Making Herb Tea and Herb Tea Pod
[0263] Wolfberries, Astragalus root, and chrysanthemum flower were
cleaned, dried, and were sterilized by heat. The mixture was then
grinded to powder, and was stored in a container.
[0264] For making herb tea pods for being used as single-serve
containers (e.g., tea cup, K-cup, tea capsule), the powder was
stored in the single-serve containers, and then the containers were
filled with nitrogen to increase storage time. The containers were
then sealed and can be used in appropriate coffee machines or tea
makers.
Example 4
[0265] Methods of Making Thermal Pads
[0266] About 200 g Cassiae semen and about 80 g borneol were added
to a cotton bag with the size of 22 cm.times.11 cm. Before using,
the bag was heated in a steamer for a sufficient period of time
(e.g., about 10 minutes). The bag should not be immersed in the
water in the steamer. After heating, the bag was left on a
container at the room temperature. When the temperature dropped to
about 46.degree. C., the bag can be placed on the eyes of a human
subject as a thermal pad.
Example 5
[0267] Testing Herb Tea and Thermal Pads in Human Subjects
[0268] The patient had dry eye syndrome for years and was being
treated with deacidified coconut oil for an extended period of
time.
[0269] During the period when the patient was administered with
deacidified coconut oil, the patient also took the herb tea for
treating dry eye disorders. Wolfberries (about 10 g), Astragalus
root (about 10 g), and chrysanthemum flower (about 10 g) were mixed
with about 2 cups of hot water. After it was cooled to appropriate
temperature, the herb tea was then orally administered to the
patient at least once per day.
[0270] The patient also received heat therapy several times a week
using the heat pad comprising Cassiae semen and borneol.
[0271] By comparing the symptoms before and after the herb tea
treatment and the heat therapy, it was determined that the herb tea
and the heat therapy significantly improved the efficacy of
decalcified coconut oil eye drops.
Example 6
[0272] Testing Deacidified Coconut Oil Eye Drops in Animal Models
for Dry Eye Syndromes
[0273] Animal models for dry eye syndromes can be placed into 4
groups. The first group is treated with artificial tear eye drops.
The second group is treated with deacidified coconut oil eye drops.
The third group is treated with deacidified coconut oil eye drops
along with artificial tear eye drops. The fourth group does not
receive any treatments, and is used as a control group.
[0274] The animal models can be any animal models for dry eye
syndromes that are known in the art. Numerous animal models for dry
eye syndromes are known in the art, and are described for example
in Barabino, et al. "Animal models of dry eye: a critical
assessment of opportunities and limitations," Investigative
ophthalmology & visual science 45.6 (2004): 1641-1646, which is
incorporated herein by reference in its entirety.
[0275] In this example, the animal model can be a rabbit model.
Because of the large exposed ocular surface in rabbits compared
with mice, standard dry eye clinical tests such as tear break-up
time and fluorescein or rose bengal staining of the ocular surface
can be much more easily performed in rabbits. An autoimmune disease
in rabbits resembling Sjogren's syndrome can be induced by
injecting into the lacrimal gland autologous peripheral blood
lymphocytes proliferated in culture with epithelial cells obtained
from the contralateral excised gland. The injection can trigger a
continuous decrease in tear production and stability.
[0276] In the first group, the artificial tear eye drops are
administered to the eyes of the animal twice per day. In the second
group, the deacidified coconut oil eye drops are administered to
the eyes of the animal twice per day. In the third group, the
artificial tear eye drops are administered to the eyes of the
animal first and then the deacidified coconut oil eye drops are
administered to the eyes of the animal. Two administrations are
performed in a day.
[0277] A Schirmer's test can be used to determine whether there is
any improvement in dry eye syndromes. The Schirmer's test
determines whether the eye produces enough tears to keep it moist.
It uses paper strips inserted into the eye for several minutes to
measure the production of tears. The exact procedure may vary
somewhat. Most often, this test includes placing a small strip of
filter paper inside the lower eyelid (inferior fornix). The eyes
are closed for 5 minutes. The paper is then removed and the amount
of moisture is measured. Sometimes a topical anesthetic is placed
into the eye before the filter paper to prevent tearing due to the
irritation from the paper. The use of the anesthetic ensures that
only basal tear secretion is being measured.
[0278] It is expected that the deacidified coconut oil eye drops
and the combination of the deacidified coconut oil eye drops and
the artificial tear eye drops can alleviate symptoms of dry eye
syndromes.
Example 7
[0279] Testing deacidified coconut oil eye drops in a clinic
trial
[0280] Human patients with dry eye syndromes can be placed into 5
groups. The first group is treated with artificial tear eye drops.
The second group is treated with deacidified coconut oil eye drops.
The third group is treated with deacidified coconut oil eye drops
along with artificial tear eye drops. For the fourth group, in
addition to be treated with the combination of deacidified coconut
oil eye drops and artificial tear eye drops, the fourth group also
receives the herb tea treatment and the heat therapy. The fifth
group is not treated with anything, and is used as a control
group.
[0281] In the first group, the artificial tear eye drops are
administered to the eyes of the subject twice per day. In the
second group, the deacidified coconut oil eye drops are
administered to the eyes of the subject twice per day. In the third
group, the artificial tear eye drops are administered to the eyes
of the subject first and then the deacidified coconut oil eye drops
are administered to the eyes or the subject twice per day. The
procedure for the fourth group is identical to the third group,
except that the subjects in the fourth group are also treated with
herb tea and heat therapy. The clinical trial can last 2 to 6
weeks.
[0282] The responses to the treatments are recorded. In some cases,
Schirmer's test can be used to provide an objective evaluation of
dry eye symptoms. In a Schirmer's test, 15 mm wetting of the paper
after 5 minutes indicates normal, 14-9 mm wetting of the paper
after 5 minutes indicates mild dry eye syndrome, 8-4 mm wetting of
the paper after 5 minutes indicates moderate dry eye syndrome, and
<4 mm wetting of the paper after 5 minutes indicates severe dry
eye syndrome.
[0283] It is expected that the deacidified coconut oil eye drops
and the combination of the deacidified coconut oil eye drops and
the artificial tear eye drops can alleviate symptoms of dry eye
syndromes, and the herb tea and heat therapy can further improve
the efficacy of the deacidified coconut oil eye drops.
Example 8
[0284] A Pilot Efficacy Study on Dry Eye in Mice
[0285] Experiments were performed to determine the efficacy of the
deacidified coconut oil composition. Biodecs001 was the deacidified
coconut oil described herein, and was used in the following
experiments.
[0286] The following materials were used in the following
examples.
[0287] C57BL/6SLAC mice were purchased from Shanghai Slack Co.,
Ltd.
[0288] Scopolamine hydrobromide was purchased from Sigma-Aldrich
(Lot Number: SLBD0119V).
[0289] Cyclosporine eye drops were obtained from Yunnan Biotech
Biotechnology Co., Ltd.
[0290] Polyvinyl alcohol eye drops (artificial tears) were
purchased from JOINN Laboratories (Suzhou) Inc. and manufactured by
Hubei Yuanda Everyday Bright Eyes Pharmaceutical Co., Ltd. (Lot
Number: 190103).
[0291] Sodium Chloride Injection was purchased from Shijiazhuang
No. 4 Pharmaceutical Co., Ltd. (Lot Number: 1805303204).
[0292] Mouse feed was purchased from Beijing Keaoxieli Feed Co.,
Ltd. (Lot Number: 19043323, 19073113).
[0293] 8.1 Experimental design Fifty-nine healthy female
C57BL/6SLAC mice were selected for experiments.
[0294] Mice that were healthy and with normal eyes were randomly
divided into age-matched control group (8 animals/group) and test
groups (51 animals) according to body weight on Day-1 (one day
before the study period). Forty mice with similar right eye corneal
fluorescein sodium staining score and tear secretion were divided
into five groups (8 animals/group) according to the right eye
corneal fluorescein sodium staining score on Day 5. Each mice was
given a unique animal number. The grouping and treatment plans of
mice are shown in the table below:
TABLE-US-00001 TABLE 1 Modelling treatment Scopolamine Test/Control
No. of No. Group Humidity hydrobromide Article Dosing animal Animal
ID.sup. 1 Age-matched D 1-D 17, -- -- -- 8 1921241~1921248 control
group normal humidity environment 2 Model control D 1-D 17, D 1-D
17, -- -- 8 1921249~1921256 group low humidity 2 time/day, 3
Negative environment 0.75 mg/eye/ Saline D 6-D 17, 2 8
1921257~1921264 control group time, 0.3 mL/ time/day, 4
Cyclosporine + eye/time, Cyclosporine + 10 .mu.L/eye, 8
1921265~1921272 artificial tears + subcutaneous artificial tears +
right eye test composition injection test composition* group* 5
Artificial tears + Artificial tears + 8 1921273~1921280 test
composition test composition# group# 6 Test composition Test
composition 8 1921281~1921288 group In Table 1, "--" means that
there was no treatment. .sup.means that the first and the last
animals of each group were involved in this study in batches.
*means that the eyes of mice in group 4 were given cyclosporine
first, 20 minutes later artificial tears were added, and one minute
later test composition were added. #means that the eyes of mice in
group 5 were given artificial tears first, one minute later test
composition were added.
[0295] Test/control compositions were dosed to the right eye by
topical instillation, at a frequency of twice daily (about 8 hours
apart) for 12 consecutive days (10 .mu.L/eye/time). 10 .mu.L
test/control compositions including artificial tears or
cyclosporine eye drop, were drawn with a pipette, and then dropped
on the exposed cornea of the mice. After at least 10 seconds,
eyelid was gently closed. Any spilled liquid was gently wiped off
with sterile gauzes. The application, dose level, frequency and
duration of dosing selected for this study was based on relevant
reference information.
[0296] All animals of all groups were observed once daily during
the study period (Day 1-Day 17). The animal's death or near-death,
mental state, behavioral activities, feeding and fecal traits were
observed by the cage. Body weights of all the animals in all groups
were obtained at the time of receipt, the end of the quarantine
period and the end of the last ophthalmic examination.
[0297] Tear secretion from the right eye of all the animals in all
groups were measured by the following method on Day-1 (one day
before the study period), Day 5, Day 9, Day 13 and Day 17. The
phenol red cotton thread was clamped by clean, toothless tweezers
and fixed in the middle of the lower eyelid conjunctival sac of
mice for 30 seconds. Under the microscope, the length of the cotton
dyeing was measured with a ruler and the picture was
photographed.
[0298] Corneal fluorescein sodium staining test of the right eye of
all the animals in all groups was performed as follows on Day-1,
Day 5, Day 9, Day 13 and Day 17. All animals were awake when
examined. The examiner described the results of each animal without
being informed of the animal's identity, and the results of the
examination was recorded by another person. About 1.5 minutes after
the sodium fluorescein solution (1.5 .mu.L, 0.5%) was dipped into
the conjunctival sac of animals, the conjunctival sacs of the mice
were washed with 1.25 ml saline every 10 seconds for 3 times, then
the liquid around the animal's eyes was wiped with paper each time.
About 5 minutes after corneal staining, the ocular surface was
observed and photos were taken by slit lamp microscope with cobalt
blue filter, and the staining content of corneal was graded by the
modified NEI fluorescent staining grading method. According to this
method, the cornea of eye is divided into 5 regions, and the
staining score of each region is up to 4. "0" indicates that
corresponding area is not clearly colored, and "1" indicates that
the dotted colored area is 1 to 25% of the corresponding area, and
"2", "3", "4" indicates percentages of 26%.about.50%,
51%.about.75%, 76%.about.100% respectively. The total score of each
eye is up to 20 (FIG. 1). The total score of corneal staining for
each eye can be calculated.
[0299] According to the protocol, during the study period, the
animals would be euthanized by excessive carbon dioxide inhalation
and thoracic opening if they developed severe eye abnormalities or
in extremis. There were no animals in this experiment with
irreversible signs and no animals were euthanized. After the last
inspection, all surviving animals were euthanized by excessive
carbon dioxide inhalation.
[0300] For the quantity of tears fluid and total score of corneal
fluorescein sodium staining of all groups at different time point,
the means and standard deviations were calculated with software
SPSS, and the group difference on each time point were analyzed by
the following statistical procedures: A Levene's test was performed
to test for variance homogeneity. If the result showed no
significance (p>0.05), a one-way analysis of variance (ANOVA)
was performed. If ANOVA showed significance (p.ltoreq.0.05), a
Dunnett's test was performed for multiple comparisons. If ANOVA
showed no significance (p>0.05), no more statistical tests were
performed. In the case of heterogeneity of variance at
p.ltoreq.0.05, a Kruskal-Wallis nonparametric test was performed.
If the Kruskal-Wallis nonparametric test showed significance
p.ltoreq.0.05), a further Mann-Whitney test was performed for
multiple comparisons. Pre- and post-modelling of the group animals
were compared using statistical methods of independent sample T
test.
[0301] 8.2 Results (Clinical observations)
[0302] During the study (Day 1-Day 17), no animal death was seen in
all groups in the dosing period, no abnormal clinical signs (other
than ocular signs) were seen. On Day 4 (before treatment) after the
modelling, animal with temporary number 3 was found dead. On Day 5
(also before treatment), animal with temporary number 59 was found
dead after corneal staining examination. In such case, the corpse
was disposed as medical waste, and no biopsy were carried out.
[0303] Four out of 8 treated eyes in the test composition group
(Animal 1921281-1921284) on Day 13-Day 17, and the other 4 treated
eyes (Animal 1921285-1921288) on Day 16-Day 17 were observed with
sparse hairing in the ocular region. Four out of 8 treated eyes
(Animal 1921273-1921276) on Day 13-Day 17, and the other 4 treated
eyes (Animal 1921277-1921280) on Day 16-Day 17 were noted also with
sparse hairing in the ocular region, and the incidence rate and
time were the same with the Test composition group. Four out of 8
treated eyes in the cyclosporine+artificial tears+test composition
group (Animal 1921269-1921272) on Day 12-Day 17, and the other 4
treated eyes (Animal 1921265.about.1921268) on Day 13-Day 17 were
all seen with eyelid bloating, reduced hairing in ocular region,
with or without peri-ocular skin redness. Other than signs
described above, the untreated eyes in the three groups mentioned
above and the age-matched control group, the model control group
and the negative control group had no abnormal ocular signs.
[0304] Peri-ocular sparse hairing was seen in all treated eyes in
the test composition group, but not seen in treated eyes in the
negative control group with the same dosing frequency, cycle, and
volume with the test composition group, thus this abnormality was
considered to be due to the overflow of excess test composition to
the peri-ocular region, which indicated the test composition could
incite irritation to the peri-ocular skin and the dosing amount
should be considered accordingly to prevent over-flow. Since the
peri-ocular sparse hairing in all treated eyes in the artificial
tears+test composition group had the same incidence rate and time
with the test composition group, giving artificial lacrimal fluid
alone might have no effect on the peri-ocular skin. Peri-ocular
hair loss, bloated eyelid, sparse hairing, with or without
periocular skin redness were noted in the treated eyes in the
cyclosporine+artificial tears+test composition group, and not seen
in the rest 6 groups. Therefore, this might be related to the
over-flow of excess cyclosporine, or both cyclosporine and test
composition to the peri-ocular skin, indicating that the
cyclosporine was significantly irritative to the peri-ocular skin
and eyelid.
[0305] 8.3 Results (Body Weight)
[0306] Body weight are summarized in FIG. 2. Before testing
(Day-1), all animals in all groups showed no statistically
significant difference in weight (p>0.05).
[0307] At the end of the observation period (Day 17), all animals
in all groups had weight loss compared to Day-1, which could be a
result of that the tested mice were all retired breeder mice, which
were in old age, and the change of feed while entering the study
(the animals ate reproduction feed before purchase and sustaining
feed after purchase). Moreover, on Day 17, the animals in
cyclosporine+artificial tears+test composition group had
significantly reduction in weight (p<0.05) compared to
time-matched negative control group, which could be due to
irritation related issues caused by cyclosporine. The rest of
animals in other groups showed no significant difference in weight
(p>0.05).
[0308] 8.4 Results (Tear Secretion)
[0309] The data of tear secretion are summarized in FIG. 3. Before
testing (Day-1), the lacrimal secretion volume in the right eyes of
all animals in all groups showed no statistical difference
(p>0.05).
[0310] After modelling treatment and before test composition
treatment (Day 5), except that the right eyes of the age-matched
control groups showed no significant difference in lacrimal
secretion volume compared to Day-1, the rest of the groups showed
significant decrease (p<0.05) in the lacrimal secretion volume
in the right eyes, which indicated low environmental humidity and
scopolamine successfully induced an reduction in the lacrimal
secretion amount, and no significant difference (p>0.05) was
seen among all modelling groups.
[0311] After treatment, the specific result of lacrimal secretion
in all groups of animal are as following:
[0312] Age-matched control group: On Day 9, Day 13, Day 17, the
lacrimal secretion of right eyes were 10.81.+-.2.36 mm,
8.75.+-.2.52 mm, 9.13.+-.1.58 mm, respectively.
[0313] Model control group: On Day 9, Day 13, Day 17, the lacrimal
secretion of the right eyes were 3.44.+-.1.59 mm, 4.00.+-.1.07 mm,
and 4.38.+-.2.57 mm, respectively, which were significantly lower
than that of the age-matched control group on the respective days
(p<0.05). The result indicated that low environmental humidity
and scopolamine induced reduction in lacrimal secretion, which was
stably maintained throughout the studyt period.
[0314] Negative control group: On Day 9, Day 13, Day 17, the
lacrimal secretion of the right eyes were 3.38.+-.1.51 mm,
3.13.+-.1.92 mm, and 3.38.+-.1.94 mm, respectively, which were all
significantly lower than that of the age-matched control group on
the respective days (p<0.05). In addition, the results showed no
statistically significant difference compared to the modelling
control group on the respective days (p>0.05), which indicated
that normal saline had no effect on the mouse lacrimal
secretion.
[0315] Test composition group (Test article group or TA group): On
Day 9, Day 13, Day 17, the lacrimal secretion of the right eyes
were respectively 3.94.+-.2.43 mm, 6.2 5.+-.1.98 mm, and
4.31.+-.3.26 mm, and the results on Day 9 and Day 13 were both
higher than those of the model control group and the negative
control group on the respective days, among which the Day 13 result
had statistically significant difference compared to that of the
negative control group (p<0.05), but the result on Day 17 showed
no statistically significant difference when compared to the
modelling control group and negative control group (p>0.05). The
result suggested that the test composition used alone effectively
improved the mouse lacrimal secretion to a certain degree on the
8th dosing day (Day 13).
[0316] Artificial tears+test composition group (AT+TA group): On
Day 9, Day 13, Day 17, the lacrimal secretion of the right eyes
were 4.81.+-.2.24 mm, 4.88.+-.1.48 mm, and 3.31.+-.1.53 mm,
respectively. The result on Day 9 and Day 13 were both higher than
those of the modelling control group and the negative control group
on the respective days, and the result on Day 17 was lower than
both the model control group and negative group on the respective
days, with no statistical significance in difference (p>0.05).
Cyclosporine+artificial tears+test composition group
(Cyclosporine+AT+TA group): On Day 9, Day 13, Day 17, the lacrimal
secretion of the right eyes were 5.13.+-.1.81 mm, 5.63.+-.1.36 mm,
and 3.81.+-.1.33 mm, respectively. Among them, the results on both
Day 9 and Day 13 were higher than the model control group and
negative control group on the respective days, in which the result
on Day 13 had significant difference compared to negative control
group (p<0.05), and the result of Day 17 showed no statistically
significant difference compared with the model control group and
negative control group on the respective days (p>0.05). The
results showed combining three drugs in treatment could at certain
level increase the lacrimal secretion of the mice on the 8th dosing
day (Day 13). Compared with the test composition group, this group
showed no significant difference among respective time points in
the right eye (p>0.05).
[0317] 8.5 Results (Corneal fluorescein sodium staining test)
[0318] Staining scores of corneal sodium fluorescein are summarized
in FIG. 4, and ratio of the staring scores are summarized in FIG.
5. Before modelling (Day-1), the corneal fluorescein sodium
staining scores of the right eyes of all animals in all groups
showed no statistically significant difference (p>0.05).
[0319] After modelling and before treatment (Day 5), except that
the staining scores of the right eyes of the age-matched control
group showed no significant difference compared to those on Day -1,
the rest of the groups showed an significant increase in the
staining scores in the right eye (p<0.05), which indicated that
low environmental humidity and scopolamine successfully induced an
increase of the mouse corneal fluorescein sodium staining scores,
and exacerbated the loss of corneal epithelium in the mice. Except
that the staining score of the negative control group was
significantly lower than that of the artificial tears+test
composition group (AT+TA group), the rest of the modelling groups
showed no significant inter-group difference (p>0.05). To reduce
the effect of inter-group difference before treatment on the
following analysis, within a particular group, each post-treatment
result was divided by the result on Day 5 (before treatment) and a
scoring value was calculated. The value could indicate the relative
ratio of the pre- and post-treatment results. As shown in FIG. 5,
no significant difference was found (p>0.05), while the average
of scoring values of each group were all 1. After treatment, the
specifics of the corneal fluorescein sodium staining scores, and
calculated scoring values are as follows:
[0320] Age-matched control group: On Day 9, Day 13, Day 17, the
staining scores of the right eyes were 11.4.+-.4.1, 13.6.+-.3.4,
and 11.6.+-.3.1, respectively, while the average of the scoring
value comparing pre-/post-treatment during the dosing period were
fluctuating in 1.0-1.2.
[0321] Model control group: On Day 9, Day 13, Day 17, the staining
scores of the right eyes were 17.3.+-.2.3, 17.1.+-.2.7, and
18.0.+-.1.7, respectively, with the average scoring value comparing
pre-/post-treatment fluctuating between 0.9-1.0. Compared to the
age-matched control group on the specific days, the staining scores
increased significantly (p<0.05), which indicated that low
environmental humidity and scopolamine induced increase in the
mouse corneal staining scores, which were stably maintained in the
whole treatment period.
[0322] Negative control group: On Day 9, Day 13, Day 17, the
staining scores of the right eyes were 19.9.+-.0.4, 18.3.+-.2.1,
and 18.0.+-.2.2, respectively. Compared to the modelling group, the
scores on Day 9 showed a significant increase, but those on Day 13
and Day 17 showed no statistical difference (p>0.05). The reason
of the difference on Day 9 was not clear, which could be derived
from systematic errors. However, considering the data on Day 13 and
Day 17, the saline solution was thought to have no significant
impact on the mouse corneal fluorescein sodium staining scores. The
average scoring value comparing pre-/post-treatment during the
dosing period fluctuated around 1.0-1.1.
[0323] Test composition group (Test Article group): On Day 9, Day
13, Day 17, the staining scores of the right eyes were 17.0.+-.2.9,
18.4.+-.2.3, and 17.5.+-.2.8, respectively. The average of staining
scoring value comparing pre-/post-treatment fluctuated around
0.9-1.0. On Day 9, the staining score and the scoring value were
both lower than those of the model control group and the negative
control group. Only in comparing with the negative control group,
statistically significant difference was found (p<0.05). At the
rest of the time points, no significant difference was observed
when compared to the model and the negative control group on
respective days (p>0.05). The results indicated that by using
test composition alone on the 4th dosing day (Day 9), the mouse
fluorescence staining scores showed a certain reducing effect.
[0324] Artificial tears+test composition group (Artificial
tears+test article group): On Day 9, Day 13, Day 17, the staining
scores of the right eyes were 18.9.+-.1.4, 19.4.+-.0.9, and
18.0.+-.4.5, respectively, while the average scoring values
comparing pre-/post-treatment were about 1.0. On Day 9 the staining
score was significantly lower than the negative control group
(p<0.05), but the average scoring value showed no significant
difference compared with the negative control group (p>0.05). In
addition, the scores together with the scoring values were all
higher than those of the model control group. On Day 13, the
staining score was significantly higher than the model control
group, but the scoring value was not significantly different to the
model control group (p>0.05). At the rest of the time points,
this group showed no statistical difference when compared to the
model control group, the negative control group, and the test
composition group at respective time points (p>0.05).
[0325] Cyclosporine+artificial tears+test composition group
(Cyclosporine+AT+TA group): On Day 9, Day 13, Day 17, the staining
scores of the right eyes were 14.8.+-.6.2, 17.9.+-.2.1,
16.5.+-.2.7, respectively, while the scoring values comparing
pre-/post-treatment fluctuated around 0.8-0.9. On Day 9, the
staining score and the average scoring value were both lower than
those of the model control group, the negative control group, the
test composition group and the artificial tears+test composition
group, but only significantly different than those of the negative
control group (p<0.05). The rest of the time points showed no
significant difference than those of the modelling groups at
respective time points (p>0.05). The results showed that by
combining the three drugs, the treatment can reduce the mouse
corneal staining score.
[0326] 8.6 Conclusion
[0327] Amount of tear secretion: During the study, on Day 5, Day 9,
Day 13, Day 17, model control group and negative control group had
a statistically significant drop (p<0.05) in tear secretion
compared to time-matched age-matched control group. On Day 13,
compared to time-matched negative control group, the test
composition group and the cyclosporine+artificial tears+test
composition group showed a rise in amount of lacrimination which is
statistically significant (p<0.05).
[0328] Corneal fluorescein sodium staining scores: During the
study, on Day 5, Day 9, Day 13, Day 17, the model control group and
negative control group showed significantly higher scores in
corneal fluorescein sodium staining than the time-matched
age-matched control group (p<0.05). On Day 9, compared to the
time-matched negative control group, the test composition group,
artificial tears+test composition group and the
cyclosporine+artificial tears+test composition group showed
significantly lower scores (p<0.05) in corneal fluorescein
sodium staining.
[0329] General clinical observation: During the study, no animal
deaths were confirmed related to the test composition, and all
animals in the age-matched control group, model control group, and
negative control group showed no abnormal clinical signs, and the
rest of the animals in other groups showed no abnormal sign besides
ocular signs. All animals in the test composition group and
artificial tears+test composition group showed decreased hairing
around the peri-ocular region in the treated eyes that might due to
the overflow of excess test composition to the peri-ocular region,
which started from Day 12 or Day 16, and lasted until the end of
the observation period. All animals in the cyclosporine+artificial
tears+test composition group showed eyelid bloating, losing hair
around ocular region, and with or without skin redness around the
ocular region in the treated eye that might due to the overflow of
excess cyclosporine or both of cyclosporine and test composition to
the peri-ocular region, which lasted from Day 12 or Day 13 until
the end of observation. All untreated eyes in animals above showed
no abnormal clinical signs.
[0330] Conclusion: Under the circumstances in this study, the test
composition Biodecs001 used alone or combined with artificial
tears, cyclosporine eye drops (twice per day, 10 .mu.L per eye in
each dose, consecutively for 12 days) could increase the lacrimal
secretion and decrease the corneal fluorescein sodium staining
scores of the dry eye mouse model induced by drying stress to a
certain degree.
Example 9
[0331] An Eye Irritation Study of Biodecs001 with 14-Day
Instillation in New Zealand White Rabbits
[0332] 9.1 Experimental design
[0333] The following materials were used in the following
examples.
[0334] New Zealand White Rabbits (SPF Grade) were purchased from
Dongfang Breeding Co., Ltd. Rabbit growth reproduction feed was
purchased from Beijing Keaoxieli Feed Co., Ltd., and Pizhou Xiaohe
Technology Development Co., Ltd. (Lot Number: 19044111,
20190325).
[0335] Within 24 hours prior to the 1st dosing (Day-1), slit lamp
microscopy and sodium fluorescein examination were applied to all
healthy animals; any animal with eye irritation, ocular defect, or
pre-existing injury was excluded. Twelve animals with normal eyes
and similar body weight were selected for experiments.
[0336] Through a computer-generated randomization procedure, the
twelve animals were randomly assigned to respective treatment
groups according to the body weight measured within 24 hours prior
to the 1.sup.st dosing (Day-1), as shown in Table 2.
TABLE-US-00002 TABLE 2 Group assignment Dosing Treatment Dosing
Number Right Volume(.mu.L/ of Group eye Left eye eye/time) animals
1 -- Saline 50 4 2 -- Biodecs001 eye drops 50 4 3 -- Artificial
tears * + 50 + 50 4 Biodecs001 eye drops (about 1 minute apart) In
Table 2, "--" means the right eye of animals was not treated with
any composition. "*" means gave the artificial tears first, and
then test composition for animals from Group 3.
[0337] Compositions were dosed to the left eye by topical
administration, at a frequency of once daily for 14 consecutive
days (50 .mu.L/eye/time). 50 .mu.L test/control compositions were
drawn with a pipette, and 100 .mu.L composition was dropped into
the conjunctiva sac by pulling out the lower eyelid of the animal.
Then, the lower eyelid was gently closed and remained for 10
seconds. The application, dose level, frequency and duration of
dosing selected for this study was based on relevant reference
information and the request of the guidance to support the
subsequent toxicity studies and/or clinical trials.
[0338] All animals of all groups were observed daily. During the
14-day dosing period, all animals were observed twice daily (before
the first instillation in the morning and one time in the
afternoon), At Day-1 (within 24 hours pre-experiment) and the
non-dosing period, all animals were observed once daily.
Observation included signs of mortality, morbidity, behavior,
respiration, secretion, excretion, pain and/or distress (e.g.
repeated pawing or rubbing of the eye, excessive blinking, and
excessive tearing) and so on. Body weight of all animals of all
groups were obtained on Day-1, Day 7, Day 14, and Day 17.
[0339] Both eyes of all animals of all groups in conscious
condition were examined by an experienced examiner with a slit lamp
microscope under observer-masked condition. The animals were
brought to the examiner at a random order and the examiner graded
each animal without knowing the animal ID and someone else was
responsible for grade recording. The examination time points are:
Day-1 (within 24 hours), prior to administration during Day 1 to
Day 14 and 1, 2, 4, 24, 48, 72 hours post-final instillation on Day
14. The following examinations were performed.
[0340] A. Slit lamp examination: The cornea, iris, conjunctiva,
edema, and ocular secretions were observed with a slit lamp and
scored according to the eye irritation response score (shown in
Table 3).
[0341] B. Sodium fluorescein examination: After examination A,
cornea epithelial change with fluorescein staining was observed
with moist sodium fluorescein indicator paper. The Corneal Staining
(% Area) was scored according to the Modified MacDonald-Shadduck
Scoring System as described in Table 4. No other abnormal eye
symptoms were found during the examination.
TABLE-US-00003 TABLE 3 Modified Draize Scoring System of Eye
Irritation Cornea Opacity: degree of density (readings should be
taken from most dense area)* No ulceration or opacity 0 Scattered
or diffuse areas of opacity (other than slight 1 dulling of normal
lustre): details of iris clearly visible Easily discernible
translucent area; details of iris slightly 2 obscured Nacrous area;
no details of iris visible; size of pupil barely 3 discernible
Opaque cornea; iris not discernible through the opacity 4 Maximum
possible: 4; * the area of corneal opacity should be noted Iris
Normal 0 Markedly deepened rugae, congestion, swelling, moderate 1
circumcorneal hyperaemia; or injection; iris reactive to light (a
sluggish reaction is considered to be an effect) Hemorrhage, gross
destruction, or no reaction to light 2 Maximum possible: 2
Conjunctivae Redness (refers to palpebral and bulbar conjunctivae;
excluding cornea and iris) Normal 0 Some blood vessels hyperaemic
(injected) 1 Diffuse, crimson colour; individual vessels not easily
2 discernible Diffuse beefy red 3 Maximum possible: 3 Chemosis
Swelling (refers to lids and/or nictating membranes) Normal 0 Some
swelling above normal 1 Obvious swelling, with partial eversion of
lids 2 Swelling, with lids about half closed 3 Swelling, with lids
more than half closed 4 Maximum possible: 4 Discharge No discharge
0 Any amount different from normal (does not include small 1
amounts observed in inner canthus of normal animals) Discharge with
moistening of lids and hairs just adjacent 2 to the lids Discharge
with moistening of lids and hairs, and considerable 3 area around
the eye Maximum possible: 3
TABLE-US-00004 TABLE 4 Modified MacDonald-Shadduck Scoring System
Corneal Staining (% Area) No area of fluorescein staining. 0 1% to
25% area of fluorescein staining. 1 26% to 50% area of fluorescein
staining. 2 51% to 75% area of fluorescein staining. 3 76% to 100%
area of fluorescein staining. 4
[0342] According to the protocol, animals with either of the
following eye lesions post-instillation should be euthanized: 1)
perforation or significant corneal ulceration including staphyloma;
2) blood in the anterior chamber of the eye; 3) grade 4 corneal
opacity; 4) absence of a light reflex (iridial response grade 2)
which persists for 72 hours.
[0343] Body weights should be obtained prior to euthanizing in
extremis. In accordance with the AVMA Guidelines for the Euthanasia
of Animals: 2013 Edition (the American Veterinary Medical
Association, 2013), rabbits could be euthanized by an intramuscular
injection of Zoletil 50 (8 mg/kg, 50 mg/mL) and Xylazine
Hydrochloride Injection (5 mg/kg, 100 mg/mL) and followed by
femoral artery exsanguinations and thoracic opening method.
[0344] There were no animals with irreversible symptoms in the
study, thus no animals was euthanized. All survival animals were
transferred back to the holding colony on Day 18.
[0345] For the body weights of all groups at different time point,
with software SPSS the means and standard deviations were
calculated, and the group difference on each time point were
analyzed by the following statistical procedures: A Levene's test
was performed to test for variance homogeneity. If the result
showed no significance (p>0.05), a one-way analysis of variance
(ANOVA) was performed. If ANOVA showed significance (p<0.05), a
Dunnett's test was performed for multiple comparisons. If ANOVA
showed no significance (p>0.05), statistical tests ended. In the
case of heterogeneity of variance at p<0.05, a Kruskal-Wallis
nonparametric test was performed. If the Kruskal-Wallis
nonparametric test showed significance (p<0.05), a further
Mann-Whitney test was performed for multiple comparisons.
[0346] The total treatment-related ocular reaction grading scores
(cornea, iris, conjunctiva, chemosis and discharge) of each
animal's each eye at each examination time point were added, the
group average scores (GAS) at each time point were calculated, and
eventually eye irritation was classified based on the method
described in Table 5.
TABLE-US-00005 TABLE 5 Criteria of Eye Irritation Scores Score
Range Classification 0-3 No Irritation 4-8 Mild Irritation 9-12
Medium Irritation 13-16 Severe Irritation
[0347] The treatment-related eye irritation effects of test
composition/vehicle were evaluated in conjunction with daily
observations, the nature and severity of lesions, and their
reversibility or lack of reversibility.
[0348] 9.2 Results (Clinical Observations)
[0349] No mortality and abnormal clinical signs were noted in any
animal throughout the study.
[0350] 9.3 Results (Body Weight) Body weight are summarized in FIG.
6. No statistically significant difference in body weights was
noted between all 3 groups on Days -1, 7, 14, and 17
(p>0.05).
[0351] 9.4 Results (Ophthalmic Observation-Slit Lamp
Examination)
[0352] The eye irritation scores in both eyes of all animals from
each group were zero throughout the study. According to the
Criteria of Eye Irritation Scores, the eye irritation of all
animals from each group at each examination time point was
classified as no irritation.
[0353] 9.5 Results (Ophthalmic Observation--Sodium Fluorescein
Examination) Staining score of corneal sodium fluorescein are
summarized in Tables 6-12.
[0354] During the 14-day dosing period and the non-dosing
observation period, some animals in the saline group, test
composition group and artificial tear+test composition group were
observed with sodium fluorescein staining in the cornea of
unilateral or bilateral eyes, and the staining scores were mostly
1, only 1 left eye of test composition group scored 2 on Day 3. And
at the end of observation, the corneal fluorescence staining score
of all eyes was 0. There were no statistical differences
(p>0.05) in the corneal fluorescence staining scores of eyes
between the 3 groups except that the left eye scores of saline
group is significantly higher than that of test composition group
and artificial tears+test composition group on Day 9 (saline group:
1.00.+-.0.00 points, test composition group: 0.25.+-.0.50 points,
artificial tears+test composition group, 0.00.+-.0.00; p<0.05).
The corneal staining difference between groups mentioned above was
lack of time-response relationship, and was considered to be not
associated with the use of test composition or the combined-use of
artificial tears and test composition.
TABLE-US-00006 TABLE 6 Summary of Corneal Sodium Fluorescein
Staining Score (Day -1 to Day 2) Dosing D -1 D 1 D 2 Group
Treatment OD OS OD OS OD OS 1 Saline Mean .+-. SD 0.00 .+-. 0.00
0.00 .+-. 0.00 0.00 .+-. 0.00 0.25 .+-. 0.50 0.00 .+-. 0.00 0.00
.+-. 0.00 n 4 4 4 4 4 4 2 Biodecs001 Mean .+-. SD 0.00 .+-. 0.00
0.00 .+-. 0.00 0.00 .+-. 0.00 0.50 .+-. 0.58 0.00 .+-. 0.00 0.50
.+-. 0.58 eye drops n 4 4 4 4 4 4 3 Artificial Mean .+-. SD 0.00
.+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-.
0.00 0.25 .+-. 0.50 tears * + n 4 4 4 4 4 4 Biodecs001 eye drops
Note: "n" means the number of animal eyes.
TABLE-US-00007 TABLE 7 Summary of Corneal Sodium Fluorescein
Staining Score (Day 3 to Day 5) Dosing D 3 D 4 D 5 Group Treatment
OD OS OD OS OD OS 1 Saline Mean .+-. SD 0.50 .+-. 0.58 0.75 .+-.
0.50 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 n
4 4 4 4 4 4 2 Biodecs001 Mean .+-. SD 0.00 .+-. 0.00 1.00 .+-. 0.82
0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.25 .+-. 0.50 eye
drops n 4 4 4 4 4 4 3 Artificial Mean .+-. SD 0.25 .+-. 0.50 0.00
.+-. 0.00 0.25 .+-. 0.50 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-.
0.00 tears * + n 4 4 4 4 4 4 Biodecs001 eye drops Note: "n" means
the number of animal eyes.
TABLE-US-00008 TABLE 8 Summary of Corneal Sodium Fluorescein
Staining Score (Day 6 to Day 8) Dosing D 6 D 7 D 8 Group Treatment
OD OS OD OS OD OS 1 Saline Mean .+-. SD 0.00 .+-. 0.00 0.00 .+-.
0.00 0.75 .+-. 0.50 0.50 .+-. 0.58 0.00 .+-. 0.00 0.00 .+-. 0.00 n
4 4 4 4 4 4 2 Biodecs001 Mean .+-. SD 0.00 .+-. 0.00 0.00 .+-. 0.00
0.25 .+-. 0.50 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 eye
drops n 4 4 4 4 4 4 3 Artificial Mean .+-. SD 0.00 .+-. 0.00 0.00
.+-. 0.00 1.00 .+-. 0.00 0.50 .+-. 0.58 0.00 .+-. 0.00 0.00 .+-.
0.00 tears * + n 4 4 4 4 4 4 Biodecs001 eye drops Note: "n" means
the number of animal eyes.
TABLE-US-00009 TABLE 9 Summary of Corneal Sodium Fluorescein
Staining Score (Day 9 to Day 11) Dosing D 9 D 10 D 11 Group
Treatment OD OS OD OS OD OS 1 Saline Mean .+-. SD 0.50 .+-. 0.58
1.00 .+-. 0.00 0.00 .+-. 0.00 0.50 .+-. 0.58 0.00 .+-. 0.00 0.75
.+-. 0.50 n 4 4 4 4 4 4 2 Biodecs001 Mean .+-. SD 0.00 .+-. 0.00
0.25 .+-. 0.50* 0.25 .+-. 0.50 0.25 .+-. 0.50 0.25 .+-. 0.50 0.25
.+-. 0.50 eye drops n 4 4 4 4 4 4 3 Artificial Mean .+-. SD 0.00
.+-. 0.00 0.00 .+-. 0.00* 0.00 .+-. 0.00 0.25 .+-. 0.50 0.00 .+-.
0.00 0.00 .+-. 0.00 tears * + n 4 4 4 4 4 4 Biodecs001 eye drops
Note: "n" means the number of animal eyes.
TABLE-US-00010 TABLE 10 Summary of Corneal Sodium Fluorescein
Staining Score (Day 12 to Day 14) Dosing D 12 D 13 D 14-before
dosing Group Treatment OD OS OD OS OD OS 1 Saline Mean .+-. SD 0.25
.+-. 0.50 0.75 .+-. 0.50 0.25 .+-. 0.50 0.50 .+-. 0.58 0.00 .+-.
0.00 0.50 .+-. 0.58 n 4 4 4 4 4 4 2 Biodecs001 Mean .+-. SD 0.00
.+-. 0.00 0.25 .+-. 0.50 0.00 .+-. 0.00 0.50 .+-. 0.58 0.50 .+-.
0.58 0.00 .+-. 0.00 eye drops n 4 4 4 4 4 4 3 Artificial Mean .+-.
SD 0.00 .+-. 0.00 0.25 .+-. 0.50 0.00 .+-. 0.00 0.25 .+-. 0.50 0.00
.+-. 0.00 0.25 .+-. 0.50 tears * + n 4 4 4 4 4 4 Biodecs001 eye
drops Note: "n" means the number of animal eyes.
TABLE-US-00011 TABLE 11 Summary of Corneal Sodium Fluorescein
Staining Score (Day 14) D 14-1 hours D 14-2 hours D 14-4 hours
Dosing after dosing after dosing after dosing Group Treatment OD OS
OD OS OD OS 1 Saline Mean .+-. SD 0.00 .+-. 0.00 0.00 .+-. 0.00
0.00 .+-. 0.00 0.50 .+-. 0.58 0.00 .+-. 0.00 0.25 .+-. 0.50 n 4 4 4
4 4 4 2 Biodecs001 Mean .+-. SD 0.00 .+-. 0.00 0.00 .+-. 0.00 0.25
.+-. 0.50 0.00 .+-. 0.00 0.00 .+-. 0.00 0.25 .+-. 0.50 eye drops n
4 4 4 4 4 4 3 Artificial Mean .+-. SD 0.00 .+-. 0.00 0.25 .+-. 0.50
0.00 .+-. 0.00 0.25 .+-. 0.50 0.00 .+-. 0.00 0.00 .+-. 0.00 tears *
+ n 4 4 4 4 4 4 Biodecs001 eye drops Note: "n" means the number of
animal eyes.
TABLE-US-00012 TABLE 12 Summary of Corneal Sodium Fluorescein
Staining Score (Day 15 to Day 17) Dosing D 15 D 16 D 17 Group
Treatment OD OS OD OS OD 0 1 Saline Mean .+-. SD 0.00 .+-. 0.00
0.25 .+-. 0.50 0.25 .+-. 0.50 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00
.+-. 0.00 n 4 4 4 4 4 4 2 Biodecs001 Mean .+-. SD 0.00 .+-. 0.00
0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00
.+-. 0.00 eye drops n 4 4 4 4 4 4 3 Artificial Mean .+-. SD 0.00
.+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-.
0.00 0.00 .+-. 0.00 tears * + n 4 4 4 4 4 4 Biodecs001 eye drops
Note: "n" means the number of animal eyes.
[0355] 9.6 Conclusion
[0356] No mortality and abnormal clinical signs were noted in any
animals throughout the study. No statistically significant
difference in body weights was noted between all 3 groups on Days
-1, 7, 14, and 17.
[0357] The eye irritation scores in both eyes of all animals from
each group were zero throughout the study. According to the
Criteria of Eye Irritation Scores, the eye irritation of all
animals from each group at each examination time point was
classified as no irritation. Sodium fluorescein staining in the
cornea of unilateral or bilateral eyes was observed in some animals
from all 3 groups during the study, The staining scores were mostly
1 and without time-response relationship, thus the corneal staining
signs were considered to be not associated with the use of
Biodecs001 eye drops or the combined-use of artificial tears and
Biodecs001 eye drops.
[0358] Under the conditions of this study, Biodecs001 topically
administered once daily to New Zealand White Rabbits for 14
consecutive days, either alone or after the instillation of
artificial tears, did not cause eye irritation.
Example 10
[0359] Metabolite Analysis of Coconut Oil Samples
[0360] 10.1 Sample preparation, instrumental parameters and data
analysis
[0361] A total of 20 coconut oil samples (divided into two groups:
unprocessed coconut oil group and the deacidified coconut oil
group) were tested by LC-MS with positive and negative ion modes,
and compared between groups according to the test data. The
analysis is designed primarily to analyze small, polar metabolites
such as amino acids, nucleic acids, sugars and small organic acids
that are typically part of primary metabolism.
[0362] The samples (100 .mu.L) were transferred into a 1.5 mL
centrifuge tube, then 300 .mu.L methanol and 10 .mu.L
dichlorophenylalanine (2.8 mg/mL) were added to the tube. Next, the
tube was vortexed for 30 seconds and incubated at -20.degree. C.
for 1 hour. Next, the tube was centrifuged at 12,000 RPM (4.degree.
C.) for 15 minutes. After the centrifugation, 200 .mu.L supernatant
was transferred to a vial for inspection.
[0363] LC-MS instrument platform (Thermo, Ultimate 3000LC, Q
Exactive) and chromatographic column Hyper gold C18 (100.times.2.1
mm 1.9 .mu.m) were used in chromatographic separation under
conditions of the follows: column temperature: 40.degree. C.; flow
rate: 0.35 mL/min; mobile phase A: water+5% acetonitrile+0.1%
formic acid; mobile phase B: acetonitrile+0.1% formic acid;
injection volume: 10 .mu.L; Automatic injector temperature:
4.degree. C. Mobile phase gradient elution procedure is shown in
Table 13.
TABLE-US-00013 TABLE 13 Mobile phase gradient elution procedure
Time Flow rate A B (min) (mL/min) (%) (%) 0 0.3 100 0 0 0.3 100 0
1.5 0.3 80 20 9.5 0.3 0 100 14.5 0.3 0 100 14.6 0.3 100 0 18 0.3
100 0
[0364] Mass spectrometry analysis was performed using the following
parameters:
[0365] ESI+: Heater Temp 300.degree. C.; Sheath Gas Flow rate,
45arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1arb; spray
voltage, 3.0 KV; Capillary Temp, 350.degree. C.; S-Lens RF Level,
30%. ESI-: Heater Temp 300.degree. C., Sheath Gas Flow rate, 45arb;
Aux Gas Flow Rate, 15arb; Sweep Gas Flow Rate, 1arb; spray voltage,
3.2 KV; Capillary Temp, 350.degree. C.; S-Lens RF Level, 60%. Scan
mode: Full Scan (m/z 70.about.1050) and data-dependent second-order
mass spectrometry scanning (dd-ms2, TopN=10); Resolution: 70,000
(MS1) & 17,500 (MS2). Collision mode: high energy collision
dissociation (HCD).
[0366] The data was analyzed by performing feature extraction and
preprocessed with compound discoverer software (Thermo), and then
normalized and edited into two-dimensional data matrix by excel
2010 software, including molecular weight, retention time (RT) and
peak intensity. The data after editing were performed Multivariate
Analysis (MVA) using SIMCA-P software (Umetrics AB, Umea,
Sweden).
[0367] 10.2 Chromatogram
[0368] The total ion chromatogram (TIC) of QC samples was
overlapped, as shown in FIGS. 7-8, which showed that the retention
time reproducibility of the instrument was good and the instrument
was stable, so the results of instrument analysis and data had high
reliability. A sample of TIC (FIGS. 9-14) for each group of samples
is listed separately.
[0369] QC sample is the mixture of different and equal samples
after sample extraction. The QC sample was injected after a certain
number of sample detections. The stability of the instrument can be
investigated through the overlapping of the QC chromatogram. (ESI+)
represents the positive ion detection mode, that is, in the
detection process, the mass analyzer only scans the positive ion
and filters out the negative ion, so as to obtain the information
of positive ion; (ESI-) represents the mode of negative ion
detection, that is, during the detection process, the mass analyzer
only scans negative charge ions and filters out positive charge
ions, so as to obtain information of negative charge ions.
[0370] 10.3 PCA analysis of all samples
[0371] Principal component analysis was carried out on the sample
reflecting the overall differences between groups of samples and
the size of the group in the degree of variation between samples.
Before using SMICA-P software to perform the analysis, the data set
was normalized in order to obtain more intuitive and reliable
results. The purpose of normalization is to make the variable scale
(a digital features, such as mean and standard deviation) on the
same level. This prevents the signals of certain ingredients that
are too high or too low from masking other differences.
[0372] In order to distinguish whether there were differences
between the two groups, the PCA modeling method was used to analyze
the samples. In this analysis, a total of 3 principal components
were obtained in the positive mode, with cumulative R.sup.2X=0.715
and Q.sup.2=0.605. In the negative mode, a total of 2 principal
components were obtained, with cumulative R.sup.2X=0.565 and
Q.sup.2=0.467. PCA Scores plot under ESL+ and ESL- modes are shown
in FIG. 15 and FIG. 16, respectively.
[0373] Principal component analysis was conducted on the Prco group
(the deacidified coconut oil group) and the Orco group (the
original coconut oil group). In this analysis, a total of 2
principal components were obtained in the positive model, with
cumulative R.sup.2X=0.718 and Q.sup.2=0.605. In the negative mode,
a total of 2 principal components were obtained, with cumulative
R.sup.2X=0.587 and Q.sup.2=0.461. PCA Scores plot under ESL+ and
ESL- modes are shown in FIG. 17 and FIG. 18, respectively.
[0374] 10.4 PLS-DA analysis
[0375] In order to obtain the ingredient information leading to
this significant difference, the supervised multidimensional
statistical method, or partial least squares discriminant analysis
(PLS-DA) was used to conduct statistical analysis on the two groups
of samples.
[0376] The model parameters were as follows: in positive mode,
there were two principal components, R.sup.2X=0.713,
R.sup.2Y=0.988, and Q.sup.2=0.97. In the negative mode, there were
two principal components, R.sup.2X=0.502, R.sup.2Y=0.917,
Q.sup.2=0.919 (FIGS. 19A-19B, 20A-20B).
[0377] The main parameters to determine the quality of the model
are R.sup.2Y (which represents the model's interpretation rate) and
Q.sup.2 (which represents the model's prediction rate). In
addition, the model was evaluated to see whether the model is
"overfitting". From the model parameters, the model was reliable in
explaining the differences between the two groups and searching for
different substances, and there was no "overfitting" in the model
from the sorting verification diagram.
[0378] The "overfitting" of the model reflects the accuracy of the
modeling. If the model is not "overfitting", it indicates that the
model can describe the sample well and can be used to find the
biomarker.
[0379] 10.5 OPLS-DA analysis
[0380] Further, the supervised method OPLS-DA was used. As a
result, one principal component and one orthogonal component were
obtained in the positive mode, R.sup.2X=0.713, R.sup.2Y=0.988, and
Q.sup.2=0.973. In the negative mode, one principal component and
one orthogonal component are obtained, R.sup.2X=0.502,
R.sup.2Y=0.979, and Q.sup.2=0.945. The model parameter R.sup.2Y
represents the model interpretation rate, and Q.sup.2 represents
the model prediction rate. Their scores are shown in FIGS.
21-22.
[0381] 10.6 Differential ingredients between groups
[0382] Qualitative analysis was conducted by using compound
discoverer and finally the information of differential ingredients
was obtained. After that, VIP (Variable Importance in the
Projection) value and t-test was combined to look for differential
expression of ingredients. ingredients with VIP value greater than
1 and p value less than 0.05 were considered as differential.
[0383] 10.7 Summary
[0384] In the positive mode, the contents of 3-hexenoic acid, 5,
8-tetradecadienoic acid and Indole in the products as compared to
the same amount of original coconut oil sample were significantly
reduced (fold change <-7). Cis-9-palmitoleic acid, PA(10:0/21:0)
and Stearamide have relatively stable changes. Campest-4-en-3-one
and Stigmastentriol contents increased significantly (fold change
>7). In general, steroidal lipids (Stigmasterol,
Stigmast-22-ene-3,6-dione, Stigmastentriol) and vitamins
(ubiquinone-4, Vitamin D3) increased in the products. Amino acids
(isolecucine, valine, glutamate, beta-alanine) were reduced after
processing.
[0385] In the negative mode, the content of sucrose, citric acid
decreased by about 7 times, and the concentration of
Piperochromenoic acid, LysoPA(a-25:0/0:0) and LysoPA(24:0/0:0) was
significantly increased (fold change>5). In general, sucrose,
mannitol, sucrose-6-glucose and glucose are lower in the product
than in the original coconut oil sample (See FIGS. 43-44 for
details).
Example 11
[0386] Lipidomics Analysis of Samples
[0387] 11.1 Sample preparation, instrumental parameters and data
analysis
[0388] A total of 20 coconut oil samples (divided into two groups:
the product group (deacidified coconut oil) and the stock solution
group (original coconut oil)) were tested by LC-MS with positive
and negative ion modes, and compared between groups according to
the test data.
[0389] The samples were dissolved in 25.degree. C. water bath pot
and transferred to a 10 mL centrifuge tube. Next, 1.5 mL
chloroform/methanol (2/1, v/v) solution was added to 100 .mu.L
samples and vortexed for 1 minute. Next, the organic phase (800
.mu.L) was transferred to a clean test tube, dried with nitrogen
gas (N2), and then dissolved with 200 .mu.L isopropanol/methanol
(1/1, v/v) for 1 minute. Next, 10 .mu.L LPC (12:0) with
concentration of 125 .mu.g/mL and 10 .mu.L TG internal standard
with concentration of 125 .mu.g/mL were added to the tube and
vortexed for 30 seconds. Next, supernatant (200 .mu.L) from the
tube was transferred to a vial for inspection.
[0390] LC-MS instrument platform (Thermo, Ultimate 3000LC, Q
Exactive) and chromatographic column Hyper gold C18 (100-2.1 mm 1.9
.mu.m) were used in chromatographic separation under conditions of
the follows: column temperature: column temperature: 50.degree. C.;
Flow rate: 0.3 mL/min; mobile phase composition A: ACN: water
(60:40, V/V), including 10 mmol/L ammonium acetate, B: ACN:
Isopropanol (10:90, V/V), including 10 mmol/L ammonium acetate;
injection volume: 5 .mu.L; automatic sampler temperature 10.degree.
C.
[0391] Mobile phase gradient elution procedure is shown in Table
14.
TABLE-US-00014 TABLE 14 Mobile phase gradient elution procedure
Time A B (min) (%) (%) 0 70 30 10.5 0 100 12.50 0 100 12.51 70 30
16 70 30
[0392] Mass spectrometry analysis was performed using the following
parameters: ESI+: Sheath gas velocity: 35 arb; Auxiliary gas
velocity: 15 arb; Purge velocity: 1 arb; Electrospray voltage: 3000
V; Capillary temperature: 350.degree. C.; Gasification temperature:
350.degree. C. S-lens RF Level, 50%. ESI-: Sheath gas velocity: 35
arb; Auxiliary gas velocity: 15 arb; Purge velocity: 1 arb;
Electrospray voltage: 2800 V; Capillary temperature: 350.degree.
C.; Gasification temperature: 350.degree. C. S-lens RF Level, 50%.
Scan mode: Full Scan (ESI+: m/z 250.about.1500; ESI-: m/z
200.about.1500) and data-dependent second-order mass spectrometry
scanning (dd-ms2, TopN=10); Resolution: 70,000 (MS1) & 17,500
(MS2). Collision mode: high energy collision dissociation
(HCD).
[0393] The data was analyzed by feature extraction and preprocessed
with Lipid Search software (Thermo), and then normalized and edited
into two-dimensional data matrix by Excel 2010 software, including
LipidIon, Class, Fatty acid chains (Fatty Acid, FA1,FA2,FA3),
CalcMz, IonFormula, Retention time (RT) and peak intensity. The
data after editing were analyzed by Multivariate Analysis (MVA)
using SIMCA-P software (Umetrics AB, Umea, Sweden).
[0394] 11.2 Chromatogram
[0395] The total ion chromatogram (TIC) of QC samples was
overlapped, as shown in FIGS. 23-24, which shows that the retention
time reproducibility of the instrument was good and the instrument
was stable, so the results of instrument analysis and data have
high reliability. A sample of TIC (FIGS. 25-30) for each group of
samples and QC is listed separately.
[0396] QC is the mixture of different and equal samples after
sample extraction. A small amount of QC was injected after
analyzing a certain number of samples. The reliability of the
instrument can be monitored through the overlapping of QC
chromatogram. ESI+represents the positive ion detection mode, that
is, in the detection process, the mass analyzer only scans the
positive ion and filters out the negative ion, so as to obtain the
information of positive ion. ESI- represents the mode of negative
ion detection, that is, during the detection process, the mass
analyzer only scans negative charge ions and filters out positive
charge ions, so as to obtain information of negative charge
ions.
[0397] 11.3 PCA analysis of all samples
[0398] Principal component analysis (PCA) was performed to analyze
the overall differences between groups of samples and the variation
between samples. Before using SMICA-P software for PCA analysis,
the dataset was normalized in order to obtain more reliable
results.
[0399] In order to distinguish whether there are differences
between the two groups, we used the PCA modeling method to analyze
the samples. In this analysis, a total of two principal components
were obtained in the positive mode, with cumulative R.sup.2X=0.316
and Q.sup.2=0.198. In the negative mode, a total of 3 principal
components were obtained, with cumulative R.sup.2X=0.605 and
Q.sup.2=0.32. PCA Scores plot under ESL+ and ESL- modes are shown
in FIG. 31 and FIG. 32, respectively.
[0400] Principal component analysis was conducted on Prco group
(the product group) and the Orco group (the original coconut oil
group). In this analysis, a total of 2 principal components were
obtained in the positive model, with cumulative R.sup.2X=0.355 and
Q.sup.2=0.16. In the negative mode, a total of 3 principal
components were obtained, with cumulative R.sup.2X=0.644 and
Q.sup.2=0.347. PCA Scores plot under ESL+ and ESL- modes are shown
in FIG. 33 and FIG. 34, respectively.
[0401] 11.4 PLS-DA analysis
[0402] In order to obtain the ingredient information leading to
this significant difference, the supervised multidimensional
statistical method, or partial least squares discriminant analysis
(PLS-DA) was used.
[0403] The model parameters are as follows: in positive mode, there
are two principal components, R.sup.2X=0.344, R.sup.2Y=0.998, and
Q2=0.962. In the negative mode, R.sup.2X=0.478,, R.sup.2Y=0.973,
Q2=0.889 (FIGS. 35A-35B, and 36A-36B).
[0404] The main parameters to determine the quality of the modeling
are R.sup.2Y (which represents the model's interpretation rate) and
Q.sup.2 (which represents the model's prediction rate). In
addition, whether the model is "overfitting" was also assessed.
From the model parameters, the model is reliable in explaining the
differences between the two groups and searching for different
substances, and there is no "overfitting" in the modeling from the
verification diagram.
[0405] The "overfitting" of the model reflects the accuracy of the
model construction. If the model is not "overfitting", it indicates
that the model can describe the sample well and can be used to find
the most relevant biomarker.
[0406] 11.5 OPLS-DA analysis
[0407] Further, the supervised method OPLS-DA was used. As a
result, one principal component and one orthogonal component were
obtained in the positive mode, R.sup.2X=0.344, R.sup.2Y=0.998, and
Q.sup.2=0.962. In the negative mode, one principal component and
one orthogonal component were obtained, R.sup.2X=0.478,
R.sup.2Y=0.973, and Q.sup.2=0.875. The model parameter R.sup.2Y
represents the model interpretation rate, and Q.sup.2 represents
the model prediction rate. Their scores are shown in FIGS.
37-38.
[0408] 11.6 Differential lipids between groups
[0409] Qualitative analysis was conducted by using Lipid search,
and finally the information of differential ingredients was
obtained, including the types of differential lipids, the change in
the chain length of each molecule of differential lipids and the
number of unsaturated bonds. After that, VIP value and t-test was
combined to look for differential expression of lipid ingredients.
Lipids with VIP value greater than 1 and p value less than 0.05
were considered as differential lipids. The data of different
ingredients are shown in FIGS. 45-46.
[0410] 11.7 Summary
[0411] In this project, 12 classes of lipids were detected at ESI+
in product samples, which are cer (Ceramides,
0.00256864604914964%), CerG1(Simple Glc series,
0.000121562094094825%), ChE(Cholesteryl Ester,
0.000027195581262643%), Co(Coenzyme, 0.000165799851752065%),
DG(diglyceride, 0.204618368597593%), LPC(lysophosphatidylcholine,
0.000691245372867429%), LPE(lysophosphatidylethanolamine,
4.39688799919827E-06%), PE(phosphatidylethanolamine,
0.0000221371777288954%), So(Sphingoshine, 0.00800073222443555%),
StE(Stigmasteryl ester, 6.13493501356116E-06%), TG (triglyceride,
99.7836547325718%), ZyE(zymosteryl, 0.000119048656356883%). In Orco
sample, 11 classes of lipids were detected, which are cer
(Ceramides, 0.00305724291322289%), CerG1(Simple Glc series,
0.000296999634909488%), ChE(Cholesteryl Ester,
0.000007562648073146%), Co(Coenzyme, 0.00106715223387679%),
DG(cdiglyceride, 0.102091925929773%), LPC(lysophosphatidylcholine,
0.00192954289175979%), LPE(lysophosphatidylethanolamine,
0.000206199217706794%), PE(phosphatidylethanolamine,
0.000994781308036394%), So(Sphingoshine, 0.00778245724664204%),
TG(triglyceride, 99.8825339050066%), ZyE(zymosteryl,
0.0000322309694142598%).
[0412] At ESI- mode, 21 classes of lipids were detected, which are
DGDG(Digalactosyldiacylglycerol, 0.00337850020676684%),
DGMG(Digalactosylmonoacylglycerol, 0.283485348778992%),
LPC(lysophosphatidylcholine, 0.278254986476203%),
LPE(lysophosphatidylethanolamine, 0.00556230615677608%),
LPG(lysophosphatidylglycerol, 0.00243740463723581%),
LPI(lysophosphatidylinositol, 0.00152071252159812%),
LPMe(lysophosphatidylmethanol, 0.0246170259125403%),
LdMePE(lysodimethylphosphatidylethanolamine, 0.0246170259125403%),
MGDG(Monogalactosyldiacylglycerol, 0.119679045099086%),
MGMG(Monogalactosylmonoacylglycerol, 2.12694214959191%),
OAHFA((O-acyl)-1-hydroxy fatty acid, 0.815004998839083%),
PA(phosphatidic acid, 21.2775572879869%), PAF(platelet-activating
factor, 52.3844436814582%), PC(phosphatidylcholine,
0.996419719716529%), PE(phosphatidylethanolamine,
0.769816735887086%), PEt(phosphatidylethanol, 5.23724828345888%),
PG(phosphatidylglycerol, 0.150818577744567%),
PI(phosphatidylinositol, 0.224349306155231%),
PMe(phosphatidylmethanol, 4.04027226374681%), cPA(cyclic
phosphatidic acid, 0.00012119001517959%),
dMePE(dimethylphosphatidylethanolamine, 0.009913724165893%).
[0413] In Orco samples, 22 classes of lipids were detected, which
are CL (Cardiolipin, 0.0205841044276305%)
DGDG(Digalactosyldiacylglycerol, 0.291159900494563%),
DGMG(Digalactosylmonoacylglycerol, 0.0960936390316132%),
LPC(lysophosphatidylcholine, 0.303192132840516%),
LPE(lysophosphatidylethanolamine, 0.104579947187398%),
LPG(lysophosphatidylglycerol, 0.00534666996716351%),
LPI(lysophosphatidylinositol, 0.0443782066618173%),
LPMe(lysophosphatidylmethanol, 0.00138869574447737%),
LdMePE(lysodimethylphosphatidylethanolamine, 4.15813005466302%),
MGDG(Monogalactosyldiacylglycerol, 1.57435383259576%),
MGMG(Monogalactosylmonoacylglycerol, 0.662589282973412%),
OAHFA((O-acyl)-1-hydroxy fatty acid, 0.864712469689971%),
PA(phosphatidic acid, 26.935329672718%), PAF(platelet-activating
factor, 18.8938270646209%), PC(phosphatidylcholine,
14.5455100823086%), PE(phosphatidylethanolamine,
7.08341135985417%), PEt(phosphatidylethanol, 10.8236884649902%),
PG(phosphatidylglycerol, 0.934358404427319%),
PI(phosphatidylinositol, 2.67410886686788%),
PMe(phosphatidylmethanol, 9.83226175604876%), cPA(cyclic
phosphatidic acid, 0.00785180275211569%),
dMePE(dimethylphosphatidylethanolamine, 0.143143589134811%).
[0414] At ESI+ mode, after been processed, the percentage of 5
classes (LPE, PE, Co, LPC, CerG1) descended of lipids in Orco
samples; meanwhile 3 lipids (Cer, TG, So) kept stable; while 4
lipid classes (DG, ChE, ZyE and StE) increased significantly. At
ESI - mode, after been processed, the percentage of 13 classes
(DGDG, cPA, LPI, LPE, PC, dMePE, MGDG, PI, PE, PG, PMe, LPG and
PEt) descended of lipids in Orco samples; meanwhile 3 lipids (PA,
LPC and OAHFA) kept stable; while 6 lipid classes (LdMePE, PAF,
DGMG, MGMG, LPMe and CL) increased significantly.
[0415] The percentage of lipid classes in the product sample
(deacidified coconut oil) at ESI+ and ESI- modes are shown in
Tables 15-16 and FIGS. 39-42. Detailed lists of each lipid class at
ESI+ and ESI- modes are shown in FIGS. 45-46.
TABLE-US-00015 TABLE 15 Percentage of lipid classes in product
sample (ESI+) Class Pcro Ocro Proc/Orco Fold change LPE 0.00000%
0.0002062% 0.02 -5.55 PE 0.00002% 0.0009948% 0.02 -5.49 Co 0.00017%
0.0010672% 0.16 -2.69 LPC 0.00069% 0.0019295% 0.36 -1.48 CerG1
0.00012% 0.0002970% 0.41 -1.29 Cer 0.00257% 0.0030572% 0.84 -0.25
TG 99.78365% 99.8825339% 1.00 0.00 So 0.00800% 0.0077825% 1.03 0.04
DG 0.20462% 0.1020919% 2.00 1.00 ChE 0.00003% 0.0000076% 3.60 1.85
ZyE 0.00012% 0.0000322% 3.69 1.89 StE 0.00001% 0.0000000% -- --
TABLE-US-00016 TABLE 16 Percentage of lipid classes in product
sample (ESI-) Class Pcro Oreo Prco/Orco Fold change DGDG 0.00%
0.291% 0.011604 -6.42928 cPA 0.00% 0.008% 0.015435 -6.01768 LPI
0.00% 0.044% 0.034267 -4.86703 LPE 0.01% 0.105% 0.053187 -4.23278
PC 1.00% 14.546% 0.068504 -3.86768 dMePE 0.01% 0.143% 0.069257
-3.85189 MGDG 0.12% 1.574% 0.076018 -3.71752 PI 0.22% 2.674%
0.083897 -3.57524 PE 0.77% 7.083% 0.108679 -3.20186 PG 0.15% 0.934%
0.161414 -2.63116 PMe 4.04% 9.832% 0.41092 -1.28307 LPG 0.00%
0.005% 0.455873 -1.13329 PEt 5.24% 10.824% 0.483869 -1.04731 PA
21.28% 26.935% 0.78995 -0.34017 LPC 0.28% 0.303% 0.917751 -0.12382
OAHFA 0.82% 0.865% 0.942516 -0.08541 LdMePE 11.25% 4.158% 2.7051
1.435682 PAF 52.38% 18.894% 2.772569 1.471223 DGMG 0.28% 0.096%
2.950095 1.560761 MGMG 2.13% 0.663% 3.210046 1.682594 LPMe 0.02%
0.001% 17.72672 4.147854 CL 0.00% 0.021% 0 --
[0416] Human meibum samples (eyelid samples) were also analyzed. 11
classes of lipids in both product samples and eyelid samples were
detected at ESI+ mode, which are DG(diglyceride), TG(triglyceride),
So(Sphingoshine), LPC(lysophosphatidylcholine), Cer(Ceramides),
ZyE(zymosteryl), LPE(lysophosphatidylethanolamine), CerG1(Simple
Glc series), ChE(Cholesteryl Ester), PE(phosphatidylethanolamine)
and StE(Stigmasteryl ester). At ESI- mode, 22 classes of lipids in
both product samples and eyelid samples were detected, which are
PE(phosphatidylethanolamine), PEt(phosphatidylethanol),
MGMG(Monogalactosyldiacylglycerol), cPA(cyclic phosphatidic acid),
PE(phosphatidylethanolamine), PI(phosphatidylinositol),
DGDG(Digalactosyldiacylglycerol),
MGDG(Monogalactosyldiacylglycerol),
LdMePE(lysodimethylphosphatidylethanolamine),
LPE(lysophosphatidylethanolamine), LPI(lysophosphatidylinositol),
LPC(lysophosphatidylcholine), LPMe(lysophosphatidylmethano),
LPG(lysophosphatidylglycerol), PG(phosphatidylglycerol),
OAHFA((O-acyl)-1-hydroxy fatty acid), PA(phosphatidic acid),
dMePE(dimethylphosphatidylethanolamine), PC(phosphatidylcholine),
DGMG(Digalactosylmonoacylglycerol), PMe(phosphatidylmethanol) and
PAF(platelet-activating factor).
Example 12
[0417] Cyclosporin Solubility Test
[0418] Solubility of cyclosporine by different organic solvents was
tested. The experiment was performed as follows.
[0419] Cyclosporin (3.0 mg, white solid powder) was added to 1.2 mL
of deacidified coconut oil. After incubation in 42.degree. C. water
bath, the white solid powder was dissolved as turbid liquid. Next,
the solution was mixed by shaking and then put in 42.degree. C.
water bath for 10 minutes, followed by sonication for 15 minutes.
As shown in FIG. 50A, cyclosporine was dissolved in the sample.
[0420] Cyclosporin (25 mg) was added to 1.0 mL of DMSO (clear and
transparent liquid). The solution was mixed by shaking, followed by
sonication for 15 minutes. As shown in FIG. 50B, cyclosporine was
dissolved in DMSO at a concentration of 2.5% as clear and
transparent liquid. In addition, cyclosporin (50 mg) was added to
1.0 mL of DMSO (clear and transparent liquid). The solution was
mixed by shaking, followed by sonication for 15 minutes. As a
result, cyclosporine was dissolved in DMSO at a concentration of 5%
as clear and transparent liquid.
[0421] Cyclosporin (25 mg) was added to 1.0 mL of olive oil (yellow
transparent liquid). The solution was mixed by shaking, followed by
sonication for 15 minutes. As shown in FIG. 50C, cyclosporine was
dissolved in olive oil at a concentration of 2.5% as clear and
transparent liquid. In addition, cyclosporin (50 mg) was added to
1.0 mL of olive oil (yellow transparent liquid). The solution was
mixed by shaking, followed by sonication for 15 minutes. As a
result, cyclosporine was dissolved in olive oil at a concentration
of 5% as clear and transparent liquid.
[0422] Cyclosporine-DMSO solution (100 .mu.l, 2.5% as described
above) was added to 900 .mu.l deacidified coconut oil. The solution
was mixed by shaking, followed by incubation in 42.degree. C. water
bath for 10 minutes. The obtained solution was clear and
transparent as shown in FIG. 51A. In addition, cyclosporine-DMSO
solution (50 .mu.l, 5% as described above) was added to 950 .mu.l
deacidified coconut oil. The solution was mixed by shaking,
followed by incubation in 42.degree. C. water bath for 10 minutes.
The obtained solution was clear and transparent as shown in FIG.
52A.
[0423] Cyclosporine-olive oil solution (100 .mu.l, 2.5% as
described above) was added to 900 .mu.l deacidified coconut oil.
The solution was mixed by shaking, followed by incubation in
42.degree. C. water bath for 10 minutes. The obtained solution was
clear and transparent as shown in FIG. 51B. In addition,
cyclosporine-DMSO solution (50 .mu.l, 5% as described above) was
added to 950 .mu.l deacidified coconut oil. The solution was mixed
by shaking, followed by incubation in 42.degree. C. water bath for
20 minutes. The obtained solution was clear and transparent as
shown in FIG. 52B.
[0424] Cyclosporine (125 mg) was added to 5 ml of decalcified
coconut oil. The solution was mixed by shaking, then incubated in
42.degree. C. water bath for 10 minutes, followed by sonication for
15 minutes. Next, the sonicated solution was sterilized with a 0.22
.mu.m PVDF filter and 2.5 ml of the filter-sterilized solution was
mixed with 22.5 ml decalcified coconut oil in a 50 ml sterile
centrifuge tube. The obtained solution was light yellow and
transparent as shown in FIGS. 53A-53B.
[0425] Cyclosporine (125 mg) was added to 5 ml of a mixed solution
that contains DMSO and decalcified coconut oil with a volume ratio
of 1:19 (250 .mu.l DMSO mixed with 4.75 ml deacidified coconut
oil). The solution was mixed by shaking, then incubated in
42.degree. C. water bath for 10 minutes, followed by sonication for
15 minutes. Next, the sonicated solution was sterilized with a 0.22
.mu.m PVDF filter and 2.5 ml of the filter-sterilized solution was
mixed with 22.5 ml deacidified coconut oil in a 50 ml sterile
centrifuge tube. The obtained solution was light yellow and
transparent as shown in FIGS. 53C-53D.
[0426] The experiments above showed that cyclosporine can be
dissolved in decalcified coconut oil.
OTHER EMBODIMENTS
[0427] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *