U.S. patent application number 16/041509 was filed with the patent office on 2019-06-27 for methods of treating chronic dry eye disease using c16:1n7-palmitoleate and derivatives thereof.
The applicant listed for this patent is Tersus Life Sciences, LLC. Invention is credited to Lochlainn O'hAimhirgin.
Application Number | 20190192465 16/041509 |
Document ID | / |
Family ID | 54141037 |
Filed Date | 2019-06-27 |
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United States Patent
Application |
20190192465 |
Kind Code |
A1 |
O'hAimhirgin; Lochlainn |
June 27, 2019 |
METHODS OF TREATING CHRONIC DRY EYE DISEASE USING
C16:1n7-PALMITOLEATE AND DERIVATIVES THEREOF
Abstract
The present technology provides methods for preventing or
treating dry eyes in a subject in need thereof. The methods include
administering to the subject an effective amount of a composition
comprising C16:1n7-palmitoleate, its derivatives, pharmaceutically
acceptable salts thereof, or a combination thereof.
Inventors: |
O'hAimhirgin; Lochlainn;
(Mentor, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tersus Life Sciences, LLC |
Bonita Springs |
FL |
US |
|
|
Family ID: |
54141037 |
Appl. No.: |
16/041509 |
Filed: |
July 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15830991 |
Dec 4, 2017 |
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16041509 |
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15583401 |
May 1, 2017 |
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15830991 |
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14731219 |
Jun 4, 2015 |
9668996 |
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15583401 |
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62007782 |
Jun 4, 2014 |
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62008489 |
Jun 5, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/20 20130101;
A61K 38/13 20130101; A61K 31/717 20130101; A61K 31/201 20130101;
A61P 27/02 20180101; A61K 45/06 20130101; A61K 31/231 20130101;
A61K 31/23 20130101; A61K 31/231 20130101; A61K 2300/00 20130101;
A61K 31/23 20130101; A61K 2300/00 20130101; A61K 31/20 20130101;
A61K 2300/00 20130101; A61K 38/13 20130101; A61K 2300/00 20130101;
A61K 31/201 20130101; A61K 2300/00 20130101; A61K 31/717 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 31/201 20060101
A61K031/201; A61K 31/231 20060101 A61K031/231; A61K 45/06 20060101
A61K045/06; A61K 31/23 20060101 A61K031/23 |
Claims
1. A method for preventing or treating dry eyes in a subject in
need thereof comprising administering to the subject an effective
amount of a composition comprising one or more of
C16:1n7-palmitoleate, a C16:1n7-palmitoleate derivative, a
pharmaceutically acceptable salt thereof, or a combination
thereof.
2. The method of claim 1, wherein the composition further comprises
a C16:0-palmitate, a C16:0-palmitate derivative, or a combination
thereof.
3. The method of claim 1, wherein the composition further comprises
a C18:1n9-oleate, a C18:1n9-oleate derivative, or a combination
thereof.
4. The method of claim 1, wherein the subject has been diagnosed
with dry eyes or dry eye syndrome.
5. The method of claim 1, wherein the subject is at risk for dry
eyes or dry eye syndrome.
6. The method of claim 5, wherein the subject is human.
7. The method of claim 1, further comprising simultaneously,
sequentially, or separately administrating at least one additional
therapeutic agent, wherein the at least one additional therapeutic
agent, is selected from the group consisting of cyclosporine
ophthalmic emulsion, antibiotics, and hydroxypropyl cellulose
insert.
8. The method of claim 1, wherein the composition is administered
orally, topically, systemically, intravenously, subcutaneously,
intraperitoneally, or intramuscularly.
9. The method of claim 1, wherein administration of the composition
prevents, treats, or ameliorates one or more signs and/or symptoms
of dry eyes.
10. The method of claim 9, wherein the signs and/or symptoms are
selected from the group consisting of stinging, burning, or
scratchy sensation in the eyes, stringy mucus in or around the
eyes, increased eye irritation from smoke or wind, eye fatigue,
sensitivity to light, eye redness, sensation of foreign object in
eyes, difficulty wearing contact lenses, periods of excessive
tearing, swollen eyes, eye discomfort, eye pain, and blurred
vision.
11. The method of claim 1, wherein the subject displays tear
osmolarity less than 308 mOsms/L.
12. The method of claim 1, wherein the composition is administered
daily for 4 weeks or more.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/830,991, filed Dec. 4, 2017, which is a continuation of U.S.
application Ser. No. 15/583,401, filed May 1, 2017, which is a
continuation of U.S. application Ser. No. 14/731,219, filed Jun. 4,
2015, which claims the benefit of and priority to U.S. Application
No. 62/007,782, filed Jun. 4, 2014, and U.S. Application No.
62/008,489, filed Jun. 5, 2014, the contents of which are
incorporated herein by reference in their entireties.
BACKGROUND
[0002] The following description is provided to assist the
understanding of the reader. None of the information provided or
references cited is admitted to be prior art.
[0003] The term "dry eyes" includes, but is not limited to, dry eye
disease, dry eye syndrome, or Chronic Dry Eye disease (CDE), is a
condition in which there are insufficient tears to lubricate and
nourish the eye. Tears are necessary for maintaining the health of
the surface of the eye and for providing clear vision. With each
blink of the eyelids, tears are spread across the front surface of
the eye, known as the cornea. Tears provide lubrication, reduce the
risk of eye infection, wash away foreign matter in the eye, and
keep the surface of the eyes smooth and clear. Excess tears in the
eyes flow into small drainage ducts, in the inner corners of the
eyelids, which drain in the back of the nose.
[0004] People with dry eyes either do not produce enough tears or
have a poor quality of tears. Without enough tears, the film
protecting the eye can break down, creating dry spots on the
surface of the eye.
[0005] Dry eyes is a chronic disease that can be caused by advanced
age, contact lens wear, certain medications, eye diseases, other
medical conditions, or environmental factors. One type of dry eyes
is caused by decreased tear production due to inflammation. Dry
eyes are a common and often chronic problem, particularly in older
adults.
SUMMARY
[0006] In one aspect, the present technology provide, a method for
preventing or treating dry eyes in a subject in need thereof
comprising administering to the subject an effective amount of a
composition comprising one or more of C16:1n7-palmitoleate, a
C16:1n7-palmitoleate derivative, a pharmaceutically acceptable salt
thereof, or a combination thereof.
[0007] In some embodiments, the composition also includes a
C16:0-palmitate, a C16:0-palmitate derivative, or a combination
thereof.
[0008] In some embodiments, the composition also includes a
C18:1n9-oleate, a C18:1n9-oleate derivative, or a combination
thereof.
[0009] In some embodiments, the subject has been diagnosed with dry
eyes or dry eye syndrome. In some embodiments, the subject is at
risk for dry eyes or dry eye syndrome. In some embodiments, the
subject is human.
[0010] In some embodiments, the method includes simultaneously,
sequentially, or separately administrating at least one additional
therapeutic agent, wherein the at least one additional therapeutic
agent, is selected from the group consisting of cyclosporine
ophthalmic emulsion, antibiotics, and hydroxypropyl cellulose
insert.
[0011] In some embodiments of the method, the composition is
administered orally, topically, systemically, intravenously,
subcutaneously, intraperitoneally, or intramuscularly.
[0012] In some embodiments of the method, administration of the
composition prevents, treats, or ameliorates one or more signs
and/or symptoms of dry eyes. In some embodiments, the signs and/or
symptoms are selected from the group consisting of stinging,
burning, or scratchy sensation in the eyes, stringy mucus in or
around the eyes, increased eye irritation from smoke or wind, eye
fatigue, sensitivity to light, eye redness, sensation of foreign
object in eyes, difficulty wearing contact lenses, periods of
excessive tearing, swollen eyes, eye discomfort, eye pain, and
blurred vision.
[0013] In some embodiments, the subject displays tear osmolarity
less than 308 mOsms/L.
[0014] In some embodiments, the composition is administered daily
for 4 weeks or more.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the patient data collection form that will be
used while conducting the study described in Example 3.
DETAILED DESCRIPTION
[0016] It is to be appreciated that certain aspects, modes,
embodiments, variations and features of the present technology are
described below in various levels of detail in order to provide a
substantial understanding of the present technology.
[0017] The definitions of certain terms as used in this
specification are provided below. Unless defined otherwise, all
technical and scientific terms used herein generally have the same
meaning as commonly understood by one of ordinary skill in the art
to which this present technology belongs.
[0018] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the content clearly dictates otherwise. For example, reference to
"a compound" includes a plurality of compounds.
[0019] As used herein, the terms "approximately" or "about" in
reference to a number are generally taken to include numbers that
fall within a range of 1%, 5%, or 10% in either direction (greater
than or less than) of the number unless otherwise stated or
otherwise evident from the context (except where such number would
be less than 0% or exceed 100% of a possible value).
[0020] As used herein, the "administration" of a composition, an
agent, or drug to a subject includes any route of introducing or
delivering to a subject a compound to perform its intended
function. Administration can be carried out by any suitable route,
including orally, intranasally, parenterally (intravenously,
intramuscularly, intraperitoneally, or subcutaneously), or
topically. Administration includes self-administration and the
administration by another.
[0021] As used herein, the term "composition" includes therapeutic
and dietary formulations including, but not limited to, a dietary
supplement, nutraceutical formulation, or pharmaceutical
formulation. Compositions comprising C16:1n7-palmitoleate include,
but are not limited to, dietary supplements, nutraceutical
formulations, and pharmaceutical compositions. "Pharmaceutically
acceptable composition" refers to a composition that is suitable
for administration to a subject, particularly, a human. Such
compositions include various excipients, diluents, carriers, and
such other inactive agents well known to the skilled artisan. In
another aspect, any of the pharmaceutical compositions, as
described in the published U.S. Patent Application US 2012/0225941,
incorporated herein by reference in its entirety, are provided
where the pharmaceutical compositions include C16:1n7-palmitoleate
or any derivatives thereof.
[0022] The methods described herein utilize compositions that
include C16:1n7-palmitoleate or any one or more derivatives thereof
as described in the published U.S. Patent Application US
2012/0225941, which is incorporated herein by reference in its
entirety. In some embodiments, the C16:1n7-palmitoleate derivative
is C16:1n7-palmitoleic acid. In some embodiments, the
C16:1n7-palmitoleate derivative is cis-C16:1n7-palmitoleic acid. In
some embodiments, the C16:1n7-palmitoleate derivative is a metal
salt (e.g., Na.sup.-, K.sup.+, or Li.sup.+) of
cis-C16:1n7-palmitoleate. In some embodiments, the
C16:1n7-palmitoleate derivative is an ester (e.g.,
(C.sub.1-C.sub.8) alkyl ester, methyl, ethyl, propyl,
monoglyceride, diglyceride, triglyceride, or a combination thereof)
of cis-C16:1n7-palmitoleate. In some embodiments, the
C16:1n7-palmitoleate derivative is a methyl ester, ethyl ester,
propyl ester of cis-C16:1n7-palmitoleate. In some embodiments, the
cis-C16:1n7-palmitoleate ester is the ethyl ester.
[0023] The methods described herein are not limited to any
particular chemical form of C16:1n7-palmitoleate and the compound
may be given to subjects either as an ester, free acid or as a
pharmaceutically acceptable salt.
[0024] As used herein, the term "effective amount" refers to a
quantity sufficient to achieve a desired therapeutic and/or
prophylactic effect, e.g., an amount which results in the
prevention of, or a decrease in, dry eyes or CDE or one or more
symptoms associated with dry eyes or CDE. In the context of
therapeutic or prophylactic applications, the amount of a
composition administered to the subject will depend on the type and
severity of the disease and on the characteristics of the
individual, such as general health, age, sex, body weight,
tolerance to drugs, the degree, severity and type of disease or
medical condition. The skilled artisan will be able to determine
appropriate dosages depending on these and other factors. In some
embodiments, the compositions are administered in combination with
one or more additional therapeutic compounds. In some embodiments,
C16:1n7-palmitoleate (or derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof) is administered to a
subject having or suspected of having one or more signs or symptoms
of dry eyes or CDE or to a subject at risk for dry eyes or CDE. By
way of example, but not by way of limitation, a "therapeutically
effective amount" of C16:1n7-palmitoleate (or derivatives,
pharmaceutically acceptable salts thereof, or a combination
thereof) means levels in which the physiological effects of the
disease or medical condition giving rise to dry eyes or CDE are, at
a minimum, ameliorated. In some embodiments, a therapeutically
effective amount can be given in one or more administrations. In
some embodiments, signs or symptoms of dry eyes or CDE include, but
are not limited to, stinging, burning, or scratchy sensation in the
eyes, stringy mucus in or around the eyes, increased eye irritation
from smoke or wind, eye fatigue, sensitivity to light, eye redness,
sensation of foreign object in eyes, difficulty wearing contact
lenses, periods of excessive tearing, swollen eyes, eye discomfort,
eye pain, and blurred vision (which worsens at the end of the day
or after focusing for a prolonged period).
[0025] As used herein, the term "monoglyceride" refers to a fatty
acid chain, such as C16:1n7-palmitoleate, covalently bonded to a
glycerol molecule through an ester linkage. As used herein, the
term "diglyceride" refers to a fatty acid chain, such as
C16:1n7-palmitoleate, covalently bonded to a glycerol molecule
through an ester linkage, wherein the glycerol molecule is further
bonded to one additional fatty acid chain, which may or may not be
C16:1n7-palmitoleate, though one additional ester linkage. As used
herein, the term "triglyceride" refers to a fatty acid chain, such
as C16:1n7-palmitoleate, covalently bonded to a glycerol molecule
through an ester linkage, wherein the glycerol molecule is further
bonded to two additional fatty acid chains, either or both of which
may or may not be C16:1n7-palmitoleate, though two additional ester
linkages.
[0026] As used herein, "prevention" or "preventing" of a disorder
or condition refers to one or more compounds that, in a statistical
sample, reduces the occurrence of the disorder or condition in the
treated sample relative to an untreated control sample, or delays
the onset or reduces the severity of one or more symptoms of the
disorder or condition relative to the untreated control sample. As
used herein, preventing dry eyes or CDE includes preventing the
onset of dry eyes or CDE.
[0027] As used herein, the term "simultaneous" therapeutic use
refers to the administration of at least two active ingredients by
the same route and at the same time or at substantially the same
time.
[0028] As used herein, the term "separate" therapeutic use refers
to an administration of at least two active ingredients at the same
time or at substantially the same time by different routes.
[0029] As used herein, the term "sequential" therapeutic use refers
to administration of at least two active ingredients at different
times, the administration route being identical or different. More
particularly, sequential use refers to the whole administration of
one of the active ingredients before administration of the other or
others commences. It is thus possible to administer one of the
active ingredients over several minutes, hours, or days before
administering the other active ingredient or ingredients. There is
no simultaneous treatment in this definition.
[0030] As used herein, the terms "treating" or "treatment" or
"alleviation" refers to both therapeutic treatment and prophylactic
or preventative measures, wherein the object is to prevent or slow
down (lessen) the targeted pathologic condition or disorder. For
example, a subject is successfully "treated" for a disease or
medical condition implicating dry eyes if, after receiving a
therapeutic amount of C16:1n7-palmitoleate (or derivatives or
pharmaceutically acceptable salts thereof) according to the methods
described herein, the subject shows observable and/or measurable
reduction in dry eyes. It is also to be appreciated that the
various modes of treatment or prevention of medical diseases and
conditions as described are intended to mean "substantial," which
includes total but also less than total treatment or prevention,
and wherein some biologically or medically relevant result is
achieved.
General
[0031] Dry eye disease is a condition in which there are
insufficient tears to lubricate and nourish the eyes. Tears are
necessary for maintaining the health of the surface of the eyes and
for providing clear vision. People with dry eyes either do not
produce enough tears or have a poor quality of tears.
[0032] With each blink of the eyelids, tears are spread across the
front surface of the eye, known as the cornea. Tears provide
lubrication, reduce the risk of eye infection, wash away foreign
matter in the eye, and keep the surface of the eyes smooth and
clear. Excess tears in the eyes flow into small drainage ducts, in
the inner corners of the eyelids, which drain in the back of the
nose. Dry eyes can result from an improper balance of tear
production and drainage.
[0033] Inadequate amount of tears: Tears are produced by several
glands in and around the eyelids. Tear production tends to diminish
with age, with various medical conditions, or as a side effect of
certain medicines. Environmental conditions such as wind and dry
climates can also affect tear volume by increasing tear
evaporation. When the normal amount of tear production decreases or
tears evaporate too quickly from the eyes and symptoms of dry eye
disease can develop..quadrature.
[0034] Poor quality of tears: Tears are made up of three layers:
oil, water, and mucus. Each component serves a function in
protecting and nourishing the front surface of the eye. A smooth
oil layer helps to prevent evaporation of the water layer, while
the mucin layer functions in spreading the tears evenly over the
surface of the eye. If the tears evaporate too quickly or do not
spread evenly over the cornea due to deficiencies with any of the
three tear layers, dry eye symptoms can develop.
[0035] The most common form of dry eyes is due to an inadequate
amount of the water layer of tears. This condition, called
keratoconjunctivitis sicca (KCS), is also referred to as dry eye
syndrome.
[0036] People with dry eyes may experience symptoms of irritated,
gritty, scratchy, or burning eyes, a feeling of something in their
eyes, excess watering, and blurred vision. Advanced dry eyes may
damage the front surface of the eye and impair vision.
[0037] Treatments for dry eyes aim to restore or maintain the
normal amount of tears in the eyes to minimize dryness and related
discomfort and to maintain eye health.
[0038] The present technology provides compositions and methods for
preventing and treating dry eyes.
C16:1n7-Palmitoleate Compositions
[0039] In some embodiments, the composition includes
C16:1n7-palmitoleate, its derivatives, pharmaceutical salts
thereof, or a combination thereof. Compositions that include
C16:1n7-palmitoleate and its derivatives to be utilized in the
methods described herein include any of those described in U.S.
Pat. No. 8,703,818, which is incorporated herein by reference in
its entirety.
[0040] By way of example, but not by way of limitation, in some
embodiments the composition is a nutraceutical, a pharmaceutical,
or a dietary supplement.
[0041] In some embodiments, the composition comprises about 1% to
about 100% of C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or a combination thereof
relative to all of the components of the composition. In some
embodiments, the composition comprises between about 5% to 20%,
between about 20% to 30%, or at least about 31%, 32%, 33%, 34%,
35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,
48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
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%, 95%, 96%, 97%, 98%, or 99%
of C16:1n7-palmitoleate, its derivatives, pharmaceutically
acceptable salts thereof, or a combination thereof relative to all
of the components of the composition.
[0042] In some embodiments, the composition comprises a
C16:1n7-palmitoleate derivative, wherein the wt % of the
C16:1n7-palmitoleate derivative exceeds the wt % of any other
single ingredient in the composition. In some embodiments, the
composition comprises at least about 50 wt % of the
C16:1n7-palmitoleate derivative. In some embodiments, the
composition comprises at least about 60 wt % of the
C16:1n7-palmitoleate derivative. In some embodiments, the
composition comprises at least about 70 wt % of the
C16:1n7-palmitoleate derivative. In some embodiments, the
composition comprises at least about 80 wt % of the
C16:1n7-palmitoleate derivative. In some embodiments, the
composition comprises at least about 90 wt % of the
C16:1n7-palmitoleate derivative.
[0043] Alternatively, or additionally, in some embodiments, the
composition comprises between about 1% to 100% of
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof relative to all of the
fatty acids and fatty acid derivatives that are present in the
composition. In some embodiments, the composition comprises between
about 5% to 20%, between about 20% to 30%, or at least about 31%,
32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,
45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 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%, 95%, 96%,
97%, 98%, or 99% of C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or a combination thereof
relative to all of the fatty acids and fatty acid derivatives that
are present in the composition.
[0044] In some embodiments, the composition comprises
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof and C16:0-palmitate, its
derivatives, pharmaceutically acceptable salts thereof, or a
combination thereof. In some embodiments, the ratio of
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof to C16:0-palmitate, its
derivatives, pharmaceutically acceptable salts thereof, or a
combination thereof is equal to or greater than 1:1. In some
embodiments, the ratio of C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or a combination thereof
to C16:0-palmitate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof (e.g.,
C16:1n7-palmitoleate: C16:0-palmitate) is about 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, 2.0:1, 2.1:1,
2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1,
3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1,
4.0:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1,
4.9:1, 5.0:1, 5.1:1, 5.2:1, 5.3:1, 5.4:1, 5.5:1, 5.6:1, 5.7:1,
5.8:1, 5.9:1, 6.0:1, 6.1:1, 6.2:1, 6.3:1, 6.4:1, 6.5:1, 6.6:1,
6.7:1, 6.8:1, 6.9:1, 7.0:1, 7.1:1, 7.2:1, 7.3:1, 7.4:1, 7.5:1,
7.6:1, 7.7:1, 7.8:1, 7.9:1, 8.0:1, 8.1:1, 8.2:1, 8.3:1, 8.4:1,
8.5:1, 8.6:1, 8.7:1, 8.8:1, 8.9:1, 9.0:1, 9.1:1, 9.2:1, 9.3:1,
9.4:1, 9.5:1, 9.6:1, 9.7:1, 9.8:1, 9.9:1, 10.0:1, 11:1, 12:1, 13:1,
14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1,
45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1,
100:1, 200:1 or a ratio between any two of the ratios above.
[0045] In some embodiments, the composition comprises a
C16:1n7-palmitoleate or its derivative and a C16:0-palmitate or its
derivative, wherein the ratio of the C16:1n7-palmitoleate or its
derivative to the C16:0-palmitate or its derivative (i.e.,
C16:1n7-palmitoleate:C16:0-palmitate) is between about 12:1 to
about 100:1; and each C16:1n7-palmitoleate or its derivative and
C16:0-palmitate or its derivative is independently selected from
the group consisting of a free acid, pharmaceutically acceptable
salt, (C.sub.1-C.sub.8) alkyl ester, monoglyceride, diglyceride,
triglyceride and a combination thereof. In some embodiments, the
ratio of the C16:1n7-palmitoleate or its derivative to the
C16:0-palmitate or its derivative is between about 15:1 to 50:1. In
some embodiments, the ratio of the C16:1n7-palmitoleate or its
derivative to the C16:0-palmitate or its derivative is between
about 50:1 to 100:1.
[0046] In some embodiments, all of the C16:1n7-palmitoleate or its
derivative and C16:0-palmitate or its derivative are
(C.sub.1-C.sub.8) alkyl esters. In some embodiments, all of the
C16:1n7-palmitoleate or its derivative and C16:0-palmitate or its
derivative are ethyl esters. In some embodiments, all of the
C16:1n7-palmitoleate or its derivative and C16:0-palmitate or its
derivatives are methyl esters. In some embodiments, all of the
C16:1n7-palmitoleate or its derivative and C16:0-palmitate or its
derivatives are propyl, butyl, pentyl, hexyl, heptyl or octyl
esters. In some embodiments, all of the C16:1n7-palmitoleate or its
derivative and C16:0-palmitate or its derivatives are free acids or
pharmaceutically acceptable salts thereof. In some embodiments, all
of the C16:1n7-palmitoleate or its derivative and C16:0-palmitate
or its derivatives are selected from the group consisting of
monoglycerides, diglycerides, triglycerides and combinations
thereof. In some embodiments of the method, the
C16:1n7-palmitoleate derivative is a cis-C16:1n7-palmitoleate
derivative.
[0047] In some embodiments, the composition comprises
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof and C18:1n9-oleate, its
derivatives, pharmaceutically acceptable salts thereof, or a
combination thereof. In some embodiments, the ratio of
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof to C18:1n9-oleate, its
derivatives, pharmaceutically acceptable salts thereof, or a
combination thereof is equal to or greater than 1:1.
[0048] In some embodiments, the ratio of C16:1n7-palmitoleate, its
derivatives, pharmaceutically acceptable salts thereof, or a
combination thereof to C18:1n9-oleate, its derivatives,
pharmaceutically acceptable salts thereof, or a combination thereof
(e.g., C16:1n7-palmitoleate: C18:1n9-oleate) is between about 1.1:1
to about 100:1. In some embodiments, the ratio of
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof to C18:1n9-oleate, its
derivatives, pharmaceutically acceptable salts thereof, or a
combination thereof is about 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, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1,
2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1,
3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4.0:1, 4.1:1, 4.2:1,
4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5.0:1, 5.1:1,
5.2:1, 5.3:1, 5.4:1, 5.5:1, 5.6:1, 5.7:1, 5.8:1, 5.9:1, 6.0:1,
6.1:1, 6.2:1, 6.3:1, 6.4:1, 6.5:1, 6.6:1, 6.7:1, 6.8:1, 6.9:1,
7.0:1, 7.1:1, 7.2:1, 7.3:1, 7.4:1, 7.5:1, 7.6:1, 7.7:1, 7.8:1,
7.9:1, 8.0:1, 8.1:1, 8.2:1, 8.3:1, 8.4:1, 8.5:1, 8.6:1, 8.7:1,
8.8:1, 8.9:1, 9.0:1, 9.1:1, 9.2:1, 9.3:1, 9.4:1, 9.5:1, 9.6:1,
9.7:1, 9.8:1, 9.9:1, 10.0:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,
17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1,
60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 200:1 or a
ratio between any two of the ratios recited above.
[0049] In some embodiments, the composition further comprises a
C18:1n9-oleate or its derivative, wherein the ratio of the
C16:1n7-palmitoleate or its derivative to the C18:1n9-oleate or its
derivative, is between about 6:1 to 100:1, and each C18:1n9-oleate
or its derivative is independently selected between the group
consisting of a free acid, pharmaceutically acceptable salt,
(C.sub.1-C.sub.8) alkyl ester, monoglyceride, diglyceride,
triglyceride and a combination thereof. In some embodiments, the
ratio of the C16:1n7-palmitoleate or its derivative to
C18:1n9-oleate or its derivative is between about 10:1 to 20:1. In
some embodiments, the ratio of the C16:1n7-palmitoleate or its
derivative to the C18:1n9-oleate or its derivative is between about
20:1 to 50:1. In some embodiments, the ratio of the
C16:1n7-palmitoleate or its derivative to C18:1n9-oleate or its
derivative is between about 50:1 to 100:1.
[0050] In some embodiments, the composition contains not more than
about 10%, not more than about 9%, not more than about 8%, not more
than about 7%, not more than about 6%, not more than about 5%, not
more than about 4%, not more than about 3%, not more than about 2%,
not more than about 1%, or not more than about 0.5%, by weight,
palmitic acid, if any. In some embodiments, the composition
contains substantially no palmitic acid. Alternatively, or
additionally, in some embodiments, a composition contains no
palmitic acid and/or derivative thereof.
[0051] C16:1n7-palmitoleate and derivatives thereof, can be
obtained from any of the sources and methods described in U.S. Pat.
No. 8,703,818, which is incorporated herein by reference in its
entirety. By way of example, but by way of limitation, in some
embodiments, C16:1n7-palmitoleate and derivatives thereof are
isolated, concentrated, and/or purified from a source selected from
the group consisting of one or more plants, animals, fish, and
microorganisms.
[0052] In some embodiments, the C16:1n7-palmitoleate moiety of the
C16:1n7-palmitoleate derivative is obtained from a source selected
from the group consisting of fish, macadamia nuts, sea buckthorn,
tallow, algae, bacteria, yeast, and a combination thereof.
[0053] In some embodiments, the C16:1n7-palmitoleate derivative
comprises a C16:1n7-palmitoleate moiety that is obtained from fish.
In some embodiments, the fish is selected from the group consisting
of anchovies, menhaden, pollock, herring, cod, salmon, smelt, tuna,
mackerel, krill and a combination thereof. In some embodiments, the
fish is an anchovy. In other embodiments, the fish is menhaden.
Methods for Preventing or Treating Dry Eyes or Chronic Dry Eye
Disease (CDE)
[0054] The present technology provides methods for preventing or
treating dry eye in a subject comprising administering to the
subject an effective amount of a composition comprising
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof. In some embodiments, the
method includes administering to the subject one or more of any one
of the above embodiments of the composition.
[0055] The compositions comprising C16:1n7-palmitoleate, its
derivatives, pharmaceutically acceptable salts thereof, or a
combination thereof described herein are useful to prevent or treat
dry eyes, chronic dry eye (CDE) disease, or dry eye syndrome. Dry
eyes, chronic dry eye (CDE) disease, or dry eye syndrome is
characterized by stinging, burning, or scratchy sensation in the
eyes, stringy mucus in or around the eyes, increased eye irritation
from smoke or wind, eye fatigue, sensitivity to light, eye redness,
sensation of foreign object in eyes, difficulty wearing contact
lenses, periods of excessive tearing, swollen eyes, eye discomfort,
eye pain, and blurred vision (which worsens at the end of the day
or after focusing for a prolonged period).
[0056] Compositions comprising C16:1n7-palmitoleate, its
derivatives, pharmaceutically acceptable salts thereof, or a
combination thereof, such as those described above, (e.g.,
C16:1n7-palmitoleate alone or C16:1n7-palmitoleate combined with
C18:1n9-oleate) are useful in treating dry eyes, chronic dry eye
(CDE) disease, or dry eye syndrome, as well as the signs, symptoms
or complications of dry eyes, chronic dry eye (CDE) disease, or dry
eye syndrome.
[0057] The disclosure also provides for both prophylactic and
therapeutic methods of treating a subject having or at risk of (or
susceptible to) dry eyes, chronic dry eye (CDE) disease, or dry eye
syndrome and related complications. Accordingly, the present
methods provide for the prevention and/or treatment of dry eyes,
chronic dry eye (CDE) disease, or dry eye syndrome in a subject by
administering an effective amount of a compositions comprising
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof to a subject in need
thereof.
[0058] Therapeutic Methods
[0059] The following discussion is presented by way of example
only, and is not intended to be limiting.
[0060] One aspect of the present technology includes methods of
treating dry eyes, chronic dry eye (CDE) disease, or dry eye
syndrome in a subject in need thereof. One aspect of the present
technology includes methods of treating dry eyes, chronic dry eye
(CDE) disease, or dry eye syndrome in a subject diagnosed as
having, suspected as having, or at risk of having, dry eyes,
chronic dry eye (CDE) disease, or dry eye syndrome. In therapeutic
applications, compositions or medicaments comprising
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof are administered to a
subject suspected of, or already suffering from dry eyes, chronic
dry eye (CDE) disease, or dry eye syndrome in an amount sufficient
to reduce the severity at least partially arrest or delay the onset
of one or more of the symptoms of the disease, including its
complications and intermediate pathological phenotypes in
development of the disease.
[0061] Subjects suffering from dry eyes, chronic dry eye (CDE)
disease, or dry eye syndrome can be identified by any or a
combination of diagnostic or prognostic assays known in the art.
For example, typical symptoms of dry eyes, chronic dry eye (CDE)
disease, or dry eye syndrome include, but are not limited to,
symptoms such as, e.g., stinging, burning, or scratchy sensation in
the eyes, stringy mucus in or around the eyes, increased eye
irritation from smoke or wind, eye fatigue, sensitivity to light,
eye redness, sensation of foreign object in the eyes, difficulty
wearing contact lenses, periods of excessive tearing, swollen eyes,
eye discomfort, eye pain, and blurred vision (which worsens at the
end of the day or after focusing for a prolonged period).
[0062] In some embodiments, dry eyes is diagnosed by tear
osmolarity test. The tear osmolarity test measures the number of
solid particles in a tear. The higher the tear osmolarity typically
indicates that the tear has less water and more particles, e.g.,
salts, proteins, lipids, and mucin. A tear osmolarity score of
below 308 mOsms/L is normal, 308-320 mOsms/L is mild dry eyes,
320-340 mOsms/L is moderate dry eyes, and above 340 mOsms/L is
severe dry eye.
[0063] Prophylactic Methods
[0064] In one aspect, the present technology provides a method for
preventing or delaying the onset of dry eyes, chronic dry eye (CDE)
disease, or dry eye syndrome or symptoms of dry eyes, chronic dry
eye (CDE) disease, or dry eye syndrome in a subject in need
thereof. Subjects at risk for dry eyes, chronic dry eye (CDE)
disease, or dry eye syndrome can be identified by, e.g., any or a
combination of diagnostic or prognostic assays known in the art. In
prophylactic applications, compositions or medicaments comprising
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or a combination thereof are administered to a
subject susceptible to, or otherwise at risk for dry eyes, chronic
dry eye (CDE) disease, or dry eye syndrome in an amount sufficient
to eliminate or reduce the risk, lessen the severity, or delay the
outset of the dry eyes, chronic dry eye (CDE) disease, or dry eye
syndrome, including biochemical, histologic and/or behavioral
symptoms of the disease or disorder, its complications and
intermediate pathological phenotypes presenting during development
of the disease. Administration of a prophylactic compositions or
medicaments comprising C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or a combination thereof
can occur prior to the manifestation of symptoms characteristic of
the disease or disorder, such that the disease or disorder is
prevented or, alternatively, delayed in its progression.
[0065] Subjects or at risk for dry eyes, chronic dry eye (CDE)
disease, or dry eye syndrome may exhibit one or more of the
following non-limiting risk factors: advanced age, wearing contact
lens, taking certain medications, eye diseases, other medical
conditions, or environmental factors.
[0066] In some embodiments the composition is administered as a
nutraceutical, a pharmaceutical, or a dietary supplement.
[0067] In some embodiments, the subject is a mammal, a reptile, or
an amphibian. In some embodiments, the mammal is any mammal,
including, for example, farm animals, such as sheep, pigs, cows,
and horses; pet animals, such as dogs and cats; laboratory animals,
such as rats, mice and rabbits. In some embodiments, the mammal is
a human.
Modes of Administration and Effective Dosages
[0068] Any method known to those in the art for contacting a cell,
organ or tissue with the compositions of the present technology
(i.e., C16:1n7-palmitoleate, its derivatives, pharmaceutically
acceptable salts thereof, or combinations thereof) may be employed.
Suitable methods include in vitro, ex vivo, or in vivo methods. In
vivo methods typically include the administration of
C16:1n7-palmitoleate or derivatives or pharmaceutically acceptable
salts thereof, such as those described above, to a mammal, suitably
a human. When used in vivo for therapy, C16:1n7-palmitoleate or
derivatives or pharmaceutically acceptable salts thereof may be
administered to the subject in effective amounts (i.e., amounts
that have desired therapeutic effects). The dose and dosage regimen
will depend upon the degree of the medical condition in the
subject, the characteristics of C16:1n7-palmitoleate or derivatives
or pharmaceutically acceptable salts thereof, e.g., its therapeutic
index, the subject, and the subject's history.
[0069] The effective amount may be determined during pre-clinical
trials and clinical trials by methods familiar to physicians and
clinicians. An effective amount of C16:1n7-palmitoleate (or
derivatives or pharmaceutically acceptable salts thereof) useful in
the methods may be administered to a mammal in need thereof by any
of a number of well-known methods for administering pharmaceutical
compounds. In some embodiments, the composition comprising
C16:1n7-palmitoleate (or its derivatives, pharmaceutically
acceptable salts thereof, or combinations thereof) may be
administered systemically or locally. In some embodiments, the
composition comprising C16:1n7-palmitoleate (or its derivatives,
pharmaceutically acceptable salts thereof, or combinations thereof)
is administered orally, topically, systemically, intravenously,
subcutaneously, intraperitoneally, or intramuscularly.
[0070] In some embodiments, C16:1n7-palmitoleate (or derivatives
thereof) may be formulated as a pharmaceutically acceptable salt.
The term "pharmaceutically acceptable salt" means a salt prepared
from a base or an acid which is acceptable for administration to a
patient, such as a mammal (e.g., salts having acceptable mammalian
safety for a given dosage regime). However, it is understood that
the salts are not required to be pharmaceutically acceptable salts,
such as salts of intermediate compounds that are not intended for
administration to a patient. Pharmaceutically acceptable salts can
be derived from pharmaceutically acceptable inorganic or organic
bases and from pharmaceutically acceptable inorganic or organic
acids. Salts derived from pharmaceutically acceptable inorganic
bases include ammonium, calcium, copper, ferric, ferrous, lithium,
magnesium, manganic, manganous, potassium, sodium, and zinc salts,
and the like. Salts derived from pharmaceutically acceptable
organic bases include salts of primary, secondary and tertiary
amines, including substituted amines, cyclic amines,
naturally-occurring amines and the like, such as arginine, betaine,
caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine,
2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,
ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,
glucosamine, histidine, hydrabamine, isopropylamine, lysine,
methylglucamine, morpholine, piperazine, piperadine, polyamine
resins, procaine, purines, theobromine, triethylamine,
trimethylamine, tripropylamine, tromethamine and the like. Salts
derived from pharmaceutically acceptable inorganic acids include
salts of boric, carbonic, hydrohalic (hydrobromic, hydrochloric,
hydrofluoric or hydroiodic), nitric, phosphoric, sulfamic and
sulfuric acids. Salts derived from pharmaceutically acceptable
organic acids include salts of aliphatic hydroxyl acids (e.g.,
citric, gluconic, glycolic, lactic, lactobionic, malic, and
tartaric acids), aliphatic monocarboxylic acids (e.g., acetic,
butyric, formic, propionic and trifluoroacetic acids), amino acids
(e.g., aspartic and glutamic acids), aromatic carboxylic acids
(e.g., benzoic, p-chlorobenzoic, diphenylacetic, gentisic,
hippuric, and triphenylacetic acids), aromatic hydroxyl acids
(e.g., o-hydroxybenzoic, p-hydroxybenzoic,
1-hydroxynaphthalene-2-carboxylic and
3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic
acids (e.g., fumaric, maleic, oxalic and succinic acids),
glucuronic, mandelic, mucic, nicotinic, orotic, pamoic,
pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,
edisylic, ethanesulfonic, isethionic, methanesulfonic,
naphthalenesulfonic, naphthalene-1,5-disulfonic,
naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic
acid, and the like. In some embodiments, the salt is an acetate,
tartrate or trifluoroacetate salt.
[0071] In some embodiments, C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or combinations thereof
described herein can be incorporated into pharmaceutical
compositions for administration, alone or in combination, to a
subject for the treatment or prevention of joint pain. Such
compositions typically include the active agent (e.g.,
C16:1n7-palmitoleate) and a pharmaceutically acceptable carrier. As
used herein the term "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. Supplementary
active compounds can also be incorporated into the
compositions.
[0072] Pharmaceutical compositions are typically formulated to be
compatible with its intended route of administration. Examples of
routes of administration include parenteral (e.g., intravenous,
intradermal, intraperitoneal or subcutaneous), oral, inhalation,
transdermal (topical), intraocular, iontophoretic, and transmucosal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic. For
convenience of the patient or treating physician, the dosing
formulation can be provided in a kit containing all necessary
equipment (e.g., vials of drug, vials of diluent, syringes and
needles) for a treatment course (e.g., 7 days of treatment).
[0073] Pharmaceutical compositions suitable for injectable use can
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, a composition for
parenteral administration must be sterile and should be fluid to
the extent that easy syringability exists. It should be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi.
[0074] Compositions containing C16:1n7-palmitoleate (or its
derivatives, pharmaceutically acceptable salts thereof, or
combinations thereof) can include a carrier, which can be a solvent
or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thiomerasol, and the like. Glutathione and other antioxidants can
be included to prevent oxidation. In many cases, isotonic agents
are included, for example, sugars, polyalcohols such as mannitol,
sorbitol, or sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, aluminum monostearate or gelatin.
[0075] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle, which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, typical methods of preparation
include vacuum drying and freeze drying, which can yield a powder
of the active compound plus any additional desired ingredient from
a previously sterile-filtered solution thereof.
[0076] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound 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.
[0077] By way of example, but not by way of limitation, in some
embodiments, administration by inhalation, C16:1n7-palmitoleate (or
its derivatives, pharmaceutically acceptable salts thereof, or
combinations thereof) is delivered in the form of an aerosol spray
from a pressurized container or dispenser, which contains a
suitable propellant, e.g., a gas such as carbon dioxide, or a
nebulizer. Such methods include those described in U.S. Pat. No.
6,468,798.
[0078] Systemic administration of C16:1n7-palmitoleate (its
derivatives, pharmaceutically acceptable salts thereof, or
combinations thereof) as described herein can also be by
transmucosal or transdermal means. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art, and include, for example, for
transmucosal administration, detergents, bile salts, and fusidic
acid derivatives. By way of example, but not by way of limitation,
in some embodiments, transmucosal administration is accomplished
through the use of nasal sprays. For transdermal administration,
the active compounds are formulated into ointments, salves, gels,
or creams as generally known in the art. In some embodiments,
transdermal administration is performed by iontophoresis.
[0079] In some embodiments, C16:1n7-palmitoleate (or its
derivatives, pharmaceutically acceptable salts thereof, or
combinations thereof) is formulated in a carrier system. In some
embodiments, the carrier is a colloidal system. By way of example,
but not by way of limitation, colloidal systems include, but is not
limited to, a liposome or a phospholipid bilayer vehicle. One
skilled in the art would appreciate that there are a variety of
methods to prepare liposomes. See Lichtenberg, et al., Methods
Biochem. Anal., 33:337-462 (1988); Anselem, et al., Liposome
Technology, CRC Press (1993)). Liposomal formulations can delay
clearance and increase cellular uptake (See Reddy, Ann.
Pharmacother., 34(7-8):915-923 (2000)). In some embodiments, an
active agent is also loaded into a particle prepared from
pharmaceutically acceptable ingredients including, but not limited
to, soluble, insoluble, permeable, impermeable, biodegradable or
gastroretentive polymers or liposomes. Such particles include, but
are not limited to, nanoparticles, biodegradable nanoparticles,
microparticles, biodegradable microparticles, nanospheres,
biodegradable nanospheres, microspheres, biodegradable
microspheres, capsules, emulsions, liposomes, micelles and viral
vector systems.
[0080] The carrier can also be a polymer, e.g., a biodegradable,
biocompatible polymer matrix. In some embodiments,
C16:1n7-palmitoleate (or its derivatives, pharmaceutically
acceptable salts thereof, or combinations thereof) is embedded in
the polymer matrix. The polymer may be natural, such as
polypeptides, proteins or polysaccharides, or synthetic, such as
poly .alpha.-hydroxy acids. Examples include carriers made of,
e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose
nitrate, polysaccharide, fibrin, gelatin, and combinations thereof.
In one embodiment, the polymer is poly-lactic acid (PLA) or copoly
lactic/glycolic acid (PGLA). The polymeric matrices can be prepared
and isolated in a variety of forms and sizes, including
microspheres and nanospheres. Polymer formulations can lead to
prolonged duration of therapeutic effect. (See Reddy, Ann.
Pharmacother., 34(7-8):915-923 (2000)).
[0081] Examples of polymer microsphere sustained release
formulations are described in PCT publication WO 99/15154 (Tracy,
et al.), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale, et
al.), PCT publication WO 96/40073 (Zale, et al.), and PCT
publication WO 00/38651 (Shah, et al.).
[0082] In some embodiments, C16:1n7-palmitoleate (or, its
derivatives, pharmaceutically acceptable salts thereof, or
combinations thereof) are prepared with carriers that will protect
C16:1n7-palmitoleate (or, its derivatives, pharmaceutically
acceptable salts thereof, or combinations thereof) against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. 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 known techniques. The materials can also be
obtained commercially, e.g., from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
C16:1n7-palmitoleate (or its derivatives, pharmaceutically
acceptable salts thereof, or combinations thereof) can also be
formulated to enhance intracellular delivery.
[0083] Dosage, toxicity and therapeutic efficacy of
C16:1n7-palmitoleate (or its derivatives, pharmaceutically
acceptable salts thereof, or combinations thereof) can be
determined by standard pharmaceutical procedures in cell cultures
or 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. In some embodiments,
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, exhibit high therapeutic
indices.
[0084] The dosage of such compounds lies 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. For
C16:1n7-palmitoleate (or its derivatives, pharmaceutically
acceptable salts thereof, or combinations thereof) used in the
methods, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to
determine useful doses in humans accurately. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0085] In some embodiments, an effective amount of
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, ranges between about
0.000001 mg per kilogram body weight per day to about 10,000 mg per
kilogram body weight per day. In some embodiments, the dosage
ranges are between about 0.0001 mg per kilogram body weight per day
to about 100 mg per kilogram body weight per day. By way of
example, but not by way of limitation, in some embodiments, dosages
are between about 1 to 10, between about 2 to 8, or between about
4-6 mg/kg body weight every day, every two days or every three
days. In some embodiments, dosages are between about 1 to 10,
between about 2 to 8, or between about 4-6 mg/kg body weight every
week, every two weeks or every three weeks.
[0086] In some embodiments, a single dosage of
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, ranges between about
0.001-10,000, between about 0.01-7,500, between about 0.1-5,000,
between about 1.0-2,500, between about 10-1,000, between about
50-750, between about 100-500, or between about 150-250 micrograms
per kg body weight. In some embodiment, concentrations of
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, in a carrier ranges between
about 0.2 to 2000, between about 0.1-1500, between about 1.0-1000,
between about 10-750, between about 50-500, between about 100-250,
or between about 125-200 micrograms per delivered milliliter.
[0087] In some embodiments, an effective amount of
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, ranges between about 1 to
1000 mg, between about 10 to 900 mg, between about 20 to 800 mg,
between about 30 to 700, between about 40 to 600 mg, between about
50 to 500 mg, between about 60 to 400, between about 70 to 300,
between about 80 to 200 mg, or between about 90 to 100 mg. In some
embodiments, an effective amount of C16:1n7-palmitoleate, its
derivatives, pharmaceutically acceptable salts thereof, or
combinations thereof is more than about 1000 mg.
[0088] By way of example, but not by way of limitation, in some
embodiments a treatment regime entails administration once per day,
once a week, twice a week, three times a week, four times a week,
five times a week, or six times a week. In some embodiments, any of
the above treatment regimes is performed for between 1 to 12 weeks,
between 2 to 11 weeks, between 3 to 10 weeks, between 4 to 9 weeks,
between 5 to 7 weeks, or more than 12 weeks. In some embodiments,
any of the above treatment regimes is performed for between 1 to 12
months, between 2 to 11 months, between 3 to 10 months, between 4
to 9 months, between 5 to 7 months, or more than 12 months.
[0089] In some embodiments, a therapeutically effective amount of
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, is defined as a
concentration of C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or combinations thereof,
at the target tissue between about 10.sup.-12 to 10.sup.-6 molar,
e.g., approximately 10.sup.-7 molar. In some embodiments,
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, is delivered by systemic
doses between about 0.001 to 100 mg/kg, or between about 0.01 to 75
mg/kg, or between about 0.1 to 50 mg/kg, or between about 1.0 to 25
mg/kg, or equivalent dose by body surface area. In some
embodiments, the schedule of doses would be optimized to maintain
the therapeutic concentration at the target tissue. In some
embodiments, the doses are administered by single daily or weekly
administration, but may also include continuous administration
(e.g., parenteral infusion or transdermal application).
[0090] In some embodiments, the dosage of C16:1n7-palmitoleate, its
derivatives, pharmaceutically acceptable salts thereof, or
combinations thereof, is provided at a "low," "mid," or "high" dose
level. In some embodiments, the low dose is provided between about
0.0001 to 0.5 mg/kg/h or between about 0.001 to 0.1 mg/kg/h. In
some embodiments, the mid-dose is provided between about 0.01 to
1.0 mg/kg/h or between about 0.01 to 0.5 mg/kg/h. In some
embodiments, the high dose is provided between about 0.5 to 10
mg/kg/h or between about 0.5 to 2 mg/kg/h.
[0091] In some embodiments, a high dosage at short intervals is
sometimes required until progression of the medical condition is
reduced or terminated, and until the subject shows partial or
complete amelioration of symptoms of the medical condition.
Thereafter, the patient is administered a prophylactic regime.
[0092] In some embodiments, C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or combinations thereof,
are administered in an amount that achieves a serum concentration
between about 100 ng/ml to 9000 ng/ml. In some embodiments,
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, are administered in an
amount that achieves a serum concentration between about 100 ng/ml
to 1000 ng/ml. In some embodiments, the serum concentration
achieved is between about 300 ng/ml to 500 ng/ml, between about 100
ng/ml to 500 ng/ml, between about 500 ng/ml to 1000 ng/ml, between
about 1000 ng/ml to 1500 ng/ml, between about 500 ng/ml to 1500
ng/ml, between about 1000 ng/ml to 2000 ng/ml, between about 1500
ng/ml to 2000 ng/ml, between about 2000 ng/ml to 3000 ng/ml,
between about 2000 ng/ml to 2500 ng/ml, between about 2500 ng/ml to
3000 ng/ml, between about 3000 ng/ml to 4000 ng/ml, between about
3000 ng/ml to 3500 ng/ml, between about 3500 ng/ml to 4000 ng/ml,
between about 4000 ng/ml to 5000 ng/ml, between about 4000 ng/ml to
4500 ng/ml, between about 4500 ng/ml to 5000 ng/ml, between about
5000 ng/ml to 6000 ng/ml, between about 5000 ng/ml to 5500 ng/ml,
between about 5500 ng/ml to 6000 ng/ml, between about 6000 ng/ml to
7000 ng/ml, between about 7000 ng/ml to 8000 ng/ml, or between
about 8000 ng/ml to 9000 ng/ml.
[0093] 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 medical
disease or condition, 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 compositions described herein can include a single
treatment or a series of treatments.
[0094] In some embodiments, C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or combinations thereof,
are formulated as a pharmaceutical composition within a soft
gelatin capsule. In some embodiments, the soft gelatin capsule
includes about 0.5 grams, about 1 gram, about 1.5 grams, or about 2
grams of the pharmaceutical composition comprising at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80% or at least 90% of C16:1n7-palmitoleate, or a
derivative or pharmaceutically acceptable salt thereof. In some
embodiments, one capsule per day is administered to a subject for
the treatment or prevention of any of the conditions, such as dry
eyes, as described herein. In some embodiments, two capsules per
day are administered to the subject. In some embodiments, two to
ten capsules per day are administered to the subject.
[0095] In some embodiments, the subject is a mammal, a reptile, or
an amphibian. In some embodiments, the mammal is any mammal,
including, for example, farm animals, such as sheep, pigs, cows,
and horses; pet animals, such as dogs and cats; laboratory animals,
such as rats, mice and rabbits. In some embodiments, the mammal is
a human.
Combination Therapy with C16:1n7-Palmitoleate and Other Therapeutic
Agents
[0096] In some embodiments, C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or combinations thereof,
may be combined with one or more additional therapeutic agents for
the prevention or treatment of dry eyes, e.g., dry eye syndrome. By
way of example, but not by way of limitation, treatment for dry
eyes, e.g., dry eye syndrome, can include, in addition to
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, but is not limited to,
Restasis.RTM. (cyclosporine ophthalmic emulsion) (Sandimmun, United
Kingdom), antibiotics, and hydroxypropyl cellulose insert (e.g.,
Lacrisert.RTM. (Valeant Pharmaceuticals, Bridgewater, N.J.)).
[0097] In some embodiments, an additional therapeutic agent is
administered to a subject in combination with C16:1n7-palmitoleate,
its derivatives, pharmaceutically acceptable salts thereof, or
combinations thereof, such that a synergistic therapeutic effect is
produced. Therefore, lower doses of one or both of the therapeutic
agents may be used in treating dry eyes, e.g., dry eye syndrome,
resulting in increased therapeutic efficacy and decreased
side-effects.
[0098] In any case, the multiple therapeutic agents may be
administered in any order, e.g., sequentially or separately or even
simultaneously. If simultaneously, the multiple therapeutic agents
may be provided in a single, unified form, or in multiple forms (by
way of example only, either as a single pill or as two separate
pills). One of the therapeutic agents may be given in multiple
doses, or both may be given as multiple doses. If not simultaneous,
the timing between the multiple doses may vary between more than
zero weeks to less than four weeks. In addition, the combination
methods, compositions and formulations are not to be limited to the
use of only two agents.
EXAMPLES
[0099] The present technology is further illustrated by the
following examples, which should not be construed as limiting in
any way. For each of the examples below, C16:1n7-palmitoleate, its
derivatives, pharmaceutically acceptable salts thereof, or
combinations thereof could be used.
Example 1
C16:1n7-Palmitoleate in Treating Dry Eyes in Human Subjects
[0100] This Example shows the treatment of dry eyes using a
composition comprising C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or combinations
thereof.
[0101] Patients diagnosed with dry eye disease (as determined by
having tear osmolarity over 308 mOsms/L, which is the clinical
minimum for dry eyes, per eye) were treated daily with a dietary
supplement containing 210 mg of C16:1n7-palmitoleate for 30
days.
[0102] Tear osmolarity for left and right eyes were scored at day 1
and day 30, see Table 1. A tear osmolarity score of below 308
mOsms/L is normal, 308-320 mOsms/L is mild dry eyes, 320-340
mOsms/L is moderate dry eyes, and above 340 mOsms/L is severe dry
eye. At day 30, patients also ranked their feeling of improvement
of dry eye on a scale of 1-10, see Table 1.
TABLE-US-00001 TABLE 1 Treatment of Dry Eyes with
C16:1n7-palmitoleate Tear Tear Symptom Osmolarity Osmolarity
Improvement Day 1 Day 30 (Scale of Patient Sex Age (mOsms/L)
(mOsms/L) 1-10) 1 Male 69 Left eye 347 Left eye 308 8 Right eye 367
Right eye 310 2 Male 54 Left eye 332 Left eye 313 3 Right eye 362
Right eye 314 3 Female 40 Left eye 294 Left eye 300 4 Right eye 305
Right eye 300 4 Female 33 Left eye 333 Left eye 302 7 Right eye 340
Right eye 308 5 Female 83 Left eye 312 Left eye 293 7 Right eye 382
Right eye 302 6 Female 56 Left eye 351 Left eye 314 6 Right eye 325
Right eye 299 7 Female 48 Left eye 322 Left eye 302 4 Right eye 341
Right eye 307 8 Female 44 Left eye 314 Left eye 301 6 Right eye 328
Right eye 367
[0103] The results show that administration of the
C16:1n7-palmitoleate dietary supplement reduced dry eyes based on
tear osmolarity score. For example, patients that initially
exhibited at least one severe dry eye, i.e., patients 1, 2, 5, and
7, exhibited normal or very mild dry eyes after 30 days of
treatment with the C16:1n7-palmitoleate, see Table 1.
[0104] Additionally, a majority of the patients reported that their
eyes felt better after the 30 day treatment with the
C16:1n7-palmitoleate nutraceutical, see Table 1.
[0105] These results show that compositions comprising
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, are useful in treating dry
eyes.
Example 2
C16:1n7-Palmitoleate Ameliorates Meibomian Gland Dysfunction in
Human Subjects
[0106] Meibomian gland dysfunction (MGD) is thought to be the
leading cause of dry eye disease. This Example demonstrates that
compositions comprising C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or combinations thereof
are useful in ameliorating or treating MGD in subjects with dry eye
symptoms.
[0107] Twenty nine patients, each diagnosed as having moderate to
severe dry eye symptoms/MGD, were selected for the study.
[0108] Patients were classified as having none, mild, or moderate
meibomian gland secretion blockage by evaluating the secretion of
the meibomian glands in the nasal, central, and temporal regions of
the lower eyelid. MGD was scored using a 0-4 grading scale, 4
correlating with severe dysfunction. A summary of the grading scale
is provided below: [0109] 1=meibomian glands in all 3 regions
secreting/cloudy [0110] 2=meibomian glands in 2 out of 3 regions
secreting/cloudy [0111] 3=meibomian glands in 1 out of 3 regions
secreting/cloudy [0112] 4=no observable meibomian gland secretion
in any of the 3 regions [0113] 2+ and 2,3 are used interchangeably
and mean more than 2/3<1.
[0114] After the initial baseline evaluation, patients received
daily doses of a dietary supplement containing 210 mg of
C16:1n7-palmitoleate for over 30 days.
[0115] Tear osmolarity (TL) and MGD for left and right eyes (LE and
RE respectively) were scored at Day 0 (baseline evaluation) and at
the follow-up visit (FV), see Table 2. A tear osmolarity score of
below 308 mOsms/L is normal, 308-320 mOsms/L is mild dry eyes,
320-340 mOsms/L is moderate dry eyes, and above 340 mOsms/L is
severe dry eye. The terms `above` and `below` refer to values above
and below the detectable range of the Tearlab diagnostic device
respectively. Patients also recorded their comments regarding
perceived improvements of their dry eye symptoms. Table 2.
TABLE-US-00002 TABLE 2 Improvement of MGD with C16:1n7-palmitoleate
Day 0 Day 0 Day 0 Day 0 FV FV FV FV Patient TL RE TL LE MGD RE MGD
LE TL RE TL LE MGD RE MGD LE Comment 1 303 323 4 4 308 306 4 1 good
2 351 327 3 3 311 303 3 3 good 3 329 314 2+ 2+ 235 299 2 2 good 4
321 311 3 2 284 317 1 1 great 5 316 307 4 4 302 298 3 3 good 6 357
349 2+ 2+ 301 303 1 1 good 7 311 312 4 4 295 291 3 3 good 8 326 307
3+ 3+ 307 310 2+ 3 good 9 329 314 3+ 3+ 304 316 2 2 good 10 383
above 2, 3 2, 3 307 314 1, 1, good 11 4 4 302 299 1 1 great 12 335
324 4 4 348 354 1 1 good 13 below 339 3 3 354 352 1 1 good 14 330
308 3 3 352 330 3 2+ poor 15 318 364 3 3 345 324 1 2 good 16 337
361 4 4 2 2 good 17 324 317 4 4 309 331 4 4 good 18 4 4 4 4 Better
19 330 above 4 4 314 327 4 4 good 20 313 314 3 3 0 0 great 21 290
364 2 2 318 300 1 1 great 22 293 302 4 4 355 above 1 1 good 23 235
326 4 4 318 305 4 4 happy 24 348 356 4 4 2 2 happy 25 310 320 3 3
below below 2 2 good 26 301 313 4 4 302 308 good 27 322 358 4 4
below below 2 2 good 28 308 349 3 3 294 323 1 3 good 29 360 317 3 3
303 311 3 3 better
[0116] The results show that administration of the
C16:1n7-palmitoleate dietary supplement resulted in the improvement
of tear osmolarity scores and/or MGD scores. For example, patients
that initially exhibited severe MGD scores, e.g., patients 1, 4-7,
11-13, 15-16, 20-22, 24 and 27, showed improvement in MG secretion
after treatment with the C16:1n7-palmitoleate, see Table 2.
[0117] Additionally, a majority of the patients reported that their
eyes felt better after treatment with the C16:1n7-palmitoleate
dietary supplement, see Table 2.
[0118] These results show that compositions comprising
C16:1n7-palmitoleate, its derivatives, pharmaceutically acceptable
salts thereof, or combinations thereof, are useful in improving MGD
scores in subjects with dry eye symptoms.
Example 3
C16:1n7-Palmitoleate Reduces Meibomian Gland Atrophy in Human
Subjects
[0119] Patient Selection: All prospective patients will be
instructed to cease using any preexisting dry eye or blepharitis
therapy prior to any baseline evaluation (e.g., punctal plugs, lid
hygiene products, warm compresses, and artificial tears). One
hundred (100) patients, each having moderate to severe dry eye
symptoms/MGD, shall be selected after being subjected to testing
for oil gland expression, lissamine green staining of lids and
conjunctiva, tear break-up time testing, Tearlab Osmolarity
analysis of both eyes, and Schirmer's aqueous testing with and
without anesthesia. These patients will also complete
questionnaires that are specifically designed for the present
study.
[0120] A standardized MGE device (MGE) will be used to classify
patients as having none, mild, or moderate meibomian gland
secretion blockage. LipiView Diagnostics (TearSciences), which
includes interferometric scanning and meibography will be used to
assess the following endpoints: Meibomian Gland Atrophy and
Structure, Lipid Layer Thickness (LLT), Blink Rate, and Blink
Closure Quality. The patients shall be separated into four groups
according to the varying degree/severity of MGD. Severe Meibomian
Gland Atrophy (Stage 4) will be excluded from the study. All
patient data will be collected using the form shown in FIG. 1.
[0121] After the initial baseline evaluation, patients will receive
daily doses of a dietary supplement containing 210 mg of
C16:1n7-palmitoleate for 90 days. The clinical parameters of each
patient will be reassessed at 3 months and 6 months following the
baseline evaluation. Multivariate analysis will be performed by
comparing meibomian gland atrophy parameters at baseline (Day 0),
with those observed at 90 days and 180 days post treatment with
C16:1n7-palmitoleate. Patient commentary regarding the subjective
improvement of dry eye symptoms and objective indicia for meibomian
gland atrophy will be analyzed.
[0122] It is predicted that at least some of the patients will show
a reduction of meibomian gland atrophy after receiving the
C16:1n7-palmitoleate dietary supplement These results will show
that compositions comprising C16:1n7-palmitoleate, its derivatives,
pharmaceutically acceptable salts thereof, or combinations thereof,
are useful in treating meibomian gland atrophy in subjects with dry
eye symptoms.
Equivalents
[0123] The present technology is not to be limited in terms of the
particular embodiments described in this application, which are
intended as single illustrations of individual aspects of the
present technology. Many modifications and variations of this
present technology can be made without departing from its spirit
and scope, as will be apparent to those skilled in the art.
Functionally equivalent methods and apparatuses within the scope of
the present technology, in addition to those enumerated herein,
will be apparent to those skilled in the art from the foregoing
descriptions. Such modifications and variations are intended to
fall within the scope of the appended claims. The present
technology is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled. It is to be understood that this present
technology is not limited to particular methods, reagents,
compounds compositions or biological systems, which can, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
[0124] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0125] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," and the like, include
the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member. Thus, for example, a group having 1-3 cells
refers to groups having 1, 2, or 3 cells. Similarly, a group having
1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so
forth.
[0126] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0127] Other embodiments are set forth within the following
claims.
* * * * *