U.S. patent application number 15/202594 was filed with the patent office on 2017-05-18 for compositions and methods for preventing or treating inflammatory bowel disease.
This patent application is currently assigned to Hill's Pet Nutrition, Inc.. The applicant listed for this patent is Hill's Pet Nutrition, Inc.. Invention is credited to Kathy Lynn Gross, Christina Khoo, William David Schoenherr.
Application Number | 20170135974 15/202594 |
Document ID | / |
Family ID | 37888012 |
Filed Date | 2017-05-18 |
United States Patent
Application |
20170135974 |
Kind Code |
A1 |
Khoo; Christina ; et
al. |
May 18, 2017 |
COMPOSITIONS AND METHODS FOR PREVENTING OR TREATING INFLAMMATORY
BOWEL DISEASE
Abstract
Compositions comprising docosahexaenoic acid (DHA) and
optionally one or more fatty acids selected from the group
consisting of eicosapentaenoic acid (EPA), arachidonic acid (ARA),
linoleic acid (LA), and .alpha.-linoleic acid (ALA) are
administered to felines for preventing or treating feline
inflammatory bowel disease (IBD).
Inventors: |
Khoo; Christina; (Duxbury,
MA) ; Schoenherr; William David; (Hoyt, KS) ;
Gross; Kathy Lynn; (Topeka, KS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hill's Pet Nutrition, Inc. |
Topeka |
KS |
US |
|
|
Assignee: |
Hill's Pet Nutrition, Inc.
Topeka
KS
|
Family ID: |
37888012 |
Appl. No.: |
15/202594 |
Filed: |
July 6, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13372251 |
Feb 13, 2012 |
9408817 |
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15202594 |
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12493685 |
Jun 29, 2009 |
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13372251 |
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11617810 |
Dec 29, 2006 |
8048922 |
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12493685 |
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60754806 |
Dec 29, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0056 20130101;
A61P 29/00 20180101; A61K 31/19 20130101; A61P 1/04 20180101; A61P
1/00 20180101; A61K 31/202 20130101 |
International
Class: |
A61K 31/202 20060101
A61K031/202 |
Claims
1-27. (canceled)
28. A method for lowering the level of CD4+ lymphocytes and
neutrophils in a feline comprising administering to the feline a
therapeutically-effective amount of docosahexaenoic acid.
Description
CROSS-REFERENCE TO PRIOR APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application No. 60/754,806 filed on Dec. 29, 2005.
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] The invention relates generally to compositions and methods
for preventing or treating inflammatory bowel disease and
particularly to the use of docosahexaenoic acid for preventing or
treating feline inflammatory bowel disease.
[0004] Description of the Related Art
[0005] The terms "inflammatory bowel disease" or "IBD" refer to a
group of chronic idiopathic gastrointestinal disorders
characterized by inflammatory infiltrates within the lamina propria
of the gastrointestinal tract. IBD encompasses segmental
granulomatous enterocolitis, lymphoplasmacytic enteritis,
eosinophilic gastroenterocolitis, lymphocytic gastroenterocolitis,
suppurative enterocolitis, and histiocytic colitis. The
lymphoplasmacytic form is probably the most common type of IBD.
These specific types of IBD are characterized based on the type of
inflammatory infiltrate found in the lamina propria. The
inflammatory infiltrates can be quite variable in terms of severity
and cell types, with lymphocytes and plasma cells being the most
common cell types. Inflammatory infiltrates may involve the
stomach, small bowel, and colon. In cats, for example, the stomach
and small bowel are affected most often. In many cases, multiple
segments of the bowel are involved and clinical signs may be mixed,
reflecting the broad distribution of mucosal lesions. The severity
of IBD varies from mild clinical signs to life-threatening
protein-losing enteropathies.
[0006] Mucosal inflammatory infiltrates are responsible for the
clinical manifestations of IBD. Mucosal inflammation disrupts
normal absorptive processes. Such disruption results in
malabsorption and osmotic diarrhea. Altered gut permeability can
result in leakage of fluid, protein, and blood into the gut lumen.
Malabsorbed fats, carbohydrates, and bile acids result in secretory
diarrhea. Inflammatory mediators may also directly trigger
intestinal secretion and mucus production by goblet cells. Mucosal
inflammatory infiltrates may alter intestinal and colonic motility
patters, a mechanism attributed to the influence of prostaglandins
and leukotrienes on smooth muscle. Inflammation of the stomach and
small bowel stimulates receptors that trigger vomiting. In cats,
for example, the most common clinical signs of IBD are chronic
vomiting, diarrhea, and weight loss.
[0007] The fundamental pathway for the development of IBD involves
hypersensitivity. Two related theories attempt to explain the
underlying cause for hypersensitivity reactions. The first theory
speculates that felines with IBD develop a defect in the intestinal
mucosal barrier. Loss of mucosal integrity results in increased gut
permeability and hypersensitivity responses to allergens that are
normally tolerated. The second theory speculates that IBD results
from aberrant immunological responses to luminal antigens. Both
potential pathways culminate in release of inflammatory mediators.
These substances may further damage the intestinal mucosal surface
and set up a cycle of inflammation and loss of barrier
function.
[0008] Essential fatty acids have specific roles in cell function
regulation. For example, the omega-3 eicosapentaenoic acid (EPA),
and the omega-6 arachidonic and gamma-linolenic acids are
precursors for the synthesis of eicosanoids which are
immunoregulatory molecules functioning as local hormones and
mediators of inflammation. The eicosanoids synthesized from
arachidonic acid (ARA) are proinflammatory compared to eicosanoids
produced from eicosapentaenoic and gamma-linolenic acids, and may
result in pathologic conditions when produced in excessive amounts.
Macrophages are a significant source of eicosanoids, and modulate
the intensity and duration of inflammatory and immune responses.
The predominant polyunsaturated fatty acid in membrane
phospholipids of macrophages and lymphocytes is ARA. Administration
of gamma-linolenic or EPA results in the replacement of ARA in the
macrophage membrane with eicosapentaenoic or gamma-linolenic acid.
The result of such replacement is the production of fewer
ARA-derived eicosanoids and more EPA-derived or gamma-linolenic
acid-derived eicosanoids, thereby reducing the immunologic response
to an inflammatory episode.
[0009] A definitive diagnosis of IBD is based on the
histopathological examination of mucosal or full-thickness
intestinal biopsy specimens collected by endoscopic or surgical
techniques. Thus, there is a need for alternative methods for
diagnosing feline IBD that are less invasive than obtaining biopsy
specimens. There is also a need for new methods and compositions
useful for preventing and treating feline IBD.
SUMMARY OF THE INVENTION
[0010] It is, therefore, an object of the invention to provide
methods for preventing or treating IBD in felines.
[0011] It is another object of the invention to provide
compositions suitable for preventing or treating IBD in
felines.
[0012] It is another object of the invention to provide methods for
determining if a feline is suffering from IBD.
[0013] It is a further object of the invention to provide articles
of manufacture in the form of kits that contain combinations of
foods, compounds, ingredients, and devices useful for preventing or
treating IBD in felines.
[0014] It is another object of the invention to provide means for
communicating information about the methods, compositions, articles
of manufacture, and benefits of the invention.
[0015] One or more of these and other objects are achieved using
novel methods for preventing and/or treating IBD in felines
susceptible to or suffering from IBD comprising administering to
the felines a therapeutically-effective amount of docosahexanoic
acid (DHA). Methods for diagnosing IBD and kits comprising
combinations of foods, compounds, ingredients, and devices useful
for preventing and/or treating IBD are also provided.
[0016] Additional objects, features, and advantages of the
invention will be apparent to those skilled in the art.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In one aspect, the invention provides methods for treating
IBD in a feline suffering from IBD. Treating IBD includes
ameliorating, suppressing, and/or eradicating IBD. Those skilled in
the art can diagnose IBD (and distinguish IBD from other
gastrointestinal diseases) utilizing diagnostic tests (e.g.,
complete blood cell count, serum biochemistry, serum thyroxine
level, immunodeficiency virus test, feline leukemia virus test,
urinalysis, fecal examinations for parasites and bacteria): dietary
trials; abdominal radiographs and/or ultrasound; and/or examination
of mucosal or full-thickness intestinal biopsy specimens. In
another aspect, the invention provides methods for preventing IBD
in a feline susceptible to developing IBD. Preventing IBD includes
reducing the risk of IBD, delaying the onset of IBD, and/or keeping
a feline from developing IBD. The methods comprise administering to
the feline a therapeutically-effective amount of docosahexaenoic
acid (DHA). A therapeutically-effective amount is an amount that
will achieve the goal of treating IBD when the feline is suffering
from IBD, or preventing IBD when the feline is susceptible to
developing IBD, or lowering the level of CD4+ lymphocytes and
neutrophils in a feline susceptible to or suffering from IBD.
Administering means introducing DHA or other compounds into the
feline in a suitable dosage form by a suitable administration route
(e.g., orally, topically, or parenterally). DHA can be
administered, for example, as pure DHA or DHA derivative (e.g., a
salt such as an ester) or as a composition comprising DHA and/or
DHA derivative(s). References herein to DHA and other fatty acids
herein include the derivatives of such compounds. A DHA-comprising
composition may also comprise one or more conventional
pharmaceutically-acceptable excipients (e.g., adjuvants, carriers,
and/or vehicles). In some embodiments, the DHA-comprising
composition may comprise a food composition.
[0018] The invention is based upon the surprising discovery that
DHA, but not EPA or other similar fatty acids, is useful for
preventing or treating IBD in felines and that DHA may have the
opposite effect in other animals. While EPA and related fatty acids
alone or in combination do not prevent or treat IBD, they are
useful for supplementing DHA in preventing or treating IBD. Thus,
the unexpected result that DHA alone or DHA in combination with EPA
and related fatty acids is effective for preventing or treating IBD
when administered to felines in a therapeutically-effective
amount.
[0019] DHA effectively lowers the level of CD4+ lymphocytes and
neutrophils in a feline, including a feline susceptible to or
suffering from IBD, when DHA is administered in a
therapeutically-effective amount, as described herein.
[0020] In some embodiments, the methods comprise administering to
the feline from about 6 to about 165 mg/kg body weight/day DHA. In
some such embodiments, from about 12 to about 65 mg/kg body
weight/day DHA is administered to the feline. In others, from about
12 to about 32 mg/kg body weight/day DHA is administered to the
feline. The daily amount of DHA can be administering in a single
dose or, alternatively, in two or more dosages that make up the
daily dose.
[0021] In some embodiments, administering DHA comprises feeding DHA
to the feline, i.e., feeding DHA or a composition comprising DHA
(including DHA derivatives).
[0022] In various embodiments, a DHA-comprising composition fed to
the feline comprises a food composition. In some embodiments, the
food composition meets the AAFCO's minimum nutrient level
requirements for reproduction or maintenance. See 2005 AAFCO
Official Publication, pages 137-40. In some embodiments, the food
composition comprises a dry food. In others, the food composition
comprises a semi-moist food. In still others, the food composition
comprises a moist food. The terms. "dry". "moist" and "semi-moist",
as used herein, are familiar to one of skill in the art. The food
composition may be a supplement, treat, snack, or partially or
fully edible toy. In some embodiments, the composition comprises a
mixture of one or more foods or a hypoallergenic food
composition.
[0023] In some embodiments, the feline is a companion feline. A
companion feline can be a feline kept as a pet. A companion feline
can also be a feline from a widely domesticated species, for
example, cats (Felis domesticus) regardless of whether or not it is
kept as a pet. In some embodiments, the feline is a growing feline.
A growing feline is one that has not reached adult size. For
example, a growing cat typically is one that is less than about one
year old. In some embodiments, the feline is an adult feline. An
adult feline is a feline of any age after juvenile growth and
development has been completed, including senior and geriatric
felines. For example, an adult cat typically is one that is from
about one year old through the remainder of its life. A senior
feline is one of an age at which it is at a risk for suffering from
an age-related disease regardless of whether or not the feline
shows obvious physical or behavioral signs of aging. For example, a
senior cat typically is a cat from about seven to about eleven
years old. A geriatric feline is a feline showing signs of advanced
age. For example, a geriatric cat typically is a cat of about
twelve years of age and beyond.
[0024] Unless otherwise stated, all percentages herein are weight
percentages on a dry matter basis. The term "dry matter basis"
means that an ingredient's concentration in a composition is
measured after any moisture in the composition is removed.
[0025] In some embodiments, the composition administered to the
feline comprises from about 0.05 to about 1% DHA. In some such
embodiments, the composition comprises from about 0.1 to about 0.4%
DHA. In others, the composition comprises from about 0.1 to about
0.2% DHA. In yet other such embodiments, the composition comprises
from about 0.05 to about 0.2% DHA. In further such embodiments, the
composition comprises from about 0.05 to about 0.15% DHA. And in
yet further such embodiments, the composition comprises from about
0.05 or about 0.1 to about 0.15, about 0.2, or about 0.4% DHA.
[0026] In additional embodiments, the composition administered to
the feline further comprises at least one fatty acid selected from
the group consisting of eicosapentaenoic acid (EPA), arachidonic
acid (ARA), linoleic acid (LA), and .alpha.-linoleic acid (ALA). In
some such embodiments, the composition comprises from about 0.05 to
about 1% of each fatty acid present in the composition. In other
such embodiments, the composition comprises from about 0.1 to about
0.5% or from 0.1 to about 0.3% of the fatty acid. In one
embodiment, the composition administered to the feline further
comprises from about 0.05 to about 1% EPA. In some such
embodiments, the composition comprises from about 0.1 to about 0.5%
EPA. In other such embodiments, the composition comprises from
about 0.1 to about 0.3% EPA. In yet other such embodiments, the
composition comprises from about 0.05 to about 0.3% EPA. In further
such embodiments, the composition comprises from about 0.15 to
about 0.3% EPA. In yet further such embodiments, the composition
comprises from about 0.05, about 0.1, or about 0.15 to about 0.2,
about 0.3, about 0.4, or about 0.5% EPA.
[0027] In some embodiments, the composition administered to the
feline comprises from about 0.05 to about 1% DHA and from about
0.05 to about 1% EPA, and the ratio of the amount of EPA present in
the composition to the amount of DHA in the composition is from
about 1 to about 2. In some such embodiments, the ratio of the
amount of EPA present in the composition to the amount of DHA
present in the composition is from about 1.2 to about 1.8. In other
such embodiments, the ratio of the amount of EPA in the composition
to the amount of DHA in the composition is from about 1.2 to about
1.5. In yet other such embodiments, the ratio of the amount of EPA
present in the composition to the amount of DHA present in the
composition is from about 1.3 to about 1.6. And in further such
embodiments, the ratio of the amount of EPA present in the
composition to the amount of DHA present in the composition is from
about 1, about, 1.2, or about 1.3 to about 1.5, about 1.6, about
1.8, or about 2.
[0028] In some embodiments, the methods of prevention and treatment
further comprise administering to the feline an anti-inflammatory
bowel disease (anti-IBD) agent in conjunction with administering
DHA or the combination of DHA and at least one fatty acid selected
from the group consisting of EPA, ARA, LA, and ALA. An anti-IBD
agent is a compound, a derivative thereof (e.g., a salt, solvate,
or hydrate of the compound), or a composition comprising such
compounds and/or derivatives that is used to prevent or treat IBD.
"In conjunction" means that an anti-IBD agent is administered to
the feline either together with DHA or separately from DHA at the
same or different frequency via the same or different
administration route and either at about the same time as DHA or
periodically. "At about at the same time" generally means that the
anti-IBD agent is either administered when DHA is administered to
the feline or within about 72 hours after administering DHA to the
feline. "Periodically" generally means that an anti-IBD agent is
administered to a feline following a dosage schedule suitable for
administering the agent while a DHA-comprising composition is fed
to the feline routinely as appropriate for that feline. Thus, the
term "in conjunction" specifically includes situations when an
anti-IBD agent is administered to a feline for a prescribed period
of time while DHA is administered to the feline for a much longer
period of time (e.g., for life). If more than one agent is
administered to a feline, the dosage form and route of
administration for each agent may vary. Those skilled in the art
would understand that one or more anti-IBD agents can be
administered to a feline while the feline is fed a single
DHA-comprising composition or, alternatively, when the feline is
fed different DHA-comprising compositions for varying time
intervals.
[0029] Suitable anti-IBD agents include, for example,
corticosteroids (e.g., prednisone, prednisolone),
immunosuppressants (e.g., azathiprine), and antibiotics (e.g.,
metronidazole, amoxicillin, tylosin). Anti-IBD agents can be
administered, for example, in the form of salts derived from
inorganic or organic acids. Depending on the particular compound, a
salt of the compound may be advantageous due to one or more of the
salt's physical properties, for example, enhanced pharmaceutical
stability in differing temperatures and humidity, or a desirable
solubility in water or oil. The salt preferably is a
pharmaceutically-acceptable salt.
[0030] The preferred total daily dose of an anti-IBD agent
(administered in either single or divided doses) is typically from
about 0.001 to about 100 mg/kg body weight, more preferably from
about 0.01 to about 30 mg/kg body weight, and even more preferably
from about 0.01 to about 10 mg/kg body weight. Dosage unit
compositions can contain such amounts and submultiples thereof to
make up the daily dose. In many instances, the administration of
the anti-IBD agent will be repeated a plurality of times. Multiple
doses per day typically may be used to increase the total daily
dose, if desired. Factors affecting the preferred dosage regimen
include, for example, the age, weight, and condition of the feline:
the severity of the disease: the route of administration:
pharmacological considerations, such as the activity, efficacy,
pharmacokinetic, and toxicology profiles of the particular anti-IBD
agent used: whether a drug delivery system is utilized; and whether
the anti-IBD agent is administered as part of a drug combination.
Thus, the dosage regimen can vary widely, and therefore, can differ
from the preferred dosage regimen discussed above.
[0031] In yet another aspect, the invention provides compositions
suitable for preventing and/or treating IBD in a feline. These
compositions are described and exemplified in the context of the
methods herein for preventing and/or treating IBD in a feline.
[0032] In a further aspect, the invention provides methods for
preparing the compositions suitable for use in methods of
prevention and treatment of IBD. Such compositions can be prepared,
for example, by mixing two or more ingredients (including food
compositions) that, when combined, yield a composition of the
invention or by mixing one or more food compositions with
additional ingredient(s) such as, for example, DHA, EPA, and/or an
anti-IBD agent. Such compositions can also be prepared by one or
more of the methods discussed in, for example, Small Animal
Nutrition, pages 127-46 (2000).
[0033] In yet further aspect, the invention provides for a use of
DHA and optionally at least one fatty acid selected from the group
consisting of EPA, ARA, LA, and ALA to prepare a composition of the
invention suitable for preventing or treating feline IBD. In some
embodiments, the invention provides a use of DHA to prepare a
composition comprising from about 0.05 to about 1% DHA. In some
such embodiments, the composition comprises from about 0.1 to about
0.4% DHA. In others, the composition comprises from about 0.1 to
about 0.2% DHA. In yet other such embodiments, the composition
comprises from about 0.05 to about 0.2% DHA. In further such
embodiments, the composition comprises from about 0.05 to about
0.15% DHA. In yet further such embodiments, the composition
comprises from about 0.05 or about 0.1 to about 0.15, about 0.2, or
about 0.4% DHA. In some embodiments, the composition comprises a
food composition.
[0034] In some embodiments, the invention provides a use of DHA and
at least one fatty acid selected from the group consisting of EPA,
ARA, LA, and ALA, preferably EPA, to prepare a composition
comprising from about 0.05 to about 1% DHA and from about 0.05 to
about 1% of each of EPA, ARA, LA, and/or ALA present in the
composition to prevent and/or treat feline IBD. In some such
embodiments, the composition comprises from about 0.1 to about 0.4%
DHA and from about 0.1 to about 0.5% of one or more of EPA, ARA,
LA, and/or ALA. In other such embodiments, the composition
comprises from about 0.1 to about 0.2% DHA and from about 0.1 to
about 0.3% of one or more of EPA, ARA, LA, and/or ALA. In yet other
such embodiments, the composition comprises from about 0.05 to
about 0.2% DHA and from about 0.05 to about 0.3% of one or more of
EPA, ARA, LA, and/or ALA. In further such embodiments, the
composition comprises from about 0.05 to about 0.15% DHA and from
about 0.15 to about 0.3% of one or more of the fatty acids. In yet
further such embodiments, the composition comprises from about 0.05
or about 0.1 to about 0.15, about 0.2, or about 0.4% DHA and from
about 0.05, about 0.1, or about 0.15 to about 0.2, about 0.3, about
0.4, or about 0.5% of one or more of the fatty acids. In some
embodiments, the composition comprises a food composition.
[0035] In some embodiments, the invention provides a use of DHA and
EPA to prepare a composition comprising from about 0.05 to about 1%
DHA and from about 0.05 to about 1% EPA wherein the ratio of the
amount of EPA present in the composition to the amount of DHA
present in the composition is from about 1 to about 2 to prevent
and/or treat feline IBD. In some such embodiments, the ratio of the
amount of EPA in the composition to the amount of DHA present in
the composition is from about 1.2 to about 1.8. In other such
embodiments, the ratio of the amount of EPA present in the
composition to the amount of DHA present in the composition is from
about 1.2 to about 1.5. In other such embodiments, the ratio of the
amount of EPA present in the composition to the amount of DHA
present in the composition is from about 1.3 to about 1.6. In
further such embodiments, the ratio of the amount of EPA present in
the composition to the amount of DHA in the composition is from
about 1, about, 1.2, or about 1.3 to about 1.5, about 1.6, about
1.8, or about 2. In some embodiments, the composition comprises a
food composition.
[0036] In a further aspect, the invention provides a method for
determining if a feline is suffering from IBD. The method comprises
dividing lymphocytes collected from blood obtained from the feline
into a first, second, and third sample comprising equal amounts of
lymphocytes; exposing the second sample to an amount of a mitogen
for a period of time; exposing the third sample to the same amount
of the same mitogen as the second sample for the same period of
time as the second sample in the presence of an amount of DHA; and
comparing the levels of lymphocyte proliferation in all samples.
The feline has IBD if the level of lymphocyte proliferation in the
second sample is higher than the level of lymphocyte proliferation
in the first sample, and the level of lymphocyte proliferation in
the third sample is lower than the level of lymphocyte
proliferation in the second sample.
[0037] Lymphocytes are collected from blood obtained from a feline
and are divided into samples comprising equal amounts of
lymphocytes. Procedures for obtaining blood from felines, isolating
the lymphocytes from that blood, and counting the lymphocytes are
known to those skilled in the art. In some embodiments, lymphocytes
can be collected as described in Example 1. The lymphocytes
isolated from a feline's blood are divided into three or more
samples with all samples comprising equal amounts of lymphocytes.
In some embodiments, the lymphocytes are divided into three samples
(i.e., a first, second, and third sample). One of those samples
(i.e., the first sample) is used as a control. One of the remaining
two samples (i.e., the second sample) is exposed to an amount of
mitogen for a period of time, and the other (i.e., the third
sample) is exposed to the same amount of mitogen as the second
sample for the same period of time as the second sample in the
presence of an amount of DHA. All samples are incubated for the
same period of time at the same temperature.
[0038] A mitogen is an agent that triggers mitosis. Any mitogen
that can trigger mitosis of feline lymphocytes is suitable for the
method of the invention. In some embodiments, the mitogen is a
polyclonal mitogen. A polyclonal mitogen is a mitogen that induces
mitosis in lymphocytes of many different specificities or clonal
origins. Mitogens suitable for the method of IBD diagnosis of the
invention include, for example, phytohemagglutinin (PHA),
concanavalin (ConA), pokeweed mitogen (PWM), lipopolysaccharide
(LPS), and anti-CD3 antibody.
[0039] Procedures for measuring lymphocyte proliferation are known
to those skilled in the art. Any procedure for measuring in vitro
lymphocyte proliferation is suitable for the method of the
invention. In vitro lymphocyte proliferation can be measured
directly (e.g., by counting cells or by determining the mitotic
index) or indirectly (e.g., by determining the rate of overall
metabolic activity in a lymphocyte population or by monitoring the
synthesis of deoxyribonucleic acid (DNA)). In some embodiments, in
vitro lymphocyte proliferation is measured as discussed in Example
1.
[0040] In some embodiments of the method of the invention,
lymphocyte proliferation is measured by monitoring DNA synthesis.
Procedures for monitoring and measuring DNA synthesis are known to
those skilled in the art. Any procedure for monitoring and
measuring DNA synthesis is suitable for the method of the
invention. DNA synthesis can be monitored and measured by, for
example, labeling the DNA of mitotically active cells with
.sup.3H-thymidine or 5-bromo-2'-deoxyuridine (BrdU) and then
determining the amount of .sup.3H-thymidine or BrdU that was
incorporated into DNA. In some embodiments. DNA synthesis is
measured as discussed in Example 1.
[0041] As discussed above, a determination if a feline is suffering
from IBD is made by comparing the levels of in vitro lymphocyte
proliferation in the first, second and third samples. If the level
of lymphocyte proliferation in the second sample (i.e., the sample
treated with a mitogen, but no DHA) is higher than the level of
lymphocyte proliferation in the first sample (i.e., the control
sample that was not treated with mitogen or DHA), then the mitogen
used in the assay has indeed stimulated in vitro lymphocyte
proliferation in the second and third sample. If that is the case,
then the level of lymphocyte proliferation in the third sample
(i.e., the sample treated with both the mitogen and DHA) is
compared to the level of lymphocyte proliferation in the second
sample. If the level of lymphocyte proliferation in the third
sample is lower than the level of lymphocyte proliferation in the
second sample, then DHA had inhibited the pro-inflammatory effect
of the mitogen indicating that the feline has IBD.
[0042] In a further aspect, the invention provides an article of
manufacture in the form of a kit suitable for preventing or
treating IBD. The kit comprises DHA or a DHA-comprising composition
of the invention. In some embodiments, the kit further comprises an
anti-IBD agent (i.e., the kit comprises one or more anti-IBD
agents). In some embodiments, the kit further comprises
instructions for one or more of (a) administering DHA to a feline,
(b) administering an anti-IBD agent to a feline in conjunction with
administering DHA to the feline, (c) preventing and/or treating IBD
in a feline by administering DHA to the feline, and (d) preventing
and/or treating IBD in a feline by administering an anti-IBD agent
in conjunction with administering DHA to the feline.
[0043] In some embodiments, the kit comprises a DHA-comprising food
composition. In some embodiments, the kit further comprises an
anti-IBD agent. In some embodiments, the kit further comprises
instructions for one or more of (a) feeding the DHA-comprising food
composition to a feline, (b) administering an anti-IBD agent to a
feline in conjunction with feeding the DHA-comprising food
composition to the feline, (c) preventing and/or treating IBD in a
feline by feeding the feline a DHA-comprising food composition, and
(d) preventing and/or treating IBD in a feline by administering to
the feline an anti-IBD agent in conjunction with feeding the feline
a DI-IA-comprising food composition.
[0044] In a further aspect, the invention provides an article of
manufacture in the form of a kit comprising two or more ingredients
that, when combined together and, optionally, with additional
ingredients that are or are not a part of the kit, yield a
DHA-comprising composition of the invention suitable for preventing
and/or treating IBD in a feline. One of the two or more ingredients
that are to be combined can be, for example, pure DHA or derivative
thereof or a composition comprising DHA. Another one of the two or
more ingredients that are to be combined can be, for example, a
food composition. If, to prepare a composition, additional
ingredients that are or are not a part of the kit are needed, the
kit provides instructions about those ingredients. In some
embodiments, the kit further comprises an anti-IBD agent. In some
embodiments, the kit further comprises instructions for one or more
of (a) preparing the composition by combining the two or more
ingredients and, optionally, additional ingredients that are or are
not a part of the kit, (b) feeding the composition to a feline to,
for example, prevent and/or treat IBD, (c) administering an
anti-IBD agent to the feline in conjunction with feeding the feline
the composition, (d) preventing and/or treating IBD in a feline by
feeding the feline the composition, and (e) preventing and/or
treating IBD in a feline by administering to the feline an anti-IBD
agent in conjunction with feeding the feline the composition.
[0045] In some embodiments, the kit comprises in separate
containers in a single package or in separate containers in a
virtual package, as appropriate for the kit component, either (A)
DHA, (B) a composition comprising DHA, or (C) two or more
ingredients that, when combined together, and, optionally, with
additional ingredients that are or are not a part of the kit, yield
a composition comprising DHA, and one or more of (1) at least one
fatty acid selected from the group consisting of EPA, ARA, LA, and
ALA, (2) a food composition suitable for consumption by a feline
susceptible to or suffering from inflammatory bowel disease, (3) an
anti-IBD agent, and (4) instructions for one or more of (a)
preparing a composition comprising DHA alone or in combination with
at least one fatty acid selected from the group consisting of EPA,
ARA, LA, and ALA, (b) preparing a food composition suitable
consumption by a feline susceptible to or suffering from
inflammatory bowel disease comprising a therapeutically-effective
amount of DHA alone or in combination with at least one fatty acid
selected from the group consisting of EPA, ARA, LA, and ALA, (c)
administering DHA alone or in combination with at least one fatty
acid selected from the group consisting of EPA, ARA, LA, and ALA to
a feline to prevent and/or treat IBD, (d) preventing and/or
treating IBD in a feline by feeding the feline a composition
comprising DHA alone or in combination with at least one fatty acid
selected from the group consisting of EPA, ARA, LA, and ALA, (e)
administering an anti-IBD agent to a feline in conjunction with
feeding the feline a composition comprising DHA alone or in
combination with at least one fatty acid selected from the group
consisting of EPA, ARA, LA, and ALA, and (f) preventing and/or
treating IBD in a feline by administering to the feline an anti-IBD
agent in conjunction with feeding the feline a composition
comprising DHA alone or in combination with at least one fatty acid
selected from the group consisting of EPA, ARA. LA, and ALA.
[0046] The term "single package" generally means that the
components of a kit are physically associated in or with one or
more containers and considered as a unit of manufacture,
distribution, sale, or use. Containers include, for example, bags,
boxes, bottles, shrink wrap packages, stapled or otherwise fixed
components, and combinations thereof. A single package can be, for
example, containers or individual food compositions physically
associated such that they are considered a unit for manufacture,
distribution, sale, or use. The term "virtual package" generally
means that the components of a kit are associated by directions on
one or more physical or virtual kit components instructing the user
how to obtain additional components. e.g., in a bag containing one
component and directions instructing the user to go to a website,
contact a recorded message, view a visual message, or contact a
caregiver to obtain instructions on how to use the kit. When the
kit comprises a virtual package, the kit is limited to instructions
in a virtual environment with one or more physical kit
components.
[0047] In a further aspect, the invention provides a means for
communicating information about or instructions for one or more of
(1) using DHA or a composition comprising DHA to prevent and/or
treat IBD in a feline, (2) preventing and/or treating IBD in a
feline by administering to the feline an anti-IBD agent in
conjunction with feeding the feline DHA or a composition comprising
DHA, and (3) using a kit of the invention for preventing and/or
treating IBD in a feline comprising a document, digital storage
media, optical storage media, audio presentation, or visual display
containing the information or instructions. In some embodiments,
the communicating means comprises a document, digital storage
media, optical storage media, audio presentation, or visual display
containing the information or instructions. Preferably, the
communication means is a displayed web site or a brochure, product
label, package insert, advertisement, or visual display containing
such information or instructions. Useful information or
instructions include, for example, (1) information and instructions
how to use a composition, method, or kit of the invention and (2)
contact information for animal caregivers if they have a question
about the invention and its uses.
[0048] In a further aspect, the present invention provides for a
use of DHA and optionally at least one fatty acid selected from the
group consisting of EPA, ARA, LA, and ALA to prepare a medicament.
In another, the invention provides for the use of a
therapeutically-effective amount of such fatty acid(s) to prepare a
medicament for preventing or treating feline IBD. Generally,
medicaments are prepared by admixing a compound or composition with
excipients, buffers, binders, plasticizers, colorants, diluents,
compressing agents, lubricants, flavorants, moistening agents, and
other ingredients known to skilled artisans to be useful for
producing medicaments and formulating medicaments that are suitable
for administration to an animal.
[0049] The invention is not limited to the particular methodology,
protocols, and reagents described herein because they may vary.
Further, the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to limit
the scope of the present invention. As used herein and in the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise.
Similarly, the words "comprise", "comprises", and "comprising" are
to be interpreted inclusively rather than exclusively.
[0050] Unless defined otherwise, all technical and scientific terms
and any acronyms used herein have the same meanings as commonly
understood by one of ordinary skill in the art in the field of the
invention. Although any methods and materials similar or equivalent
to those described herein can be used in the practice of the
present invention, the preferred methods, devices, and materials
are described herein.
[0051] All patents, patent applications, and publications mentioned
herein are incorporated herein by reference to the extent allowed
by law for the purpose of describing and disclosing the compounds,
processes, techniques, procedures, technology, articles, and other
compositions and methods disclosed therein that might be used with
the present invention. However, nothing herein is to be construed
as an admission that the invention is not entitled to antedate such
disclosure by virtue of prior invention.
EXAMPLES
[0052] The invention can be further illustrated by the following
examples, although it will be understood that the examples are
included merely for purposes of illustration and are not intended
to limit the scope of the invention unless otherwise specifically
indicated.
Example 1
[0053] This example illustrates the effect of DHA and EPA on in
vitro lymphocyte proliferation of lymphocytes obtained from the
blood of healthy cats and cats with IBD.
[0054] The study utilizes eleven healthy cats and eleven cats with
IBD. The cats with IBD are diagnosed by an intestinal biopsy, and
have a history of chronic diarrhea. For six weeks, all cats are fed
a nutritionally adequate dry food indicated for gastrointestinal
distress. Blood is drawn from all cats at four, five, and six
weeks. Samples with equal amounts of lymphocytes are used in an in
vitro lymphocyte proliferation assay.
[0055] More specifically, 4.5 ml blood is mixed with 4.5 ml HBSS
(Hank's Balanced Salt Solution)+25 mM HEPES. 4 ml of Ficoll-Paque
Plus (Amersham) are slowly injected under the diluted blood. The
mixture is centrifuged for twenty minutes at 500 g at 25.degree. C.
The upper layer is discarded. The lymphocytes are transferred into
a clean tube, resuspended in 6 ml of HBSS+25 mM HEPES, and then
centrifuged for ten minutes at 200 g at 25.degree. C. The
supernatant is discarded, and the lymphocytes are washed with 6 ml
of HBSS. This centrifugation and wash step is repeated two more
times. The washed lymphocytes are then resuspended in 5 ml of AIM
medium (Invitrogen). Samples with 200.000 lymphocytes in 100 .mu.l
of AIM medium are used for the lymphocyte proliferation assay.
[0056] The lymphocyte proliferation assay is performed utilizing
Amersham's Biotrak cell proliferation ELISA system, version 2.
Lymphocyte samples are incubated in a 96-well plate with 7 .mu.g/ml
PHA (phytohemagglutinin) in the absence or presence of 12.5 .mu.M
and 25 .mu.M DHA or EPA in a 37.degree. C. incubator with 5%
CO.sub.2 and 90% humidity for forty hours. After that, 20 .mu.l of
00 .mu.M BrdU is added, and the samples are incubated for two hours
at 37.degree. C. The plates are then removed from the incubator and
centrifuged at 300 g for ten minutes at 25.degree. C. The medium is
removed by tapping and blotting, and the cells are dried in a
chemical fume hood for at least fifteen minutes. 200 .mu.l of
fixative is added to every well and the plate is incubated for
thirty minutes at room temperature. The cell fixative is removed by
tapping and blotting. 200 .mu.l of blocking buffer are added to
every well, and the plate is incubated for thirty minutes at room
temperature. The blocking buffer is removed by tapping and
blotting, and 100 .mu.l of anti-BrdU working solution is added to
each well and incubated for ninety minutes at room temperature. The
anti-BrdU solution is removed by tapping and blotting, and each
well is rinsed three times with 300 .mu.l washing solution. 100
.mu.l 3,3'5,5'-tetamethylbenzidine is added to each well and
incubated for ten minutes or until color intensity is achieved. 25
.mu.l 1M sulfuric acid is added to stop the reaction, and the plate
is read on a fusion microplate reader (PerkinElmer) within five
minutes. The results from the in vitro proliferation assay are
presented in tables 1 and 2.
TABLE-US-00001 TABLE 1 Effect of DHA on Lymphocyte Proliferation
PHA + 12.5 .mu.M PHA + 25 .mu.M no PHA PHA DHA DHA Healthy 0.05
.+-. 0.05 0.32 .+-. 0.21 0.28 .+-. 0.19 0.25 .+-. 0.17 cats Cats
0.09 .+-. 0.11 0.58 .+-. 0.26 0.49 .+-. 0.15 0.45 .+-. 0.18 with
IBD
TABLE-US-00002 TABLE 2 Effect of EPA on Lymphocyte Proliferation
PHA + 12.5 .mu.M PHA + 25 .mu.M no PHA PHA EPA EPA Healthy 0.05
.+-. 0.05 0.32 .+-. 0.21 0.38 .+-. 0.23 0.37 .+-. 0.23 cats Cats
0.09 .+-. 0.11 0.58 .+-. 0.26 0.62 .+-. 0.27 0.63 .+-. 0.29 with
IBD
[0057] As can be seen from Tables 1 and 2, the proliferation
activity of the lymphocytes obtained from the blood of the cats
with IBD is higher than the proliferation activity of the
lymphocytes obtained from the blood of the healthy cats. Incubation
of lymphocytes from the cats with IBD with both 12.5 and 25 .mu.M
DHA results in a decrease in lymphocyte proliferation. Incubation
of lymphocytes from the cats with IBD with both 12.5 and 25 .mu.M
EPA does not result in a decrease in lymphocyte proliferation.
Example 2
[0058] This example illustrates the effect of DHA and EPA on the
cytokine profile of lymphocytes obtained from the blood of healthy
cats and cats with IBD.
[0059] Lymphocytes are obtained from the blood of healthy cats and
cats with IBD as described in Example 1 and then treated with DHA
or EPA also as described in Example 1. Ribonucleic acid (RNA) is
extracted from all samples, and real time polymerase chain reaction
(RT-PCR) is performed to examine the changes in the level of
expression of the following cytokines: interleukins 1.alpha.,
1.beta., 2, 6, and 10 (IL-1.alpha., IL-1.beta., IL-2, IL-6, and
IL-10), macrophage inhibitory factor (MIF), interferon gamma
(IFN-.gamma.), and transforming growth factor beta (TGF-.beta.).
The results from the cytokine PCR analysis are presented in Table
3.
TABLE-US-00003 TABLE 3 Effect of DHA and EPA on Cytokine Expression
IL- IL- IL- IFN- IL- IL- TGF- 1.alpha. 6 MIF 2 .gamma. 1.beta. 10
.beta. Healthy cats 1 1 1 1 1 1 1 1 (no PHA) Healthy cats 0.82 3.55
12.25 1.03 5.95 1.03 0.65 1.1 (PHA) Healthy cats 0.86 5.75 16.9
21.05 37.6 0.87 0.95 3.55 (PHA + DHA) Healthy cats 1.05 10.6 14.05
59.4 130.9 1.17 1.2 4.7 (PHA + EPA) Cats with 0.355 * 0.56 39.25
106 0.53 1.4 6.9 IBD (no PHA) Cats with 0.57 5.1 10.45 7.9 23.75
0.63 0.37 6.95 IBD (PHA) Cats with 0.57 13.9 6.55 3 4.9 1.85 0.45
1.45 IBD (PHA + DHA) Cats with 0.46 6.45 4.45 1.89 4.45 1.34 0.34
2.4 IBD (PHA + EPA) * = undetected DHA = 12.5 .mu.M DHA EPA = 12.5
.mu.M EPA
[0060] Referring to Table 3, the level of expression of the
proinflammatory interleukins IL-2 and IFN-.gamma. is much higher in
cats with IBD compared to healthy cats, indicating the presence of
inflammation. The level of expression of the anti-inflammatory
cytokine TGF-.beta. is slightly higher in cats with IBD compared to
healthy cats, probably to counteract inflammation. DHA decreases
the level of expression of IL-2 and IFN-.gamma. in cats with IBD to
a level similar to the level of expression of IL-2 and IFN-.gamma.
in the healthy cats stimulated with PHA in the absence of DHA. The
levels of expression of cytokines in lymphocytes from cats with IBD
treated with PHA and DHA are similar to the levels of expression of
cytokines in lymphocytes from healthy cats treated with PHA in the
absence of DHA, suggesting that DHA normalizes the response of the
lymphocytes from the cats with IBD to mitogen stimulation.
[0061] EPA also decreases the level of expression of IL-2 and
IFN-.gamma. in cats with IBD to a level similar to the level of
expression of IL-2 and IFN-.gamma. in the healthy cats stimulated
with PHA in the absence of EPA. The levels of expression of
cytokines in lymphocytes from cats with IBD treated with PHA and
EPA are similar to the levels of expression of cytokines in
lymphocytes from healthy cats treated with PHA in the absence of
EPA, suggesting that EPA, like DHA, also normalizes the response of
the lymphocytes from the cats with IBD to mitogen stimulation
(although, as shown in Example 1. EPA does not decrease the level
of lymphocyte proliferation in vitro).
[0062] In the specification, there have been disclosed typical
preferred embodiments of the invention and, although specific terms
are employed, they are used in a generic and descriptive sense only
and not for purposes of limitation, the scope of the invention
being set forth in the claims. Obviously many modifications and
variations of the invention are possible in light of the above
teachings. It is therefore to be understood that within the scope
of the appended claims the invention may be practiced otherwise
than as specifically described.
* * * * *