U.S. patent application number 14/129708 was filed with the patent office on 2015-01-01 for methods for treating ocular inflammatory disorders.
This patent application is currently assigned to MASSACHUSETTS EYE AND EAR INFIRMARY. The applicant listed for this patent is Demetrios G. Vavvas. Invention is credited to Demetrios G. Vavvas.
Application Number | 20150005254 14/129708 |
Document ID | / |
Family ID | 46640089 |
Filed Date | 2015-01-01 |
United States Patent
Application |
20150005254 |
Kind Code |
A1 |
Vavvas; Demetrios G. |
January 1, 2015 |
METHODS FOR TREATING OCULAR INFLAMMATORY DISORDERS
Abstract
Provided are methods for treating ocular inflammatory disorders,
including macular edema, using an AMP kinase activator, e.g.,
5-aminoimidazole-4-carboxamide-1-.beta.-d-ribofuranoside (AICAR).
The method reduces inflammation, thereby minimizing the loss of
vision or visual function associated with these ocular
disorders.
Inventors: |
Vavvas; Demetrios G.;
(Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vavvas; Demetrios G. |
Boston |
MA |
US |
|
|
Assignee: |
MASSACHUSETTS EYE AND EAR
INFIRMARY
Boston
MA
|
Family ID: |
46640089 |
Appl. No.: |
14/129708 |
Filed: |
June 27, 2012 |
PCT Filed: |
June 27, 2012 |
PCT NO: |
PCT/US2012/044409 |
371 Date: |
August 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61597258 |
Feb 10, 2012 |
|
|
|
61501586 |
Jun 27, 2011 |
|
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|
Current U.S.
Class: |
514/52 |
Current CPC
Class: |
A61K 31/7056 20130101;
A61P 27/02 20180101 |
Class at
Publication: |
514/52 |
International
Class: |
A61K 31/7056 20060101
A61K031/7056 |
Claims
1. A method of treating macular edema in a subject in need thereof,
the method comprising: administering AICAR or a pharmaceutically
acceptable salt, ester or prodrug thereof to the subject in an
amount sufficient to ameliorate a symptom of the macular edema.
2. The method of claim 1, wherein the macular edema occurs as a
result of age-related macular degeneration, cataract surgery,
diabetes, drug toxicity, eye injury, retinal vein occlusion, or
other inflammatory eye diseases.
3. The method of claim 1, wherein the macular edema occurs as a
result of cataract surgery or diabetes.
4. A method of treating autoimmune uveitis or uveitis associated
with type II, type III, type IV, or type V hypersensitivity
reactions in a subject in need thereof, the method comprising:
administering AICAR or a pharmaceutically acceptable salt, ester or
prodrug thereof to the subject in an amount sufficient to
ameliorate a symptom of the uveitis.
5. A method of treating endophthalmitis in a subject in need
thereof, the method comprising: administering
5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or a
pharmaceutically acceptable salt, ester or prodrug thereof to the
subject in an amount sufficient to ameliorate a symptom of the
endophthalmitis.
6. The method of claim 5, wherein the endophthalmitis is the
exogenous form.
7. The method of claim 5, wherein the endophthalmitis is the
endogenous form.
8. The method of claim 1, wherein from about 0.01 mg/kg to about
500 mg/kg of AICAR or a pharmaceutically acceptable salt, ester or
prodrug thereof is administered.
9. The method of claim 1, wherein from about 0.01 mg/kg to about
200 mg/kg of AICAR or a pharmaceutically acceptable salt, ester or
prodrug thereof is administered.
10. The method of claim 1, wherein about 0.5 mg/kg to about 100
mg/kg of AICAR or a pharmaceutically acceptable salt, ester or
prodrug thereof is administered.
11. The method of claim 1, wherein about 1 mg/kg to about 10 mg/kg
of AICAR or a pharmaceutically acceptable salt, ester or prodrug
thereof is administered.
12. The method of claim 1, wherein the AICAR or the
pharmaceutically acceptable salt, ester or prodrug thereof is
administered to the eye.
13. The method of claim 1, wherein the AICAR or the
pharmaceutically acceptable salt, ester or prodrug thereof is
administered by intraocular injection.
14. The method of claim 1, wherein the AICAR or the
pharmaceutically acceptable salt, ester or prodrug thereof is
administered intravitreally.
15. The method of claim 1, wherein the AICAR or the
pharmaceutically acceptable salt, ester or prodrug thereof is
administered systemically.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional Patent Application No. 61/501,586, filed Jun. 27, 2011,
and U.S. Provisional Patent Application No. 61/597,258, filed Feb.
10, 2012, the contents of each of which are hereby incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
ocular inflammatory disorders. More particularly, the invention
relates to methods for treating such disorders using an AMP kinase
activator, e.g.,
5-aminoimidazole-4-carboxamide-1-.beta.-d-ribofuranoside
(AICAR).
BACKGROUND OF THE INVENTION
[0003] There are a variety of ocular inflammatory disorders, which,
if untreated, may lead to partial or even complete vision loss. One
prominent ocular inflammatory disorder is uveitis, which is
estimated to be responsible for approximately 10% of the blindness
in the United States. Uveitis is an inflammatory eye disorder of
the uveal tract and contiguous structures including the vascular
coat of the eye composed of the iris, ciliary body and choroid
(Durrani et al. (2004) BR. J OPHTHALMOL. 88:1159-1162; Durrani et
al. (2004) OPHTHALMOLOGICA 218:223-236). Other examples of ocular
inflammatory disorders include endophthalmitis (e.g., the
endogenous form and the exogenous form), macular edema (e.g.,
macular edema that occurs as a result of age-related macular
degeneration, cataract surgery, diabetes, drug toxicity, eye
injury, or retinal vein occlusion), conjunctivitis, episcleritis,
keratitis, optic neuritis, orbital pseudotumor, retinal vasculitis,
and scleritis. Each of these disorders, if left untreated, can
cause permanent vision loss.
[0004] Available treatments for uveitis and other ocular
inflammatory disorders are limited. Corticosteroids are the main
drugs used for its treatment but they have numerous ocular
(cataract and secondary glaucoma) and non-ocular adverse effects.
Prolonged systemic steroid use can also suppress musculoskeletal
growth, cause impaired wound healing, and result in increased
susceptibility to infections. In addition to corticosteroids,
antimetabolites, cycloplegics, and biologics are also often used to
control the inflammatory process. However, there are patients who
do not respond or cannot tolerate these agents (Galor et al. (2008)
OPHTHALMOLOGY 115:1826-1832; Imrie et al. (2007) CURR. OPIN.
OPHTHALMOL. 18:481-486).
[0005] Thus, there is still an ongoing need for methods of
preventing the onset of ocular inflammatory disorders, and once
established, the treatment of such disorders.
SUMMARY OF THE INVENTION
[0006] The invention is based, in part, on the discovery that an
AMP kinase activator, e.g.,
5-aminoimidazole-4-carboxamide-1-.beta.-d-ribofuranoside (AICAR)
can be used to prevent and/or treat ocular inflammatory disorders,
such as macular edema, uveitis (e.g., autoimmune uveitis and
uveitis associated with type II, type III, type IV, or type V
hypersensitivity reactions), and endophthalmitis. The disclosed
methods comprise administering AICAR or a pharmaceutically
acceptable salt, ester, prodrug, or polymorph thereof to a subject
in need thereof in an amount sufficient to ameliorate a symptom of
the disorder, e.g., to reduce ocular inflammation in the affected
eye or part of the eye. In exemplary embodiments, the AICAR is
administered to the eye, e.g., by intraocular injection or by
topical administration to the eye.
[0007] The foregoing aspects and embodiments of the invention may
be more fully understood by reference to the following figures,
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The objects and features of the invention may be more fully
understood by reference to the drawings described herein.
[0009] FIGS. 1A-C depict the effect of AICAR on clinical
endotoxin-induced uveitis (EIU). FIG. 1A is a graph showing the
clinical inflammation scores of EIU in Lewis rats in the absence
and presence of AICAR determined at 24 hours after
lipopolysaccharide (LPS) injection. (Normal: n=6, EIU: n=15,
LPS+AICAR 50 mg/kg: n=15, LPS+AICAR 100 mg/kg: n=12). Data are
shown as mean.+-.SD; *p<0.05 and ***p<0.001. FIG. 1B provides
photographs indicating that fibrin formation and posterior
synechiae were seen less in AICAR-pretreated EIU rats. FIG. 1C
provides histopathologic findings of EIU rat's eyes which received
LPS with and without 50 mg/kg of AICAR pretreatment. The number of
leukocytes infiltrated around the iris-ciliary body was suppressed
in AICAR pretreated rats. Sections were visualized at
200.times..
[0010] FIGS. 2A-B depict the effect of AICAR on cellular
infiltration and protein leakage in the aqueous humor. FIG. 2A is a
graph depicting the number of infiltrated cells as determined by
trypan blue exclusion cell counting. FIG. 2B is a graph depicting
the total protein concentration in the aqueous humor (Normal: n=7,
AICAR: n=6, LPS: n=11, LPS with AICAR: n=11). LPS induced cellular
infiltration and protein leakage in the aqueous humor were
significantly suppressed by AICAR pretreatment. Data are shown as
mean.+-.SD; **p<0.01.
[0011] FIGS. 3A-B depict the effect of AICAR on leukocyte adhesion
in retinal vessels. FIG. 3A is a graph showing the number of
retinal adherent leukocytes (Normal: n=5, AICAR: n=6, LPS: n=8, LPS
with AICAR: n=11). AICAR-pretreated EIU rats showed significantly
fewer adherent leukocytes than PBS-treated rats. FIG. 3B provides
photographs of flatmounted retinas from normal control rats treated
with AICAR, LPS only and LPS with AICAR pretreatment. EIU rats
revealed a significant number of leukocyte adhesion (arrows)
compared to AICAR pretreated rats. Data are shown as mean.+-.SD;
**p<0.01.
[0012] FIG. 4 depicts the effect of AICAR on NF-.kappa.B activity.
NF-.kappa.B P65 levels in the retinal nuclear extract from normal
control (n=6), LPS (n=9), LPS+AICAR (n=9) were determined by ELISA.
Data are shown as mean.+-.SD; *p<0.05, **p<0.01.
[0013] FIGS. 5A-B depict the effect of AICAR on CD14 expression.
FIG. 5A is a graph showing CD14 mRNA expression as measured by
ELISA (Normal: n=6, AICAR: n=6, LPS: n=9, LPS with AICAR: n=9).
Data are shown as mean.+-.SD; *p<0.05 and **p<0.01. FIG. 5B
depicts photographs showing protein levels of CD14 as determined by
Western blot analysis. Data are representative of three independent
experiments with similar results.
[0014] FIGS. 6A-F depict the effect of AICAR on experimental
autoimmune uveitis (EAU). FIG. 6A is a graph showing EAU clinical
score as assessed by funduscopic examination at 21 days after
immunization. (Controls: open circles, n=17; AICAR 100 mg/kg:
closed circles, n=10; AICAR 200 mg/kg: closed triangles, n=15).
FIG. 6B is a graph showing histopathologic score as assessed with
hematoxylin and eosin (H&E) sections. Mean scores are indicated
by horizontal bars. FIGS. 6C-F show representative fundus
photographs and histopathological findings of vehicle-treated EAU
mice (FIGS. 6C and E) and AICAR-treated (200 mg/kg) mice (FIGS. 6D
and F). Clinical papilledema and vasculitis (FIG. 6C) as well as
histopathological cellular infiltration, papilledema and retinal
folds (FIG. 6E) were seen in vehicle-treated EAU mice. Data are
shown as mean.+-.SD; ***p<0.001. Results were combined from
three separate experiments.
[0015] FIGS. 7A-B depict the effect of AICAR on retinal
inflammation. FIG. 7A provides graphs showing TNF-.alpha., IL-6 and
IFN-.gamma. gene expression in the retina as measured by real-time
PCR. Relative expression was normalized to beta-actin (naive
wildtype mice: n=3, control untreated EAU mice: n=5, AICAR treated
EAU mice: n=5). FIG. 7B provides graphs showing TNF-.alpha., IL-6
and IFN-.gamma. protein levels in the retina as assessed by ELISA
(control: n=10, AICAR: n=10). Data are shown as mean.+-.SD and is
representative of two to three independent experiments. *p<0.05,
**p<0.01. N.D.=not detectable.
[0016] FIGS. 8A-H depict the effect of AICAR on the development of
IRBP-reactive T cells in vivo. Lymph node (LN) cells from control
(open circles) and AICAR-treated mice (closed squares) were
stimulated with IRBP (FIG. 8A) and anti-CD3 (FIG. 8B).
Proliferative response was measured with BrdU incorporation. FIGS.
8C-F depict graphs showing production of IFN-.gamma., IL-17, IL-4,
and IL-10, respectively, as measured by ELISA. FIGS. 8G and 8H
depict graphs showing T-bet and ROR.gamma.t mRNA expression,
respectively, in CD4 T cells, as measured by real-time PCR.
Relative expression was normalized to beta-actin (n=6 to 8). Data
are expressed as mean.+-.SD and representative of two to three
independent experiments. *p<0.05, **p<0.01.
[0017] FIG. 9 depicts the effect of AICAR on the T.sub.reg
population. LN cells from controls and AICAR-treated mice were
collected at 21 days after immunization (n=5). The number of
FoxP3.sup.+CD25.sup.+CD4.sup.+ T cells was analyzed by
fluorescence-activated cell sorting (FACS). Data are representative
of two independent experiments.
[0018] FIGS. 10A-E depict the effect of AICAR on IRBP-reactive T
cells in vitro. LN cells from untreated EAU mice (day 14) were
cultured with or without AICAR in the presence of IRBP.
Proliferative response (FIG. 10A) and production of IFN-.gamma.
(FIG. 10B), IL-17 (FIG. 10C), IL-4 (FIG. 10D) and IL-10 (FIG. 10E)
were measured. Data are expressed as mean.+-.SD and representative
of three independent experiments. **p<0.01.
[0019] FIG. 11 depicts the effect of AICAR on dendritic cell (DC)
maturation in vivo. Spleen cells from naive, control EAU and
AICAR-treated EAU mice (n=4 to 5 for each group) were separated.
The number of CD11c-gated CD40, CD80, CD86 and I-A.sup.b positive
cells were examined by FACS. Data are representative of two
independent experiments.
[0020] FIGS. 12A-B depict the effect of AICAR on bone marrow
derived dendritic cell (BMDC) maturation Immature BMDCs were
stimulated with LPS for 24 hours in the presence or absence of
AICAR. FIG. 12A provides graphs showing the number of CD 11c-gated
CD40, CD80, CD86 and I-A.sup.b positive cells as measured by FACS.
Bars represent mean.+-.SD from three independent experiments. FIG.
12B provides graphs showing IL-6, IL-12/23 p40 and TNF-.alpha.
production as measured by ELISA. Data are expressed as mean.+-.SD
and representative of two independent experiments. *p<0.05,
**p<0.01.
[0021] FIG. 13 depicts western blots showing AMPK expression in WT
and AMPK.alpha.1 KO mice. Lysates of BMDC, liver and spleen were
analyzed by Western blot using antibodies against AMPK.alpha.1 and
.alpha.2. WT BMDCs only express AMPK.alpha.1. AMPK.alpha.1KO BMDCs
do not express detectable amounts of AMPK.
[0022] FIGS. 14A-B depict the effect of AMPK.alpha.1 knock down and
AICAR on BMDC maturation. BMDCs derived from AMPK.alpha.1KO mice
were stimulated with LPS with or without AICAR. FIG. 14A provides
graphs showing the number of CD11c-gated CD40, CD80, CD86 and
I-A.sup.b positive cells as measured by FACS. Bars represent
mean.+-.SD from 3 independent experiments. FIG. 14B depicts graphs
showing IL-6, IL-12/23 p40 and TNF-.alpha. production as measured
by ELISA. Data are expressed as mean.+-.SD and representative of
two independent experiments. *p<0.05, **p<0.01.
[0023] FIG. 15 depicts the combined effect of AICAR and a
nucleoside transporter or an adenosine kinase inhibitor on BMDC
maturation. WT BMDCs were stimulated with LPS and 1 mM AICAR in the
presence or absence of 0.1 mM IODO or 1 .mu.M DPY. The number of
CD11c-gated CD40, CD80, CD86 and I-A.sup.b positive cells was
measured by FACS. Bars represent mean.+-.SD from three independent
experiments.
DETAILED DESCRIPTION
[0024] The invention relates to methods for treating and/or
preventing ocular inflammatory disorders using AMP kinase (AMPK)
activators, e.g.,
5-aminoimidazole-4-carboxamide-1-.beta.-d-ribofuranoside (AICAR).
The invention is based, at least in part, on the discovery that AMP
kinase activators, e.g., AICAR, suppress ocular inflammation, e.g.,
uveitis-related ocular inflammation.
[0025] For convenience, certain terms in the specification,
examples, and appended claims are collected in this section.
[0026] As used herein, "AMP kinase activator" or "adenosine
monophosphate (AMP) kinase activator" refers to compounds that
activate, increase, or stimulate AMP kinase activity. AMP kinase
activators include
5-aminoimidazole-4-carboxamide-1-.beta.-d-ribofuranoside (AICAR) or
pharmaceutically acceptable salts, esters, or polymorphs thereof,
AICAR analogs and/or AICAR prodrugs. For example, AICAR is an
analog of AMP.
[0027] One exemplary AMP kinase activator is AICAR or a
pharmaceutically acceptable salt thereof. As used herein, "AICAR"
means 5-aminoimidazole-4-carboxamide-1-.beta.-d-ribofuranoside and
having the following chemical structure (I):
##STR00001##
including pharmaceutically acceptable salts, solvates, solvates of
a pharmaceutically acceptable salt, esters, or polymorphs thereof.
See, for example, U.S. Pat. No. 4,575,498.
[0028] In another embodiment, the AMP kinase activator is an AICAR
prodrug. A "prodrug" includes compounds that are transformed in
vivo to yield a compound of Formula (I) or a pharmaceutically
acceptable salt, ester, or polymorph of the compound. The
transformation may occur by various mechanisms, such as through
hydrolysis in blood. For example, where the compound of Formula (I)
contains an alcohol functional group, a prodrug can be formed by
the replacement of the hydrogen atom of the alcohol group with a
group such as (C.sub.1-C.sub.6)alkanoyloxymethyl,
1-((C.sub.1-C.sub.6)alkanoyloxy)ethyl,
1-methyl-1-((C.sub.1-C.sub.6)alkanoyloxy)ethyl
(C.sub.1-C.sub.6)alkoxycarbonyloxymethyl,
N--(C.sub.1-C.sub.6)alkoxycarbonylaminomethyl, succinoyl,
(C.sub.1-C.sub.6)alkanoyl, .alpha.-amino(C.sub.1-C.sub.4)alkanoyl,
arylacyl and .alpha.-aminoacyl, or
.alpha.-aminoacyl-.alpha.-aminoacyl, where each .alpha.-aminoacyl
group is independently selected from the naturally occurring
L-amino acids, P(O)(OH).sub.2,
--P(O)(O(C.sub.1-C.sub.6)alkyl).sub.2 or glycosyl (the radical
resulting from the removal of a hydroxyl group of the hemiacetal
form of a carbohydrate). Further, for example, where the compound
of Formula (I) incorporates an amine functional group, a prodrug
can be formed by the replacement of a hydrogen atom in the amine
group with a group such as R-carbonyl, RO-carbonyl, NRR'-carbonyl
where R and R' are each independently (C.sub.1-C.sub.10)alkyl,
(C.sub.3-C.sub.7)cycloalkyl, benzyl, or R-carbonyl is a natural
.alpha.-aminoacyl or natural .alpha.-aminoacyl-natural
.alpha.-aminoacyl, --C(OH)C(O)OY.sup.1 wherein Y.sup.1 is H,
(C.sub.1-C.sub.6)alkyl or benzyl, --C(OY.sup.2)Y.sup.3 wherein
Y.sup.2 is (C.sub.1-C.sub.4)alkyl and Y.sup.3 is
(C.sub.1-C.sub.6)alkyl, carboxy(C.sub.1-C.sub.6)alkyl,
amino(C.sub.1-C.sub.4)alkyl or mono-N-- or
di-N,N--(C.sub.1-C.sub.6)alkylaminoalkyl, --C(Y.sup.4)Y.sup.5
wherein Y.sup.4 is H or methyl and Y.sup.5 is mono-N-- or
di-N,N--(C.sub.1-C.sub.6)alkylamino, morpholino, piperidin-1-yl or
pyrrolidin-1-yl. Exemplary AICAR prodrugs include the generic
and/or specific compounds listed in U.S. Pat. No. 5,082,829, which
is incorporated herein by reference in its entirety.
[0029] It is also contemplated herein that generic and/or specific
compounds listed in U.S. Pat. No. 5,777,100 and PCT Publication No.
WO01/93873, which are incorporated herein by reference in their
entirety, may be used in the methods described herein.
[0030] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0031] As used herein, the term "pharmaceutically acceptable salt"
refers to any pharmaceutically acceptable salt (e.g., acid or base)
of a compound of the present invention which, upon administration
to a subject, is capable of providing a compound of this invention
or an active metabolite or residue thereof. As is known to those of
skill in the art, "salts" of the compounds of the present invention
may be derived from inorganic or organic acids and bases. Examples
of acids include, but are not limited to, hydrochloric,
hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic,
phosphoric, glycolic, lactic, salicylic, succinic,
toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,
ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,
benzenesulfonic acid, and the like. Other acids, such as oxalic,
while not in themselves pharmaceutically acceptable, may be
employed in the preparation of salts useful as intermediates in
obtaining the compounds of the invention and their pharmaceutically
acceptable acid addition salts or esters.
[0032] Examples of bases include, but are not limited to, alkali
metals (e.g., sodium) hydroxides, alkaline earth metals (e.g.,
magnesium), hydroxides, ammonia, and compounds of formula
NW.sub.4.sup.+, wherein W is C.sub.1-4 alkyl, and the like.
[0033] Examples of salts include, but are not limited to: acetate,
adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
palmoate, pectinate, persulfate, phenylpropionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate,
undecanoate, and the like. Other examples of salts include anions
of the compounds of the present invention compounded with a
suitable cation such as Na.sup.+, NH.sub.4.sup.+, and
NW.sub.4.sup.+ (wherein W is a C.sub.1-4 alkyl group), and the
like.
[0034] For therapeutic use, salts of the compounds of the present
invention are contemplated as being pharmaceutically acceptable.
However, salts of acids and bases that are non-pharmaceutically
acceptable may also find use, for example, in the preparation or
purification of a pharmaceutically acceptable compound.
[0035] As used herein, the term "therapeutically effective amount"
is understood to mean the amount of an active ingredient, for
example, AICAR, that is sufficient to reduce, ameliorate, or treat
a symptom associated with certain ocular inflammatory disorders
described herein, e.g., a symptom of the condition affecting any
part of the eye or surrounding tissue. The compounds of the
invention are administered in amounts effective at, e.g., reducing
or preventing inflammation in at least part of an affect eye or the
surrounding tissues, preserving vision, improving vision, and/or
preventing vision loss.
[0036] Disclosed herein is a method of treating ocular inflammatory
disorders using AMP kinase activators, e.g.,
5-aminoimidazole-4-carboxamide-.beta.-d-ribofuranoside (AICAR) or a
pharmaceutically acceptable salt thereof, an AICAR analog, and/or
an AICAR prodrug. Ocular inflammatory conditions that may be
treated with AICAR include, but are not limited to endophthalmitis
(e.g., the endogenous form and the exogenous form), macular edema
(e.g., macular edema that occurs as a result of age-related macular
degeneration, cataract surgery, diabetes, drug toxicity, eye
injury, retinal vein occlusion, or other inflammatory eye
diseases), conjunctivitis, episcleritis, keratitis, optic neuritis,
orbital pseudotumor, retinal vasculitis, scleritis, and uveitis
(e.g., (i) uveitis associated with sepsis (e.g., LPS-induced
uveitis); (ii) autoimmune uveitis (e.g., uveitis associated with
lupus); or (iii) uveitis associated with type II, type III, type
IV, or type V hypersensitivity reactions).
[0037] It is contemplated herein that the AICAR may be administered
after diagnosis of certain ocular inflammatory conditions described
herein, at the time of diagnosing certain ocular inflammatory
conditions described herein or, if the subject is determined to be
a risk of developing certain ocular inflammatory conditions as
described herein, the AICAR may be administered prior to diagnosis
of the condition.
[0038] In certain embodiments, the method comprises administering
AICAR to the eye of a subject in which a region of eye has been
affected by inflammation. The AICAR may be administered in an
amount sufficient to give a final concentration of AICAR in the eye
in the range from about 1 .mu.M to about 2500 .mu.M, from about 1
.mu.M to about 2000 .mu.M, from about 1 .mu.M to about 1500 .mu.M,
from about 1 .mu.M to about 1000 .mu.M, from about 10 .mu.M to
about 900 .mu.M, from about 100 .mu.M to about 900 .mu.M, from
about 200 .mu.M to about 800 .mu.M, or from about 300 .mu.M to
about 500 .mu.M.
[0039] In view of the fact that the volume of the eye in a given
subject is known (for example, typical human eye contains 4 to 6 mL
of fluid (humor)) it is within the skill in the art to calculate
the dosage of the AICAR to be administered to give the
therapeutically effective concentrations noted above. In other
embodiments, an effective amount of AICAR may be in the range of
from about 0.01 mg/kg to about 500 mg/kg, optionally from about
0.01 mg/kg to about 250 mg/kg, optionally from about 0.01 mg/kg to
about 200 mg/kg, optionally from about 1.0 mg/kg to about 100
mg/kg, optionally from about 1 mg/kg to about 10 mg/kg, of body
weight.
[0040] In therapeutic uses for treating ocular disorders, the
active ingredients typically are administered orally, parenterally
and/or topically at a dosage to obtain and maintain a concentration
that is therapeutically effective in the eye. In certain
circumstances, a therapeutically effective dose of AICAR prevents
or reduces inflammation in at least part of an affected eye. The
amount administered likely will depend on such variables as the
type and extent of disease or indication to be treated, the overall
health status of the particular patient, the relative biological
efficacy of the compound delivered, the formulation of the drug,
the presence and types of excipients in the formulation, and the
route of administration. Also, it is to be understood that the
initial dosage administered may be increased beyond the above upper
level in order to rapidly achieve the desired blood-level or tissue
level, or the initial dosage may be smaller than the optimum and
the daily dosage may be progressively increased during the course
of treatment depending on the particular situation. If desired, the
daily dose may also be divided into multiple doses for
administration, for example, two to four times per day. For
example, AICAR may be administered once a day, twice a day or more
frequently. In other embodiments, AICAR may be administered every
other day, three times a week, twice a week, or once a week.
[0041] In an exemplary embodiment, the AICAR may be administered
locally to the eye, for example, by intravitreal, intraocular,
intraorbital, periorbital, subconjuctival, subretinal, subtenons or
transscleral routes. In an exemplary embodiment, the AICAR may be
administered locally to the eye by intravitreal injection. Without
wishing to be bound by theory, local modes of administration can
reduce or eliminate the incidence of potential toxic side effects
that may occur. In another embodiment, local administration to the
eye is by topical administration.
[0042] Alternatively, the AICAR may be administered systemically,
e.g., by oral or parenteral routes. Parenteral routes include, for
example, intravenous, intrarterial, intramuscular, intradermal,
subcutaneous, intranasal, intraperitoneal and transdermal
routes.
[0043] Administration may be provided as a periodic bolus (for
example, intravitreally or intravenously) or as continuous infusion
from an internal reservoir (for example, from an implant disposed
at an intra- or extra-ocular location (see, U.S. Pat. Nos.
5,443,505 and 5,766,242)) or from an external reservoir (for
example, from an intravenous bag, or a contact lens slow release
formulation system). The AICAR may be administered locally, for
example, by continuous release from a sustained release drug
delivery device immobilized to an inner wall of the eye or via
targeted transscleral controlled release into the choroid (see, for
example, PCT/US00/00207, PCT/US02/14279, Ambati et al. (2000)
INVEST. OPHTHALMOL. VIS. SCI. 41:1181-1185, and Ambati et al.
(2000) INVEST. OPHTHALMOL. VIS. SCI. 41:1186-1191). A variety of
devices suitable for administering agents locally to the inside of
the eye are known in the art. See, for example, U.S. Pat. Nos.
6,251,090, 6,299,895, 6,416,777, 6,413,540, and 6,375,972, and
PCT/US00/28187.
[0044] The formulations, both for human and for veterinary medical
use, typically include AICAR in association with a pharmaceutically
acceptable carrier or excipient.
[0045] The AICAR may be solubilized in a carrier, for example, a
viscoelastic carrier, that is introduced locally into the eye. The
AICAR also may be administered in a pharmaceutically acceptable
carrier or vehicle so that administration does not otherwise
adversely affect the recipient's electrolyte and/or volume balance.
The carrier may comprise, for example, physiologic saline or other
buffer system. In exemplary embodiments, the AICAR may be
solubilized in PBS or another aqueous buffer by sonication.
Alternatively, the AICAR may be solubilized using conventional
solvent or solubilization systems, for example, dimethyl sulfoxide
(DMSO), dimethoxyethane (DME), dimethylformamide (DMF),
cyclodextran, micelles, liposomes, liposomal agents, and other
solvents known in the art to aid in the solubilization and
administration of hydrophobic agents.
[0046] In other embodiments, the AICAR may be solubilized in a
liposome or microsphere. Methods for delivery of a drug or
combination of drugs in liposomes and/or microspheres are
well-known in the art.
[0047] In addition, it is contemplated that the AICAR may be
formulated so as to permit release of the agent over a prolonged
period of time. A release system can include a matrix of a
biodegradable material or a material, which releases the
incorporated agent by diffusion. The AICAR can be homogeneously or
heterogeneously distributed within a release system. A variety of
release systems may be useful in the practice of the invention,
however, the choice of the appropriate system will depend upon the
rate of release required by a particular drug regime. Both
non-degradable and degradable release systems can be used. Suitable
release systems include polymers and polymeric matrices,
non-polymeric matrices, or inorganic and organic excipients and
diluents such as, but not limited to, calcium carbonate and sugar
(for example, trehalose). Release systems may be natural or
synthetic. However, under certain circumstances, synthetic release
systems are preferred because generally they are more reliable,
more reproducible and produce more defined release profiles. The
release system material can be selected so that inhibitors having
different molecular weights are released by diffusion through or
degradation of the material.
[0048] Representative synthetic, biodegradable polymers include,
for example: polyamides such as poly(amino acids) and
poly(peptides); polyesters such as poly(lactic acid), poly(glycolic
acid), poly(lactic-co-glycolic acid), and poly(caprolactone);
poly(anhydrides); polyorthoesters; polycarbonates; and chemical
derivatives thereof (substitutions, additions of chemical groups,
for example, alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art),
copolymers and mixtures thereof. Representative synthetic,
non-degradable polymers include, for example: polyethers such as
poly(ethylene oxide), poly(ethylene glycol), and
poly(tetramethylene oxide); vinyl polymers-polyacrylates and
polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl
methacrylate, acrylic and methacrylic acids, and others such as
poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl
acetate); poly(urethanes); cellulose and its derivatives such as
alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various
cellulose acetates; polysiloxanes; and any chemical derivatives
thereof (substitutions, additions of chemical groups, for example,
alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art),
copolymers and mixtures thereof.
[0049] One of the primary vehicles currently being developed for
the delivery of ocular pharmacological agents is the
poly(lactide-co-glycolide) microsphere for intraocular injection.
The microspheres are composed of a polymer of lactic acid and
glycolic acid, which are structured to form hollow spheres. These
spheres can be approximately 15-30 .mu.m in diameter and can be
loaded with a variety of compounds varying in size from simple
molecules to high molecular weight proteins such as antibodies. The
biocompatibility of these microspheres is well established (see,
Sintzel et al. (1996) EUR. J. PHARM. BIOPHARM. 42:358-372), and
microspheres have been used to deliver a wide variety of
pharmacological agents in numerous biological systems. After
injection, poly(lactide-co-glycolide) microspheres are hydrolyzed
by the surrounding tissues, which cause the release of the contents
of the microspheres (Zhu et al. (2000) NAT. BIOTECH. 18:52-57). As
will be appreciated, the in vivo half-life of a microsphere can be
adjusted depending on the specific needs of the system.
[0050] Formulations suitable for topical administration, including
eye treatment, include liquid or semi-liquid preparations such as
liniments, lotions, gels, applicants, oil-in-water or water-in-oil
emulsions such as creams, ointments or pastes; or solutions or
suspensions such as drops.
[0051] Formulations suitable for oral or parenteral administration
may be in the form of discrete units such as capsules, gelatin
capsules, sachets, tablets, troches, or lozenges, each containing a
predetermined amount of the active agent; a powder or granular
composition; a solution or a suspension in an aqueous liquid or
non-aqueous liquid; or an oil-in-water emulsion or a water-in-oil
emulsion. Formulations suitable for injectable use 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 ELTM (BASF, Parsippany, N.J.) or phosphate buffered
saline (PBS). 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 filter sterilization.
Formulations suitable for intraarticular administration may be in
the form of a sterile aqueous preparation of the drug which may be
in microcrystalline form, for example, in the form of an aqueous
microcrystalline suspension. Liposomal formulations or
biodegradable polymer systems may also be used to present the drug
for intraarticular administration. Formulations for topical
administration to the skin surface can be prepared by dispersing
the drug with a dermatologically acceptable carrier such as a
lotion, cream, ointment or soap. For intranasal or inhalation
treatments, inhalation of powder (self-propelling or spray
formulations) dispensed with a spray can, a nebulizer, or an
atomizer can be used. Such formulations can be in the form of a
fine powder for pulmonary administration from a powder inhalation
device or self-propelling powder-dispensing formulations.
EXAMPLES
[0052] The invention is further illustrated by the following
examples, which are provided for illustrative purposes only, and
should not be construed as limiting the scope or content of the
invention in any way.
[0053] In the examples described herein, all animal experiments
adhered to the Association for Research in Vision and Ophthalmology
Statement for the Use of Animals in Ophthalmic and Vision Research,
and protocols were approved by the Animal Care Committee of the
Massachusetts Eye and Ear Infirmary.
[0054] All results were expressed as mean.+-.SD. EIU and EAU scores
were compared by the Mann-Whitney test. Continuous variables from
the other experiment were analyzed with the unpaired student's t
test. Differences were considered significant at P<0.05.
Example 1
AICAR Treatment Suppresses Ocular Inflammation in a Rat EIU
Model
[0055] Endotoxin-induced uveitis (EIU) is a rodent model of human
uveitis, induced by a single systemic injection of endotoxin or
lipopolysaccharide (LPS) (Rosenbaum et al., (1980) NATURE
286:611-613). Injected animals develop acute bilateral anterior
inflammation, characterized by a breakdown of the blood-ocular
barrier and accumulation of inflammatory cells. EIU has been used
to investigate the pathogenesis of uveitis and to evaluate the
therapeutic effect of several drugs (Herbort et al. (1989) EXP. EYE
RES. 48:693-705; Avunduk et al. (2004) EXP. EYE RES. 79:357-365;
Chang et al. (2006) BR. J. OPHTHALMOL. 90:103-108; Wakefield et al.
(2010) BR. J. OPHTHALMOL. 94:271-274). The effect of AICAR on
ocular inflammation associated with uveitis was assessed using the
EIU rat model.
[0056] Male Lewis rats ranging from 6-8 weeks old (Charles River,
Wilmington, Mass.) were used for these studies. To study the
effects of AICAR pretreatment, AICAR and PBS (for placebo
treatment) were delivered via intraperitoneal injection of AICAR
(50 mg/kg or 100 mg/kg body weight; Sigma) diluted in 150 .mu.l PBS
or equal volume of PBS at 6 hours before and at the same time as
LPS injection. Four experimental groups of rats were compared: (1)
Normal: no treatment (2) AICAR: normal rats treated with AICAR 6
hours before examination; (3) LPS: EIU rats treated with PBS; (4)
LPS+AICAR: EIU rats treated with AICAR. Two additional groups of
rats were used to study the effects of AICAR (50 mg/kg) given
either at the same time as LPS injection or at 6 hours after LPS
injection.
[0057] Clinical scoring of EIU was performed as previously
described (Pouvreau et al. (1998) J. NEUROIMMUNOL. 86:171-181).
Slit lamp examination was conducted 24 hours after LPS injection in
a masked fashion. The severity of EIU was graded from 0 to 4 by a
masked investigator, using the following scale: 0=no inflammatory
reaction; 1=discrete inflammation of the iris and conjunctival
vessels; 2=dilation of the iris and conjunctival vessels with
moderate flare in the anterior chamber; 3=hyperemia in iris
associated with Tyndall effect in the anterior chamber; and 4=same
clinical signs as 3 plus the presence of fibrin or synechiae.
[0058] For histopathologic evaluations, EIU rats were euthanized 24
hours after LPS injection. The eyes were enucleated immediately and
stored in 10% formalin solution. The eyes were then embedded in
paraffin, and 10 .mu.m sagittal sections were cut and stained with
hematoxylin and eosin. For histopathologic evaluation, the anterior
chamber and posterior chamber around the iris-ciliary body complex
were examined with light microscopy.
[0059] As depicted in FIG. 1A, clinical inflammation scores were
significantly reduced in EIU rats pretreated with AICAR at either
the 50 mg/kg dosage (2.2.+-.0.75, n=15) or at the 100 mg/kg dosage
(2.7.+-.1.4, n=12) when compared with PBS-treated EIU rats
(3.6.+-.0.7, n=15) (p=0.00008 and 0.045, respectively). No
inflammation was detected in age-matched normal rats that received
AICAR alone (n=6). Further, fibrin formation and posterior
synechiae were rarely seen in AICAR-pretreated EIU animals when
compared to PBS-treated EIU rats or normal rats that received AICAR
alone (FIG. 1B). Histopathological findings also revealed severe
leukocyte infiltration in EIU rats compared to rats pretreated with
AICAR (FIG. 1C).
[0060] The effects of a single dose of AICAR administered at the
time of LPS injection or at 6 hours after LPS injection were also
studied. With these alternative timing regimens, there was no
suppression of inflammation compared with controls. EIU clinical
scores were 3.7.+-.0.5 (n=6) and 3.9.+-.0.2 (n=6), respectively.
Since pretreatment with AICAR at the 100 mg/kg dosage was not more
effective in reducing inflammation than the 50 mg/kg dosage (no
statistically significant difference, p=0.347), the remainder of
the experiments were conducted with the 50 mg/kg dose.
[0061] Together, these data indicate that AICAR administration
suppresses ocular inflammation in the rat EIU model as measured by
slit lamp assessment and histopathology.
Example 2
AICAR Treatment Reduces Cell Infiltration into the Aqueous
Humor
[0062] Aqueous humor was collected by anterior chamber puncture
with a 30-gauge needle 24 hours after LPS injection. For cell
counting, 1 .mu.l of aqueous humor was diluted with an equal amount
of Trypan-blue solution, and the cells were counted with a
hematocytometer under a light microscope. A separate sample of
aqueous humor was centrifuged at 2500 rpm for 5 minutes at
4.degree. C. and the total protein concentration was measured by
Lowry methods using a Bio-Rad protein assay kit (Hercules, Calif.).
Aqueous samples were stored on ice until used; cell counts and
total protein concentrations were measured on the day of sample
collection.
[0063] FIGS. 2A-B depict the anti-inflammatory effect of AICAR on
cellular infiltration and protein concentration in the aqueous
humor. LPS injection induced severe cell infiltration into the
aqueous humor (22.0.times.10.sup.5 cells/ml, range 2.5-53.0
cells/ml, n=11), whereas the number of infiltrating cells was
reduced with AICAR pretreatment (1.4.times.10.sup.5 cells/ml, range
0-4.5 cells/ml, n=11, p=0.001). There was also an increase in total
protein concentration in the aqueous humor of EIU rats (18.3 mg/ml,
range 8.2-37.8 cells/ml, n=11), which was inhibited in
AICAR-pretreated EIU rats (7.9 mg/ml, range 0-17.8 cells/ml, n=11,
p=0.006). Normal control rats (n=6) and AICAR-only-treated rats
(n=6) did not show any significant infiltration of cells nor a
significant increase in protein levels in the aqueous humor.
[0064] These data indicate that AICAR treatment reduces cell
infiltration into the aqueous humor.
Example 3
AICAR Treatment Suppresses Leukocyte Adhesion
[0065] Inflammation in the posterior segment of the eye is seen in
EIU with the adherence of recruited leukocytes to the retinal
vascular endothelium (Koizumi et al. (2003) INVEST. OPHTHALMOL.
VIS. SCI. 44:2184-2191). Leukocyte adhesion to the retinal vessels
was evaluated at 24 hours after EIU induction by using the
Concanavalin A (Con A) lectin staining technique (Smith et al.
(1994) INVEST. OPHTHALMOL. VIS. SCI. 35:101-111). After deep
anesthesia, the chest cavities of rats were opened, and a 20-gauge
perfusion cannula was introduced into the aorta. Rats were then
perfused with 20 ml of PBS to remove erythrocytes and nonadherent
leukocytes, followed by 20 ml of fluorescein-isothiocyanate
(FITC)-coupled Con A (Vector Laboratories, Burlington, Calif.) in
PBS for staining the vascular endothelium and firmly adherent
leukocytes. Subsequently, residual unbound Con A was removed with
20 ml of PBS. The eyes were subsequently enucleated and the retinas
were carefully flatmounted. The flatmounts were imaged using an
epifluorescence microscope (DM RXA; Leica) and the total number of
Con A-stained adherent leukocytes per retina was counted.
[0066] EIU-associated leukocyte adhesion to retinal vascular
endothelium was reduced with AICAR pretreatment (FIGS. 3A and 3B).
Normal control rats (n=5) and AICAR-only-treated rats (n=6) showed
few adherent leukocytes. In contrast, EIU rats revealed significant
numbers of adherent leukocytes at 24 hours after LPS injection
(1163.+-.244 cells, n=8). AICAR pretreatment of EIU rats resulted
in significant suppression of leukocyte adhesion (576.+-.249 cells,
n=11, p=0.001).
Example 4
AICAR Treatment Suppresses Vascular Leakage
[0067] Protein leakage from retinal vessels was assessed with a
biotinylated bovine serum albumin (bBSA) assay as previously
described (Trichonas et al. (2010) INVEST. OPHTHALMOL. VIS. SCI.
51:1677-1682). At 23 hours after LPS injection, rats were
anesthetized and 0.14 ml of 43.7 mg/ml bBSA (Santa-Cruz
Biotechnology, Santa Cruz, Calif.) was injected through the femoral
vein. After one hour, the chest cavities of rats were opened, and
the rats were perfused with lactated Ringer's solution via the left
ventricle for 6 minutes. Subsequently, the eyes were enucleated and
retinas were carefully removed and placed in cold PBS. The retinas
were then placed in 500 .mu.l of lysis buffer containing protease
inhibitor (Complete Protease Inhibitor Cocktail Tablets, Roche
Diagnostics Corp, Indianapolis, Ind.), sonicated, and centrifuged
at 13,000 rpm for 10 minutes. Supernatant was collected, and bBSA
concentration was measured by enzyme-linked immunosorbent assay
(ELISA). The degree of vascular leakage was estimated by measuring
the protein concentration in each retina.
[0068] EIU rats had a higher amount of protein leakage from the
retinal vessels as measured with bBSA assay (30.5.+-.21.2 ng/mg
total retinal protein, n=8) when compared to AICAR-pretreated EIU
rats (11.2.+-.7.2 ng/mg total retinal protein, n=8, p=0.03).
Example 5
AICAR Treatment Inhibits NF-.kappa.B Activity
[0069] To investigate the LPS-induced signal transduction pathway,
NF-.kappa.B P65 protein levels translocated into the nuclei of
retinal cell extracts were examined 3 hours after LPS injection.
For the measurement of NF-.kappa.B activity, pooled retinas from
six normal rats, nine EIU PBS-treated rats, and nine EIU
AICAR-treated rats were analyzed. Retinas were homogenized and
nuclear extracts were prepared with ProteoJet Cytoplasmic and
Nuclear Protein Extraction Kit (Fermentas inc, Burlington, Canada).
The amount of NF-.kappa.B in the nuclear extracts was analyzed by
measuring the p65 protein levels with a NF-.kappa.B detection kit
(p65 Transcription Factor Assay kit; Active Motif, Carlsbad,
Calif.). NF-.kappa.B concentration was calculated and corrected for
protein concentration.
[0070] As shown in FIG. 4, protein levels in the nuclear extracts
were significantly elevated in PBS-treated EIU rats (27.3.+-.11.0
ng/mg total protein, n=9) compared to control rats (8.3.+-.8.6
ng/mg total protein, n=6, p=0.004), whereas AICAR pretreatment
significantly suppressed the upregulation (18.2.+-.3.9 ng/mg total
protein, n=9, p=0.03).
[0071] Stimulation by various signals, including exposure to LPS,
lead to NF-.kappa.B activation and its movement into the nucleus
where it triggers transcription of various pro-inflammatory genes
including MCP-1, TNF-.alpha. and ICAM-1 (Baeuerle et al., (1994)
ANNU REV IMMUNOL, 12:141-179; Baldwin et al., (1996) ANNU REV
IMMUNOL, 14:649-683). In the retinal samples, LPS-induced
NF-.kappa.B translocation to nuclei was significantly reduced in
AICAR-pretreated rats. These results suggest that the subsequent
diminished expression of inflammatory mediators in the eye could be
the result of inhibition of NF-.kappa.B activity.
Example 6
AICAR Treatment Alters Intra-Ocular Cytokine and Chemokine
Levels
[0072] Under deep anesthesia, experimental rats were sacrificed and
the retinas were carefully removed 24 hours after LPS injection,
placed into 200 .mu.l of lysis buffer and then sonicated. The
lysate was centrifuged at 13,000 rpm for 10 minutes at 4.degree. C.
and supernatant was isolated. Three hours after LPS injection,
blood samples were collected from the heart and subsequently
centrifuged at 3000 rpm for 30 minutes at 4.degree. C., and serum
samples were separated. Cytokine and chemokine levels in the serum,
aqueous humor, and retina were assessed using the rat CCL2/MCP-1
kit (Invitrogen, Camarillo, Calif.), the rat TNF-.alpha. kit
(R&D system, Minneapolis, Minn.) and the rat ICAM-1 kit
(R&D systems) by ELISA.
[0073] Protein expression of CCL2/MCP-1, TNF-.alpha. and ICAM-1 in
the aqueous humor (n=12) and retina (n=14) were measured (Table 1).
When compared to PBS-treated EIU rats, AICAR-pretreated EIU rats
had significantly lower aqueous humor levels of CCL2/MCP-1
(p=0.005), TNF-.alpha. (p=0.002) and ICAM-1 (p=0.04). Similarly,
expression of CCL2/MCP-1 and ICAM-1 in the retina was suppressed in
the AICAR-pretreated group as compared to the PBS-treated group
(p=0.004 and p=0.02, respectively). TNF-.alpha. protein was not
detected in any retina.
[0074] Since AICAR was administered systemically, its suppressing
effects on intraocular inflammation could have been mediated
locally or systemically. To investigate the systemic effects of
AICAR during EIU development, the serum cytokine levels at 3 hours
after LPS injection (n=11) were also measured. Both CCL2/MCP-1 and
ICAM-1 levels were suppressed in AICAR-pretreated EIU rats compared
with PBS-treated rats (CCL2/MCP-1: 0.4.+-.0.2 ng/ml vs 6.8.+-.0.5
ng/ml, p=0.002 and ICAM-1: 21.6.+-.5.0 ng/ml vs 28.7.+-.8.1 ng/ml,
p=0.02).
TABLE-US-00001 TABLE 1 Cytokine and chemokine production in aqueous
humor, retina and serum LPS LPS + AICAR p value Aqueous humor (n =
12) MCP-1 (ng/ml) 4.4 .+-. 3.9 0.8 .+-. 1.0 0.005 TNF-.alpha.
(pg/ml) 277.8 .+-. 123.9 92.5 .+-. 140.5 0.002 ICAM-1 (ng/ml) 8.7
.+-. 4.4 4.4 .+-. 3.3 0.037 Retina (n = 14, pg/total retinal
protein) MCP-1 18.7 .+-. 11.3 8.8 .+-. 2.9 0.004 TNF-.alpha. N.D.
N.D. ICAM-1 316.4 .+-. 132.3 202.9 .+-. 113.4 0.021 Serum (n = 11)
MCP-1 (ng/ml) 6.8 .+-. 0.5 0.4 .+-. 0.2 0.002 TNF-.alpha. (pg/ml)
147.2 .+-. 337.1 57.8 .+-. 143.1 0.475 ICAM-1 (ng/ml) 28.7 .+-. 8.1
21.6 .+-. 5.0 0.023 N.D. = not detectable
[0075] Leukocyte adhesion to the retinal vessel is a well
documented finding in EIU and expression of adhesion molecules such
as ICAM-1 play a pivotal role in the pathogenesis of this finding
(Yang et al. (2005) BLOOD 106:584-592). It has been previously
noted that ICAM-1 is expressed on vascular endothelial cell of the
iris and the ciliary body and that an antibody to ICAM-1 reduces
ocular inflammation (Becker et al. (2001) INVEST. OPHTHALMOL. VIS.
SCI. 42:2563-2566). As seen in Example 3, the number of adherent
leukocytes in retinal vessels of EIU rats was reduced by AICAR
pretreatment. It is contemplated that this may be explained at
least in part by the observed reduction in retinal expression of
ICAM-1 and MCP-1, which is also involved in leukocyte recruitment.
Moreover, as seen in Example 4, retinal vascular impermeability was
well maintained in the AICAR-pretreated rats. This can also be at
least partially attributed to the reduced expression of
inflammatory mediators observed in this study. These results
suggest that AICAR could be effective for the posterior segment
manifestation of inflammation, such as cystoid macular edema, which
is a significant cause of vision loss in human uveitis.
Example 7
AICAR Treatment Suppresses CD14 Expression
[0076] CD14 is a co-receptor for LPS and expressed mainly on
monocytes, macrophages and neutrophils, and its association with
Toll-like receptor 4 leads to activation of transcriptional
factors, including NF-.kappa.B (Takeda et al. (2003) ANNU. REV.
IMMUNOL. 21:335-376). There have been reports that suppression of
CD14 expression has a potent therapeutic effect on reducing
LPS-induced inflammation including EIU (Sanchez-Lemus et al. (2009)
AM. J. PHYSIOL. REGUL. INTEGR. COMP. PHYSIOL. 296:R1376-1384;
Poulaki et al. (2007) FASEB. J. 21:2113-2123). Thus, the effects of
AICAR treatment on CD14 mRNA and protein expression were assessed
by real time PCR and Western Blot, respectively.
[0077] For preparation of peripheral blood mononuclear cell (PBMC)
samples, blood samples were collected at three hours after LPS
injection, and PBMC samples were isolated by density gradient
centrifugation with Histopaque 1083 (Sigma). The red blood cells
were lysed with Red Blood Cell Lysing Buffer (Sigma).
[0078] For Western blot analysis, pooled PBMCs from three rats were
homogenized with lysis buffer (Roche Diagnostics Corp) and
centrifuged at 13,000 rpm for 10 minutes at 4.degree. C. Thirty
.mu.g of protein per sample was electrophoresed in a 4-20% gradient
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (Invitrogen) and electroblotted to polyvinylidene
fluoride membrane (Millipore). After blocking with 5% skim milk,
the membranes were incubated with a rabbit polyclonal antibody
against CD14 (1:200, Santa Cruz Biotechnology) or GAPDH antibody
(1:1000, Cell Signaling, Danvers, Mass.) at room temperature for
one hour. The membranes were washed three times (five minutes each
time) with TBS/Tween (TBST) and incubated for 30 minutes at room
temperature with horseradish peroxidase-labeled anti-rabbit
secondary antibody (1:20,000; Jackson ImmunoResearch, West Grove,
Pa.). The membranes were then washed three times (five min each
time) in TBST, and the proteins were visualized by ECL plus (GE
Healthcare).
[0079] For measurement of CD14 mRNA expression by real time PCR,
total RNA of PBMC was harvested from cells using the RNeasy kit
(Qiagen, Valencia, Calif.), and complementary DNA (cDNA) was
generated with the OligodT primer (Invitrogen) and Superscript II
(Invitrogen) according to the manufacturer's instructions.
Real-time PCR was carried out using the following TaqMan gene
expression assays (Applied Biosystems): CD14 (Rn00572656_g1) and
actin (Rn00667869_m1). Quantitative expression data were acquired
and analyzed with a Step One Plus real-time PCR system (Applied
Biosystems).
[0080] As depicted in FIGS. 5A and 5B, at three hours after LPS
injection, CD14 mRNA expression in PBMCs from EIU rats was
significantly increased (3.8.+-.0.7, n=9) compared to normal rats
(0.7.+-.0.3, n=6, p=0.009). AICAR pretreatment significantly
suppressed CD14 mRNA levels (1.7.+-.1.0, n=9, p=0.03). Similarly,
protein expression of CD14 was increased in EIU rats but decreased
in AICAR-pretreated rats.
[0081] Collectively, these results demonstrate that both protein
and mRNA expression of CD14 were increased after LPS stimulation
and pretreatment with AICAR significantly suppressed these
elevations. It has been reported that LPS stimulation decreases
AMPK activity in macrophages and increases their production of
inflammatory cytokines, while AICAR may upregulate AMPK activity
and suppress cytokine production. It is contemplated that one of
the treatment effects of AICAR is to decrease LPS susceptibility by
suppressing CD14 expression.
Example 8
AICAR Treatment Suppresses Uveitis in Experimental Autoimmune
Uveitis (EAU)
[0082] The ability of AICAR to suppress autoimmune-mediated
intraocular inflammation was investigated in a model of
experimental autoimmune uveitis (EAU). EAU is a rodent model of
human autoimmune uveitis and has been used for studying the
mechanism of autoimmune uveitis and for developing therapeutic
strategies (Luger et al. (2008) SEMIN. IMMUNOPATHOL. 30:135-143).
In this model, EAU is induced by immunizing mice with retinal
antigens such as interphotoreceptor retinoid-binding protein (IRBP)
(Rizzo et al. (1996) J. IMMUNOL. 156:1654-1660; Sanui et al. (1989)
J. EXP. MED. 169:1947-1960).
[0083] Female C57BL/6 (WT) mice ranging from 6-8 weeks old (Charles
River, Wilmington, Mass.) were used for these studies.
[0084] To induce EAU, WT mice were immunized subcutaneously with
200 .mu.g of human interphotoreceptor retinoid-binding protein
(hIRBP) 1-20 (GPTHLFQPSLVLDMAKVLLD) (SEQ ID NO:1) (Biomatik,
Wilmington, Del.) emulsified in CFA (1:1 v/v) containing 2.5 mg/ml
M. tuberculosis (Difco, Detroit, Mich.). As an additional adjuvant,
0.1 g of purified Bordetella pertussis toxin (PTX, Sigma, St.
Louis, Mo.) was also injected intraperitoneally. To study the
effect of AICAR on EAU, AICAR (100 or 200 mg/kg body weight,
Toronto Research Chemicals, Ontario, Canada) was diluted in 0.15 ml
phosphate-buffered saline (PBS) and administered daily via
intraperitoneal injection from day 0 to 21 after immunization.
Control animals were injected with PBS.
[0085] Clinical scoring of EAU was performed by funduscopic
examination in a masked fashion as previously described (Pouvreau
et al. (1998) J. NEUROIMMUNOL. 86:171-181). On day 21 after
immunization, vascular dilation, white focal vascular lesions,
white linear vascular lesions, retinal hemorrhage and retinal
detachment were evaluated and the severity of EAU was graded on
scale of 0-4 as described by Thurau et al. (1997) CLIN. EXP.
IMMUNOL. 109:370-376. For histological assessment, eyes were
enucleated on day 21 and immediately frozen in optimal cutting
temperature compound (Sakura Finetek, Torrance, Calif.). Ten
.mu.m-thick sections were cut near the optic nerve head, air-dried,
and fixed in 4% paraformaldehyde and stained with hematoxylin and
eosin. The severity of EAU in each eye was scored on a scale of 0-4
based on the number, type, and size of lesions as previously
described (Caspri et al. (1988) J. IMMUNOL. 140:1490-1495).
[0086] As depicted in FIGS. 6A, C and D, fundus examination showed
that AICAR treatment suppressed clinical inflammation in a
dose-dependent manner. More specifically, mean clinical scores were
significantly reduced in mice treated with 200 mg/kg of AICAR
(1.32.+-.0.95, n=15, p=0.0002) when compared with the control
animals (2.6.+-.0.70, n=17). Histological examination also
confirmed the ability of AICAR to suppress ocular inflammation. As
shown in FIGS. 6B, E and F, mean pathologic scores were reduced in
mice treated with 200 mg/kg of AICAR (0.53.+-.0.73, p=0.0008) when
compared to control mice (1.65.+-.0.68, p=0.0008). Since 200 mg/kg
of AICAR showed the most robust results compared to controls, all
other experiments using the EAU model were conducted with this
dose.
[0087] Altogether, these data indicate that AICAR administration
has an anti-inflammatory effect on EAU as demonstrated by clinical
and histological findings.
Example 9
AICAR Suppresses Intraocular Inflammation
[0088] To investigate the effect of AICAR on retinal inflammation,
the mRNA and protein levels of various inflammatory cytokines were
assessed. Total RNA from the retina was harvested using the RNeasy
kit (Qiagen, Valencia, Calif.). Complementary DNA (cDNA) was
generated with OligodT primer (Invitrogen, Camarillo, Calif.) and
Superscript II (Invitrogen) according to manufacturer's
instructions. Real-time PCR was carried out using the following
TaqMan gene expression assays (Applied Biosystems, Foster City,
Calif.): IL-6 (Mm99999064_m1), IFN-.gamma. (Mm01168134_m1), TNF
(Mm99999068_m1), and actin (Mm00607939_s1). Quantitative expression
data were acquired and analyzed with a Step One Plus real-time PCR
system (Applied Biosystems).
[0089] For the retinal cytokine analysis, 8 to 10 retinas were
removed 21 days after immunization and placed into 500 .mu.l of
lysis buffer (Complete Protease Inhibitor Cocktail Tablets, Roche
Diagnostics Corp, Indianapolis, Ind.) containing protease
inhibitor. After sonication, the lysate was centrifuged at 13,000
rpm for 10 minutes at 4.degree. C. and supernatant was collected.
Cytokine levels were measured by ELISA using the Quantikine mouse
IFN-.gamma., IL-6 and TNF-.alpha. kits (R&D system,
Minneapolis, Minn.). The amount of cytokine present was normalized
to total protein concentration in each retina.
[0090] As shown in FIG. 7A, EAU (i.e., control) mice showed
elevated levels of TNF-.alpha., IL-6 and IFN-.gamma. mRNA when
compared to wildtype (i.e., naive) mice. AICAR treatment
significantly reduced the levels of TNF-.alpha., IL-6 and
IFN-.gamma. mRNA. As shown in FIG. 7B, AICAR treatment also
suppressed the protein levels of IL-6 when compared to naive and
control mice. Together these data indicate that AICAR suppresses
intraocular inflammatory cytokine production and retinal
inflammation.
Example 10
AICAR Suppresses Proliferation and Cytokine Production by Lymph
Node (LN) Cells
[0091] In the EAU model, activated and sensitized Th1 and Th17
cells are considered to play a major role in the initiation and
maintenance of intraocular inflammation (Yoshimura et al. (2008)
INT. IMMUNOL. 20:209-214; Amadi-Obi et al. (2007) NAT. MED.
13:711-718). Specifically, Th1 and Th17 cells respond to retinal
antigen resulting in intraocular inflammation, posterior uveitis,
disc edema, retinal exudates, vasculitis, and retinal detachment
(Luger et al. (2008) SEMIN. IMMUNOPATHOL. 30:135-143; Caspi (2008)
IMMUNOL. RES. 42:41-50). Thus, to determine the mechanism by which
AICAR suppresses intraocular inflammation and uveitis, the effect
of AICAR on T cell response was investigated. More specifically,
IRBP-specific T cell responses and cytokine profiles were examined
in lymph node (LN) cells.
[0092] To extract LN cells, draining lymph nodes from 6 to 8 mice
were isolated at sixteen days after immunization and pooled. Single
cell suspensions were made using a cell strainer (BD, Franklin
Lakes, N.J.). CD4 T cell-enriched fractions were prepared using CD4
Microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), and
purity of the suspensions was determined by staining with anti-CD4
antibody (GK1.5, Biolegend, San Diego, Calif.) and flow cytometry.
All experimental samples of CD4 T cell-enriched fractions were at
least 95% CD4-positive.
[0093] To measure cell proliferation, LN cells were resuspended at
5.times.10.sup.5 cells per 200 .mu.l of medium in 96-well
flat-bottom plates. Cells were incubated for 72 hours and
proliferation during the last 12 hours was measured by using a
bromodeoxyuridine (BrdU) cell proliferation assay kit (Millipore,
Billerica, Mass.). Supernatant in the culture medium was collected
at 48 hours and cytokine production in the supernatant was measured
by ELISA using the Quantikine mouse IFN-.gamma., IL-4, IL-10 and
IL-17 kits (R&D system, Minneapolis, Minn.).
[0094] To measure the expression of T-bet and ROR.gamma.t,
real-time PCR was carried out as described above using the
following TaqMan gene expression assays: T-bet (Mm00450960_m1) and
ROR.gamma.t (Mm01261022_m1). Quantitative expression data were
acquired and analyzed with a Step One Plus real-time PCR system
(Applied Biosystems).
[0095] In order to assess the in vivo effect of AICAR on the
development of IRBP-reactive T cells, LN cells from control and
AICAR-treated mice were stimulated with either IRBP or a
non-specific T cell stimulator, anti-CD3. Proliferative response
was measured with BrdU incorporation. As shown in FIG. 8A, AICAR
treatment suppressed antigen-specific T cell proliferation in a
dose-dependent manner when compared to untreated mice. In
comparison, when the cells were treated with anti-CD3, there was no
difference in T cell proliferation between the AICAR treated and
control mice (FIG. 8B).
[0096] As shown in FIGS. 8C-F, AICAR significantly suppressed the
production of IFN-.gamma., IL-17, and IL-10 by LN cells. Further,
AICAR treatment also suppressed the expressions of T-bet and
RoR.gamma.t, which are transcription factors for Th1 and Th17 cells
(FIGS. 8G and H, respectively).
[0097] Together, these results suggest that AICAR suppresses Th1
and Th17 cell proliferation and cytokine production.
Example 11
Effect of AICAR on Fox-P3 Expression (Treg) on LN Cells
[0098] In the EAU model, it was previously demonstrated that a
shift towards a Th2 response and an increase in the Treg population
and in regulatory cytokine production occur as a result of Th1 and
Th17 cell suppression (Sun et al. (2010) INVEST. OPTHALMOL. VIS.
SCI. 51:383-389; Keino et al. (2007) BR. J. OPTHALMOL. 91:105-110).
It has been further reported that the Th2 response is related to
the resolution of EAU (Takeuchi et al. (2001) JPN. J. OPTHALMOL.
45:463-469) and that this response increases in AICAR-treated
experimental autoimmune encephalomyelitis mice during the late
phase (Nath et al. (2005) J. IMMUNOL. 175:566-574).
[0099] In the current EAU model, a Th2 response was not detected
during the inflammatory stage and could not be induced by in vitro
treatment with AICAR. Further, as was shown in Example 10, AICAR
suppressed the production of IL-10, which is a regulatory cytokine
produced by Type-1 T regulatory (Tr1) cell (Pot et al. (2011)
SEMIN. IMMUNOL. 23:202-208) and Treg cells.
[0100] The effect of AICAR on the regulatory T (Treg) cell
population in the EAU mice was also directly assessed.
Specifically, LN cells from five mice were harvested at 21 days
after immunization and stained with a mouse regulatory T cell
staining kit #2 (eBioscience) according to manufacturer's
instructions. CD4.sup.+CD25.sup.+Foxp3.sup.+ Treg populations were
subsequently detected by flow cytometry. As shown in FIG. 9, the
CD4.sup.+CD25.sup.+Foxp3.sup.+ Treg population was not
significantly different between control and AICAR-treated mice.
These results suggest that AICAR may mediate its effects
independent of any effects on Th2, Tr1 and Treg cells.
Example 12
AICAR Suppressed EAU During the Effector Phase
[0101] To examine whether AICAR might have an effect on the
effector phase of EAU, AICAR or PBS were administered from day 8 to
21 after immunization. Both clinical and histopathological findings
revealed that AICAR treatment administered during the effector
phase significantly suppressed EAU (mean clinical score:
2.44.+-.0.56 in controls vs. 1.50.+-.0.86 in AICAR-treated mice,
p=0.031; mean pathologic score: 1.03.+-.0.75 in controls vs.
0.13.+-.0.27 in AICAR-treated mice, p=0.004, n=8).
[0102] To assess the effect of AICAR on already developed
IRBP-specific T cells, LN cells from untreated EAU mice were
cultured with IRBP peptide in the presence of AICAR (FIG. 10).
Specifically, LN cells were resuspended at 5.times.10.sup.5 cells
per 200 .mu.l of medium in 96-well flat-bottom plates. Triplicate
samples of cells were stimulated with or without IRBP at the
indicated concentrations. AICAR was added at the indicated
concentrations. Cell proliferation and cytokine production were
measured as previously described in Example 10.
[0103] As shown in FIG. 10, in vitro treatment with AICAR
suppressed T cell proliferation and IFN-.gamma., IL-17 and IL-10
production. IL-4 production was not significantly induced.
[0104] Given that AICAR suppressed inflammation and T cell
proliferation as well as cytokine production during the effector
phase of EAU, it is contemplated that AICAR may be effective in
treating ongoing human uveitis.
Example 13
AICAR Affects Dendritic Cell (DC) Phenotype
[0105] It is contemplated that AICAR may impair the interaction
between T cells and antigen presenting cells (APCs), more
specifically, dendritic cells (DCs) which are known as professional
APCs. It has been reported that AMPK negatively regulates DC
maturation by affecting their energy production pathway (Krawczyk
et al. (2010) BLOOD 115:4742-4749). Further, it is know that
co-stimulatory signals are involved in the course of EAU and
blockade of these signals ameliorates intraocular inflammation
(Bagenstose et al. (2005) J. IMMUNOL. 175:124-130; Namba et al.
(2000) J. IMMUNOL. 165:2962-2969; Fukai et al. (1999) GRAEFES.
ARCH. CLIN. EXP. OPTHALMOL. 237:928-933). For antigen specific T
cell proliferation, naive CD4 T cells interacts with antigen
presenting cells (APC) which express co-stimulatory molecules
(second signals) such as CD40, CD80 and CD86 in addition to MHC
molecules (first signals) (Jenkins (1994) IMMUNITY 1:443-446;
Janeway et al. (1994) CELL 76:275-285).
[0106] To investigate whether DC maturation was affected by AICAR,
splenic DCs were isolated from EAU mice and analyzed for
co-stimulatory molecule expression. Specifically, spleen cells from
4 to 5 EAU mice were pooled at twelve days after immunization and
incubated with the following monoclonal antibodies: anti-CD11c
(N418), CD40 (3/23), CD80 (16-10A1), CD86 (GL-1), I-A.sup.b
(AF6-120.1) (Biolegend). Spleen cells were gated on the basis of
forward and side scatter profile and propidium iodide or DAPI
exclusion. Samples of 1.times.10.sup.6 cells were analyzed by LSR
(Becton Dickinson, Franklin Lakes, N.J.).
[0107] As indicated in FIG. 11, the expression of CD80 and I-Ab was
elevated whereas CD40 and CD86 were not changed after IRBP
immunization. There was no difference between the AICAR-treated and
non-treated groups, which may be due to the heterogeneity of the DC
population in vivo. Thus, experiments using in vitro cultured bone
marrow-derived DCs (BMDCS) were performed to examine the potential
role of AICAR on DC maturation.
[0108] BMDCs were generated as previously described (Wang et al.
(2005) IMMUNOL. LETT. 98:123-130). Briefly, bone marrow was flushed
from the femurs and tibias of naive mice (6 to 8-week-old WT or
AMPK.alpha.1 KO mice). The red blood cells were lysed using Red
Blood Cell Lysing Buffer (Sigma). Approximately 2.times.10.sup.6
cells were cultured in complete medium (RPMI 1640 medium containing
10% FBS, 50 mM 2-ME, 10 mM HEPES [pH 7.4], 2 mM glutamine, 100 U/ml
penicillin, and 100 mg/ml streptomycin) including 10 ng/ml GM-CSF
(Pepro Tech, London, England). Fresh media was added to the cells
on day 4 of culture, and nonadherent cells and loosely adherent
cells were collected as immature DCs on day 7. Immature DCs were
stimulated with 100 ng/ml LPS (Salmonella typhimurium; Sigma) for
24 hours to obtain mature DCs. In some experiments, AICAR was added
to the culture. As shown in FIG. 12A, the expressions of CD40,
CD80, CD86 and I-A.sup.b were markedly elevated after stimulation
of BMDCs with LPS. AICAR significantly suppressed these elevations
in a dose-dependent manner.
[0109] After LPS stimulation for 24 hours, supernatant of the BMDC
culture was collected and IL-6, TNF-.alpha. and IL12/23 p40
concentrations were measured (R&D system). As indicated in FIG.
12B, AICAR significantly suppressed the production of IL-6,
IL-12/23 p40 and TNF-.alpha. in BMDCs.
[0110] Altogether, these data suggest that at least part of the
effect of AICAR in EAU is mediated through its effect on DC
maturation and subsequent T cell proliferation and
differentiation.
Example 14
AICAR Affects DC Maturation and AMPK
[0111] In these experiments, AMPK.alpha.1 KO mice were provided as
previously described (Jorgensen et al. (2004) J. BIOL. CHEM.
279:1070-1079).
[0112] AICAR is a cell permeable activator of AMP-activated protein
kinase (AMPK), which is a Serine/Threonine kinase that senses
cellular energy status. Once inside the cell, AICAR is
phosphorylated by adenosine kinase to the monophosphorylated form
(ZMP), which mimics AMP and activates AMPK. AMPK is also implicated
in the inflammatory response. Activation of AMPK changes macrophage
function to an anti-inflammatory phenotype (Sag et al. (2008) J.
IMMUNOL. 181:8633-8641) and inhibits dendritic cell (DC) maturation
(Krawczyk et al. (2010) BLOOD 155:4742-4749), which is essential to
induce lymphocyte activation.
[0113] AMPK is a heterotrimeric complex and the catalytic subunits
of AMPK.alpha. consists of .alpha.1 and .alpha.2 and regulatory
.beta. and .gamma. subunits (Hardy et al. (2003) FEBS LETT.
546:113-120). Western blot analysis was performed to determine the
AMPK.alpha.1 and AMPK.alpha.2 expression in mice. Specifically, 20
.mu.g of lysate from BMDC, liver and spleen of WT and AMPK.alpha.1
KO mice were electrophoresed in a 4-20% gradient sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen)
and electroblotted to polyvinylidene fluoride membrane (Millipore).
After blocking with blocking buffer (Thermo scientific, Rockford,
Ill.), the membranes were incubated with a rabbit polyclonal
antibody against AMPK.alpha.1, AMPK.alpha.2 (1:1000, Abcam,
Cambridge, Mass.) or GAPDH antibody (1:1000, Cell Signaling,
Danvers, Mass.). The membranes were washed three times (5 minutes
each time) with TBS/tween (TBST) and incubated for 30 minutes at
room temperature with horseradish peroxidase-labeled anti-rabbit
secondary antibody (1:20,000; Jackson ImmunoResearch, West Grove,
Pa.). The membranes were washed again three times (5 minutes each
time) in TBST, and the proteins were visualized by ECL plus (GE
Healthcare, Piscataway, N.J.). As shown in FIG. 13, BMDCs expressed
only the AMPK.alpha.1 subunit.
[0114] Since BMDCs expressed only AMPK.alpha.1, BMDCs were
extracted from AMPK.alpha.1 KO mouse and used to determine the role
of AMPK in the anti-inflammatory effects of AICAR. As shown in
FIGS. 14A and B, AICAR suppressed the maturation of AMPK.alpha.1 KO
BMDCs. Specifically, AICAR significantly suppressed the expression
of CD40, CD80, CD86 and I-A.sup.b in AMPK.alpha.1 KO BMDCs after
LPS stimulation (FIG. 14A). Further, AICAR also suppressed the
production of IL-6, TNF-.alpha. and IL12/23 p40 in AMPK.alpha.1 KO
BMDCs after LPS stimulation (FIG. 14B).
[0115] To further study AICAR's suppressive effect, an adenosine
kinase inhibitor (IODO) was used to inhibit AICAR conversion to
ZMP. In addition, an inhibitor of nucleoside transporter (DPY) was
used to block AICAR translocation into cells. As demonstrated in
FIG. 15, IODO did not affect the downregulation of co-stimulatory
molecule expression by AICAR, suggesting that AICAR suppresses DC
maturation mainly through an AMPK-independent pathway. However, DPY
reversed the AICAR-mediated suppression of co-stimulatory molecule
expression, indicating that the effects of AICAR are mediated via
intracellular pathways.
INCORPORATION BY REFERENCE
[0116] The entire disclosure of each of the patent documents and
scientific articles cited herein are incorporated by reference in
their entirety for all purposes.
EQUIVALENTS
[0117] The invention can be embodied in other specific forms with
departing from the essential characteristics thereof. The foregoing
embodiments therefore are to be considered illustrative rather than
limiting on the invention described herein. The scope of the
invention is indicated by the appended claims rather than by the
foregoing description, and all changes that come within the meaning
and range of equivalency of the claims are intended to be embraced
therein.
* * * * *