Note: Descriptions are shown in the official language in which they were submitted.
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LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
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THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.
CA 02640986 2010-02-08
. "
THERAPEUTIC COMPOSITIONS FOR TREATMENT OF INFLAMMATION OF
OCULAR AND ADNEXAL TISSUES
RELATED APPLICATIONS
[01]
FIELD OF THE INVENTION
[02] This invention relates generally to the field of ophthalmology.
BACKGROUND OF THE INVENTION
[03] Inflammation of the ocular and adnexal tissues can occur by a variety of
mechanisms and
is associated, either primarily or secondarily, with a large number of disease
conditions. Current
treatments for inflammation of these tissues involve the systemic
administration of antibiotics,
steroids, and immune-system inhibitors. The difficulty of using these systemic
drugs becomes
apparent through damaging long-term side effects in the ease of steroids, long-
term drug
resistance in the case of antibiotics, or insufficient long-term persistence
at the target site in the
case of signaling inhibitors. Moreover, the systemic inhibition of signaling
within the immune
system can have deleterious outcomes for individuals already afflicted with
disease, whose
susceptibility to additional complications is increased as a result of the
systemic use of these
treatments.
SUMMARY OF THE INVENTION
[04] The present invention overcomes these obstacles by administering a
topical composition
comprising one or more antagonists of IL-1 function, or a combination of IL-1
and other
inflammatory antagonists, to locally decrease or prevent inflammation of the
ocular and adnexal
tissues.
[05] A method for inhibiting or reducing the severity of an ocular
inflanunatory disorder is
carried out by locally administering to an ocular or adnexal tissue of a
subject a composition that
inhibits an activity of an inflammatory interleuldn-1 cytokine such as binding
of an
inflammatory IL-1 cytoldne to an IL-1 receptor. The subject is identified as
suffering from a
ocular inflammatory disorder by detecting a sign or symptom selected from the
group consisting
1
W020091025763 CA 02640986 2010-02-08 PCT/US2008/009776
of epithelial overexpression of an inflammatory cytolcine, vascular
hyperplasia or thickening of
lid margin, neovascularization of lid margin or corneal periphery, increase of
leukocytes at an
ocular or adnexal tissue, or overexpression of a matrix metalloprotease at an
ocular or adnexal
tissue. The method of therapy inhibits or reduces the severity of at least one
of these signs or
symptoms. For example, the inflammatory disorder is blepharitis. The method
comprises
administration of a compound that inhibits binding of an inflammatory IL-1
cytolcine to an IL-1
receptor. Optionally, the composition also contains an antibiotic compound.
The composition
does not comprise tetracycline alone, e.g. in the absence of a functional
antagonist that
specifically targets IL-1. For example, the composition comprises an
antibiotic composition
administered in combination with a functional antagonist specifically
targeting IL-1.
[06] The composition that inhibits binding of an inflammatory IL-1 cytokine to
an IL-1
receptor comprises the amino acid sequence of SEQ ID NO: 16. For example, the
composition
is present in a concentration range of 0.1-10%, with preferred ranges between
1-5%, or 2-2.5% =
(mg/ml). Exemplary liquid formulations for eye drops contain 2-2.5% (mg/ml) of
the
composition. Preferred formulations are in the form of a solid, a paste, an
ointment, a gel, a
liquid, an aerosol, a mist, a polymer, a film, an emulsion, or a suspension.
The formulations are
administered topically, e.g., the composition is delivered to an ocular or
adnexal tissue to directly
contact that tissue. The method does not involve systemic administration or
substantial
dissemination of the composition to non-ocular or non-adnexal tissue. For
example,
subcutaneous injection of Kineret (see SEQ ID NO: 15 and 16) at 1-2 mg/kg
results in an =
estimated peak blood serum concentration of about 1200-1500 ng/ml 7 hours post-
injection.
Topical administration of Kineret, as disclosed herein, temporally and
spatially restricts
absorption of the drug to a much greater degree than subcutaneous injection.
The systemic
dissemination of a topically administered Kineret composition contributes
significantly less drug
to the blood serum concentration than a subcutaneous injection.
[07] Optionally, the composition further contains a compound selected from the
group
consisting of a physiological acceptable salt, poloxamer analogs with
carbopol,
carbopol/hydroxypropyl methyl cellulose (HPMC), carbopol-methyl cellulose,
carboxymethylcellulose (CMC), hyaluronic acid, cyclodextrin, and petroleum.
[08] The invention comprises a composition that inhibits an activity of an
inflammatory
interleulcin-1 cytoldne, the composition being in the form of a solid, a
paste, an ointment, a gel, a
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W02009/025763 CA 02640986 2010-02-08 PCT/US2008/009776
liquid, an aerosol, a mist, a polymer, a film, an emulsion, or a suspension.
The composition is
present at a concentration of 0.1 - 10% (mg/ml). An exemplary composition
includes a
polypeptide comprising the amino acid sequence of SEQ ID NO: 16.
[09] A method for inhibiting or reducing the severity of an ocular
inflammatory disorder is
also carried out by locally administering to an ocular or adnexal tissue of a
subject a composition
comprising a polynucleotide, a polypeptide, an antibody, a compound, or a
small molecule that
inhibits the transcription, transcript stability, translation, modification,
localization, secretion, or
function of a polynucleotide or polypeptide encoding an inflammatory
interleukin-1 cytokine
(IL-la, SEQ ID NO: 1 and 2, or IL-lb, SEQ ID NO: 3 and 4), an IL-1 receptor
(type 1, SEQ ID
NO: 17 and 18, or type 2, SEQ ID NO: 17-21), an IL-1R binding protein (IL-
1RAP, SEQ ID
NO: 24-27), or an IL-1R downstream signaling effector (IRAK1, SEQ ID NO: 28-
33).
[10] Alternatively, the composition inhibits or enhances the transcription,
transcript stability,
translation, modification, localization, secretion, or function of a
polynucleotide or polypeptide
encoding the IL-1 receptor, type 2 (IL-1R2). IL-1R2 binds IL-1 and can inhibit
the function of
IL-1R1. Thus, in one embodiment, enhancement of IL-1R2 function provides
another
mechanism by which IL-1R1 activity is inhibited. In this same embodiment,
inhibition of an
antagonist of IL-1R2, specifically, IL-1Ra3, inhibits IL-1R1 function. Thus,
the composition
alone, or in combination with an enhancer of IL-1R2, inhibits the
transcription, transcript
stability, translation, modification, localization, secretion, or function of
a polynucleotide or
polypeptide encoding IL-1Ra3, SEQ ID NO: 22 or 23. Alternatively, in an
embodiment wherein
IL-1R2 receptor function augments the activity of IL-1R1, the composition
contains one or more
regions of a polynucleotide or polypeptide encoding IL-1Ra3 to augment IL-1R2
inhibition.
Furthermore, the composition of this embodiment comprises the whole
polynucleotide or
polypeptide encoding IL-1Ra3.
=
[11] The composition comprises a polynucleotide, a polypeptide, an antibody, a
compound, or
=
a small molecule with means to inhibit the transcription, transcript
stability, translation,
modification, localization, secretion, or function of a polynucleotide or
polypeptide encoding an
accessory protein of an IL-1 Receptor. For example, this IL-1 receptor
accessory protein is IL-
1RAP, which directly binds IL-1 and IL-1R1, and is defined by the
polynucleotide sequence of
SEQ ID NO: 24 or 26 and the polypeptide sequence of SEQ ID NO: 25 or 27. IL-
1RAP belongs
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W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
to a signaling complex that is required for signal transduction from IL-1R1.
Thus, inhibition of
IL-1RAP antagonizes IL-1R1 function.
[12] In another embodiment, the composition comprises a polynucleotide, a
polypeptide, an
antibody, a compound, or a small molecule with means to inhibit the
transcription, transcript
stability, translation, modification, localization, secretion, or function of
a polynucleotide or
polypeptide encoding an associated kinase to an IL-1 receptor. For example, IL-
1 receptor-
associated ldnase is IRAK1. IRAK1 is a downstream signaling effector that
leads to
= transcriptional events associated with escalating inflammatory responses
and is defined by the
polynucleotide sequence of SEQ ID NO: 28, 30, or 32 and the polypeptide
sequence of SEQ ID
= NO: 29, 31, or 33. Upon IL-1 receptor binding by IL-1, IRAK1 is recruited
to the receptor
=
complex, becomes hyperphosphorylated, and participates in the formation of a
new protein
complex consisting of hyperphosphorylated IRAK1 and TRAF6. The formation of
this
IRAK1/TRAF6 complex is a prerequisite for tumor necrosis factor (TNF)
associated factor 6
= (TRAF6)-mediated activation of nuclear factor-KB (NF-KB) and subsequent
induction of an
inflammatory response. Thus, the inhibition of IRAK1 expression and/or
function provides an
additional mechanism for inhibiting an IL-1-mediated immune response.
= [13] The composition comprises a polynucleotide, a polypeptide, an
antibody, or a small
molecule that binds or modifies the function of IL-la, IL-lb, IL-1R1, IL-1R2,
IL-1Ra3, IL-
1RAP, or IRAK1. Moreover the composition comprises morpholino antisense
oligonucleotides,
= microRNAs (miRNAs), short hairpin RNA (shRNA), or short interfering RNA
(siRNA) to
silence gene expression. Exemplary compounds to be adapted for topical
administration include,
but are not limited to, anakinra/Kineret (recombinant human IL-1Ra, rhIL-1Ra,
and SEQ ID
= NO: 15 and 16), IL-1R antisense oligomers (U.S. Patent No. 2005033694),
IL-1Ra-like nucleic
acid molecule (Amgen, U.S. Patent No. 2001041792), and polynucleotide encoding
a soluble IL-
1R accessory molecule (Human Genome Sciences, U.S. Issued Patent No. 6974682).
[14] The composition comprises microRNA molecules adapted for topical
administration to
ocular or adnexal tissues in order to silence gene expression. Exemplary
miRNAs that bind to
human IL-la include, but are not limited to, miR-30c (SEQ ID NO: 34), miR-30b
(SEQ ID NO:
= 35), miR-30a-5p (SEQ ID NO: 36), and nR-24 (SEQ ID NO: 37). Exemplary
miRNAs (and
= -corresponding sequences) that bind to human IL-1R1 include, but are not
limited to, miR-135b
(SEQ ID NO: 38), miR-326 (SEQ ID NO: 39), miR-184 (SEQ ID NO: 40), miR-214
(SEQ ID
4
W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
a
NO: 41), miR-203 (SEQ ID NO: 42), miR-331 (SEQ ID NO: 43), and miR-205 (SEQ ID
NO:
44).
[15] Exemplary polypeptides to be adapted for topical administration to ocular
or adnexal
tissues include, but are not limited to, analdnra/Kineret (recombinant human
IL-1Ra, rhIL-1Ra,
and SEQ ID NO: 15 and 16), AF12198 (binds human IL-1R1, Ac-FEWTPGWYQJYALPL-
NH2 where J represents the unnatural amino acid, 2-azetidine-1 -carboxylic
acid, SEQ ID NO:
45), IL-1R and IL-1RAP peptide antagonists (U.S. Patent No. 20060094663), IL-
1R accessory
molecule polypeptides (U.S. Patent No. 20050171337), IL-1Ra peptides (U.S.
Patent No.
2005105830), and IL-1Ra-related peptides (Amgen, U.S. Patent No. 2001042304).
[16] Exemplary antibodies to be adapted for topical administration to ocular
or adnexal tissues
include, but are not limited to, IL-1 TRAP Online fusion double chain protein
of IL1R-gp130
= . with hIgGFc, Regeneron, U.S. Issued Patent No. 6,927,044), anti-
IL-la (U.S. Patent No.
20030026806), anti-IL-1f3 (U.S. Patent No. 20030026806 and Yamasaki et al.
Stroke. 1995;
26:676-681), and humanized monoclonal anti-IL-1R (Amgen, U.S. Patent No.
2004022718 and .
Roche, U.S. Patent No. 2005023872).
[17] Small molecules are organic or inorganic. Exemplary organic small
molecules include,
= but are not limited to, aliphatic hydrocarbons, alcohols, aldehydes,
ketones, organic acids, esters,
mono- and disaccharides; aromatic hydrocarbons, amino acids, and lipids.
Exemplary inorganic
small molecules comprise trace minerals, ions, free radicals, and metabolites.
Alternatively,
small molecule inhibitors can be synthetically engineered to consist of a
fragment, or small
' = portion, or a longer amino acid chain to fill a binding pocket of an
enzyme. Typically small
molecules are less than one kilodalton. An exemplary small molecule to be
adapted for topical
administration to ocular or adnexal tissues is ZnPP (IL-1 blocker zinc
protoporphyrin, naturally-
occurring metabolite, Yamasaki et al. Stroke. 1995; 26:676-681).
[18] The composition does or, alternatively, does not comprise one or more
antibiotic
compositions to be used in combination with an antagonist of IL-1 function.
The antibiotic and
IL-1 antagonist compositions are administered simultaneously or sequentially.
Exemplary
antibiotic compositions used for combination-therapy with antagonists of HA -
mediated
inflammation include but are not limited to, amikacin, gentamicin, kanamycin,
neomycin,
netilmicin, streptomycin, tobramycin, teicoplanin, vancomycin, azithromycin,
clarithromycin,
W02009/025763 CA 02640986 2010-02-08 PCT/US2008/009776
clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin,
amoxicillin,
ampicillin, azlocillin, carbenicillin, clozacillin, dicloxacillin,
flucozacillin, mezlocillin, nafcillin,
penicillin, piperacillin, ticarcillin, bacitracin, colistin, polymyxin B,
ciprofloxacin, enoxacin,
gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,
oflazacin, trovafloxacin,
mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole,
trimethoprim,
cotrimoxazole, demeclocycline, soxycycline, minocycline, oxytetracycline, or
tetracycline.
[19] The composition comprises an antagonist of an IL-1 cytokine or an IL-1
receptor,
administered simultaneously or sequentially with a second immunosuppressive
composition. The
immunosuppressive compound comprises cyclosporin A or analogs thereof a
concentration of
0.05 - 4.0 % (mg/ml). Alternatively, or in addition, the immunosuppressive
composition
comprises a glucocorticoid, a cytostatic agent, an alkylating agent (nitrogen
mustards/cyclophosphamide, nitrosoureas, platinum compounds), an antimetabolic
agent
(methotrexate, any folic acid analog, azathioprine, mercaptopurine, any purine
analog, any
pyrimidine analog, any inhibitor of protein synthesis), a cytotoxic antibiotic
(dactinomycin, an
anthracycline, mitomycin C, bleomycin, mithramycin), a polyclonal antibody
(Atgatne, . =
.Thympglobuline , any antibody against the antilymphocyte or antithymocyte
antigens), a
'monoclonal antibody (OKT30, any antibody against the T-cell receptor, any
antibody against
= IL-2, basiliximab/Simulect , declizumab/Zenapax0),
Tacrolimus/PrografTWFK506,
Siroliiims/Rapamunerm/Rapamycin, interferon beta, interferon gamma, an opioid,
a TNFa
binding protein, mycophenolate, or FTY720.
[20] The composition comprises a polynucleotide, a polypeptide, an antibody,
or a small . =
molecule that binds or modifies the function of IL-la, IL-lb, IL-1Ra,.IL-1R1,
IL-1R2, I1-1Ra3,
IL-1 RAP, or MAKI , administered topically with a pharmaceutically appropriate
carrier.
Delivery methods for polynucleotide compositions include, but are not limited
to, liposomes,
= receptor-mediated delivery systems, naked DNA, and engineered viral
vectors such as herpes
viruses, retroviruses, adenoviruses and adeno-associated viruses, among
others. Polynucleotide
compositions are administered topically with a pharmaceutically acceptable
liquid carrier, e.g., a
liquid carrier, which is aqueous or partly aqueous. Alternatively,
polynucleotide sequences
within the composition are associated with a liposome (e.g., a cationic or
anionic liposome).
[21] A number of methods have been developed for delivering short DNA or RNA
sequences
into cells; e.g., polynucleotide molecules can be contacted directly onto the
tissue site, or
6
=
W020091025763 CA 02640986 2010-02-08 PCT/US2008/009776
modified polynucleotide molecules, designed to specifically target desired
cell types (e.g.,
sequences linked to peptides or antibodies that specifically bind receptors or
antigens expressed
on the target cell surface).
[22] A preferred approach uses a recombinant DNA construct in which the short
polynucleotide sequence is placed under the control of a strong polymerase III
or polymerase II
promoter. The use of such a construct will result in the transcription of
sufficient amounts of
polynucleotide that will form complementary base pairs with the endogenous
transcripts of
nucleic acids of the invention and thereby prevent translation of endogenous
mRNA transcripts.
The invention encompasses the construction of a short polynucleotide using the
complementary
= strand as a template. For example, a vector can be introduced in vivO
such that it is taken up by a
cell and directs the transcription of an interfering RNA or precursor to a
double stranded RNA
molecule: Alternatively, the template for the short polynucleotide transcript
is placed under the
transcriptional control of a cell-type specific promoter or other regulatory
element. Thus,
diffusion or absorption of a topically administered composition beyond the
intended ocular or
= adnexal target tissue does not cause deleterious or systemic side
effects. The vector remains
episomal or becomes chromosomally integrated, as long as it can be transcribed
to produce the
desired polynucleotide.
=
[23] Vectors are constructed by recombinant DNA technology methods standard in
the art.
Vectors can be plasmid, viral, or others known in the art, used for
replication and expression in .
mammalian cells. Expression of the sequence encoding the short polynucleotide
can be placed
under the control of any promoter known in the art to act in mammalian,
preferably human cells.
Promoters are inducible or constitutive. Exemplary promoters include, but are
not limited to: the
SV40 early promoter region (Bernoist et al., Nature 290:304, 1981); the
promoter contained in
the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell,
22:787-797, 1988); the ,
herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA,
78:1441, 1981);
or the regulatory sequences of the metallothionein gene (Brinster et al.,
Nature, 296:39, 1988).
[24] Polypeptide compositions are associated with liposomes alone or in
combination with
receptor-mediated delivery systems, to enable transport across the plasma
membrane.
Polypeptide compositions are soluble or membrane-bound. An exemplary receptor-
mediated
delivery system involves fusion of a low-density or very-low-density
lipoprotein containing
7
CA 02640986 2010-02-08
particle or vesicle to the low-density lipoprotein (LDL) receptor (LDLR) as
observed with
Hepatitis C Virus (HCV) infection and HCV-mediated drug delivery methods.
[25] Compositions comprise one or more extracellular or intracellular
antibodies, also called
intrabodies, raised against one or more of the following: IL-la, IL-lb, IL-
1Ra, IL-1R1, IL-1R2,
I1-1Ra3, IL-1 RAP, or IRAK1. Extracellular antibodies are topically
administered with a
pharmacologically appropriate aqueous or non-aqueous carrier. Sequences
encoding intracellular
antibodies are subcloned into a Viral or mammalian expression vector, packed
in a lipophilic
device to facilitate transport across the plasma membrane, and topically
administered to ocular or
adnexal tissue with a pharmacologically appropriate aqueous or non-aqueous
carrier. Once inside
the plasma membrane, host cell machinery transcribes, translates; and
processes the intrabody
code to generate an intracellular function-blocking antibody targeted against
IL-la, IL-Ih; IL-
1Ra, IL-1R1, IL-1R2,11-1Ra3, IL-1RAP, or MAKI. In the case of secreted
molecules,
intracellular antibodies prevent post-translational modification or secretion
of the target protein.
In the case of membrane-bound molecules, intracellular antibodies prevent
intracellular signaling
events upon receptor engagement by IL-1 cytokines.
[26] The composition comprises an antagonist of IL-1 and/or IL-1R function in
combination
with other inhibitory elements. Antagonists of IL-1 and/or IL-1R and other
inhibitory elements
are administered simultaneously or sequentially. In one embodiment, the
composition comprises
an antagonist of IL-1 and/or IL-1R function and an antagonist of tumor
necrosis factor alpha
(TNFa). Exemplary functional blockers of INFa include, but are not limited to,
recombinant
and/or soluble TNFa receptors, monoclonal antibodies, and small molecule
antagonists and/or
inverse agonists. One or more commercially-available TNF-a blocking agents are
reformulated
for topical administration in this embodiment. Exemplary commercial 1NF-a
blocking agents
used for reformulation include, but are not limited to, etanercept/Enbrerm,
infliximab/Remicadelm,
and adalimumab/Humiralm. Alternatively, the composition comprises an
antagonist of IL-1 and/or
IL-1R function and antagonist(s) of one or more interleukin cytoldnes.
Exemplary cytoldnes
include, but are not limited to, IL-2, IL-4, IL-5, IL-6, IL-8, 1L-12, IL-17,
1L-18, and 1L-23. In
another embodiment, the composition comprises an antagonist of IL-1 and/or IL-
1R function and
antagonist (s) of one or more member(s) of the vascular epithelial growth
factor (VEGF) family
composed of growth factors and receptors (VEGFR). Exemplary members include,
but are not
limited to, VEGF-A, VEGF-C, VEGFR-2, and 'VEGFR-3. In another embodiment, the
composition
comprises an antagonist of IL-1 and/or IL-1R function and an antagonist of
8
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W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
interferon-gamma. In another embodiment, the composition comprises an
antagonist of IL-1
and/or IL-1R function and antagonist(s) of one or more chemokines and their
receptors.
Exemplary chemokines and receptors comprised by the composition of this
embodiment include,
but are not limited to, chemokine (C-C motif) receptor 1 (CCR1), chemokine (C-
C motif)
receptor 2 (CCR2), chemokine (C-C motif) receptor 5 (CCR5), chemokine (C-C
motif) receptor
7 (CCR7), and chemokine (C-X-C motif) receptor 3 (CXCR3).
[27] In embodiments wherein the composition comprises an antagonist of IL-1
and/or IL-1R
function and antagonist(s) of one or more inflammatory species, the respective
doses of the IL-1
antagonist to the other inflammatory antagonist(s) is a ratio between 1:10 and
10:1
(mass/weight). Alternatively, the ratio is 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3,
1:2, 1:1, 2:1, 3:1, 4:1;
= 5:1, 6:1, 7:1, 8:1, or 9:1. =.
= [28] The invention also comprises a contact lens device consisting of a
composition that
= inhibits an activity of an inflammatory interleukin-1 cytokine and a
pharmaceutically compatible
polymer. This composition also comprises a combination of antagonists of IL-1
or IL-1R =
function as well as antagonists of other inflammatory agents. For example, the
composition is
incorporated into or coated onto said lens. The composition is either
chemically bound or =
physically entrapped by the contact lens polymer. The contact lens is either
hydrophobic or
.; .. hydrophilic.
=
[29] The invention comprises a drug-delivery device consisting of a
composition that inhibits
= - an activity of an inflammatory interleulcin-1 cytokine and a
pharmaceutically compatible
= polymer. This composition also comprises a combination of antagonists of
IL-1 or IL-1R
= function as well as antagonists of other inflammatory agents. For
example, the composition is
incorporated into or coated onto said polymer. The composition is either
chemically bound or
= physically entrapped by the polymer. The polymer is either hydrophobic or
hydrophilic. The
polymer device comprises multiple physical arrangements. Exemplary physical
forms of the
polymer device include, but are not limited to, a film, a scaffold, a chamber,
a sphere, a
microsphere, a stent, or other structure. The polymer device has internal and
external surfaces. =
The device has one or more internal chambers. These chambers contain one or
more
. compositions. The device contains polymers of one or more chemically-
differentiable
.= monomers. The subunits or monomers of the device polymerize in vitro or in
vivo.
9
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PCT/US2008/009776
[30] Exemplary mucoadhesive polyanionic natural or semi-synthethic polymers
from which
the device is formed include, but are not limited to, polygalacturonic acid,
hyaluronic acid,
carboxymethylamylose, carboxymethylchitin, chondroitin sulfate, heparin
sulfate, and
mesoglycan. In one embodiment, the device comprises a biocompatible polymer
matrix that may
optionally be biodegradable in whole or in part. A hydrogel is one example of
a suitable
polymer matrix material. Examples of materials which can form hydrogels
include polylactic
= acid, polyglycolic acid, PLGA polymers, alginates and alginate
derivatives, gelatin, collagen,
agarose, natural and synthetic polysaccharides, polyamino acids such as
polypeptides
particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-
.epsilon.-
caprolactone, polyanhydrides; polyphosphazines, poly(vinyl alcohols),
poly(allcylene oxides)
particularly poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates),
modified styrene
= polymers such as poly(4-azninomethylstyrene), pluronic polyols,
polyoxamers, poly(uronic
acids), poly(vinylpyrrolidone) and copolymers of the above, including graft
copolymers. In
= another embodiment, the scaffolds may be fabricated from a variety of
synthetic polymers and :
naturally-occurring polymers such as, but not limited to, collagen, fibrin,
hyaluronic acid,
agarose, and laminin-rich gels.
=
=
=
[31] One preferred material for the hydrogel is alginate or modified alginate
material.
= Alginate molecules are comprised of (1-4)-linked P-D-mannuronic acid (M
units) and a L-
= .
guluronic acid (G units) monomers which vary in proportion and sequential
distribution along
the polymer chain. Alginate polysaccharides are polyelect-olyte systems which
have a strong
affinity for divalent cations (e.g. Ca+2, Mg+2, Ba+2) and form stable
hydrogels when exposed to =
these molecules. See Martinsen A., et al., Biotech. & Bioeng., 33 (1989) 79-
89. =
[32] An embodiment of the invention utilizes an alginate or other
polysaccharide of a lower
molecular weight, preferably of size which, after dissolution, is at the renal
threshold for =
clearance by humans. Polymeric devices are located topically or
subcutaneously, though very
superficially, wherein either a composition chemically bound or physically
entrapped by the
= polymeric device or the device itself, degrades and must be cleared from
the body. For a
biodegradable polymeric device, it is preferred that the alginate or
polysaccharide is reduced to a
molecular weight of 1000 to 80,000 daltons, more preferably 1000 to 60,000
daltons, particularly
preferably 1000 to 50,000 daltons. It is also useful to use an alginate
material of high guluronate .
W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
content since the guluronate units, as opposed to the mannuronate units,
provide sites for ionic
crosslinking through divalent cations to gel the polymer.
[33] Internal and external surfaces optionally contain pores. Pores are either
created prior to
administration into a subject or result from the inclusion of pore-forming
agents within the
device that perforate surfaces upon administration to a subject. Exemplary
pore forming agents
include, but are not limited to, water soluble compounds such as inorganic
salts and sugars. Pore
forming agents are added as particulates and comprise between one and thirty
percent
(weight/weight of polymer). Pore size is sufficient for diffusion of proteins
but not large enough
cell migration into or out of the device.
=
[34] The device is administered topically, subconjuntively, or in the
episcleral space,
= subcutaneously, or intraductally. Specifically, the device is placed on
or just below the surface if
an ocular or adnexal tissue. Alternatively, the device is placed inside a tear
duct or gland. The
composition incorporated into or onto the polymer is released or diffuses from
the device.
[35] The invention comprises a method of contacting a composition, with means
to inhibit IL-
1R activity, to an ocular or adnexal tissue surface of a subject whO presents
symptoms or.
= associated conditions of posterior blepharitis. Unlike all other
treatments for this condition, this =
. method comprises a composition that is topically administered to the subject
and affects local
= disease mechanisms without the side effects present with systemically
administered treatments.
The composition is not only effective immediately, but also safe for long-term
administration.
[36] The invention comprises a method of treating non-infectious eye disease,
wherein a
composition containing an inhibitor of IL-1 or IL-1R function alleviates,
prevents, or attenuates
a symptom, cause, or mechanism of said disease by contacting said composition
to the ocular
= surface of an affected subject. Exemplary disease mechanisms comprise
inflammation,
= hyperplasia, neovascularization, leukocyte recruitment, or cytokine
production within superficial
eye structures and those structures juxtaposed to the ocular surface. For
instance, non-infectious
eye disease is caused by obstruction, thickening, inflammation,
neovascularization, or atrophy of .
the meibomian glands. Alternatively, non-infectious eye disease is caused by
damage to
structures other than the meibomian glands. Exemplary alternative causes
include, but are not
limited to; oily tear film, papillary hypertrophy of the tarsal conjunctiva,
corneal punctate
epitheliopathy, vernal keratoconjunctivitis, atopic keratocojunctivitis,
chemical burn, trachoma,
11
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pterygium, pemphigoid, corneal angiogenesis(growth of new blood vessels),
psoriasis, icthyosis,
erythema mutiforme, anhydrotic ectodermal dysplasia, systemic retinoid
therapy, or exposure to
polychlorinated byphenols. Finally, non-infectious eye disease can also result
from a deficient
tear lipid layer, an increase in tear evaporation, or the occurrence of an
evaporative dry eye. The
methods treat non-infectious eye disease that is or is not coincident with the
presentation of
dermatoses comprising acne rosacea, seborrhoeic dermatitis, or atopic
dermatitis. Furthermore,
methods are applicable to treat a primary or secondary non-infectious eye
disease that is
= coincident with chalazia, pannus, phlyctenules, recurrent conjunctivitis,
ocular surface damage,
ocular rosacea, corneal ulceration, corneal perforation or secondary
complications of ocular
infection.
=
=
=
[37] The methods described herein are not intended to treat eye disease that
results from an
immune response that consequently arises following the transplantation of
foreign tissue onto or
into an ocular or adnexal tissue. For instance, inflammatory responses occur
following host.
rejection of corneal transplant or as a consequence of infection following
surgical procedures. =
The instant application is not intended to treat eye conditions that result
directly from tissue
= rejection or infectious disease. However, immune responses to foreign
tissues or to infectious
= agents can lead to protean secondary complications, such as growth of
blood or lymph vessels,
or overexpression of molecules that cause tissue injury; in such cases the
methods described in
the present application are useful as a treatment for reducing inflammation
associated with such
= secondary complications. Furthermore, the methods. and devises comprising
the instant .
application are not intended to treat conditions arising from autoinunune
responses, e.g. immune
responses raised against healthy host tissues.
=
:
[38] In a preferred embodiment, the non-infectious eye disease comprises
blepharitis and/or
posterior blepharitis that is or is not coincident with inflammation of the
posterior lid margin, = .
inflammation of the ocular surface, burning, irritation, or patient
discomfort. Alternatively, the
present invention is intended to treat blepharitis that is coincident with
meibomian gland
dysfunction.
[39] The methods alleviate symptoms of non-infectious eye disease. Exemplary
symptoms
include, but are not limited to, dryness, discomfort, burning, itching,
irritation, inflammation,
. skin abnormalities surrounding the eye, photophobia, blurred vision, and
contact lens
intolerance.
= 12
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[40] The present invention comprises a composition with variable physical and
chemical
forms; however, the composition is topically administered and contacts an
ocular or adnexal
tissue directly. The composition is administered as a solid, a paste, an
ointment, a gel, a liquid,
an aerosol, a mist, a polymer, a film, an emulsion, or a suspension.
Furthermore, the composition
is incorporated into or coated onto a contact lens, from which one or more
molecules diffuse
away from the lens or are released in a temporally-controlled manner. In this
embodiment, the
contact lens composition either remains on the ocular surface, e.g. if the
lens is required for
vision correction, or the contact lens dissolves as a function of time
simultaneously releasing the
composition into closely juxtaposed tissues.
[41] In one preferred embodiment, the present invention comprises a
composition with means
to inhibit the transcription, transcript stability, translation, modification,
localization, secretion,
or receptor binding of IL-la, IL-113, or a combination of both cytolcines. In
one embodiment, the
composition comprises a polynucleotide capable of binding to a region of the
IL-la mRNA
transcript, defined by SEQ ID NO: 1. In another embodiment, the composition
comprises a =
=
polynucleotide capable of binding to a region of the IL-113 mRNA transcript,
defined by SEQ ID
NO: 3.
[42] In another embodiment, the composition is capable of increasing the
abundance of the
naturally-occuring IL-1 Receptor antagonist (IL-1Ra). The composition
comprises a
polynucleotide, a polypeptide, an antibody, a compound, or a small molecule
that binds to a
region of the IL-1Ra gene, mRNA transcript defined by SEQ ID NO: 5, 7, 9, 11,
or 13, a
polypeptide isoform of IL-1Ra defined by SEQ ID NO: 6, 8, 10, 12, or 14, or a
recombinant IL-
.
1Ra protein defined by SEQ ID NO: 16. Alternatively, the composition contains
mRNA
transcripts or polypeptides encoding a region or the entirety of the IL-1Ra
gene.
1431 The composition comprises an antagonist or inverse agonist of a receptor
for IL-la or
IL-113, specifically, IL-1R1. In this embodiment an antagonist is defined as a
binding partner, or
ligand, of an IL-1R that inhibits the function of an agonist, IL-1, or inyerse
agonist by blocking
its binding to the receptor. An inverse agonist is defined as a molecule which
binds to the same
IL-1R binding-site as an agonist, for instance, IL-1, but exerts the opposite
pharmacological
effect. The-composition contains a polynucleotide, a polypeptide, an antibody,
a compound, or a
small molecule that binds .to a region of the IL-1R1 defined by the
polynucleotide and
13
W02009/025763 CA 02640986 2010-02-08
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polypeptide sequences SEQ ID NO: 17-21. In an alternative embodiment, the
composition
comprises a molecule with means to inhibit IL-1R transcription, transcript
stability, translation,
modification, localization, secretion, ligand binding, or association with an
accessory protein of
an IL-1R (IL-1RAP). IL-1RAP is defined by the polynucleotide sequence of SEQ
ID NO: 24 or
26 and the amino acid sequence of SEQ ID NO: 25 or 27.
[44] The composition comprises a molecule with means to inhibit IL-lct- or IL-
13-mediated
modulation of matrix metalloproteinase (MMP) overexpression, which degrades
collagen
matrices within tissues and retards wound healing.
= [45] In another preferred embodiment, the composition comprises a human
recombinant IL- .
1R antagonist either in pure form, or as a component of a mixture. The human
recombinant IL-
1R antagonist is combined with balanced saline, carboxymethylcellulose (CMC),
or hyaluronic
acid (HA), or other vehicles prior to the composition contacting the ocular or
lid surface. Within =
these mixtures, the human recombinant IL-1R antagonist comprises at least
0.1%, 2.0%, 2.5%,
5%, or at most 10% of the total volume administered. Preferred aqueous
formulations contain 2-
2.5% of the purified antagonist. Purified is defined as the antagonist in the
absence of unrelated
= polynucleotides, polypeptides, cellular organelles, or lipids. Purified
is defines a degree of
sterility that is safe for administration to a human subject, e.g. lacking
infectious or toxic agents.
=
=
[46] Affected subjects to which this invention is administered are identified
by a variety of
methods. The following examinations are used individually or combinatorially
to assess the
=
presence or absence of non-infectious eye disease (for explanation of
examinations, see
= Examples). Subjects are identified by a break-up time of less than 10
seconds following a
= standard Tear Film Break-up Time (TFBT) test. Subjects are identified by
a significant corneal
fluorescein staining of tear film and/or conjunctival lissamine green or rose
bengal staining.
Subjects are identified by a result of lOmm or less for the Schrimer test with
or without
anesthesia. Subjects are identified by significantly viscous tear excreta
and/or a decrease in the
number of meibomian glands or meibomian gland orifices capable of excreting
tears. Subjects
= are identified by shades of red discoloration of the lid margin,
palpebral conjunctiva, and/or
bulbar conjunctiva indicating increasing extreme vascular injection
(erythema). Subjects are also
classified as presenting increasingly severe non-infectious eye disease with
the increasing
abundance and severity of the above factors.
14
CA 02640986 2010-02-08
[47] The compositions and methods provided herein are used modify intraocular
pressure. In
one aspect of the invention, the compositions and methods provided herein are
used to decrease
intraocular pressure. Inhibitors or antagonists of IL-1 cytolcines and/ or IL-
1 receptors are used.
alone or in combination with other compounds to modify intraocular pressure.
In a preferred
embodiment of the invention, inhibitors or antagonists of IL-1 cyiokines and/
or IL-1 receptors
are used alone or in combination with other compounds to decrease intraocular
pressure.
Alternatively, or in addition, inhibitors or antagonists of IL-1 cytokines
and/ or IL-1 receptors
are used alone or in combination with other compounds to treat ocular
hypertension.
1[48] Exemplary compounds to be used in combination with IL-1 cytokines and/
or IL-1
receptors include, but are not limited to, prostaglandin analogs (such as
latanoprost (XalatannI),
bimatoprost (Lumigair) and travoprost (TravatanTh) which increase uveoscleral
or trabecular
outflow of aqueous humor); topical beta-adrenergic receptor antagonists (such
as timolorrm,
levobunolor (Betaganni), and betaxolol, which decrease aqueous humor
production by the
ciliary body); Alpha2-adrenergic agonists (such as brimonidine (Alphagair),
which work by a
dual mechanism, decreasing aqueous production and increasing uveo-scleral
outflow);
less-selective sympathomimetics (such as epinephrine and dipivefriir
(Propinelm), which
increase outflow of aqueous humor through trabecular meshwork and possibly
through
uveoscleral outflow pathway, probably by a beta2-agonist action); miotic
agents
(parasympathomimetics) (such as pilocarpine, which work by contraction of the
ciliary muscle,
tightening the trabecular meshwork and allowing increased outflow of the
aqueous humour);
carbonic anhydrase inhibitors (such as dorzolamide (Trusopr), brinzolamide
(Azoptni),
acetazolamide (Diamoxn"), which lower secretion of aqueous humor by inhibiting
carbonic
anhydrase in the ciliary body); physostigmine which is also used to treat
glaucoma and delayed
gastric emptying; fish oil; omega 3 fatty acids, bilberries, vitamin E,
cannabinoids, camitine,
coenzyme Q10, curcurmin, Salvia miltiorrhiza, dark chocolate, erythropoietin,
folic acid,
Ginkgo biloba, Ginseng, L-glutathione, grape seed extract, green tea,
magnesium, melatonin,
methylcobalamin, N-acetyl-L cysteine, pycnogenols, resveratrol, quercetin, and
fludrocortisone.
[49] The invention also provides compositions and methods for treating
individuals or
subjects with ocylarhypertension including administrating to these subjects a
composition
comprising an IL-1 or IL-1R inhibitor or antagonist. In one aspect orthe
invention, the IL-1 or
IL-1R inhibitor or antagonist ia administered topically to the ocular surface
as a liquid. In
W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
another aspect of the invention, the IL-1 or IL-1R inhibitor or antagonist is
administered
intraocularly by injection.
[50] The invention also provides compositions and methods for preventing
glaucoma in
individuals or subjects with ocular hypertension including administrating to
these subjects a
composition comprising an IL-1 or IL-1R inhibitor or antagonist. In one aspect
of the invention,
the IL-1 or IL-1R inhibitor or antagonist is administered topically to the
ocular surface as a
liquid. In another aspect of the invention, the IL-1 or IL-1R inhibitor or
antagonist is
administered intraocularly by injection.
[51] The invention provides a method for reducing intraocular pressure,
including identifying
a subject suffering from or at risk of a condition associated with above-
normal intra-ocular
pressure and locally administering to the subject a composition that inhibits
an activity of an
inflammatory interleuldn-1 cytoldne. In one preferred aspect of the invention,
the condition is
. glaucoma.
=
[52] In one aspect of the invention, subjects are identified by measuring
their intraocular
= pressure and determining if the measured intraocular pressure is
elevated above normal levels. =.
As used herein, the term "normal level" is meant to describe value within an
acceptable range of
values that one of ordinary skill in the art and/or a medical professional
would expect a healthy
subject of similar physical characteristics and medical history to have. For
example; normal
=
= intraocular pressure (TOP) is defined as IOP in the range of 10 mmHg to
21 mmHg.
=
[53] In another aspect of the invention, subjects are identified as those
individuals who are at
= risk for developing elevated intraocular pressure based upon non-limiting
factors such as medical
history (for instance, diabetes), side effects of medications, lifestyle
and/or diet, medical
intervention (such as surgery to the eye), trauma/injury, hormone changes, and
aging.
Compositions of the invention are administered to these subjects for
preventative means.
=
. [54] All polynucleotides and polypeptides of the invention are purified
and/or isolated. As used
herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide,
polypeptide, or protein,
can be substantially free of other cellular material, or culture medium when
produced by
recombinant techniques, or chemical precursors or other chemicals when
chemically
synthesized. =
16
= -
CA 02640986 2010-02-08
[55]
BRIEF DESCRIPTION OF THE DRAWINGS
[56] Figure 1 is.the Ocular Surface Disease Index (OSDI) 12-item
questionnaire.
[57] Figure 2 the Oxford Schema for grading corneal and conjunctival staining.
[58] Figure 3 is a graph representing the effect of IL-la on intraocular
pressure (I0P).
[59] Figure 4 is a schematic representation of signaling pathways that are
transduced from the
IL-1R and the downstream effectors involved in carrying these intracellular
signals (drawing
reproduced from BioCarta website).
[60] Figure 5 is a questionnaire given to subjects of a study to diagnose
Meibomian Gland
Dysfunction (Posterior Blepharitis).
[61] Figure 6 is a flow diagram showing the sequence of clinical tests
performed on study
subjects during the screening process described in Example 8.
DETAILED DESCRIPTION
Posterior Blepharitis
[62] Posterior blepharitis is a common chronic eyelid condition that is
described as
generalized inflammation of the posterior lid margin and associated with
inflammation of the
ocular surface and with symptoms of burning, irritation, and discomfort:
Posterior blepharitis is
associated with various disorders of the meibomian glands, known collectively
as meibomian
gland dysfunction (MGD). It is associated either with obstruction and
inflammation of the
meibomian glands or, less commonly, atrophy of the meibomian glands (Foulks,
G. et al. 2003.
Ocul Surf 107-26; Bron, A.J. et al. 2004. Ocul Surf. 2:149-65).
17
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[63] Clinically, MGD often presents with inspissated meibomian glands, oily
tear film, as well
as inflammation and vascularization of the meibomian gland orifices. Papillary
hypertrophy of
the tarsal conjunctiva and corneal punctate epitheliopathy are often present,
and there are
prominent associations with derrnatoses, such as acne rosacea, seborrhoeic
dermatitis, and atopic
dermatitis. Other causes and associations of MGD are: vernal
keratoconjunctivitis, atopic
keratoconjunctivitis, chemical bums, trachoma, pemphigoid, psoriasis,
icthyosis, erythema
multiforme, anhydrotic ectodermal dysplasia, and systemic retinoid therapy.
Additionally,
exposure to polychlorinated biphenyls, through ingestion of contaminated
cooking oils, causes a
chronic form of MGD with gross and extensive acneiform skin changes (Fu, Y.A.
1984. Am J
Ind Med. 5:127-32).
[64] MGD of sufficient extent and degree is associated with a deficient tear
lipid layer, an
increase in tear evaporation, and the occurrence of an evaporative dry eye. In
fact MGD is
considered to be the most common cause of evaporative dry eye (Foulks, G. et
al. 2003. Ocul
Surf. 107-26). Individuals with MGD often complain of significant discomfort,
including
burning, itching, irritation, and photophobia. They may also have other
associated symptoms of
= = dry eye and may be plagued by blurred vision, gradual contact
lens intolerance. Furthermore,
= these patients may become functionally handicapped by the negative impact
of dry eye on their
crucial daily activities such as working, reading, using computer, and driving
(Goto, E. et al.
2002. Am J Opthalmol. 133:181-186; Miljanovic, B. et al. 2007. Am J Opthalmol.
143: 409-15). =
[65] In addition to dry eye, other sequelae of MGD that confront the
ophthalmologist include
chalazia, pannus, phlyctenules, and recurrent conjunctivitis which increase
risk of ocular surface
damage, ocular infection, corneal ulceration, and perforation.
[66] Despite the high incidence of posterior blepharitis, there is currently
no consistently
effective treatment for this condition and it still remains a therapeutic
challenge. Posterior
blepharitis has traditionally been managed with eyelid hygiene, topical
antibiotics (erythromycin
or bacitracin ointments), oral tetracyclines (tetracycline, doxycycline, or
minocycline) and
corticosteroids which are often time consuming, frustrating, and frequently
ineffective or
variably effective.
[67] The current treatments for posterior blepharitis are limited, in part, by
their inability to
target the underlying pathophysiologic processes. There is ample evidence that
posterior
blepharitis is the result of an underlying cytolcine and inflammatory-mediated
process affecting
18
W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
both the meibomian glands and the ocular surface (Kocak-Altintas, A.G. et al.
2003. Eur J
Opthalmol. 13:351-359; McCulley, J.P. et al. 2000. Cornea. 19:650-658). In an
experimental
blepharoconjunctivitis, it has been shown that T-cells are the prime
orchestrator driving the
infiltration of other inflammatory cells into the conjunctiva, as well as the
upregulation of
chemoldnes (Fukushima, A. et al. 2003. Invest. Opthalmol. Vis. Sci. 44:4366-
4374).
= [68] In ocular rosacea which belongs to the large pathologic group of
blepharitis and MGD,
inflammation of the ocular surface was clearly demonstrated with an increase
of inflammatory
mediators in tears such as interleuldn (IL)-la (Barton, K. et al. 1997.
Opthalmology. 104:1868-
1874). Flow cytometry and impression cytology of conjunctival epithelium in
ocular rosacea has
demonstrated a significantly higher level of intercellular adhesion molecule-1
(ICAM-1) which
is known to play a significant role in inflammation associated with dry eye
disease. This
expression of ICAM-1 could be due to the liberation of one or more pro-
inflammatory. cytokines
= such as IL-1I3 or interferon-y (Pisella, P.J. et al. 2000. Opthalmology.
107:1841-9).
[69] Importance of inflammation in the pathogenesis of posterior blepharitis
is underscored by
reports that the signs and symptoms of MGD markedly improve with anti-
inflammatory
therapies such as topical steroids (Marsh, P. et al. 1999.
Opthalmology.106:811-816).
[70] Systemically administered tetracycline antibiotics have long been
recognized as effective
therapies for ocular surface inflammatory diseases. The semisynthetic
tetracycline, doxycycline,
has been reported to successfully treat the common dry eye condition acne
rosacea. One of the
mechanisms of action of doxycycline in MGD and dry eye may be the
downregulation of the IL-
1¨mediated inflammatory cascade in the corneal epithelium (Solomon, A. et al.
2000. Invent.
Opthalmol. Vis. Sci. 41:2544-57).
[71] In addition, findings from several studies indicate that MGD is also
associated with
aqueous deficiency dry eye patients, especially those with Sjogren syndrome
(SS) (McCulley,
J.P. et al.1977. A, J Opthalmol. 84:788-93; Shimazaki, J. et al.
1998.0pthalmology. 105: 1485-
1488). In these studies, at least 35% of patients with aqueous tear deficiency
had MGD.
Classically, SS affects the exocrine glands, not the sebaceous glands such as
the meibomian
glands. Underlying inflammation as well as desiccation and keratinization of
ocular surface
epithelia occurs in chronic dry eye disease and plays a role in pathogenesis
of MGD.
[72] Consistent with the concept that inflammation is a key feature in the
pathophysiology of
dry eye syndrome is the finding that both aqueous tear deficiency and
meibomian gland disease
19
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are effectively treated with the corticosteroid methylprednisolone (Marsh, P.
et al. 1999.
Opthalmology. 107:967-74). Unfortunately the long-term use of topical
corticosteroids is limited
by potential sight-threatening side effects, such as glaucoma and cataracts.
Therefore, there is a
clinical need for nontoxic steroid-sparing anti-inflammatory therapies that
target the underlying
inflammatory environment of the ocular surface in this common condition.
[73] Based on the concept that inflammation is a key component of the
pathogenesis of MGD
and dry eye, therapies targeting the underlying inflammatory environment of
the ocular surface
represent a major improvement in the management of these conditions and will
have a major
clinical impact. Given the facts that IL-1¨mediated inflammatory activities
play a critical role on
the ocular surface in patients with MOD and dry eye, therefore, local blockade
of IL-1 activity
by IL-1Ra, is Used to reduce one or more symptoms of dry eye syndrome..
Ocular and Adnexal Tissues:
[74] Ocular tissues or compartments that contact the compositions comprised by
the present
invention include, but are not limited to, the cornea, aqueous humor, iris,
and sclera. The term
= "adnexal" is defined in general terms as the appendages of an organ. In
the present invention,
adnexal. defines a number of tissues or surfaces that are in immediate contact
with the ocular
surface but are not, by definition, comprised by the ocular surface. Exemplary
adnexal tissues
= include, but are not limited to, the eyelids, lacrimal glands, and
extraocular muscles. Topical
administration of the presently invented compositions contact the following
tissues and
structures. within the eyelid: skin, subcutaneous tissue, orbicularis oculi,
orbital septum, tarsal
plates, palpebral conjuntiva, and meibomian glands. The adnexal tissues
comprise all
subdivisions of the lacrimal glands, including the orbital and palpebral
portions, as well as all
tissues contacted by these glands. Extraocular muscles belonging to this
category of adnexal
tissues include, but are not limited to, the superior and inferior rectus,
lateral and medial rectus,
and superior and inferior oblique muscles. Compositions comprised by the
present invention are
applied topically and contact these tissues either alone, or in combination
with ocular tissues.
Intraocular Pressure
[75] Intraocular pressure is maintained by the liquid aqueous humor, which is
produced by the
ciliary body of the eye. Aqueous humor normally does not go into the posterior
segment of the
eye; it is kept out of this area by the lens and the Zonule of Zinn. Instead,
it stays only in the
anterior segment, which is divided into the anterior and posterior chambers.
While the anterior
. .
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PCT/US2008/009776
and posterior chambers are very similarly named to the anterior and posterior
segments, they are
not synonymous. The anterior and posterior chambers are both parts of the
anterior segment.
[76] When the ciliary bodies produce the aqueous humor, it first flows into
the posterior
chamber (bounded by the lens and the iris). It then flows through the pupil of
the iris into the
anterior chamber (bounded by the iris and the cornea). From here, it flows
through a structure
known as the trabecular meshwork to enter the normal body circulation. Thus,
the two main
mechanisms of ocular hypertension are an increased production of aqueous
humor, or a
decreased outflow of aqueous humor.
[77] Ocular hypertension (OHT) is intraocular pressure higher than normal in
the absence of
optic nerve damage or visual field loss. Current consensus in ophthalmology
defines normal
intraocular pressure (TOP) as that between 10 mmHg and 21 mmHg. Intraocular
pressure is
measured with a tonometer. Elevated IOP is the most important risk factor for
glaucoma, so
those with ocular hypertension are frequently considered to have a greater
chance of developing
= the condition. Intraocular pressure can increase when a patient lies
down. There is evidence that
some glaucoma patients (e.g., normal tension glaucoma patients) with normal
IOP while sitting
or standing may have intraocular pressure that is elevated enough to cause
problems when they
are lying down.
=
=
[78] Differences in pressure between the two eyes is often clinically
significant, and
= potentially associated with certain types of glaucoma, as well as iritis
or retinal detachment.
[79] Because of the effect of corneal thickness and rigidity on measured value
of intraocular
pressure, some forms of refractive surgery (such as photorefractive
keratectomy) can cause
traditional intraocular pressure measurements to appear normal when in fact
the pressure may be
abnormally high.
[80] = Intraocular pressure may become elevated due to anatomical problems,
inflammation of
the eye, genetic factors, as a side-effect from medication, or during
exercise. Intraocular pressure
usually increases with age and is genetically influenced.
Hypotony, or ocular hypotony, is typically defined as intraocular pressure
equal to or less than 5
mmHg. Such low intraocular pressure could indicate fluid leakage and deflation
of the eyeball.
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Glaucoma
[81] Glaucoma is a leading cause of irreversible blindness. A primary risk
factor for glaucoma
is elevated intraocular pressure (IOP), which can contribute to significant
optic nerve damage
and vision loss. Elevated IOP due to reduction in aqueous outflow facility is
a major causal risk
factor. The main aqueous outflow pathway of the eye consists of a series of
endothelial-cell¨
lined channels in the angle of the anterior chamber comprising the trabecular
meshwork (TM),
Schlemm's canal, the collector channels and the episcleral venous system.
[82] Glaucoma is a group of diseases of the optic nerve involving loss of
retinal ganglion cells
= in a characteristic pattern of optic neuropathy. Although raised
intraocular pressure is a
significant risk factor for developing glaucoma, there is no set threshold for
intraocular pressure
= that causes glaucoma. Untreated glaucoma leads to pennanent damage of the
optic nerve and
resultant visual field loss, which can progress to blindness. Loss of visual
field often occurs
gradually over a long time and may only be recognized when it is already quite
advanced. Once
lost, this damaged visual field can never be recovered. Worldwide, it is the
second leading cause
of blindness. Glaucoma affects one in two hundred people aged fifty and
younger, and one in ten
= over the age of eighty.
= [83] Ocular hypertension is the largest risk factor in most glaucomas.
Though, in some
populations only 50% of patients with primary open angle glaucoma have
elevated ocular
pressure. Diabetics and those of African descent are three times more likely
to develop primary
= open angle glaucoma. Higher age, thinner corneal thickness, and myopia
are also risk factors for
= primary open angle glaucoma. People with a family history of glaucoma
have about a six percent
chance of developing glaucoma. Asians are prone to develop angle-closure
glaucoma, and Inuit
= have a twenty to forty times higher risk than Caucasians of developing
primary angle closure
glaucoma. Women are three times more likely than men to develop acute angle-
closure
glaucoma due to their shallower anterior chambers. Use of steroids can also
cause glaucoma.
[84] primary open angle glaucoma (POAG) has been found to be associated with
mutations in
genes at several loci. Normal tension, glaucoma, which comprises one third of
POAG, is
associated with genetic mutations.
[85] There is increasing evidence of ocular blood flow to be involved in the
pathogenesis of
glaucoma. Current data indicate that fluctuations in blood flow are more
harmful in
=
22 =
> ¨ -
¨
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glaucomatous optic neuropathy than steady reductions. Unstable blood pressure
and dips are
linked to optic nerve head damage and correlate with visual field
deterioration.
[86] A number of studies also suggest that there is a correlation, not
necessarily causal,
between glaucoma and systemic hypertension (i.e. high blood pressure). In
normal tension
glaucoma, nocturnal hypotension may play a significant role. On the other hand
there is no clear
evidence that vitamin deficiencies cause glaucoma in humans, nor that oral
vitamin
supplementation is useful in glaucoma treatment.
[87] Various rare congenital/genetic eye malformations are associated with
glaucoma.
Occasionally, failure of the normal third trimester gestational atrophy of the
hyaloid canal and
=
= the tunica vasculosa lentis is associated with other anomalies. Angle
closure induced ocular
hypertension and glaucomatous optic neuropathy may also occur with these
anomalies. These
rare developmental causes of glaucoma are modeled in mice.
=
[88] Only a few glaucomas are associated with visible cellular inflammation in
the ocular
compartments; however, Interleukin-1 (IL-1) is consistently up-regulated by
glaucomatous TM
= = cells and acts as a key factor in accelerated TM cell injury.
This condition is mediated by both
IL-1 effect on the TM cells functioning as Well as their expression of
adhesion factors that can =
affect outflow facility. The net effect is that IL-1 can compromises the
function of the trabecular
meshwork, causing reduced outflow facility in the open angle glaucoma.
Interleukin-1 receptor.
= antagonist (IL-1Re) is a naturally occurring IL-1 isoform that with high-
affinity binds to the IL-1
receptors and blocks its effects. Therefore, topical application of IL-1Ra,
such as human
recombinant forms (anakinra/Kineret) suppresses IL-1-mediated cell injury in
TM, enhances
outflow facility, and reduces TOP in glaucomatous eyes.
[89] This mechanism of action is in contrast with the other views that IL-1
itself increases
matrix metalloproteinase expression in the TM and increases outflow facility.
Therefore, others
believed that inhibition of action of IL-1 by an IL-1 neutralizing antibody or
with IL-1 receptor
antagonist causes reduced outflow facility in trebecular meshwork.
=
Interleukin-1 (IL-1):
[90] The IL-1 family is a group of cytokines. that function as major mediators
of inflammation
and immune response (Dinarello, C.A. 1996. Blood. 15:2095-2147). This family
is composed of
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=
three forms: two proinflammatory forms, IL-la and IL-1B, each having a
precursor form, and an
anti-inflammatory form, IL-1 receptor antagonist (IL-1Ra). The
proinflanunatory cytolcine IL-1
plays an important role in inflammation and immunity by increasing chemokine
production,
adhesion factors, macrophage infiltration and activity, and lymphocyte
proliferation. IL-1 has
been implicated in the pathogenesis of human inflammatory diseases, such as
rheumatoid
arthritis, septic shock, and periodontitis (Jiang, Y. et al. 2000. Arthritis
Rheum. 43:1001-1009;
Okusawa, S. etal. 1988: J Clin Invest. 81: 1162-1172; McDevitt, M.J. et al.
2000. J. Periodontol.
71:156-163). =
[91] The IL-1 cytolcines play an important role in the regulation of
inflammation and wound
= healing in the corneal and ocular surface diseases (Fabre, E.J. et al.
1991. Cliff Eye Res. 10:585-
592; Rosenbaum, J.T. et al. 1995. Invest Opthalmol Vis Sci. 36: 2151-2155).
Both IL-la and IL-
.
1B have been found to modulate matrix metalloproteinase (MMP) expression by
corneal stromal
fibroblasts (Fini, M.E. et al. 1990. Invest Opthalmol Vis Sci. 31:1779-1788).
Increased levels of
IL-la and IL-1B have been shown in patients with SjOgren syndrome and ocular
rosacea =
(Pflugfelder; S.C. et al. 1999. Curr Eye Res. 19:201-211; Solomon, A. et al.
2001. Invest
= =
Opthalmol Vis Sci. 42:2283-2292). Levels of proinflanunatory forms of IL-1 are
directly
correlated with the intensity of corneal fluorescein staining and are
inversely correlated with
conjunctival goblet cell density. In both types of dry eye syndrome,
evaporative and aqueous
= deficiency, as tear clearance from the ocular surface decreases, the
concentrations of both
isoforms of the proinflammatory cytolcine IL-1 increase in the tear fluid
(Solomon, A. et al.
2001. Invest Opthalmol Vis=Sci. 42:2283-2292). The results of a study on the
effects of
= doxycycline on the expression patterns of the IL-1 gene family in the
human limbal epithelium
= in response to a strong inflammatory stimulus, have demonstrated an
inhibitory effect of
doxycycline on the expression of the inflammatory cytokine IL-10, with a
concomitant
upregulation of the anti-inflammatory IL-1Ra (Solomon, A. et al. 2000. Invest
Opthalmol Vis
Sci. 41:2544-57). These results imply that some of the clinically proven
benefits of tetracycline =
compounds (tetracycline, doxycycline, and minocycline) in treating the ocular
surface disease of
MGD and dry eye may be mediated through their regulatory effects on the
synthesis, processing,
or release of IL-1.
[92] IL-1 induces ocular surface disease, e.g., the chronic subclinical ocular
surface
= inflammation of MGD and dry eye. The compositions and methods described
herein inhibit the
activity of human IL-la and/or IL-113, as defined by the ability to induce
signal transduction or
=
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initiate/activate a downstream signaling cascade from an IL-1 receptor.
Compositions that
contain an inhibitor of human IL-la or IL-113 function antagonize the activity
of an IL-1
receptor. The composition comprises a polynucleotide, a polypeptide, an
antibody, a compound,
or a small molecule with means to inhibit the transcription, transcript
stability, translation,
modification, localization, secretion, or function of a polynucleotide or
polypeptide encoding
human IL-la or IL-113. Moreover, the inhibitory polynucleotide or polypeptide
composition
binds to one or more region(s) of IL-la or IL-113 comprised by SEQ ID NO: 1
and SEQ ID NO: =
= 2 (IL-1a) or SEQ ID NO: 3 and SEQ ID NO: 4 (IL-113). The inhibitory
polynucleotide or
polypeptide composition binds to one or more fragments of IL-la or IL-1f3
comprised by SEQ
ID NO: 1 and SEQ ID NO: 2 (IL-1a) or SEQ ID NO: 3 and SEQ ID NO: 4 (IL-113).
[93] A fragment, in the case of these sequences and all others provided
herein, is defined as a
part of the whole that is less than the whole. Moreover, a fragment ranges in
size from a single
= nucleotide or amino acid within a polynucleotide or polypeptide sequence
to one fewer
nucleotide or amino acid than the entire polynucleotide or polypeptide
sequence. Finally, a
fragment is defined as any portion of a complete polynucleotide or polypeptide
sequence that is
intermediate between the extremes defined above.
[94]
Human IL-la is encoded by the following mRNA sequence (NCBI Accession No.
NM_000575 and SEQ ID NO: 1): (For all mRNA transcripts incorporated into the
present =
application, the initiator methionine, encoded by the codon "atg," is bolded
and capitalized to
delineate the start of the coding region.) = =
=
=
=
accaggcaacaccattgaaggctcatatgtaaaaatccatgccttcctttctcccaatctccattcccaa
= acttagccactggcttctggctgaggccttacgcatacctcccggggcttgcacacaccttcttctacag
aagacacaccttgggcatatcctacagaagaccaggcttctctctggtccttggtagagggctactttac
tgtaacagggccagggtggagagttctctcctgaagctccatcccctctataggaaatgtgttgacaata
ttcagaagagtaagaggatcaagacttctttgtgctcaaataccactgttctcttctctaccctgcccta
accaggagcttgtcaccccaaactctgaggtgatttatgccttaatcaagcaaacttccctcttcagaaa
agatggctcattttccctcaaaagttgccaggagctgccaagtattctgccaattcaccctggagcacaa
tcaacaaattcagccagaacacaactacagctactattagaactattattattaataaattcctctccaa
atctagccccttgacttcggatttcacgatttcteccttcctcctagaaacttgataagtttcccgcgct
tccctttttctaaga.ctacatgtttgtcatcttataaagcaaaggggtgaataaatgaaccaaatcaata
acttctggaatatctgcaaacaacaataatatcagctatgccatctttcactattttagccagtatcgag
ttgaatgaacatagaaaaatacaaaactgaattcttccctgtaaattccccgttttgacgacgcacttgt
agccacgtagccacgcctacttaagacaattacaaaaggcgaagaagactgactcaggcttaagctgcca
gccagagagggagtcatttcattggcgtttgagtcagcaaagaagtcaagATGgccaaagttccagacat
gtttgaagacctgaagaactgttacagtgaaaatgaagaagacagttcctccattgatcatctgtctctg
aatcagaaatccttctatcatgtaagctatggcccactccatgaaggctgcatggatcaatctgtgtctc
tgagtatctctgaaacctctaaaacatccaagcttaccttcaaggagagcatggtggtagtagcaaccaa .
cgggaaggttctgaagaagagacggttgagtttaagccaatccatcactgatgatgacctggaggccatc
gccaatgactcagaggaagaaatcatcaagcctaggtcagcaccttttagcttcctgagcaatgtgaaat
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acaactttatgaggatcatcaaatacgaattcatcctgaatgacgccctcaatcaaagtataattcgagc
caatgatcagtacctcacggctgctgcattacataatctggatgaagcagtgaaatttgacatgggtgct
tataagtcatcaaaggatgatgctaaaattaccgtgattctaagaatctcaaaaactcaattgtatgtga
ctgcccaagatgaagaccaaccagtgctgctgaaggagatgcctgagatacccaaaaccatcacaggtag
tgagaccaacctcctcttcttctgggaaactcacggcactaagaactatttcacatcagttgcccatcca
aacttgtttattgccacaaagcaagactactgggtgtgcttggcaggggggccaccctctatcactgact
ttcagatactggaaaaccaggcgtaggtctggagtctcacttgtctcacttgtgcagtgttgacagttca
tatgtaccatgtacatgaagaagctaaatcctttactgttagtcatttgctgagcatgtactgagccttg
taattctaaatgaatgtttacactctttgtaagagtggaaccaacactaacatataatgttgttatttaa
agaacaccctatattttgcatagtaccaatcattttaattattattcttcataacaattttaggaggacc
agagctactgactatggctaccaaaaagactctacccatattacagatgggcaaattaaggcataagaaa
actaagaaatatgcacaatagcagttgaaacaagaagccacagacctaggatttcatgatttcatttcaa
ctgtttgccttctacttttaagttgctgatgaactcttaatcaaatagcataagtttctgggacctcagt
tttatcattttcaaaatggagggaataatacctaagccttcctgccgcaacagttttttatgctaatcag
ggaggtcattttggtaaaatacttcttgaagccgagcctcaagatgaaggcaaagcacgaaatgttattt
tttaattattatttatatatgtatttataaatatatttaagataattataatatactatatttatgggaa
ccccttcatcctctgagtgtgaccaggcatcctccacaatagcagacagtgttttctgggataagtaagt
ttgatttcattaatacagggcattttggtccaagttgtgcttatcccatagccaggaaactctgcattct
agtacttgggagacctgtaatcatataataaatgtacattaattaccttgagccagtaattggtccgatc
= tttgactcttttgccattaaacttacctgggcattcttgtttcaattccacctgcaatcaagtcctacaa
gctaaaattagatgaactcaactttgacaaccatgagaccactgttatcaaaactttcttttctggaatg
taatcaatgtttcttctaggttctaaaaattgtgatcagaccataatgttacattattatcaacaatagt
gattgatagagtgttatcagtcataactaaataaagcttgcaacaaaattctctgacaaaaaaaaaaaaa
aaa.
[95] Human IL-la is encoded by the following amino acid sequence
(NCBI Accession No.
NM_000575 and SEQ ID NO: 2):
MAKVPDMFEDLICNCYSENEEDSSSIDHLSLNQKSFYHVSYGPLHDSEEEIIKPRSAPFSFL
= SNVKYNFMRIIKYEFILNDALNQSIIRANDQYLTAAALHNLDEAVICFDMGAYKSSICDDA
KITVILRISKTQLYVTAQDEDQPVLLICEMPEIPKTITGSETNLLFFWETHGTICNYFTSVAH
PNLFIATKQDYWVCLAGGPPSITDFQILENQA.
=
[96]
= Human IL-l13 is encoded by the following mRNA sequence (NCBI Accession
No. =
NM_000576 and SEQ ID NO: 3):
accaaacctcttcgaggcacaaggcacaacaggctgctctgggattctcttcagccaatcttcattgctc
= aagtgtctgaagcagccATCscagaagtacctgagctcgccagtgaaatgatggcttattacagtggcaa .
tgaggatgacttgttctttgaagctgatggccctaaacagatgaagtgctccttccaggacctggacctc
tgccctctggatggcggcatccagctacgaatctccgaccaccactacagcaagggcttcaggcaggccg
=
=
cgtcagttgttgtggccatggacaagctgaggaagatgctggttccctgcccacagaccttccaggagaa
= tgacctgagcaccttctttcccttcatctttgaagaagaacctatcttcttcgacacatgggataacgag
gcttatgtgcacgatgcacctgtacgatcactgaactgcacgctccgggactcacagcaaaaaagcttgg
tgatgtctggtccatatgaactgaaagctctccacctccagggacaggatatggagcaacaagtggtgtt
ctccatgtcctttgtacaaggagaagaaagtaatgacaaaatacctgtggccttgggcctcaaggaaaag
aatctgtacctgtcctgcgtgttgaaagatgataagcccactctacagctggagagtgtagatcccaaaa
attacccaaagaagaagatggaaaagcgatttgtcttcaacaagatagaaatcaataacaagctggaatt
tgagtctgcccagttccccaactggtacatcagcacctctcaagcagaaaacatgcccgtcttcctggga
gggaccaaaggcggccaggatataactgacttcaccatgcaatttgtgtcttcctaaagagagctgtacc
cagagagtcctgtgctgaatgtggactcaatccctagggctggcagaaagggaacagaaaggtttttgag
tacggctatagcctggactttcctgttgtctacaccaatgcccaactgcctgccttagggtagtgctaag
aggatctcctgtccatcagccaggacagtcagctctctcctttcagggccaatccccagcccttttgttg .
agccaggcctctctcacctctcctactcacttaaagcccgcctgacagaaaccacggccacatttggttc
taagaaaccctctgtcattcgctcccacattctgatgagcaaccgcttccctatttatttatttatttgt
ttgtttgttttattcattggtctaatttattcaaagggggcaagaagtagcagtgtctgtaaaagagcct
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agtttttaatagctatggaatcaattcaatttggactggtgtgctctctttaaatcaagtcctttaatta
agactgaaaatatataagctcagattatttaaatgggaatatttataaatgagcaaatatcatactgttc
aatggttctgaaataaacttcactgaag .
[97] Human IL-113 is encoded by the following amino acid sequence (NCBI
Accession No.
NM_000576 and SEQ ID NO: 4):
MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYS
KGFRQAASVVVAMDICLR1CMLVPCPQTFQENDLSTFFPFIFEEEPLFFDTWDNEAYVHDA
PVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVA
LGLKEKNLYLSCVLKDDICPTLQLESVDPICNYPKICKMEKRFVFNKIEINNICLEFESAQFP
NWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS.
=
Interleukin-1 Receptor (type 1) antagonist (IL-1Ra):
[98] IL-1Ra is an endogenous receptor antagonist which is primarily produced
by activated
. monocytes and tissue macrophages, inhibits the activities of the
proinflammatory forms of IL-1
by competitively binding to IL-1 receptor. (Gabay, C. et al. 1997. 159: 5905-
5913). IL-1Ra is an
= inducible gene that is typically upregulated in inflammatory conditions,
such as rheumatoid
arthritis (Arend, W.P. 1993. Adv Immunol. 54: 167-223).
[99] Normal tear fluid has been found to contain high concentrations of IL-1Ra
in
= concentrations 25,000 and 40,000 times greater than both proinflanunatory
forms of IL-1
(Solomon, A. etal. 2001. Invest Opthalmol Vis Sci. 42: 2283-2292). The high
concentration of
IL-1Ra in the tear fluid may be a natural homeostatic mechanism for preventing
inappropriate
. activation of IL-1¨mediated inflammatory events on the.ocular surface
(Dinarello, C.A. 1996.
Blood. 15:2095-2147).
= [100]. An increased concentration of IL-1Ra has been detected in the tear
fluid of patients with
dry eye and in the conjunctival epithelium of patients with dry eye syndrome
(Solomon, A. et al.
2001. Invest Opthalmol Vis Sci. 42: 2283-2292). Despite this increased level
of expression, the
ratio of IL-1Ra to proinflammatory forms IL-1 dry-eye was significantly lower
than in normal
subjects. Reports of placebo-controlled clinical trials in which IL-1Ra was
administered to =
patients with rheumatoid arthritis have noted that increasing the ratio of IL-
1Ra to the
= proihflammatory forms significantly improves clinical symptoms
(Dinarello, C.A. 2000. N Engl
= J Med. 343:732-734).
[101] In the present invention, compositions comprise one or more regions of
IL-1Ra -
transcripts 1, 2, 3, or 4, intacellular IL-1Ra (icIL-1Ra), or their
corresponding polypeptide
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isoforms. Alternatively, compositions comprise the entirety of IL-1Ra
transcripts 1, 2, 3, or 4,
intacellular IL-1Ra (icIL-1Ra), or their corresponding polypeptide isoforms.
Compositions
comprising any form of human IL-1Ra, or fragments thereof, inhibit the
function of IL-1R1.
These polynucleotides and polypeptides are defined by the following sequences.
ROA
Human IL-1Ra, transcript 1, is encoded by the following mRNA sequence
(NCBI
Accession No. NM_173842 and SEQ ID NO: 5):
= atttctttataaaccacaactctgggcccgcaatggcagtccactgccttgctgcagtcacagaATGgaa
atctgcagaggcctccgcagtcacctaatcactctcctcatcttcctgttccatteagagacgatctgcc
gaccctctgggagaaaatccagcaagatgcaagccttcagaatctgggatgttaaccagaagaccttcta
tctgaggaacaaccaactagttgctggatacttgcaaggaccaaatgtcaatttagaagaaaagatagat
gtggtacccattgagcctcatgctctgttcttgggaatccatggagggaagatgtgcctgtcctgtgtca
=
=
agtctggtgatgagaccagactccagctggaggcagttaacatcactgacctgagcgagaacagaaagca
ggacaagcgcttcgccttcatccgctcagacagcggccccaccaccagttttgagtctgccgcctgcccc
= ggttggttcctctgcacagcgatggaagctgaccagcccgtcagcctcaccaatatgcctgacgaaggcg
tcatggtcaccaaattctacttccaggaggacgagtagtactgcccaggcctgcctgttcccattcttgc
= atggcaaggactgcagggactgccagtccccctgccccagggctcccggctatgggggcactgaggacca
= gccattgaggggtggaccctcagaaggcgtcacaagaacctggtcacaggactctgcctcctcttcaact
= gaccagcctccatgctgcctccagaatggtctttctaatgtgtgaatcagagcacagbagcccagcaca
= aagcccttccatgtcgcctctgcattcaggatcaaaccccgaccacctgcccaacctgctctcctcttgc
cactgcctcttcctccctcattccaccttcccatgccctggatccatcaggccacttgatgacccccaac
=
caagtggctcccacaccctgttttacaaaaaagaaaagaccagtccatgagggaggttt-ttaagggtttg
=
= tggaaaatgaaaattaggatttcatgatttttttttttcagtccccgtgaaggagagcccttcatttgga
- .
=
gattatgttctttcggggagaggctgaggacttaaaatattcctgcatttgtgaaatgatggtgaaagta
= =
agtggtagctttccettctttttcttctttttttgtgatgtcccaacttgtaaaaattaaaagttatgg
tactatgttagccccataattttttttttccttttaaaacacttccataatctggactcctctgtccagg
cactgctgcccagcctccaagctccatctccactccagattttttacagctgcctgcagtactttacctc
ctatcagaagtttctcagctcccaaggctctgagcaaatgtggctcctgggggttctttcttcctctgct
= gaaggaataaattgctccttgacattgtagagcttctggcacttggagacttgtatgaaagatggctgtg
= cctctgcctgtctcccccaccgggctgggagctctgcagagcaggaaacatgactcgtatatgtctcagg =
tccctgcagggccaagcacctagcctcgctcttggcaggtactcagcgaatgaatgctgtatatgttggg
= tgcaaagttccctacttcctgtgacttcagctctgttttacaataaaatcttgaaaatgcctaaaaaaaa
aaaaaaaaaa . =
[103] Human IL-1Ra, transcript 1, is encoded by the following amino acid
sequence (NCBI .
Accession No. NM_173842 and SEQ ID NO: 6):
= =
MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYL
= QGPNVNLEEICIDWPIEPHALFLGIHGGICMCLSCVKSGDETRLQLEAVNITDLSENRKQD
= KRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE.
=
[104] Human IL-1Ra, transcript 2, is encoded by the following mRNA sequence
(NCBI
Accession No. NM_173841 and SEQ ID NO: 7):
gggcagctccaccctgggagggactgtggcccaggtactgcccgggtgctactttatgggcagcagctca
gttgagttagagtctggaagacctcagaagacctcctgtcctatgaggccctccccATOgctttagctga
cEtgtatgaagaaggaggtggaggaggaggagaaggtgaagacaatgctgactcaaaggagacgatctgc
cgaccctctgggagaaaatccagcaagatgcaagccttcagaatctgggatgttaaccagaagaccttct
atctgaggaacaaccaactagttgctggatacttgcaaggaccaaatgtcaatttagaagaaaagataga
= tgtggtacccattgagcctcatgctctgttcttgggaatccatggagggaagatgtgcctgtcctstgtc
= 28
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aagtctggtgatgagaccagactccagctggaggcagttaacatcactgacctgagcgagaacagaaagc
aggacaagcgcttcgccttcatccgctcagacagcggccccaccaccagttttgagtctgccgcctgccc
cggttggttcctctgcacagcgatggaagctgaccagcccgtcagcctcaccaatatgcctgacgaaggc
gtcatggtcaccaaattctacttccaggaggacgagtagtactgcccaggcctgcctgttcccattcttg
catggcaaggactgcagggactgccagtccccctgccccagggctcccggctatgggggcactgaggacc
agccattgaggggtggaccctcagaaggcgtcacaagaacctggtcacaggactctgcctcctcttcaac
tgaccagcctccatgctgcctccagaatggtctttctaatgtgtgaatcagagcacagcagcccctgcac
aaagcccttceatgtcgcctctgcattcaggatcaaaccccgaccacctgcccaacctgctctcctcttg
ccactgcctcttcctccctcattccaccttcccatgccctggatccatcaggccacttgatgacccccaa
ccaagtggctcccacaccctgttttacaaaaaagaaaagaccagtccatgagggaggtttttaagggttt
gtggaaaatgaaaattaggatttcatgatttttttttttcagtccccgtgaaggagagcccttcatttgg
agattatgttctttcggggagaggctgaggacttaaaatattcctgcatttgtgaaatgatggtgaaagt
aagtggtagcttttcccttctttttcttctttttttgtgatgtcccaacttgtaaaaattaaaagttatg
gtactatgttagccccataattttttttttccttttaaaacacttccataatctggactcctctgtccag
= gcactgctgcccagcctccaagctccatctccactccagattttttacagCtgcctgcagtactttacct
= cctatcagaagtttctcagctcccaaggctctgagcaaatgtggctcctgggggttctttcttcctctgc
tgaaggaataaattgctccttgacattgtagagettctggcacttggagacttgtatgaaagatggctgt
gcctctgcctgtctcccccaccgggctgggagctctgcagagcaggaaacatgactcgtatatgtctcag
gtccctgcagggccaagcacctagcctcgctcttggcaggtactcagcgaatgaatgctgtatatgttgg
= gtgcaaagttccctacttcctgtgacttcagctctgttttacaataaaatcttgaaaatgcctaaaaaaa
- aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa .
pumq Human IL-1Ra, transcript 2, is encoded by the following amino acid
sequence (NCBI= = =
Accession No. NM_173841 and SEQ ID NO: 8):
== MALADLYEEGGGGGGEGEDNADSKETICRPSGRKSS1CMQAFRIWD'VNQKTFYLRNNQ
= LVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGICMCLSCVICSGDETRLQLEAVNITDLS
ENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFY .
FQEDE. -
.
=
.= [106] Human IL-1Ra, transcript 3, is encoded by the following mRNA
sequence (NCBI
= Accession No. NM_000577 and SEQ ID NO: 9):
gggcagctccaccctgggagggactgtggcccaggtactgcccgggtgctactttatgggcagcagctca
. gttgagttagagtctggaagacctcagaagacctcctgtcctatgaggccctccccATOgctttagagac
= gatctgccgaccctctgggagaaaatccagcaagatgcaagccttcagaatctgggatgttaaccagaag
accttctatctgaggaacaaccaactagttgctggatacttgcaaggaccaaatgtcaatttagaagaaa
agatagatgtggtacccattgagcctcatgctctgttcttgggaatccatggagggiagatgtgcctgtc
= ctgtgtcaagtctggtgatgagaccagactccagctggaggcagttaacatcactgacctgagcgagaac
agaaagcaggacaagcgcttcgccttcatccgctcagacagcggccccaccaccagttttgagtctgccg
cctgccccggttggttcctctgcacagcgatggaagctgaccagcccgtcagcctcaccaatatgcctga
cgaaggcgtcatggtcaccaaattctacttccaggaggacgagtagtactgcccaggcctgcctgttccc
attcttgcatggcaaggactgcagggactgccagtccccctgccccagggctcccggctatgggggcact
gaggaccagccattgaggggtggaccctcagaaggcgtcacaagaacctggtcacaggactctgcctcct
cttcaactgaccagcctccatgctgcctccagaatggtctttdtaatgtgtgaatcagagcacagcagcc
= cctgcacaaagcccttccatgtcgcctctgcattcaggatcaaaccccgaccacctgcccaacctgctct
cctcttgccactgcctcttcctccctcattccaccttcccatgccctggatccatcaggccacttgatga
cccccaaccaagtggctcccacaccctgttttacaaaaaagaaaagaccagtccatgagggaggttttta
agggtttgtggaaaatgaaaattaggatttcatgatttttttttttcagtccccgtgaaggagagccctt
= catttggagattatgttctttcggggagaggctgaggacttaaaatattcctgcatttgtgaaatgatgg
tgaaagtaagtggtagcttttcccttctttttcttctttttttgtgatgtcccaacttgtaaaaattaaa
= agttatggtactatgttagccccataattttttttttccttttaaaacacttccataatctggactcctc
= tgtccaggcactgctgcccagcctccaagctccat ctccactccagattt t t
tacagctgcctgcagtac
tttacctcctatcagaagtttctcagctcccaaggctctgagcaaatgtggctcctgggggttctttctt
cctctgctgaaggaataaattgctccttgacattgtagagcttctggcacttggagacttgtatgaaaga
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tggctgtgcctctgcctgtctcccccaccgggctgggagctctgcagagcaggaaacatgactcgtatat
gtetcaggtccctgcagggccaagcacctagcctcgctcttggcaggtactcagcgaatgaatgctgtat
atgttgggtgcaaagttccctacttcctgtgacttcagctctgttttacaataaaatcttgaaaatgcct
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa .
[107] Human IL-1Ra, transcript 3, is encoded by the following amino acid
sequence (NCBI
Accession No. NM_000577 and SEQ ID NO: 10):
MALETICRPSGRKSSICMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEICIDVVPIE
PHALFLGIHGGICMCLSCVKSGDETRLQLEAVNITDLSENRKQDICRFAFIRSDSGPTTSFE
SAACPGWFLCTAMEADQPVSLTNMPDEGVNIVTKFYFQEDE.
[108] Human IL-1Ra, transcript 4, is encoded by the following mRNA sequence
(NCBI
Accession No. NM_173843 and SEQ ID NO: 11):
gggcagctccaccctgggagggactgtggcccaggtactgcccgggtgctactttatgggcagcagctca
gt tgagt t agagtc tggaagac ctcagaagac c t cctgtcc ta tgaggc cct cc cca tggc t
ttaggggg
attataaaactaatcatcaaagccaagaaggcaagagcaagcatgtaccgctgaaaacacaagataactg
cataagtaatgactttcagtgcagattcatagctaacccataaactgctggggcaaaaatcatcttggaa
ggctctgaacctcagaaaggattcacaagacgatctgccgaccctctgggagaaaatccagcaagATOca
= =
agccttcagaatctgggatgttaaccagaagaccttctatctgaggaacaaccaactagttgctggatac
ttgcaaggaccaaatgtcaatttagaagaaaagatagatgtggtacccattgagcctcatgctctgttct
tgggaatccatggagggaagatgtgcctgtcctgtgtcaagtctggtgatgagaccagactccagctgga
ggcagttaacatcactgacctgagcgagaacagaaagcaggacaagcgcttcgccttcatccgctcagac
agcggccccaccaccagttttgagtctgccgcctgccccggttggttcctctgcacagcgatggaagctg
accagcccgtcagcctcaccaatatgcctgacgaaggcgtcatggtcaccaaattctacttccaggagga
= cgagtagtactgcccaggcctgcctgttcccattcttgcatggcaaggactgcagggactgccagtcccc
= ctgccccagggctcccggctatgggggcactgaggaccagccattgaggggtggaccctcagaaggcgtc
acaagaacctggtcacaggactctgcctcctcttcaactgaccagcctccatgctgcctccagaatggtc
tttctaatgtgtgaatcagagcacagcagcccctgcacaaagcccttccatgtcgcctctgcattcagga
tcaaaccccgaccacctgcccaacctgctctcctcttgccactgcctcttcctccctcattccaccttcc
catgccctggatccatcaggccacttgatgacccccaaccaagtggctcccacaccctgttttacaaaaa
agaaaagaccagtccatgagggaggtttttaagggtttgtggaaaatgaaaattaggatttcatgatttt
tttttttcagtccccgtgaaggagagcccttcatttggagattatgttctttcggggagaggctgaggac
ttaaaatattcctgcatttgtgaaatgatggtgaaagtaagtggtagcttttcccttctttttcttcttt
ttttgtgatgtcccaacttgtaaaaattaaaagttatggtactatgttagccccataattttttttttcc
ttttaaaacacttccataatctggactcctctgtccaggcactgctgcccagcctccaagctccatctcc
= actecagattttttacagctgcctgcagtactttacctectatcagaagtttctcagctcccaaggctct
= gagcaaatgtggctcctgggggttctttcttc.ctctgctgaaggaataaattgctccttgacattgtaga
gcttctggcacttggagacttgtatgaaagatggctgtgcctctgcctgtctcccccaccgggctgggag
ctctgcagagcaggaaacatgactcgtatatgtctcaggtccctgcagggccaagcacctagcctcgctc
ttggcaggtactcagcgaatgaatgctgtatatgttgggtgcaaagttccctacttcctgtgacttcagc
tctgttttacaataaaatcttgaaaatgcctaaaaaaaaaaaaaaaaaaaaaaaaiaaaaaaaaaaaaaa
= aaaaaaaaaaaaa.
[109] Human IL-1Ra, transcript 4, is encoded by the following amino acid
sequence (NCBI
Accession No. NM_173843 and SEQ ID NO: 12):
MQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGICMCL
SCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEA
= DQPYSLTNMPDEGVMVTKFYFQEDE.
=
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[110] Human intracellular IL-1Ra, icIL-1Ra, is encoded by the following mRNA
sequence
(NCBI Accession No. M55646 and SEQ ID NO: 13):
agctccaccctgggagggactgtggcccaggtactgcccgggtgctactttatgggcagcagctcagttg
agttagagtctggaagacctcagaagacctcctgtcctatgaggccctccccATGgctttagagacgatc
tgccgaccctctgggagaaaatccagcaagatgcaagccttcagaatctgggatgttaaccagaagacct
tctatctgaggaacaaccaactagttgctggatacttgcaaggaccaaatgtcaatttagaagaaaagat
agatgtggtacccattgagcctcatgctctgttcttgggaatccatggagggaagatgtgcctgtcctgt
gtcaagtctggtgatgagaccagactccagctggaggcagttaacatcactgacctgagcgagaacagaa
agcaggacaagcgcttcgccttcatccgctcagacagtggccccaccaccagttttgagtctgccgcctg
ccccggttggttcctctgcacagcgatggaagctgaccagcccgtcagcctcaccaatatgcctgacgaa
ggcgtcatggtcaccaaattctacttccaggaggacgagtag.
= [111] Human intracellular IL-1Ra, ic1L-1Ra, is encoded by the following
amino acid sequence
(NCBI Accession No. M55646 and SEQ ID NO: 14): =
= MALETICRPSGRKSSICMQAFRIWDVNQKTFYLRNNQLVAGYLQGPIWNLEEKIDVVPIE
= PHALFLGIHGGICMCLSCVICSGDETRLQLEAVNITDLSENRKQDKRFAF1RSDSGPTTSFE
= SAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE.
= =
=
=
=
Human Recombinant IL-1Ra:
[112] A recombinant form of human IL-1Ra (rHuLL-1Ra) was developed and tested
in animal
models for arthritis. This form of rHuIL-1Ra is also known as Anakinra or
Kineret differs from
=
= the native nonglycosylated IL-1Ra by the addition of an N-terminal
methionine. It binds to IL-
.
1R type I with the same affinity as IL-18. Kineret consists of 153 amino
acids and has a
molecular weight of 17.3 kilodaltons. It is produced by recombinant DNA
technology using an
= E. coli bacterial expression system.
[113] Anakinra has been investigated in several conditions considered mediated
at least in part.
via IL-1. Some evidence suggests involvement of IL-1 in the pathogenesis of
rheumatoid
= arthritis and septic shock (Jiang, Y. et al. 2000. Arthritis Rheum.
43:1001-1009; Fisher, C.J. et
al.1994. JAMA.271:1836-1843; Okusawa, S. et al. 1988. J Clin Invest. 81:1162-
1172;
=
=
Bresnihan; B. et al. 1998. Rheum Dis Clin North Am. 24(3):615-628; Dayer, J.M.
et al. 2001.
Curr Opin Rheutnatol. 13:170-176; Edwards, C.K. 2001. J Clin Rheumatol. 7:S17-
S24).
Anakinra has been approved by the FDA for the reduction in signs and symptoms
of moderately
to severely active rheumatoid arthritis in patients 18 years of age or older
who have failed one or
more disease-modifying antirheumatic drugs. Considering its high safety
profile, administration
of Anakinra has also been used in the treatment of arthritis in patients with
Juvenile rheumatoid
=
arthritis (Reiff, A. 2005. Curr Rheumatol Rep. 7:434-40). Other indications
include prevention
of graft-versus-host disease (GVHD) after bone marrow transplantation (Antin,
J.H. et al. 1994.
31
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Blood.84:1342-8), uveitis (Teoh, S.C. et al. 2007. Br J Opthalmol. 91: 263-4)
osteoarthritis
(Caron, J.P. et al. 1996. Arthritis Rheum. 39:1535-44), asthma, inflammatory
bowel disease,
acute pancreatitis (Hynninen, M. et al. 1999. J Crit Care. 14:63-8),
psoriasis, and type II diabetes
mellitus (Larsen, C.M. et al. 2007. N Engl J. Med. 356:1517-26). The systemic
safety profile of
IL-1Ra is extremely favorable, especially in comparison to other
immunosuppressive treatments
such as TNF-a blockers, cytotoxic agents, or even steroids.
=
=
[114] Topical human recombinant IL-1Ra has been successfully used for
prevention of corneal
transplant rejection (Yamada, J. et al. 2000. Invest Opthalmol Vis Sci.
41:4203-8) and allergic
conjunctivitis (Keane-Myers, A.M. et al. 1999. Invest Opthalmol Vis Sci.
40:3041-6) in
=
experimental animal models. Similarly, using topical=IL-1Ra significantly
decreases corneal
=.= inflammation and leads to enhanced corneal transparency in the rat
model of corneal alkali injury
(Yamada, J. et al. 2003. Exp Eye Res. 76:161-7).
=
- = =
=
[115] A recombinant form of human IL-1Ra (rHuIL-1Ra) was developed and
approved for use
in humans by subcutaneous injection for the treatment of arthritis. This form
of rHuIL-1Ra, also
=
known as Analdnra or Kineret (Amgen Inc.), differs from the native
nonglycosylated IL-1Ra
. = by the addition of an N-terminal methionine. It binds to human IL-
1R, type 1, (IL-1R1) with the
same affinity as IL-1 B. Kineret consists of 153 amino acids (see SEQ ID NO:
16) and has a
molecular weight of 17.3 kilodaltons. It is produced by recombinant DNA
technology using an E =
coli bacterial expression system. This composition comprises one or more
regions of SEQ ID .=
NO: 15 or SEQ ID NO: 16. Furthermore, this composition comprises the entire
sequence of,
=
either SEQ ID NO: 15 or SEQ ID NO: 16.
=
=
=
= ===
= =
[116] Anakinra/Kineret is encoded by the following mRNA sequence (NCBI
Accession No.
M55646 and SEQ NO:.15):=
= ==
= agctccaccctgggagggactgtggcccaggtactgcccgggtgctactttatgggcagcagctcagttg
agttagagtctggaagacctcagaagacctcctgtcctatgaggccctccccATGgctttagagacgatc
tgccgaccctctgggagaaaatccagcaagatgcaagccttcagaatctgggatgttaaccagaagacct
tctatctgaggaacaaccaactagttgctggaticttgcaaggaccaaatgtcaatttagaagaaaagat
agatgtggtacccattgagcctcatgctctgttcttgggaatccatggagggaagatgtgcctgtcctgt
gtcaagtctggtgatgagaccagactccagctggaggcagttaacatcactgacctgagcgagaacagaa
agcaggacaagcgcttcgccttcatccgctcagacagtggccccaccaccagttttgagtctgccgcctg
ccccggttggttcctctgcacagcgatggaagctgaccagccagtcagcctcaccaatatgcctgacgaa
= ggcgtcatggtcaccaaattctacttccaggaggacgagtag.
= = - - - - - - - - - -
-
=
32
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[117] Anakinra/Kineret is encoded by the following polypeptide sequence
(DrugBank
= = Accession No. BTD00060 and SEQ ID NO: 16):
MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALF
LGIHGGICMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACP
GWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE
=
.= IL-1 Receptors:
[118] The composition of the present invention comprises a polynucleotide, a
polypeptide, an
antibody, a compound, or a small molecule with means to inhibit the
transcription, transcript
stability, translation, modification, localization, secretion, or function of
a polynucleotide or
=
polypeptide encoding the IL-1 receptor, either type 1 or 2. In the
present application the IL-1 .
Receptor, type 1 (IL-1R1), is defined by the polynucleotide sequence of SEQ ID
NO: 17 or the
=
polypeptide sequence of SEQ ID NO: 18. In the present application the IL-1
Receptor, type 2
(IL-1R2), transcript variants 1 and 2, are defined by the polynucleotide
sequences of SEQ ID
NO: 19 and 20; or the polypeptide sequence of SEQ ID NO: 21. IL-1R2 can
function as A
"decoy" receptor which binds IL-1 cytolcines and inhibits IL-1R1.
Polynucleotide or
=
polypeptide compositions bind to one or more region(s) of IL-1R1 or IL-1R2,
and associated =
isoforms, comprised by SEQ ID NO: 17-21. = "
=
=
= =
= [119] =IL-1R1 is encoded by the following mlINA sequence (NCBI Accession
No. NM_000877
and SEQ ID NO:. 17):
=
= tagacgcaccctctgaagatggtgactccctcctgagaagctggacccct tggtaaaagabaaggccttc
-tccaagaagaatATGaaagtgttactcagacttatttgtttcatagctctactgatttcttctctggagg
=
ctgataaatgcaaggaacgtgaagaaaaaataattttagtgtcatctgcaaatgaaattgatgttcgtcc
= ctgtcctcttaacccaaatgaacacaaaggcactataacttggtataaagatgacagcaagacacctgta
= tctacagaacaagcctccaggattcatcaacacaaagagaaactttggtttgttcctgctaaggtggagg
attcaggacattactattgcgtggtaagaaattcatcttactgcctcagaattaaaataagtgcaaaatt
tstsgagaatgagcctaacttatstt,ataatscacaagccatatttaascagaaactacccgttscagga
gacggaggacttgtgtgcccttatatggagttttttaaaaatgaaaataatgagttacctaaattacagt
ggtataaggattgcaaacctctacttcttgacaatatacactttagtggagtcaaagataggctcatcgt
.= gatgaatgtggctgaaaagcatagagggaactatacttgtcatgcatcctacacatacttgggcaagcaa
tatcctattacccgggtaatagaatttattactctagaggaaaacaaacccacaaggcctgtgattgtga
= gcccagctaatgagacaatggaagtagacttgggatcccagatacaattgatctgtaatgtcaccggcca
= gttgagtga'cattgcttactggaagtggaatgggtcagtaattgatgaagatgacccagtgetaggggaa
= gactattacagtgtggaaaatcctgcaaacaaaagaaggagtaccctcatcacagtgcttaatatatcgg
aaattgaaagtagattttataaacatccatttacctgttttgccaagaatadacatggtatagatgcagc
atatatccagttaatatatccagtcactaatttccagaagcacatgattggtatatgtgtcacgttgaca
gtcataattgtgtgttctgttttcatctataaaatcttcaagattgacattgtgctttggtacagggatt
= cctgctatgattttctcccaataaaagcttcagatggaaagacctatgacgcatatatactgtatccaaa
= gactgttggggaagggtctacctctgactgtgata.tttttgtgtttaaagtcttgcctgaggtcttggaa
aiacagtgtggatataagctgttcatttatggaagggatgactacgtt gggaagacattgttgaggtca
. ttaatgaaaacgtaaagaaaagcagaagactgattatcattttagtcagagaaacatcaggcttcagctg =
gctgggtggttcatctgaagagcaaatagccatgtataatgctcttgttcaggatggaattaaagttgtc
ctgcttgagctggagaaaatccaagactatgagaaaatgccagaatcgattaaattcattaagcagaaac
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atggggctatccgctggtcaggggactttacacagggaccacagtctgcaaagacaaggttctggaagaa
tgtcaggtaccacatgccagtccagcgacggtcaccttcatctaaacaccagttactgtcaccagccact
aaggagaaactgcaaagagaggctcacgtgcctctcgggtagcatggagaagttgccaagagttctttag
gtgcctcctgtcttatggcgttgcaggccaggttatgcctcatgctgacttgcagagttcatggaatgta
actatatcatcctttatccctgaggtcacctggaatcagattattaagggaataagccatgacgtcaata
gcagcccagggcacttcagagtagagggcttgggaagatcttttaaaaaggcagtaggcccggtgtggtg
= gctcacgcctataatcccagcactttgggaggctgaagtgggtggatcaccagaggtcaggagttcgaga
ccagcccagccaacatggcaaaaccccatctctactaaaaatacaaaaatgagctaggcatggtggcaca
= cgcctgtaatcccagctacacctgaggctgaggcaggagaattgcttgaaccggggagacggaggttgca
= gtgagccgagtttgggccactgcactctagcctggcaacagagcaagactccgtctcaaaaaaagggcaa
= taaatgccctctctgaatgtttgaactgccaagaaaaggcatggagacagcgaactagaagaaagggcaa
gaaggaaatagccaccgtctacagatggcttagttaagtcatccacagcccaagggeggggctatgcctt
= gtctggggaccctgtagagtcactgaccctggagcggctctcctgagaggtgctgcaggcaaagtgagac
tgacacctcactgaggaagggagacatattcttggagaactttccatctgcttgtattttccatacacat
ccccagccagaagttagtgtccgaagaccgaattttattttacagagcttgaaaactcacttcaatgaac
= aaagggattctccaggattccaaagttttgaagtcatcttagctttccacaggagggagagaacttaaaa
. aagcaacagtagcagggaattgatccacttcttaatgctttcctccctggcatgaccatcctgtcctttg
ttattatectgcattttacgtctttggaggaacagetcCctagtggcttcctccgtctgcaatgtccctt
gcacagcccacacatgaaccatccttcccatgatgccgctcttctgtcatcccgctcctgctgaaacacc
tcccaggggctccacctgttcaggagctgaagcccatgctttcccaccagcatgtcactcccagaccacc
= tccctgccctgtcctccagcttcccctcgctgtectgctgtgtgaattcccaggttggcctggtggccat
gtcgcctgcccbcagcactcctctgtctctgctcttgcctcgacccttcctcctcctttgcctaggaggc
= cttctcgcattttctctagctgatcagaattttaccaaaattcagaacatcctccaattccacagtctct
=
gggagactttccctaagaggcgacttcctctccagccttctctctctggtcaggcccactgcagagatgg= .
tggtgagcacatctgggaggctggtctccctccagctggaattgctgctctctgagggagaggctgtggt
ggctgtctctgtccctcactgccttccaggagcaatttgcacatgtaacatagatttatgtaatgcttta
tgtttaaaaacattccccaattatcttattta.atttitgcaattattctaattttatatatagagaaagt
gacctattttttaaaaaaatcacactctaagttctattgaacctaggacttgagcctccatttctggctt
ctagtctggtgttctgagtacttgatttcaggtcaataacggteccccctcactccacactggcacgttt
gtgagaagaaatgacattttgctaggaagtgaccgagtctaggaatgcttttattcaagacaccaaattc
= caaacttctaaatgttggaattttcaaaaattgtgtttagattttatgaaaaactcttctactttcatct
attctttccctagaggcaaacatttcttaaaatgtttcattttcattaaaaatgaaagccaaatttatat
* = gccaccgattgcaggacacaagcacagttttaagagttgtatgaacatggagaggacttttggtttttat
= .atttctcgtatttaatatgggtgaacaccaacttttatttggaataataattttcctcctaaacaaaaac
- = acattgagtttaagtctctgactcttgcctttccacctgctttctcctgggcccgctttgcctgcttgaa
ggaacagtgctgttctggagctgctgttccaacagacagggcctagctttcatttgacacacagactaca
= gccagaagcccatggagcagggatgtcacgtcttgaaaagcctattagatgttttacaaatttaattttg .
cagattattttagtctgtcatccagaaaatgtgtcagcatgcatagtgctaagaaagca4gccaatttgg
aaacttaggttagtgacaaattggccagagagtgggggtgatgatgaccaagaattacaagtagaatgg
cagctggaatttaaggagggacaagaatcaatggataagcgtgggtggaggaagatccaaacagaaaagt
' gcaaagttattccccatcttccaagggttgaattctggaggaagaagacacattcctagttccccgtgaa
= cttectttgacttattgtccccactaaaacaaaacaaaaaacttttaatgccttccacattaattagatt
ttcttgcagtttttttatggcatttttttaaagatgccctaagtgttgaagaagagtttgcaaatgcaac
aaaatatttaatticcggttgttaaaactggtttagdacaatttatattttccctctcttgcctttctta
tttgcaataaaaggtattgagccattttttaaatgacatttttgataaattatgtttgtactagttgatg
aaggagttttttttaacctgtttatataattttgcagcagaagccaaattttttgtatattaaagcacca- =-
aattcatgtacagcatgctcacggatcaatagactgtacttattttccaataaaattttcaaactttgt =
= actgttaaa.
= pumq IL-1R1 is encoded by the following amino acid sequence (NCBI
Accession No.
NM_000877 and SEQ ID NO: 18):
= MKVLLRLICHALLISSLEADKCIC.EREEKIILVSSANEEDVRPCPLNPNEHKGTTIWYKDDS = , . .
KTPVSTEQASRIHQHKEICLWFVPAKVEDSGHYYCVVRNSSYCLRIKISAKFVENEPNLC
YNAQAIFKQICLPVAGDGGLVCPYM.EFFKNENNELPKLQWY1CDCKPLILDNIHFSGVKD
34 =
=
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PCT/US2008/009776
RLIVMNVAEKHRGNYTCHASYTYLGKQYPITRVIEFITLEENKYIRPVIVSPANETMEVD
LGSQIQLICNVTGQLSDIAYWKWNGSVIDEDDPVLGEDYYSVENPANICRRSTLITVLNIS
EIESRFYICHPFTCFAKNTHGIDAAYIQUYPVTNFQICHIv1IGICVTLTVIIVCSVFIYKIFKIDI
VLWYRDSCYDFLPIKASDGKTYDAYILYPKTVGEGSTSDCDIFVFKVLPEVLEKQCGYK
LFIYGRDDYVGEDIVEVINENVKKSRRLIIILVRETSGF SWLGGSSEEQIAMYNALVQDGI
KVVLLELEKIQDYEICMPESIICFIKQKHGAIRWSGDFTQGPQSAKTRFWICNVRYHMPVQ
RRSPSSKHQLLSPATKEKLQREAHVPLG.
= [121] IL-1R2, transcript variant 1, is encoded by the following mRNA
sequence (NCBI
=
Accession No. NM_004633 and SEQ ID NO: 19):
= cccgtgaggaggaaaaggtgtgtccgctgccacccagtgtgagcgggtgacaccacccggttaggaaatc
ccagctcccaagagggtataaatccctgct ttactgctgagctcctgctggaggtgaaagtctggcctgg
cagcdttccccaggtgagcagcaacaaggccacgtgctgctgggtctcagtcctccacttcccgtgtcct
ctggaagttgtcaggagcaATGttgcgcttgtacgtgttggtaatgggagtttctgccttcacccttcag
cctgcggcacadacaggggctgccagaagctgccggtttcgtgggaggcattacaagcgggagttcaggc
= tggaaggggagcctgtagccctgaggtgcccccaggtgccctactggttgtgggcctctgtcagcccccg
catcaacctgacatggcataaaaatgactctgctaggacggtcccaggagaagaagagacacggatgtgg
= gcccaggacggtgctctgtggcttctgccagccttgcaggaggactctggcacctacgtctgcactacta
= =
gaaatgcttcttactgtgacaaaatgtccattgagctcagagtttttgagaatacagatgctttcctgcc
gttcatctcatacccgcaaattttaaccttgtcaacctctggggtattagtatgccctgacctgagtgaa
ttcacccgtgacaaaactgacgtgaagattcaatggtacaaggattctcttcttttggataaagacaatg
agaaatttctaagtgtgagggggaccactcacttactcgtacacgatgtggccctggaagatgctggcta .
= =
ttaccgctgtgtectgacatttgcccatgaaggccagcaatacaacatcactaggagtattgagctacgc
atcaagaaaaaaaaagaagagaccattcctgtgatcatttcccccctcaagaccatatcagcttctctgg.
ggtcaagactgacaatcccgtgtaaggtgtttctgggaaccggcacacccttaaccaccatgctgtggtg-
gacggocaatgacacccacataqagagcgcctacccgggaggccgcgtgaccgaggggccacgccaggaa
tattcagaaaataatgagaactacattgaagtgccattgatttttgatcctgtcacaagagaggatttgc
=.acatggattttaaatgtgttgtccataataccatgagttttcagacactacgcaccacagtcaaggaagc =
,
ctactccacgttctcctggggcattgtgctggccccactttcactggccttcttggttttggggggaata
tggatgcacagacggtgcaaacacagaactggaaaagcagatggtctgactgtgctatggcctcatcatc
= -aagactttcaatcctatcccaagtgaaataaatggaatgaataattcaaacacaaaaaaaaaaaaaaaa
= aaaaa.aaaaaaaaa .
= =
[122] IL-1R2, transcript valiant 2, is encoded by the following mRNA sequence
(NCBI
=
, .
." 'Accession No. NIVI_173343 and SEQ ID NO: 20):
gggatgggagatactgtt.gtggtcacctctggaaaatacattctgctactcttaaaaactagtgacgctc
atacaaatcaacagaaagagcttctgaaggaagactttaaagctgcttctgccacgtgctgctgggtctc
. agtcctccacttcccgtgtcctctggaagttgtcaggagcaATGttgcgcttgtacgtgttggtaatggg
= agtttctgccttcacccttcagcctgcggcacacacaggggctgccagaagctgccggtttcgtgggagg
= = =cattacaagcgggagttcaggctggaaggggagcctgtagccctgaggtgcccccaggtgccctactggt
tgtgggcctctgtcagcccccgdatcaacctgacatggcataaaaatgactctgctaggacggtcccagg
,agaagaagagacacggatgtgggcccaggacggtgctctgtggcttctgccagccttgcaggaggactct
ggcacctacgtctgcactactagaaatgcttcttactgtgacaaaatgtccattgagctcagagtttttg
= agaatacagatgctttcctgccgttcatctcatacccgcaaattttaaccttgtcaacctctggggtatt
agtatgccctgacctgagtgaattcacccgtgacaaaactgacgtgaagattcaatggtacaaggattct
= cttcttttggataaagacaatgagaaatttctaagtgtgagggggaccactcacttactcgtacacgatg
= tggccctggaagatgctggctattaccgctgtgtcctgacatttgcccatgaaggccagcaatacaacat
cactaggagtattgagctacgcatcaagaaaaaaaaagaagagaccattactgtgatcatttcccccctc
aagaccatatcagcttctctggggtcaagactgacaatcccgtgtaaggtgtttctgggaaccggcacac
ccttaaccaccatgctgtggtggacggccaatgacacccacatagagagcgcctacccgggaggccgcgt
= -= gaccgaggggccacgccaggaatat
tcagaaaataatgagaactacattgaagtgccattgatttttgat =
.cctgtcacaagagaggatttgcacatggattttaaatgtgttgtccataataccctgagttttcagacac
ticgcaccacagtcaaggaagcctcctccacgttctcctggggcattgtgctggccccactttcactggc
= 35
=
¨ ,õ
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PCT/US2008/009776
cttcttggttttggggggaatatggatgcacagacggtgcaaacacagaactggaaaagcagatggtctg
actgtgctatggcctcatcatcaagactttcaatcctatcccaagtgaaataaatggaatgaaataattc
aaacacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa .
[123] IL-1R2, transcript variants 1 and 2, are encoded by the following amino
acid sequence
(NCBI Accession No. NM_004633, NM_173343, and SEQ ID NO: 21):
MLRLYVLVMGVSAFTLQPAAHTGAARSCRFRGRHYKREFRLEGEPVALRCPQVPYWL
WASVSPRINLTWHICNDSARTVPGEEETRMWAQDGALWLLPALQEDSGTYVCTTRNAS
YCDICMSIELRVFENTDAFLPFISYPQILTLSTSGVLVCPDLSEFTRDKTDVICIQWYKDSLL
LDICDNEKFLSVRGTTHLLVHDVALEDAGYYRCVLTFAHEGQQYNITRSIELRIKKICKEE
TIPVIISPLKTISASLGSRLTIPCKVFLGTGTPLITMLWWTANDTHIESAYPGGR'VTEGPRQ
= EYSENNENYIEVPLIFDPVTREDLHMDFKCVVHNTLSFQTLRTTVICEASSTFSWGIVLAP
LSLAFLVLGGIVIMHRRCKHRTGICADGLTVLIATPHHQDFQSYPK.
Interleuldn-1 Receptor (type 2) antagonist (IL-1Ra31:
[124] The present invention comprises compositions with means to inhibit or
enhance the
activity of the human IL-1R2. Compositions that comprise the IL-1R2
antagonist, IL-1Ra3, have
= either agonist or antagonist activity regarding the efficacy of IL-1R1
function. The composition
comprises a polynucleotide, a polypeptide, an antibody, a compound, or a small
molecule with .
= means to inhibit the transcription, transcript stability, translation,
modification, localization,
secretion, or function of a polynucleotide or polypeptide encoding IL-1Ra3.
The inhibitory
polynucleotide or polypeptide composition binds to one or more region(s) of IL-
1Ra3 comprised
= by SEQ ID NO: 22 and SEQ ID NO: 23.
= [125] IL-1Ra3 is encoded by a region or the entirety of the following
mRNA sequence (NCBI
=
=
Accession No. AF_057168 and SEQ ID NO: 22): (for this sequence, the bolded and
capitalized
. .
coclon does not encode methionine, but rather represents the codon that
encodes the first amino
acid of the corresponding polypeptide)
=
cagaigacctcctgtcctatgaggccctccccatggctttaggtaagctecttcoactctcattttttca
catgagaaatgagagaggaaaatgtctacaattggtgtttatcaaatgctttcaggctctggtgagcaag
cgtccaggaaaatgt caagcgcatggagct ccaggcctgt ctgggggat c tgggcacggggaggcatcca
tgggagaccatgcaggcactctgaggcaggggctgcaagctagtgcctgctggggcagcaggtgaacaga
gaggtgtaactgctgtgacaga.agtcatggagtccttggagtgtgagggtcattttccactgttgataga
=
atagggaaattggtgaaa.tagccctgttaaatgagagaaagaacagtgtgagctcaatgagaaatactaa
= tagaatgtggcactgagccacaaggtctgagggttgattgataaggaagggtggggactgtggagaatta
agggcttggcacaggtcagttccaccagttgtcacaagagaatgcaggctcaggtggccagaacttctcg
cttttccagaagagtccgatattctgatttcattatatatagtattctgattaaaccagacaataaagca
agcagataaaatatttaaagtataagctgccagtttgcaacctccggttaggatttgtgtggggCaaaga
= aaaaaactctcaggatcattggtatgtagactctaattttaagtttctaatttaaaattggcccctgagg
= ctgggcgtggtggctcacacctgtaatcccagcattttgggaggccaaggtgggtggatctcttgaggtc =
aagagttcaaggcctgcctggccaacatggtgaaaccctgtctctattaaaaatacaaaaattagctggg
catggtggtgcatgtctgcaatcttagctacttgggtagctaaggcaggagaattgctggaacccgggag
gtagaggttgcagtgaatggagatcacaccactgcactccagtctgggcaatagagagagacgctctctc
taaaaaaaaatatgtaaagataaataaaatgaaataaaataggcctctaatgagcaggccattctccttt
ctgggtcttactttccttgcactcctttctgggtgttaagaggaggtctagaggaagctggacaactctt
36
=,M1.00-A=r+
W02009/025763 CA 02640986 2010-02-08 PCT/US2008/009776
agcttgtagtaagcacagtggaagtatcagctcttaatgggtcatggacacgttacgaagctaggcgccg
tgctgagcactttacatggtttatcccactgaaccctctcaataaccctatgaggaagggctattattgc
tcacattttcagaagaggaaatggatatagagagattagataatttgcccatggccagacagctagtata
agaggaggaggtggattgactgcagacattctgtcttcaaaccactacactatgctatggaggcacagag
acttaatgaaatcatggagaggggaattgctttgtcaaccacaagcagttattccgggggcagcagatcc
tcccctgtcccccagtggtacaatggtccctggtgggttgtgctacaatgttagcccatggtettatgtg
tttttcaaatgtgtaaagtaggatgctggaaccactcttagaaccagataccaatacattgtgaagaaat
aaatctctgtgcttaaaactggttcatcccaaaatattttgaactgacacacaataggtgctaaataaat
gtgtgttaacttgaattggattgaattcgggaaaaaagtgcaataagcttagtgaagacaccatgttccc
tgggtagaggaaccacattctccatctaaggccaggagtatgggaggtatcaatgtttgcccagcacaga
acagggtgccaagaagagaaaagttgacggggtgcatactctgactggaaactggaagggtgagaacaga
gggtaaaggatagagatggaaccatgtgcatacactttgtgttaccttggacaagtcattcatttctctg
gacctctgctttctctctacacaatggggtcccaccacttcccttacagctgacttgtatgaagaaggag
gtggaggaggaggagaaggtgaagacaatgCTGactcaaagggtaaattatttttaggatccaagtttga
aaacaattttaggctactagatatgaacaacatcttgattatgtagttgaaggaaattaaagatgaatgg
tttaattaaaaattaatcagaatgaaaacgattgattactaatatatctgcaatggtttattttcctgag
tggcagactcactaaggtttttgaatactcctgtgtgattgctctatgtatgtatgtatgtatgtatgta
tgcatgtatctatctatctgttgtctaatagaatggatcacatctctgctaataaaaacactacactggc
agggtacaattataatcattaactgtgcctggaatttgcagcagcagccaccagaggtaccagtgccctt
taagggttcataatttagaataatccaattatctgagtttttcagggactgaggggtttggcaaggtgta
gaactttcagtaataaagtcaagaaagtcctggacaaaccaaggtagttggtcactctagtccataacca
ggtaaagagctttccctgtaacctgtgtaaggttttagaatcatttctttccttattaccaaaaatcctc
cccaaattttcaagaaattatgaactaaatagttactctatgagataggagttcagcccaaaagaaacac
cataagaacaaatataattcttgcttatgttaaccatgcaatgaagcagagagaaaaagtcagtggcctc
tttaggaggactgtagtgtgggaagaaataactaaactgggtttcaatcctggcctggccaggatctgga
gcaagtgagttaatctttctaagccttgagtagtttataaaagaatggccactccatagacagagtagcc
tgaaccttgagttcttctataaagtcactatgaatttatactcattttgaaagtgggtgtcaatatgtct
gtccactttgcacagctgttatgtggacaaaaggagatctgtgtgaaagtgtaacacagagcctaaacta
taacaggtaagcaacacagttgtccct .
[126] One or more isoforms of IL-1Ra3 comprise the following amino acid
sequence (NCBI
Accession No. AF_057168 and SEQ ID NO: 23):
DLYEEGGGGGGEGEDNADSK.
Interleuldn-1 Receptor Accessory Protein (IL-1 RAP):
[127] Compositions that inhibit the activity of human IL-1RAP inhibit IL-1RAP
binding to an
cytokine or an IL-1 receptor, and subsequent transduction of downstream
intracellular
signals. Compositions that comprise an inhibitor of IL-1RAP function
antagonize the activity of
an IL-1 receptor. The composition comprises a polynucleotide, a polypeptide,
an antibody, a
compound, or a small molecule with means to inhibit the transcription,
transcript stability,
translation, modification, localization, secretion, or function of a
polynucleotide or polypeptide
encoding IL-1RAP. The inhibitory polynucleotide or polypeptide composition
binds to one or
more region(s) of IL-1RAP, and associated isoforms, comprised by SEQ ID NO: 24-
27.
[128] IL-1 RAP, transcript variant 1, is encoded by the following mRNA
sequence (NCBI
Accession No. NM 002182 and SEQ ID NO: 24):
37
õ
,
W020091025763 CA 02640986 2010-02-08
PCT/US2008/009776
tgccgggatccaggtctccggggtccgctttggccagaggcgcggaaggaagcagtgcccggcgacactg
cacccatcccggctgcttttgctgcgccctctcagcttcccaagaaaggcatcgtcatgtgatcatcacc
taagaactagaacatcagcaggccctagaagcctcactcttgcccctccctttaatatctcaaaggATGa
cacttctgtggtgtgtagtgagtctctacttttatggaatcctgcaaagtgatgcctcagaacgctgcga
tgactggggactagacaccatgaggcaaatccaagtgtttgaagatgagccagctcgcatcaagtgccca
ctctttgaacacttcttgaaattcaactacagcacagcccattcagctggccttactctgatctggtatt
ggactaggcaggaccgggaccttgaggagccaattaacttccgcctccccgagaaccgcattagtaagga
gaaagatgtgctgtggttccggcccactctcctcaatgacactggcaactatacctgcatgttaaggaac
actacatattgcagcaaagttgcatttcccttggaagttgttcaaaaagacagctgtttcaattccccca
tgaaactcccagtgcataaactgtatatagaatatggcattcagaggatcacttgtccaaatgtagatgg
atattttccttccagtgtcaaaccgactatcacttggtatatgggctgttataaaatacagaattttaat
aatgtaatacccgaaggtatgaacttgagtttcctcattgccttaatttcaaataatggaaattacacat
gtgttgttacatatccagaaaatggacgtacgtttcatctcaccaggactctgactgtaaaggtagtagg
= ctctccaaaaaatgcagtgccccctgtgatccattcacctaatgatcatgtggtctatgagaaagaacca
ggagaggagctactcattccctgtacggtctattttagttttctgatggattctcgcaatgaggtttggt
ggaccattgatggaaaaaaacctgatgacatcactattgatgtcaccattaacgaaagtataagtcatag
tagaacagaagatgaaacaagaactcagattttgagcatcaagaaagttacctctgaggatctcaagcgc
agctatgtctgtcatgctagaagtgccaaaggcgaagttccaaagcagccaaggtgaagcagaaagtgcc
agctccaagatacacagtggaactggcttgtggttttggagccacagtcctgctagtggtgattctcatt
gttgtttaccatgtttactggctagagatggtcctattttaccgggctcattttggaacagatgaaacca
= ttttagatggaaaagagtatgatatttatgtatcctatgcaaggaatgcggaagaagaagaatttgtatt
= actgaccctccgtggagttttggagaatgaatttggatacaagctgtgcatctttgaccgagacagtctg
= ='cctgggggaattgtcacagatgagactttgagcttcattcagaaaagcagacgcctectggttgttctaa
gccccaactacgtgctccagggaacccaagccctcctggagctcaaggctggcctagaaaatatggcctc
tcggggcaacatcaacgtcattttagtacagtacaaagctgtgaaggaaacgaaggtgaaagagctgaag
agggctaagacggtgctcacggtcattaaatggaaaggggaaaaatccaagtatccacagggcaggttct
ggaagcagctgcaggtggccatgccagtgaagaaaagtcccaggcggtctagcagtgatgagcagggcct
= ctcgtattcatctttgaaaaatgtatgaaaggaataatgaaaagggtaaaaagaacaaggggtgctccag
gaagaaagagtccccccagtcttcattcgcagtttatggtttcataggcaaaaataatggtctaagcctc
= =
ccaatagggataaatttagggtgactgtgtggctgactattctgcttcctcaggcaacactaaagtttag
aaagatatcatcaacgttctgtcaccagtctctgatgccactatgttctttgcaggcaaagacttgttca
= atgcgaatttccccttctacattgtctatccctgtttttatatgtctccattctttttaaaatcttaaca
tatggagcagcctttcctatgaatttaaatatgcctttaaaataagtcactgttgacagggtcatgagtt
tccgagtatagttttctttttatcttatttttactcgtccgttgaaaagataatcaaggcctacatttta
= gctgaggataatgaacttttttcctcattcggctgtataatacataaccacagcaagactgacatccact
taggatgatacaaagcagtgtaactgaaaatgtttc.ttttaattgatttaaaggacttgtcttctatacc
acccttgtcctcatctcaggtaatttatgaaatctatgtaaacttgaaaaatatttcttaatttttgttt
ttgctccagtcaattcctgattatccacaggtcaacccacattttttcattccttctccctatctgctta
tatcgcattgctcatttagagtttgcaggaggctccatactaggttcagtctgaaagaaatctcctaatg
gtgctatagagagggaggtaacagaaagactcttttagggcatttttctgactcatgaaaagagcacaga
==
aaaggatgtttggcaatttgtcttttaagtcttaaccttgctaatgtgaatactgggaaagtgatttttt
= ctcactcgtttttgttgctccattgtaaagggcggaggtcagtcttagtggccttgagagttgcttttgg
cattaatattctaagagaattaactgtatttcctgtcacctattcactagtgcaggaaatatacttgctc
caaataagtcagtatgagaagtcactgtcaatgaaagttgttttgtttgttttcagtaatattttgctgt
ttttaagacttggaaaactaagtgcagagtttacagatggtaaatatctatgttacatgtagattatac
atatatatacacacgtgtatatgagatatatatcttatatctccacaaacacaaattatatatatacata
= tccacacacatacattacatatatctgtgtatataaatccacatgcacatgaaatatatatatatatata
= atttgtgtgtgtgtatgtgtatgtatatgactttaaatagctatgggtacaatattaaaaaccactggaa
ctcttgtccagtttttaaattatgtttttactggaatgtttttgtgtcagtgttttctgtacatattatt
tgttaattcacagctcacagagtgatagttgtcatagttcttgccttccctaagtttatataaataactt
=
aagtattgctacagtttatctaggttgcagtggcatctgctgtgcacagagcttccatggtcactgctaa
gcagtagccagccatcgggcattaattgatttcctactatattcccagcagacacatttagaaactaagc
tatgttaacctcagtgctcaactatttgaactgttgagtgataaaggaaacaaatataactgtaaatgaa
= tcttggtatcctgtgaaacagaataattcgtaatttaagaaagcccttatcccggtaacatgaatgttga
tgaacaaatgtaaaattatatcctatatttaagtaccCataataaatcatttccctctataagtgttitt
.gattattttaaattgaaaaaagtttcacttggatgaaaaaagtagaaaagtaggtcattcttggatctac
ttttttttagccttattaatatttttccctattagaaaccacaattactccctctattaacccttcactt
38
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actagaccagaaaagaacttattccagataagctttgaatatcaattcttacataaactttaggcaaaca
gggaatagtctagtcaccaaaggaccattctcttgccaatgctgcattccttttgcacttttggattcca
tatttatcccaaatgctgttgggcacccctagaaataccttgatgttttttctatttatatgcctgcctt
tggtacttaattttacaaatgctgtaatataaagcatatcaagtttatgtgatacgtatcattgcaagag
aatttgtttcaagatttttttttaatgttccagaagatggccaatagagaacattcaagggaaatgggga
aacataatttagagaacaagaacaaaccatgtctcaaatttttttaaaaaaaattaatggttttaaatat
atgctatagggacgttccatgcccaggttaacaaagaactgtgatatatagagtgtctaattacaaaatc
atatacgatttatttaattctcttctgtattgtaacttagatgattcccaaggactctaataaaaaatca
= cttcattgtatttggaaacaaaaacatcattcattaattacttattttctttccataggttttaatattt
tgagagtgtcttttttatttcattcatgaacttttgtatttttcatttttcatttgatttgtaaatttac
ttatgttaaaaataaaccatttattttcagctttg .
[129] IL-IRAP, transcript variant 1, is encoded by the following amino acid
sequence (NCBI
= Accession No. NM_002182 and SEQ ID NO: 25):
MTLLWCVVSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLICFNYS
= TAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISICEKDVLWFRPTLLNDTGNYTCMLRN
=TTYCSKVAFPLEVVQKDSCFNSPMICLPVHICLYIEYGIQRITCPNVDGYFPSSVKPTITWY
MGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSP
KNAVPPVIHSPNDHVVYEICEPGEELLIPCTVYFSFLMDSRNEVWWTIDGICKPDDITIDVT
= INESISHSRTEDETRTQILSIICKVTSEDLICRSYVCHARSAKGEVAICAAICVKQK'VPAPRYT
= VELACGFGATVLLVVILIVVYHVYWLEMVLFYRAHFGTDETILDGICEYDIYVSYARNAE
EEEFVLLTLRGVLENEFGYICLCIFDRDSLPGGIVTDETLSFIQKSRRLLVVLSPNYVLQGT
QALLELICAGLENMASRGNINVILVQYICAVICETKVICELKRAICTVLTV1KWKGEKSKYPQ
GRF'WKQLQVAIV1PVKICSPRRSSSDEQGLSYSSLICNV.
[130] IL-1RAP, transcript variant 2, is encoded by the following mRNA sequence
(NCBI
Accession No. NM_134470 and SEQ ID NO: 26):
= tgccgggatccaggtctccggggtccgctttggccagaggcgcggaaggaagcagtgcccggcgacactg
=. = cacccatcccggctgcttttgctgcgccctctcagcttcccaagaaaggcatcgtcatgtgatcatcacc
taagaactagaacatcagcaggccctagaagcctcactcttgcccctccctttaatatctcaaaggATGa
. . =
cacttctgtggtgtgtagtgagtctctacttttatggaatcctgcaaagtgatgcctcagaacgctgcga =
== tgactggggactagacaccatgaggcaaatccaagtgtttgaagatgagccagctcgcatcaagtgccca
ctctttgaacacttcttgaaattcaactacagcacagcccattcagctggccttactctgatctggtatt . =
= ggactaggcaggaccgggaccttgaggagccaattaacttccgcctccccgagaaccgcattagtaagga
= =
gaaagatgtgctgtggttccggcccactctcctcaatgacactggcaactatacctgcatgttaaggaac
actacatattgcagcaaagttgcatttcccttggaagttgttcaaaaagacagctgtttcaattccecca
= tgaaactcccagtgcataaactgtatatagaatatggcattcagaggatcacttgtccaaatgtagatgg
atattttccttccagtgtcaaaccgactatcacttggtatatgggctgttataaaatacagaattttaat
aatgtaatacccgaaggtatgaacttgagtttcctcattgccttaatttcaaataatggaaattacacat
= gtgttgttacatatccagaaaatggacgtacgtttcatctcaccaggactctgactgtaaaggtagtagg
= ctctccaaaaaatgcagtgccc.cctgtgatccattcacctaatgatcatgtggtctatgagaaagaacca
ggagaggagctactcattccctgtacggtctattttagttttctgatggattctcgcaatgaggtttggt
= ggaccattgatggaaaaaaacctgatgacatcactattgatgtcaccattaacgaaagtataagtcatag
tagaacagaagatgaaacaagaactcagattttgagcatcaagaaagttacctctgaggatctcaagcgc
== agctatgtctgtcatgctagaagtgccaaaggcgaagttgccaaagcagccaaggtgaagcagaaaggta
= atagatgcggtcagtgatgaatctctcagctccaaattaacattgtggtgaataaggacaaaaggagaga
ttgagaacaagagagctccagcacctagcccgacggcatctaacccatagtaatgaatcaaacttaaatg
aaaaatatgaaagttttcatctatgtaagatactcaaaatattgtttctgatattgttagtaccgtaatg
= cccaantgtagctaaaaaaatcgacgtgagtacagtgagacacaattttgtgtctgtacaattatgaaaa
at taaaaacaaagaaaatattcaaagctaccaaagatagaaaaaactggtagagccacatattgttggtg
= aattattaagacccttttaaanatcattcatggtagagtttaagagtcataaaaaagattgcatcatctg
acctaagactttcggaatttttcctgaacaaataacagaaagggaattatataccttttaatattattag
= aagcattatctgtagttgtaaaacattattaatagcagccatccaattgtatgcaactaattaaggtatt
==.
39
= =
W020091025763 CA 02640986 2010-02-08
PCT/US2008/009776
gaatgtttattttccaaaaatgcataattataatattattttaaacactatgtatcaatatttaagcagg
tttataatataccagcagccacaattgctaaaatgaaaatcatttaaattatgattttaaatggtataaa
catgatttctatgttgatagtactatattattctacaataaatggaaattataaagccttcttgtcagaa
gtgctgctcctaaaaaaaaaaaaaaaaaaaaaa.
[131] IL-1RAP, transcript variant 2, is encoded by the following amino acid
sequence (NCBI
Accession No. NM_134470 and SEQ ID NO: 27):
MTLLWCWSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLICFNYS
= TAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEICDVLWFRPTLLNDTGNYTCMLRN
TTYCSKVAFPLEWQKDSCFNSPMKLPVHICLYIEYGIQRITCPNVDGYFPSSVICPTITWY
MGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCWTYPENGRTFHLTRTLTVKWGSP
ICNAVPPVIHSPNDHVVYEICEPGEELLIPCTVYFSFLMDSRNEVWWTIDGICICPDDITIDVT
INESISHSRTEDETRTQILSIKKVTSEDLICRSYVCHARSAKGEVAICAAKVKQKGNRCGQ.
Interleukin-1 Receptor Associated Kinase 1 (IRAK1):
= [132] The invention also comprises compositions and methods to inhibit
the activity of human
= IRAK1, defined as the ability of this protein to bind an IL-1 receptor
following ligation of this
receptor with IL-1, as well as to traniduce downstream signals leading to an
inflammatory
response. Compositions that comprise an inhibitor of IRAK1 antagonize
downstream signaling
from an ILrl receptor. The composition comprises a polynucleotide, a
polypeptide, an antibody,
a compound, or a small molecule with means to inhibit the transcription,
transcript stability,
translation, modification, localization, secretion, or function of a
polynucleotide or polypeptide
encoding IRAK1. The inhibitory polynucleotide or polypeptide composition binds
to one or
more region(s) of IRAK1, and associated isoforms, comprised by SEQ ID NO: 28-
33.
[133] IRAK1, transcript variant 1, is encoded by the following mRNA sequence
(NCBI
Accession.No. NM_001569 and SEQ ID NO: 28):
cgcggacccggccggcccaggcccgcgcccgccgcggccctgagaggccccggcaggtcccggcccggcg
=.
gcggcagccATGgccggggggccgggcccgggggagcccgcagcccccggcgcccagcacttcttgt acg
aggtgccgccctgggtcatgtgccgcttctacaaagtgatggacgccctggagctcgccgactggtgcca
gttcgccgccctgatcgtgcgcgaccagaccgagctgcggctgtgcgagcgctccgggcagcgcacggcc
= agcgtcctgtggccctggatcaaccgcaacgcccgtgtggccgacctcgtgcacatcctcacgcacctgc
agctgctccgtgcgcgggacatcatcacagcctggcaccctcccgccccgcttccgtccccaggcaccac
tgccccgaggcccagcagcatccctgcacccgccgaggccgaggcctggagcccccggaagttgccatcc
tcagcctccaccttcctctccccagcttttccaggctcccagacccattcagggcctgagctcggcctgg
tcccaagccctgcttccctgtggcctccaccgccatctccagccccttcttctaccaagccaggcccaga
gagctcagtgtccctcctgcagggagcccgcccctttccgttttgctggcccctctgtgagatttcccgg
ggcacccacaacttctcggaggagctcaagatcggggagggtggctttgggtgcgtgtaccgggcggtga
tgaggaacacggtgtatgctgtgaagaggctgaaggagaacgctgacctggagtggactgcagtgaagca
gagcttcctgaccgaggtggagcagctgtccaggtttcgtcacccaiacattgtggactttgctggctac
tgtgctcagaacggct.tctactgcctggtgtacggcttcctgcccaacggctccctggaggaccgtctcc
actgccagacccaggcctgcccacctctctcctggcctcagcgactggacatccttctgggtacagcccg
ggcaattcagtttctacatcaggacagccccagcctcatccatggagacatcaagagttccaacgtcctt
ctggatgagaggctgacacccaagctgggagactttggcctggcccggttcagccgctttgccgggtcca
. .
= gccccagccagagcagcatggtggcccggacacagacagtgcggggcaccctggcctacctgcccgagga
gtacatcaagacgggaaggctggctgtggacacggacaccttcagctttggggtggtagtgctagagacc
= 40
_
IV
10 :0IsI cn bas Pug zvzszcsioo-m =oN uo!ssooav
IeD1.0 aouanbas visnitu Bwmouo; oq cqpapoouas `z ;tiepin Idgosuau µDivui NEU
=
= =
baaasaadotmatmolonssssiabiscrivoaaKprrxbiutublivaNmemaassssv
ssoivloanvicifisociosomssapvicobcoovanavanaosarmmsrivvsimi
saasandbmoubqbawbvsvosayvadbmavvosHvInissiusmabasactosvva
SHOdADVAAWY1ITIIIHAA61IAIdcIIIMIVIIIIHIODDYIbUIDID'Iadd3d0cIIIKIIIDDI
AibwviavvivivavviovOusbisrivnovaadaaaKICUIA.31111VORDIAVITODYI
iaanAnalsalainAvniamAaacridwuminibilpinmissbscissovalsamoda
=
omummannisissmaoHnsasabinabrvIrvi.uniumbamsucovbibmilma
isotsundoitnioAdombvpAovaannskuntmsqbanandstouviAkaiavmayna
AVAAII=IIIIAIAVIIAADDJ0133DIXIMSaRILIDIISIgaIlMalcILDIVOOTISASSacIDDI
= issavasaaaamisvasanmaaaosaLbsociavasuisvssammisAvvavavavassa
xdVJ-LDdScriddddHAWIHC[IIVIITIZYIRL'IIHNICWAIIVNIINIMcIAVIASV.L11ODSIIaal
InaLbaunvvabom.avaawawnxikalownmadnaKHHOvocivvdaododooviAl
= uopsoopv
:(6z :ON bas Puv 69S I OCIAIN -ToN
= mats) =limbos mu oupuu Suptiollo; alp q papooua s '1 luegun ldposuari
Dpirui [I]
=
.Revegeeeveeeeeweevl
eepfiliSqegfineyeeeqvin6e61433161165voolbau61561qe3666qp6ey6q6poopoBlepe
4oppeoq5e616336polq645glqwe6q6voeolq066epeoSeq6qo665yopo5p1Seeveop4616
0614013016.46e6povelbleolq166eolbeeofty6yoqqp164voyogel6zeop6eeee666eol
qggeoeve666peS66yoeSvp6Sgeobe6661q6eeee6e366ee661popoSep5e616voy6smope
.416eqopv5yeoppMeelpEeelogffiev000vqovoo6566v6v356q612y6qoro65e33355poqop
Svogoftypepo6.6e66341366voq66epo6y6poqp6e6pv5eoeoo5o665qoppleopS6enoBee
ofiqoa6S466q6leeqpoffiqp5e5D5qope5566qoe111355eolloq516y33666olopywoqoo
voopeofto6e6345yeebepoqopqe6y6133qee5p6o5vefteoBeloolftogq66qeoq336eeq
613651eq62e6qbeeey.666qoppeerrySeevreolq16g000rBeez6eBypeSq6654=6epolo
gobweftel6qwebleolfie6q5e3Sq366e63166y6661106qinglyou.66y66y362v6go66e
6586106w6epooq66q6qop6opoSo6v456q6offilooSellelpeewqqqqqqeguree336qpqo
qbwooe6p5ge61yovvo656epoSepoeSee3166eqftpooft651363.4v5EmbEep6SyypoEsby
= 6564143e3612oopqev461poSpeow66166p6q66633661.6q6evueffilqqueovq436qopygo
owoo666v1=667e5pyBoo55564evoepEsq6powo6616epe66qqwwlvopoBqqww5y36
=
epffiqolooffivoopEpeo6q3163666e36qepo6y5e6qoppoqp65135e666eq5s661616666ep
6q64o5436666e6e66e5666e6q6pope6voeo61366q36q366e6661161ea63661066v66e6
qbeopleoqp366qp.6612e66eep66666qoqvoftoo6lwoqop6l000ev66q6e61.366e36.6eqv
o666q6low66eqoqq613613=66266v6See626v66566o533361epoo6666616665636vo
66335lowvoftowol6e6ovo6166123103q6vv6ep166qeolo.416yeepl6ele6600lsgepo =
oppqr5e3666qopeolq6451e6136eBeoqq12v6qe615elgreBveSpoo6666w66w26.6epee6
'6w35561436SepoolopowEmpol5a4136wEsep6qooSeov66303365561e56rEopl6loo
offiwBesSepol654e6evEmpeSpoofilopoveolvoqvq4v6epappeopEmBeal6pq6olvoqep
6q3136e0661433366weB6e266q6336vowoo663=1255s.33365q6E56665q36v6olve6
E662366peos.66656eoloolBw5.63366e6ffieowoopeofiepoov66lowoofiqoftepoloe6
qwyo66133106361=36w6lololoo56366eqopEetheSoySq6e5r6616opoBrooveopoo5
Se6e6e36106eo6p6e35eoffieopp516vooft66voqeop63623661oopo6v3661voolo6136
656q6eogoop6e6e3661ovp6woq616oeqooloveSvESvo6000pqq0000poqvofiqoftop5o
066266311ro566330636655636646615vo66yoBlofte5r6egoSEmbe5ovq53661mpae5q
egooqop66e56eveepp665=Eopeo610361o5w5433664D6e33666qop6561=666qp6e6
qopypop6qopp6.663=55eopooeSSlopeoSee6eepegoTe6e36qypoSolyppolo61355633
o64e6e36136513166eobeeo6wepeo6e6y000epeeeftfilq1365q6e66qoffieffie6306612
66efte65466loor5yre61D4226epopeffivoo5365peo5pefteSq5qp.666y6y34664355q4
9LL600/800ZSI1/I3d 80-ZO-OTOZ 98609Z0 3
9LSZO/600Z OM
- -
ZI
=
=
:(i cu bas puu zvzszotoci¨m =oist uops933-v =
NoN) Gouanbas pp u oupug Supnolloj oq q papinua s `z iunpvit ldposuan `Dpilil
MAJ
=yyPeevyvvee2eevveezevoSqq6leq6oeeeveleeq6u6qq3315;q65voolEov6q65qqgo6
66436z26.45=33354weqovoraq6v6q.633623446q64141v6q623eoggo6BypeoSeq6436
6BypooftqfiegyepoqB4636gloqopq6q6e6poye161v314166eogfteoftebeoggogfiqppy
3qvq6leovEmpee.5.66e3llqlepepe655e.666eop6.2366qe36e666qqBeeve5e366ve6E:q3.
3o36ee5e646we6ep33rqq6eq33e6er33o66v.406el3q86ee3p3el3vo36556e6u356q6q
leblow65epooffiepq636vow6euaepoffieffioqq365epq66120o6e5polo6e6ovEmpeop6
36663333423366eop6yep6gooffig566qeeqooffiqobv636133e66661ov1113661m1134.
6q6epoSSEsoloovenoqoorpoorp6go6p6o16Evv6vooqooqeftSqoqqeeEp6o6vvEmvp6eq
= poq6pollffiqppqn3Svel.6qoffiqvq6peSqbever6664opoweesvSeveevolq46qoppefteo-
. Stevoe6q66610362oploeoSqoe611a1611eS4ep.46e646w6w66e6o466e666qqp6leggq
solyMe6SygSveSloSbeSESSqo6-436epoogSpqbqopeovp5o6eq65q5365qop573.4.4egovi .
241111queuevepoSqoloqbwooe6v63p6qt.peep6SEmpoSepovSeep.466ygEmpoo5y561D .
Eoge.66e66e365repp66e666qqwvoSropoqeel6=Speoqo661.6605q6663366q5q5eev
v65qqqeepeggp6qopeqopqopp66623366y6pe63066861evoeobl000vo366q5voeffiq4
loqpleoop6qqlogoEmobeoffiqogoo66epooSso6qpq63656u363Epoft5s6qopoolo66qp.
= Em.666e16u6.616q6655e36q6q36136665e6vS6e5666v6.46poosemovo51365w5qp65e55
6q161=63561p66v66e6qEmoozeoqeoffiqp66veffievo66656qpqw6vooBlepowoblop
= ove6.616e6y366eo66eqe356546w4offirwqq6436qopp66166e66ve6v6e66666363336
qgoop56666q6566646e356=54owvoftogooqEmEipeo5166qvoloqqfte5eogffiqeowq
=
=
qfteeplEsee5Boolvve000pogebeo666gooyoq.46q6qe613.6e6soqqqeySge616evr6ev6o
336666e066epeffigoee65=66644066epoolopoloftopq6pg6w6w6w6gooSepe661 .
36366564m6Sebov16130366136ev6vool661v6ev6vovEopo6loopevoweoqvqqv6v3633
eooftEmoq5pq6olvoleo6q3135w661q3=66q0E.66EvEs6q6poSepepoo66oppqm56yopo
66q5e56666135e6olveSe66yoMovos.56665eowa46.4366=56e666voqopoovoSeppoy.
663plopoSqp6eppogoe541peoffiloow636woofiq56qowloo6636621336e5e6oe616e
5e65q5pooftoogeopoo5ftem5v35q36eoftevobea65voop6q5epoftWeaqeopEoSeo68
w0006e356geoploSq36666q5eoeppoSefto65qovoftopq5q6ovqopwee6e6Sepope5.4
vqoploo66e6Beeee336663DEopeoBwo61361p5=66q36y=6661=666q33656436e6.
woepoo64=666opoffieopoovffilopeoftefteovqoqeSe361,DoSoqypopqp6w66633
oSgeBeoBw6513q6EseobeyofigovoepftEmpopeo6e2Sy6qq3366q6e663356y6Sy6q3662
66e5ve6.6q661pogftee6qolvlBegoov66e=62663w6oeftv6q610566v6soq66q066qq
ovefie6v335q6eq6616666qqqp6eolqopeor6Speoe66364366q366ev6663e6evoqeovq6 = =
e66v63336qo3el3366q333w65553516y3y6we3e6603365q6Bleo6e36v6e336e33336
yopq65533614w6op6p3q356opo551=5614we6e655335eepope3p61365eee6le55g3
gwolEspegooqq6efteoqeDe6e551.vooqeogooft0000Sepe6Seolepegoqqq6eoggerp56
Spooftoe1656qoqqopleoe6613e5o6voqopS6goologolopeopo6=66epooeSvoobloy
oploq6opv65256qopowMoveopoEslooggoffiovq6166qopfiqoulolloWoev6e0435.45q
pego66q361113e6Sq61qeoweeoppeolboq3166epoq5qp6e36e56q66y5opE6qoplw6e5
upSee6q6e364oe66z6e66433e6goboyebeffive6loffisEppe6q5w6qeq64.66oepue66efiq
=
66poollq26e64613;3303661o6q14q6pollwoop6opoSe666e35qooloopq5q5ww5e6
eftoop56eopbegopewlloqq0000ftooqolvoo6powoqop5645=3313.6qopoSeepooq
661=663w6v6=665poqqeoppeftoopqn6SepoggqloSepoopwloolqoogooloo6poq
opqypo6-416.erWoopoo6e6613366e6=66e6306opoyoblopolenEmoSepooffieb000p6q
ovopeoffipoopoq63311o6poopEopploppeoffiqopftovoqeolvor66536o6gBoolo5136v
06qopRo6ovolooqepuo6q6pqopeSpoS51616apobogypEopeyole66133366-46-4031636y
po66320636w566oplo6p6e6o6q6136636q36p5opeSepoP63636q6pqv61333600634q6
goo6q561olthopEsoopSe5.54popEorB63g6q6sieeovqolgoSpo6gEsqw1666133353351662
Bovq5113q1oeoftopp6356pooppEmoSpooft66566=666336656663360/yooSso6535
6o55=66303166e36633=66e5v513336636opEopo6o633366epoo660365poole6636o
9LL600/800ZS11/13c1 80-ZO-OTOZ 98609Z0 VD
9LSZO/600Z OM
= Et
33e6ee36e6voe6q666goo6epogoe3613e6w46qqe6groq6e6q6e35.1366e6og66e666qq
=
364ellgeoge66e66eq6ev6q366e6666q36136e333366q6q3363e3636e166163661336e
gleweee11411leeeeee335.3343.461333e6e6qe6qeDego666e336e3De6ee3.466e16e=
36e66q363qe66e66e366er3366e655q113eD6eopoqre1643363e31366q66361666o366
1646eeee66qqqeepe14351Doeq3313=666eq3366e63e5336656lee3e361333v336616 . .
eoe6611qoqa42333611qolo6e36e3661343366epoo6e3613q63666eD6qe=6e6e6woo
343661D6e666e16e66q6q5666e3616q36q36666e6e66e6666e6q63Doe6epeo6g366436
1366e666q361e36366q366266e6q6epolvoqe3661366ge66ve366666qoqe36e336ge33
q3361333ee6646e6e366e366e1e366646134356eqo416q36433366166266ee6e6e6666
6363336z233366666166666q6e36633613q3e36e3qopl6e63e361661goq3qq6re6epq6
612313116erepq6ee6633qeerooppole6e3666133e3q1616ge6136e6e3q4ler6qe616e
er6pe63336666e366epe66eoveb6q33666q1366e3331333136e33463.46136q36e36133
6e3e6613336666qe66e63eq6133366136ee6e33166.4e6ve6eoeboopfiqoporeoqeoqell
e6e3633e336e6.834634634e34e361D436e3664.433366goe66ev6616D36e3e33D66333.4
==e66e33366q6e66666436e634ee6e66e3663voe66666e3433.45go66D356e666eogooppe
=
36epooe6643.43336.436eepoloe5413e366.4334363543336436g3gog3366366e4336e6e
63e6q6e6e56q53336eopeeD33365e6e6e36135e35e5e35ea55e3336=45e336e65e34e33
65 66556
=
e3633331433Dopoleo6136e3353366e6634qe36663336q6666636616615e366eD6g36e
=
e6e6ego66e6263eq6q66131e46eeope66e3361663e36De6ee6q6q3666e6e3166q366q1
= = ope6e6eq36q6ig661666611136vol133vor663e3e664613661366ee666orbeeogeoeg6
=
e65e63336q36e13366q333e366663616e3e6e3e3e6633366166qeo6e36e6e336epooD6
'
e33q666336qq136336e3qq6633366q33661143e6e666136ev333eDe6q366e6e6qe6613
q133163eepolq6e6eepleoe6e661e33qe3.4336e33336roe66eoqepe131q16eoggre366
63p35e3e1665134.4331epe66132636e313366gooqoqolooe33361336.6e333e6e336q3e
=
.
op131633e66e66133pq356Dev3336q331.43663eq6q65q336qoeqolq3663ee6e3g36.16.4
3eq366q3611q3e66q6gleaeveopoeoq6oggq66e33q6136e36e66166e633e6133qq36r6
e36ee6q6e361oe66q6e66133e6436Dee6e66ve61366e6vg6q5136geq61663epee66e51
=
=
e6166366633e164636166611q3661666e6666oqe6ee3136e66e663131weeproppeo65
65o33glge6e6q6q3q33D3664351qqq633111333363335e666e36.433q3331616e3136e6
e6e33366eopfieepoeqoqq31q33336e33131e33633epowD5646gooplgo613336eeppog
66133663436e6g33666231qepooe6e3331366e3oggq1D6e3333.43qoolgopeo3q336eog
poqe336116ev66333336e66q3366e6D366e6336333e3613331e36e36e33366e6333361
aeope366e3333q633qq36333363331333e365.4o36e3e3qeoqe3e6663636=4633135136e=
=
36133e363e3w3lepeo6g63q33e63o66161633363ee3633evole6613o356q6133.4636e
30663e3636e36.6633136o6e636q6436636q36e6ope6e3oe63636g6ole6q33363363q16
vo36466q3e63353336e66q33363e6612616gee3ewlq36336151e3166613336336166e
63e161q3413eD6epoo6366Dopoo6e363336e6666633366633666666036Dyypo6e36636
6D66333663o3166e366333366e6e6q333663633636o63633366epoo663366333e66363
=
:(Z :ort m bas Rue EVZSZOI00 J,sJJ ON uolsso33v
HIDN) =limbos vNum Stqmolloj alp Aq mom s! µE lue!Jun *mum) ix-vm Lai
==sbaaasaaapOwatmoTodissss-nbisa
TvoaaKivixbAmbuvaNnibadassssvssoTvioanviausodosomssaovinob
coovalnavda-mosarmAksrivvnomsaasambmamenbavvbvsvosavvld
OisnavvostivuoissiusistabisuanDrvInuropovittoimoiachodocruaaimni
AZINVIdWAVICIVVIDWYTISOISITTVADvaaVa3aNICI311AXLIIVORDIAVIODYI
ITIAAADdSdICLIXIAVIIIDINIAncrIAVILDIIAIMIIVAINSSOSdSSOVIIISRIVIOKI
DIMIERIaCITIANISSXICE91-111ScISCOITHOIVIIVIDTIKIMINIMS'IddaVoib3H1114a3
1SONcrIADANIDA.IONOVDADVACIANd1111311S-1baAa1-l3SWIAVIIATICEVNHYRIN
AVAAINUNIAVITAAD9309aDDFI3aSANILLOIISIHD1cLa3lcladlIVNYTISASSadDal
ISSaVdSdddclAVISVdSdAlgladOSILLoSNAWS-1,1ISVSSd-DRIdStAindaVdVdISSd
IldVLIDdSd1ddcidfltAYBURIVIITIZYIHrlIHA'ICIVAIIVNIINIAVIAVIASVIIIODS11301
InaibcaunvvAbotnavanvmAinmaolAamaanaivulibvpavvagodoapovw
9LL600/800ZSaLlad 80-ZO-OTOZ 98609Z0 VD
C9LSZO/600Z OAA
W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
a
ctgtttcaaaaagaaaaaccctgggaaaagtgaagtatggctgtaagtctcatggttcagtcctagcaag
aagcgagaattctgagatcctccagaaagtcgagcagcacccacctccaacctcgggccagtgtcttcag
gctttactggggacctgcgagctggcctaatgtggtggcctgcaagccaggccatccctgggcgccacag
acgagctccgagccaggtcaggcttcggaggccacaagctcagcctcaggcccaggcactgattgtggca
gaggggccactacccaaggtctagctaggcccaagacctagttacccagacagtgagaagcccctggaag
gcagaaaagttgggagcatggcagacagggaagggaaacattttcagggaaaagacatgtatcacatgtc
ttcagaagcaagtcaggtttcatgtaaccgagtgtcctcttgcgtgtccaaaagtagcccagggctgtag
cacaggcttcacagtgattttgtgttcagccgtgagtcacactacatgcccccgtgaagctgggcattgg
tgacgtccaggttgtccttgagtaataaaaacgtatgttgcaataaaaaaaaaaaaaaaaaa .
[138]
IRAK1, transcript variant 3, is encoded by the following amino acid
sequence (NCBI
Accession No. NM_ 001025243 and SEQ ID NO: 33):
MAGGPGPGEPAAPGAQHFLYEVPPWVMCRFYKVMDALEPADWCQFAALIVRDQTELR
= LCERSGQRTASVLWPWINRNARVADLVHILTHLQLLRARDIITAWHPPAPLPSPGTTAPR
PSSIPAPAEAEAWSPRICLPSSASTFLSPAFPGSQTHSGPELGLVPSPASLWPPPPSPAPSST
KPGPESSVSLLQGARPFPFCWPLCEISRGTHNFSEELKIGEGGFGCVYRAVMRNTVYAV
= KRLKENADLEWTAVKQSFLTEVEQLSRFRHPNIVDFAGYCAQNGFYCLVYGFLPNGSL
EDRLHCQTQACPPLSWPQRLDILLGTARAIQFLHQDSPSLIHGDIKSSNVLLDERLTPICLG
DFGLARFSRFAGSSPSQSSMVARTQTVRGTLAYLPEEYIKTGRLAVDTDTFSFGVVVLET
LAGQRAVICTHGARTKYLVYERLEKLQAVVAGVPGHSEAASCIPPSPQENSYVSSTGRA
HSGAAPWQPLAAPSGASAQAAEQLQRGPNQPVESDESLGGLSAALRSWHLTPSCPLDP =
APLREAGCPQGDTAGESSWGSGPGSRPTAVEGLALGSSASSSSEPPQIIINPARQKMVQK
LALYEDGALDSLQLLSSSSLPGLGLEQDRQGPEESDEFQS.
Silencing Expression with MicroRNAs
= [139] The present invention comprises compositions with means to inhibit
the activity of IL-
la, IL-lb, IL-1R1, IL-1R2, I1-1Ra3, IL-1RAP, or IRAK1, by delivering microRNA
(miRNA)
molecules to an ocular or adnexal tissue with an appropriate pharmaceutical
carrier.
Compositions that comprise an miRNA targeted to either IL-la, IL-lb, IL-1R1,
IL-1R2, II-
=
=
1Ra3,.IL-1RAP, or IRAK1 antagonize the function of IL-1R1. The
composition comprises one .
= or more miRNA(s) that bind to one or more regions of IL-la, IL-lb, IL-
1R1, IL-1R2, 11-1Ra3,
= IL-1RAP, or IRAK1. The following table contains exemplary miRNAs that
have been shown to
= partially or completely silence the expression of human IL-la or IL-1R1.
Table 1: Summary of miRNAs, their human target genes, nucleotide sequences,
and their
sequence identifier numbers.
= Target Gene miRNA Polynucleotide
sequence SEQ ID NO:
(5' to 3')
IL-la miR-30c UGUAAACAUCCUACACUCUCAGC 34
IL-la miR-30b UGUAAACAUCCUACACUCAGC 35
=
= IL- 1 a miR-30a-5p UGUAAACAUCCUCGACUGGAAGC 36
IL-la miR-24 UGGCUCAGUUCAGCAGGAACAG 37
IL-1R1 miR-135b UAUGGCUUUUCAUUCCUAUGUG = 38
IL-1R1 = miR-326 CCUCUGGGCCCUUCCUCCAG 39
IL-1R1 miR-184 UGGACGGAGAACUGAUAAGGGU 40 =
IL-1R1 miR-214 ACAGCAGGCACAGACAGGCAG 41
44
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IL-1R1 miR-203 GUGAAAUGUUUAGGACCACUAG 42
IL-1R1 miR-331 GCCCCUGGGCCUAUCCUAGAA 43
IL-1R1 miR-205 UCCUUCAUUCCACCGGAGUCUG 44
IL-1 and IL-1R-Mediated Signaling
[140] As used herein, the term "inhibit an activity of an inflammatory
interleukin-1 cytokine" is
meant to describe the inhibition, prevention, diminution, reduction, decrease,
repression, or
interruption intracellular signaling initiated, communicated, or transduced
from an IL-1 receptor.
In one aspect of the invention, inhibition, prevention, diminution, reduction,
decreases,
repression, or interruption of intracellular signaling initiated,
communicated, or transduced from
an IL-1 receptor is achieved by preventing or decreasing binding of an IL-1
cytokine to an IL-
1R. Alternatively, or in addition, transduction of intracellular signaling
from an IL-1R is
= prevented by removing, silencing, or mutating a downstream effector or
target within a signaling
cascade. The expression and/or function or activity of downstream effectors
and/or targets are
removed (e.g. deleted, knocked-out, sequestered, denatured, degraded, etc.),
silenced (degraded,
transcriptionally or translationally repressed), or mutated (nucleotide or
amino acid sequence
encoding the active product is altered to encode a non-functional product) by
genetic
modification or administration of a therapeutic compound.
[141] Exemplary downstream effectors and/or targets include, but are not
limited to, one or
more isoforms or homologs of an IL-1 (interleukin 1), an IL-la (interleukin 1
alpha), an IL-
113 (interleukin 1 beta), an IL-1R (interleukin 1 receptor; type I), an IL-1Ra
(11L-1R antagonist),
an IL-1RAcP (IL-1R accessory protein), a TOLLIP (TOLL interacting protein), an
MAKI (IL-
1R associated kinase 1), an IRAK2 (IL-1R associated kinase 2), an IRAK 3 (IL-
1R associated
kinase 3), a MYD88 (myeloid differentiation primary response gene 88), an
ECSIT
(evolutionarily conserved signaling, intermediate in Toll pathways), a TRAF6
(TNF-receptor
associated factor 6), a MEKK1 (MAP ERK kinase kinase 1), a TAB1 (TAK1 binding
protein 1),
a TAK1 (transforming growth factor b activated kinase 1), a NIK (NFkB Inducing
Kinase), a
RKIP (Raf ldnase inhibitor protein), a MEK3 (Mitogen-Activated Protein Kinase
Kinase 3;
MEK3 or MKIC3), a MEK6 (Mitogen-Activated Protein Kinase Kinase 6; MEK6 or
MICK6), a
MAPK14 (mitogen activated protein kinase 14), a MAPK8 (mitogen activated
protein kinase 8),
a MEKK1 (mitogen activated protein kinase kinase kinase 1), a MAP3K14 (mitogen
activated
protein kinase kinase kinase 14), a MEKK7 (mitogen activated protein kinase
kinase kinase 7 or
_
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MICK7), a MAP3K7IP1 (mitogen activated protein kinase kinase kinase 7
interacting protein 1),
a JNK (Jun N-terminal kinase), p38 (also known as p38 MAPK or p38 mitogen
activated protein
kinase), cJUN (Jun oncogene), AP-1 (activator protein 1; transcription
factor), IL-6 (interleukin
6, also known as interferon beta 2), TNFa ( tumor necrosis factor-alpha), a
TNF (tumor necrosis
factor superfamily member), an IFNcc (interferon alpha, interferon alpha 1),
an IFNI3 (interferon
beta, interferon beta 1), a TGF131 (transforming growth factor beta 1), a
TGFI32 (transforming
growth factor beta 2), a TGFP3 (transforming growth factor beta 3), an IICKa
(inhibitor of kappa
light polypeptide gene enhancer in B-cells, kinase alpha), an IKICI3
(inhibitor of kappa light
polypeptide gene enhancer in B-cells, kinase beta), a IxBa (nuclear factor of
kappa light
polypeptide gene enhancer in B-cells inhibitor, alpha), a Chulc (conserved
helix-loop-helix
ubiquitous kinase), and a NFicB (nuclear factor of kappa light polypeptide
gene enhancer in B-
cells 1; also known as p105). Additional signaling molecules and relationships
are defined by
O'Neill, L. A. J. and Greene, C. 1998. Journal of Leukocyte Biology. 63: 650-
657.
[142] The inhibition of an activity of an inflammatory interleuldn-1 cytokine
is determined by
sampling an ocular or adnexal tissue or fluid and determining the abundance of
a polynucleotide
or polypeptide which encodes for component of an IL-1R signaling cascade. An
increase or
decrease in the abundance of a polynucleotide or polypeptide which encodes for
component of
= an IL-1R signaling cascade following administration of a therapeutic
composition of the
=
invention compared to the abundance of the component of an IL-1R signaling
cascade prior to
the administration indicates inhibition of an activity of an inflammatory
interleukin-1 cytokine.
[143] Specifically, Figure 4 shows the functional interrelationships between
components of two
exemplary signaling cascades. The arrows between components in this figure
indicate that the
component preceding the arrow activates the component following the arrow.
Conversely, the
blunted lines indicated that the component preceding the blunted line inhibits
the activity or
function of the component following the blunted line.
[144] Briefly, the IL-1R, type I, binds IL-l13, however, IL-1R requires the IL-
1 receptor
accessory protein (IL-1RAcP) to transduce a signal. IL-1 binding causes
activation of two
kinases, IRAK-1 and IRAK-2, associated with the IL-1 receptor complex. IRAK-1
(IL-1
Receptor Associated Kinase) activates and recruits 1'RAF6 to the IL-1 receptor
complex. TRAF6
activates two pathways, one leading to NF-IcB activation and another leading
to c-jun activation.
The TRAF associated protein ECSIT leads to c-Jun activation through the Map
kinase/JNK
46
=
-
W020091025763 CA 02640986 2010-02-08
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signaling system. TRAF6 also signals through the TAB1/TAK1 kinases to trigger
the
degradation of I-IcB, and activation of NF-IcB.
=
[145] For instance, in certain embodiments of the invention, a decrease in the
abundance or
absence of the processed form of MEKK1, a decrease in the abundance or absence
of
phosphorylated hcBa, a decrease in the abundance or absence of phosphorylated
c-JUN, a
decrease in the 'abundance or absence of ICAM-1, or a decrease in the
abundance or absence of
IL-6, TNFa, IFNa, IFNf3, TGFil in an ocular or adnexal tissue or fluid is
indicative of inhibition
of an inflammatory interleuldn-1 cytokine. Similarly, a decrease or absence of
activity or
function of any of the above-listed components is indicative of inhibition of
an inflammatory
= interleulcin-1 cytokine.
=
=
Pharmaceutically-Appropriate Carriers
[146] Exemplary compounds incorporated.to facilitate and expedite transdermal
delivery of
. topical compositions into ocular or adnexal tissues include, but are not
limited to, alcohol .
(ethanol, propanol, and nonanol), fatty alcohol (lauryl alcohol), fatty acid
(valeric acid, caproic
acid and capric acid), fatty acid ester (isopropyl myristate and isopropyl n-
hexanoate), alkyl
= ester (ethyl acetate and butyl acetate), polyol (propylene glycol,
propanedione and hexanetriol),
sulfoicide .(dimethylsulfoxide and decylmethylsulfoxide), amide (urea,
dimethylacetainide=and
pyrrolidone derivatives), surfactant (sodium lauryl sulfate,
cetyltrimethylammonium bromide,
polaxamers, spans, tweens, bile salts and lecithin), terpene (d-limonene,
alpha-terpeneol, 1,8-
cineole and menthone), and alkanone (N-heptane and N-nonane). Moreover,
topically-
administered compositions comprise surface adhesion Molecule modulating agents
including, but
not limited to, a cadherin antagonist, a selectin antagonist, and an integrin
antagonist.
= [147] Optionally, the composition further contains a compound selected
from the group
= =
consisting of a physiological acceptable salt; poloxamer analogs with
carbopol,
. carbopol/hydroxypropyl methyl cellulose (IIIPMC), carbopol-methyl cellulose,
carboxymethylcellulose (CMC), hyaluronic acid, cyclodextrin, and petroleum.
Drug Delivery by Contact Lens
[148] The invention comprises a contact lens and a composition that inhibits
an activity. of an
inflammatory interleuldn-1 cytokine. For example, the composition is
incorporated into or
coated onto said lens. The composition is chemically bound or physically
entrapped by the
47 =
,r1
W020091025763 CA 02640986 2010-02-08
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=
contact lens polymer. Alternatively, a color additive is chemically bound or
physically entrapped
by the polymer composition that is released at the same rate as the
therapeutic drug composition,
such that changes in the intensity of the color additive indicate changes in
the amount or dose of
therapeutic drug composition remaining bound or entrapped within the polymer.
Alternatively,
or in addition, an ultraviolet (UV) absorber is chemically bound or physically
entrapped within
the contact lens polymer. The contact lens is either hydrophobic or
hydrophilic.
[149] Exemplary materials used to fabricate a hydrophobic lens with means to
deliver the
compositions of the present invention include, but are not limited to,
amefocon A, amsilfocon A,
aquilafocon A, arfocon A, cabufoccm A, cabufocon B; carbosilfocon A, crilfocon
A, crilfocon B,
= dimefocon A, enflufocon A, enflofocon B, erifocon A, flurofocon A,
flusilfocon A, flusilfocon
= =
B, flusilfocon C, flusilfocon D, flusilfocon E, hexafocon A, hofocon A,
hybufocon A,
itabisfluorofocon A, itafluorofocon A, itafocon A, itafocon B, kolfocon A,
kolfocon B, kolfocon
= C, kolfocon D, lotifocon A, lotifocon B, lotifocon C, melafocon A,
migafocon A, nefocon A,
nefocon B, nefocon C, onsifocon A, oprifocon A, oxyfluflocon A; paflufocon B,
paflufocon C,
paflufocon D, paflufocon E, paflufocon F., pasifocon A, pasifocon B, pasifocon
C, pasifocon D,
pasifocon E, pemnfocon A, porofocon A, porofocon B, roflufocon A, roflufocon
B, roflufocon
= C, roflufocon D, roflufocon E, rosilfocon A, satafocon A, siflufocon A,
silafocon A, sterafocon
=.A, sulfocon A, sulfocon B, telafocon A, tisilfocon A, tolofocon A, trifocon
A, unifocon A,
vinafocon A, and wilofocon A.
[150] Exemplary materials used to fabricate a hydrophilic lens with means to
deliver the
compositions of the present invention include, but are not limited to;
abafikon A, acofilcon A,
acofikon B, acquafilcon A, alofilcon A, alphafilcon A, amfilcon A, astifilcon
A, atlafilcon A,
balafilcon A, bisfilcon A, bufilcon A, comfilcon A, crofilcon A, cyclofildon
A, darfilcon A,
deltafikon A, deltafilcon B, dimefilcon A, droxfilcon A, elastofikon A,
epsilfilcon A,
esterifilcon A, etafilcon A, focofilcon A, galyfilcon A, genfilcon A,
govafilcon A, hefilcon A,
hefilcon B, hefilcon C, hilafilcon A, hilafilcon B, hioxifilcon A, hioxifilcon
B, hioxifilcon C,
hydrofilcon A, lenefilcon A, licryfilcon A, licryfilcon B, lidofilcon A,
lidofilcon B, lotrafikon A,
lotrafilcon B, mafilcon A, mesafilcon A, methafilcon 13; mipafikon A,
nelfilcon A, netrafikon
A, ocufilcon A, ocufilcon B, C, ocufilcon D, ocufilcon E, ofilcon A, omafilcon
A, oxyfilcon A,
pentafilcon A, perfilcon A, pevafilcon A, phemfikon A, polymacon, senofilcon
A, silafilcon A,
= siloxyfilcon A, surfilcon A, tefilcon A, tetrafilcon A, trilfilcon A,
vifilcon A, vifilcon B, and
xylofilcon A.
48
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EXAMPLES
EXAMPLE 1: The Ocular Surface Disease Index (OSDI)
[151] The Ocular Surface Disease Index (OSDI) is a 12-item questionnaire that
provides a
rapid assessment of the symptoms of ocular irritation consistent with ocular
surface disease,
including posterior blepharitis and dry eye disease, and their impact on
vision-related
functioning (Figure 1). The 12 items of the OSDI questionnaire are graded on a
scale of 0 to 4,
where 0 indicates none of the time; 1, some of the time; 2, half of the time;
3, most of the time;
and 4, all of the time. The total OSDI score is then calculated on the basis
of the following
formula: OSDI=[(sum of scores for all questions answered) x 100]/[(total
number .0 questions
answered) x 4]. Thus, the OSDI is scored on a scale of 0 to 100, with higher
scores representing
greater disability. A negative change from baseline indicates an improvement
in vision-related
function and the ocular inflammatory disorders described herein. For the
therapeutic method
=
described herein, treatment is considered more effective than control
(vehicle) as indicated by a
mean change (decrease) from baseline for the OSDI of >10 units compared to
control.
= [152] Therapeutic treatment is considered more effective than the vehicle
as indicated by a
= mean change from baseline of average score (0 -100) for the Ocular
Surface 'Disease Index
(OSDI) of >10 units better than vehicle.
EXAMPLE 2: Tear Film Break-up Time
=
[153] The standard TBUT measurement is performed by moistening a fluorescein
strip with
sterile non-preserved saline and applying it to the inferior tarsal
conjunctiva. After several
blinks, the tear film is examined using a broad beam of the slit lamp with a
blue filter. The time
lapse between the last blink and the appearance of the first randomly
distributed dark
discontinuity in the fluorescein stained tear film is measured three times and
the mean value of
the measurements is calculated. The tear break-up time is evaluated prior to
the instillation of
any eye drops and before the eyelids are manipulated in any way. Break-up
times less than 10
seconds are considered abnormal. A positive change from baseline indicates
improvement in
= symptoms of the ocular inflammatory disorders described herein. The
treatment described
herein, leads to an improvement in TBUT significantly greater than that
observed from treatment
with vehicle alone. =
EXAMPLE 3: Corneal and Conjunctival Staining
49
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[154] Corneal staining is a measure of epithelial disease, or break in the
epithelial barrier of the
ocular surface, typically seen with ocular surface disorders such as posterior
blepharitis and dry
eye, among others. Importantly, corneal staining can exist even without
clinically evident dry
eye, if there is significant lid disease, such as posterior blepharitis.
Corneal staining is highly
correlated with ocular discomfort in many, though not all patients; in general
corneal staining is
associated with high scores in the OSDI, as described above. For corneal
fluorescein staining,
saline-moistened fluorescein strips or I% sodium fluorescein solution are used
to stain the tear
film. The entire cornea is then examined using slit-lamp evaluation with a
yellow barrier filter
(#12 Wratten) and cobalt blue illumination (staining is more intense when it
is observed with a . =
yellow filter). Staining is graded according to the Oxford Schema (Figure 2).
[155] Conjunctival staining is a measure of epithelial disease or break in the
epithelial barrier
of the ocular surface, typically seen with ocular surface disorders such as
posterior blepharitis
and dry eye, among others. Importantly, conjunctival staining, similar to
corneal staining, can
exist even without clinically evident dry eye, if there is significant lid
disease, such as posterior =
blepharitis. Conjunctival staining can also correlate with symptoms of oaular
irritation and high
OSDI scores as described above. Conjunctival staining is performed under the
slit-lamp using
lissamine green. Saline-moistened strip or 1% lissamine green solution is used
to. stain the tear
film, and interpalpebral conjunctival staining is evaluated more than 30
seconds, but less than 2
minutes, later. Using white light of moderate intensity, only the
interpalpebral region of the nasal
and temporal conjunctival staining is graded using the Oxford Schema (above).
The treatment
=
described herein leads to decreases in ocular staining scores beyond what is
observed with the
vehicle alone.
[156] Therapeutic treatment is considered more effective than vehicle as
indicated by a mean
change from baseline in average score (0-5 scale) for corneal and conjunctival
staining of > 1
unit better than vehicle, e.g. as detected using the Oxford Schema.
EXAMPLE 4: Schirmer Test
[157] The Schirmer test is performed in the presence and in the absence of
anesthesia by
placing a narrow filter-paper strip (5 x 3 5mm strip of Whatman #41 filter
paper) in the inferior
cul-de-sac. This test is conducted in a dimly lit room. The patient gently
closes his/her eyes until
five minutes have elapsed and the strips are removed. Because the tear front
will continue
= advancing a few millimeters after it has been removed from the eyes, the
tear front is marked
with a ball-point pen at precisely five minutes. Aqueous tear production is
measured by the
W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
length in millimeters that the strip wets during 5 minutes. Results of 10 mm
or less for the
Schirmer test without anesthesia and 5 mm or less for the Schirmer test with
anesthesia are
considered abnormal. A positive change from baseline indicates improvement of
one or more
symptoms of an ocular inflammatory disorder described herein.
EXAMPLE 5: Meibomian Gland Evaluation
[158] In the center of the lower lid, 10 adjacent central glands are located
on both sides and the
glands are expressed by applying a firm digital pressure at the base of the
glands. The number of
glands expressed for each eye is documented. The quality of secretion is
described as follows:
= Clear excreta or clear with small particles (0)
= Opaque excreta with normal viscosity (1)
= Opaque excreta with increased viscosity (2)
= Secretions retain shape after expression (3)
[159] Posterior blepharitis is associated with lid inflammation and
alterations in the quantity
and/or quality of the meibomian gland secretions, with severe disease
associated with quality
=
= grades 2-3, as described above. The treatment described herein leads to
improvement in =
= meibomian secretion characterized by a decrease in this score; for
example, from 3 to 2, or from
= 2 to 1. An improvement is indicated by a mean change from baseline (0-3
scale) for meibomian
gland secretion quality of > 1 unit better than vehicle.
EXAMPLE 6: Lid and Lid Margin Erythema
= [160] Lid margin vascular injection (erythema) is defined as a red
discoloration, compared to
the surrounding eyelid skin and is graded as follows:
None (0): none
Mild (1): redness localized to a small region of the lid
margin(s) or skin
= Moderate (2): redness of most of the lid margin(s)
Severe (3): redness of most or all the lid margin(s) and skin
= =
Very Severe (4): marked diffuse redness of both lid margins and skin
The presence or absence of tarsal telangiectasis is also noted. Lid
telangiectasia is defined as the
= presence of at least two blood vessels along the eyelid margin.
EXAMPLE 7: Conjunctiva Hyperemia
[161] Bulbar conjunctival hyperemia is graded as follows:
=
None (0): none
Mild (1): slight localized injection
Moderate (2): pink color, confined to palpebral or bulbar
conjunctiva
Severe (3): red color of the palpebral and/or bulbar
conjunctiva
51
,
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Very Severe (4): marked dark redness of the palpebral and/or bulbar
conjunctiva
The presence. or absence of tarsal papillary hypertrophy is also noted.
EXAMPLE 8: Topical Administration of IL-1Ra for Treating Posterior Blepharitis
[162] The following study evaluates the therapeutic benefit of topically
administering a
solution comprising a known, and commercially-available, recombinant IL-1
receptor antagonist,
Ana. kinra (Cineret0), versus vehicle to subjects with inflammatory conditions
affecting one or
more ocular and/or adnexal tissues.
[163] The following is a prospective, single-center, randomized, double-
masked, vehicle-
controlled, parallel-group clinical study. There is one active treatment group
and one vehicle
treated group. Patients who meet the requirements of the inclusion/exclusion
criteria at the
screening visit are separated into a moderate stratum and a severe stratum
based on the
meibomian gland secretion quality. Within each stratum, patients are
randomized to either 2.5%
topical human recombinant IL-1Ra or vehicle in even allocations. There are a
minimum of 20
= patients in each treatment group in the moderate stratum and a minimum of
10 patients in each
=
treatment group in the severe stratum. This study consists of 6
scheduled visits over four months =
= (Table 2).
=
=
[164] Subjects who present signs or reported symptoms consistent with
inflammatory disease '
affecting an Ocular or adnexal tissue are further evaluated prior to treatment
using the above-
'
= described Ocular Surface Disease Index (OSDI). Exemplary subjects are
Male and female
= .
= patients with signs and symptoms of posterior blephaiitis (provided that
not more than 7 glands
are not expressible) with or without aqueous deficiency excluding those
patients with end-stage . . .
=
lacrimal gland (Schirmer reading without anesthesia < 3 min/5 min
or if their dry eye disease is '
the result of destruction of conjunctival goblet cells or scarring). A subject
is included in the =
= following study if he or she meets the following criteria: male or
female; at least 18 years of age; =
has not worn contact lenses for at least 2 weeks prior to the study and agrees
to not wear contact
lenses during study.; patient is in generally good &stable overall health.
patient must have a =
diagnosis of posterior blepharitis as defined in Figure 5; a negative urine
pregnancy test result =
for women of childbearing potential; women of childbearing potential must
agree to use
adequate contraception (hormonal or barrier method of birth control) prior to
study entry and for
= the duration of study participation; normal licl=position and closure;
ability to understand and
=
=
52
g,
W02009/025763 CA 02640986 2010-02-08 PCT/US2008/009776
provide informed consent to participate in this study; and willingness to
follow study instructions
and likely to complete all required visits.
[165] Subjects are excluded if they had used topical steroids or the
commercially-available
drug, Restasis, within the past 2 weeks as well as tetracycline compounds
(tetracycline,
doxycycline, and minocycline) within the last month or isotretinoin (Accutane)
within the past 6
months. Subjects who report any previous treatment with Analdnra (Kinerete) or
any
therapeutic agent targeted at IL-1 blockade are similarly excluded.
Furthermore, a subject is
excluded from the following study if he or she: has a history of Stevens-
Johnson syndrome or
ocular pemphigoid; has a history of eyelid surgery; has had intra-ocular
surgery or ocular laser
surgery within 3 months; has a history of microbial keratitis, including
herpes; has active ocular
= allergies; has a corneal epithelial defect > 1mm2; has used topical
steroids or Restasis within the
past 2 weeks; has experienced any change in dosage of tetracycline compounds
(tetracycline,
doxycycline, and minocycline) within the last month; has used isotretinoin
(Accutane) within the
past 6 months; has had any previous treatment with Analcinra (Kineret0) or any
therapeutic
agent targeted at IL-1 blockade; is a pregnant or lactating woman; has signs
of current infection,
= including fever and current treatment with antibiotics; has liver, renal,
or hematologic disease;
has a history of cancer; has used any other investigational drug.
[166] Under certain circumstances, subjects are withdrawn from the following
study. The
following criteria are used to determine when a subject must be removed or
permitted to leave =
the study. Individuals are discontinued 'early from the study due to the
following reasons, =
including, but not limited to: protocol violations, adverse events, lack of
efficacy, pregnancy, and
administrative reasons (e.g., inability to continue, lost to follow-up).
Screening Visit
[167] Prospective patients as defined by inclusion/exclusion criteria are
considered for entry
into this study. The study design and treatment regimen are discussed with
each patient. Those
wishing to participate are examined for entry into the study with the
following exams:
= Medical and ophthalmic histories
= Patient questionnaire (OSDI) (see Example 1 and Figure 1)
= Best corrected visual acuity (BCVA) (Snellen chart)
= Fluorescein tear break-up time (TBUT) (see Example 2)
= Cornea and conjunctival staining (see Example 3
=
Schirmer test without anesthesia (see Example4) =
= Schirmer test with anesthesia (see Example 4)
= Meibomian gland evaluation (see Example 5)
53
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W02009/025763 CA 02640986 2010-02-08 PCT/US2008/009776
= Biomicroscopy
= Intraocular pressure
= Fundus examination
[168] To avoid the influence of one procedure on another, dry eye tests and
ocular surface
evaluation are done in the following sequence because the Schirmer test can
disrupt tear film
stability and cause false-positive ocular surface dye staining: measurement of
tear film break-up
time (TBUT), ocular surface dye staining pattern (fluorescein and lissamine
green staining),
Schirmer's test without and with anesthesia, and meibomian gland evaluation
(Figure 6).
[169] After the eye examinations, each patient who qualifies to continue in
the study (according
to the inclusion/exclusion criteria) is instructed to instill one drop of the
randomly assigned
masked treatment three times a day in both eyes. The topical solution of 2.5%
(25 mg/ml)
concentration of Kineret is formulated and prepared from commercially
available preservative-
free solution of Kineret (Amgen, Thousand Oaks, CA) by the MEEI hospital
pharmacy using .
aseptic technique. For the control group, vehicle [Refresh Liquigel (1%
carboxymethylcellulose)] is used three times a day in both eyes. During the
treatment phase,
patients are instructed to instill 1 drop of study medication three times a
day for 3 months; 1 drop =
in each eye upon waking in the morning, at noon, and at bedtime.
[170] To reduce the chance of systemic absorption of Kineret, patients are
asked to put digital
pressure on lacrimal ducts for five minutes. Patients are advised not use
artificial tears or other . =
topical medications 30 minutes before or 30 minutes after instilling the study
medication to
prevent dilution of the study medication. This study does not include blood
sampling or any
pharmacolcinetic measures.
=
Compliance Visit
[171] Each patient returns for a compliance visit at 2 weeks (Table 2). At
this visit, the
investigator asks direct questions about patient compliance with the study
treatment and adverse
events. Patients can be discontinued from the study because of adverse events
and protocol
violations, but every effort is made to enhance compliance and maintain
subjects on the
treatment protocol.
Follow-up Visits
[172] To determine the safety and efficacy of topical IL-1Ra for management of
posterior
blepharitis, 5 follow-up visits (eye exam) are scheduled at day 1, week 2,6,
12, and 16 (see
=
54
owe
..... .
W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
,
Table 2). Patients are instructed to strictly follow the study visit schedule.
In each follow-up
visit, the patient is asked to fill out the OSDI questionnaires (Figure 1). In
addition to a
comprehensive eye examination, tear function and ocular surface tests
including TBUT, cornea
and conjunctival staining tests, meibomian gland evaluation, and Schirmer
tests, are performed.
Table 2: Schedule of Events and Procedures
Visit 1 2 3 4 5
6
Screening Day 1 Week 2
Time Period Visit (Safety (Compliance Week 6 Week 12
Week 16
Visit) Visit)
Obtain Informed
X
=
Consent
Inclusion/exclusion
X
criteria =
.
OSDI questionnaire X X X X
X
Medical & = =
X
Ophthalmic History _
Pregnancy Test X
.
BCVA = X X X = X =
X _
TBUT X X X
X
Cornea & Conj. =
=
Staining X X X
X =
= = X =
Schirmer without
X X . X X
X
Anesthesia
Schirmer with
X X X
Anesthesia =
= Meibomian Gland = .
X X = X X
X
Evaluation
Biomicroscopy X X X X X
X
Intraocular Pressure X . X X = X
X
Funduscopy X X X X
X
_
-
=
Compliance Check X X X X
-
Adverse Event Query
X X X X
X
=
Concurrent Therapies
[173] Concurrent enrollment in another clinical investigational drug or device
study is
- prohibited.. The use of any concurrent medication, prescription or over-the-
counter, is recorded
along with the reason the medication is taken. During the study, all
concomitant medication
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W02009/025763 CA 02640986 2010-02-08 PCT/US2008/009776
treatment regimens, ocular hygiene treatments (i.e., lid scrubs and warm
compresses), or
insertion of punctal plugs are kept as constant as permitted by accepted
medical practice.
[174] Using artificial tears is permissible at screening visits and during the
study. However,
concomitant use of artificial tears is monitored. At each study visit,
patients are queried about the
average number of times they used artificial tears each day during the past
week and the number
of days during the past week when they did not use any artificial tears.
[175] Systemic and topical ophthalmic medications that could interfere with
the response to
study medications or the interpretation of the study results are prohibited
during the study. This
includes any systemic or topical steroid, cyclosporine, and tetracycline
compound.
[176] When necessary for the treatment of patients, administration of a
prohibited therapy is
done with the safety of the study participant as the primary consideration.
The administration of
a prohibited medication or procedure is considered a protocol violation and
the patient may be
discontinued from the study.
[177] During the course of study, if the patient complains of mild to moderate
form of ocular
surface symptoms, more frequent use of artificial tears (Refresh ) is
encouraged without
unmasking the patient. If treatment with artificial tears is inadequate, or
the patient develops a
severe form of ocular surface disease and dry eye, for the safety and proper
treatment of the
subject, the investigator can unmask the subject's treatment assignment to
determine which
treatment has been assigned and institute appropriate follow-up care. However,
the patient is
kept in the study so that an "intention to treat" analysis can be performed.
Efficacy Measures
[178] Several signs of the efficacy of administration of therapeutic compounds
of the invention
are monitored. The objective signs are meibomian gland secretion quality,
meibomian gland
occlusion, tear break-up time, corneal and conjunctival staining, and Schirmer
test (with and
without anesthesia). The subjective endpoints are the OSDI questionnaire
score. All these
variables are carefully measured at baseline and all visits toward the end of
follow up (Table 2).
[179] The following are non-limiting examples of primary efficacy variables:
Meibomian gland
secretion quality; Tear Break-up time; Cornea and Conjunctival staining score;
and the OSDI
56
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W02009/025763 CA 02640986 2010-02-08
PCT/US2008/009776
questionnaire score. Alternatively, or in addition, secondary efficacy
variables are used to
determine the therapeutic value of administration of compositions of the
invention. Nonlimiting
examples of secondary efficacy variables include: Meibomian gland occlusion;
Schirmer test
without anesthesia; Schirmer test with anesthesia. Primary and/or secondary
efficacy variables
are considered.
[180] In this study, the treatment with topical IL-1Ra is considered
efficacious if the 2.5%
solution shows a mean change from baseline (0-3 scale) for meibomian gland
secretion quality
of > 1 unit better than vehicle at the week-12 visit. Alternatively, the
treatment with topical IL-
1Ra is considered efficacious if the 2.5% solution shows a mean change from
baseline in average
= score (0-5 scale) for corneal and conjunctival staining of > 1 unit
better than vehicle at the week- ,
12 visit.
Safety Measures
[181] The primary safety variable Monitored is the occurrence of adverse
events. The severity
of each adverse event observed (ocular and systemic) is rated from mild
(awareness of sign or
symptom, but easily tolerated) to severe (incapacitating with inability to
work or do usual
=
activity). The relationship of the event to the study medication is assessed
by the investigator as =
none, unlikely, possible, probable, or definite. Safety variables are
evaluated at baseline and at
=
all study visits.
[182] At each visit throughout the study, the investigator begins querying for
adverse events by
= aaking each patient a general, non-directed question such as "How have
you been feeling since
= the last visit?" Directed questioning and examination is then done as
appropriate. The
investigator asks questions to subjects at each visit to determine if they
have had any changes to
the use of concomitant medications since the previous visit. A comprehensive
eye examination
including best-corrected visual acuity, measuring intraocular pressure,
evaluation of the
condition of the lid/lashes, conjunctiva, cornea, anterior chamber,
iris/pupil, lens, vitreous; =
macula and optic nerve is performed. Any changes in the study eye from the
baseline visit is
recorded.
=
57
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W020091025763 CA 02640986 2010-02-08
PCT/US2008/009776
STATISTICAL PROCEDURES
Power and Sample Size Considerations:
Table 3. Estimated Prevalence for Antici . = ted Ordered Cate l . ries of
Meibomian Quality Scores
ft -Ai
l'=-ttf,11 r6t-Ti1,1, ft_(?,z=
0.20 0.28 0.40 0
0.28 0.32 0.28 1
0.32 0.25 0.20 2
0.20 0.20 0.12 3
Mean (SD) = 1.52 Mean (SD) = 1.32 Mean (SD) = 1.04
(1.02) (1.09) (1.04)
[183] In order to estimate the power of the study, it is assumed that 50% of
the subjects are
randomized to topical ILl-Ra and 50% to vehicle. A conservative estimate can
then be
=
constructed of study power, according to Sullivan and D'Agostino
(Sullivan, L.M. and =
D'Agostino, R.B. 2003. Stat Med. 22(8):1317-34), using a t-test comparison of
the predicted
distribution of primary endpoints within ordered categories. Power estimates
using this method
are conservative because the actual analysis uses data from two eyes while
appropriately ,
accounting for their correlation, which has been demonstrated to have greater
power as
compared to methods based on using the person as the unit of analysis.
=
[184] The work of Sullivan and D'Agostino indicates that the t-test performs
well for the
comparison of ordinal scales with 3 to 5 ordinal categories. Estimates of
power are based on
three scenarios (high, moderate, and low) for the anticipated distribution of
scores-in the placebo
group. As displayed in Table 3 above, a mean score between 1.04 and 1.52 is
anticipated, with a
standard deviation between 1.02 and 1.09. A clinically meaningful difference
is a reduction of 1 -
in the mean score, or approximately 1 standard deviation.
[185] Based on the two-sample t-test, with Bonferonni adjustment for multiple
comparisons
(four primary outcome variables) an observer would have 280% power to detect a
mean
difference of 1. standard deviation for sample sizes of 28, 30, and 28, per
group, under the high,
moderate, and low scenarios, respectively. As noted, actual power would be
enhanced through
use of scores from both eyes of study participants, with consideration of the
correlation between
fellow eyes in the analysis. Therefore, the sample size of N=30 per group was
chosen.
Statistical Analysis:
[186] For efficacy variables and any other variables except for safety, all
subjects are analyzed
with the treatment to which they were randomized (the intent-to-treat
population). For safety
variables, subjects are analyzed with the treatment actually received (the
safety population). =
58
...VW...A ¨%
.nNad, õ ,=46.,A
_
W020091025763 CA 02640986 2010-02-08 PCT/US2008/009776
[187] The primary analysis for the proposed study is based on a standard
intention-to-treat
analysis with each study participant analyzed with respect to the randomized
treatment
assignment, regardless of eventual compliance. A secondary analysis may
include imputation of
missing data for select variables. A per-protocol analysis, disqualifying
patients or patient visits,
might also be done, but is not planned because observational analyses of
actual treatment use
could introduce bias if the pattern of use is in some way related to the
outcome.
[188] Despite the randomized nature of the treatment assignments, in this
relatively small
sample of study subjects there may be imbalances with regard to potential
confounding
variables. Thus, as an initial step in the analysis, those assigned to active
treatment versus those
randomized to vehicle are compared with regard to demographic characteristics
and potential .
confounding variables, using the non-parametric Wilcoxon rank sum test for
continuous or
ordinal variables, and chi-square or Fisher's exact tests for categorical
variables.
[189] To assess the effect of topical IL-1Ra treatment, the distribution of a)
meibomian gland
secretion score, and b) corneal and conjunctival staining scores between
patients assigned to
topical IL-1Ra and those assigned to vehicle are compared using the stratified
Wilcoxon rank
sum test with variance correction for clustering effects as recently developed
by Rosner and
Glynn (Rosner, et al. 2003. Biometrics. 59(4): 1089-98). Based on simulation
studies, analyses
of ophthalmologic data that use the information from two eyes and
appropriately account for
their correlation have greater power than methods based on a single eye or the
average of the two -
eyes (Rosner, et al. 2003. Biometrics. 59(4): 1089-98; Glynn, R.J. and Rosner,
B. 1994. Stat
Med. 13(10): 1023-36). This method uses large sample theory to incorporate
clustering effects
to ordinal outcomes such as the clinical scoring scales used here. It can be
implemented with
standard software (e.g. SAS PROC RANK), and provides a valid test for either
balanced or
unbalanced clustered data in the presence or absence of tied rankings in
datasets in which there
are >20 clusters per group. The test may be used in place of the cluster-mean
procedure (e.g. as
would result from analysis using SAS PROC MIXED), and provides unbiased p-
values where
the standard Wilcoxon test is inappropriate. These analyses are then extended
to control for
potential confounding by variables using the stratified version of the test. A
logical cut-point for
stratification of continuous variables would be the median level among the
vehicle treated group.
[190] Prior to the development of these methods, studies have generally used
the person as the
unit of analysis, using either a composite score or data from one eye per
subject. However,
59
W020091025763 CA 02640986 2010-02-08 PCT/US2008/009776
information is potentially lost by collapsing eye-specific grades into a
single person-specific
grade.
[191] In secondary analyses, the effect of topical IL-1Ra on other outcomes
such as the OSDI
score and tear break up time is also explored. Similarly, these analyses use
the stratified
Wilcoxon test with variance correction.
[192] In general, a two-sided test with p-value less than or equal to 0.0125
is considered
statistically significant. This level of significance was arrived at by
dividing the overall type 1
error rate of 0.05 by 4, which is the number of comparisons in the Primary
analysis.
[193] Although patients are evaluated at multiple time points throughout the
study, the primary
endpoint is on the last observation while on treatment during the 3-month
study period.
EXAMPLE 9: Effect of IL-1Ra on Intraocular Pressure (I0P)
[194] IL-1Ra was administered to one eye of wild type BALB/c mice and the mean
intraocular
pressure (I0P) of the both the treated and the non-treated (contralateral eye)
were measured.
Data from these experiments show that administration of IL-1Ra is sufficient
to reduce IOP by a
statistically significant amount over the course of one day, and particularly,
over the duration of
one night (Table 4, below, and Figure 3). IOP is a risk factor for the
development of glaucoma.
Importantly, the compositions and methods used were effective on wild type, or
normal, =
subjects. As such, this example is proof-of-concept that the compositions and
methods of the
invention are effective for treating elevated intraocular pressure or ocular
hypertension that has
been caused by a variety of mechanisms, including, but not limited to IL-1-
mediated
inflammation.
W020091025763 CA 02640986 2010-02-08 PCT/US2008/009776
Table 4. Effect of one drop of topical IL-1Ra (2.5%) on Intraocular pressure
(I0P) during the
Day (after 6 hours) and Overnight (after 12 hours) in BALB/c mice. Data are
expressed as the
mean IOP (mm Hg) SEM. P value is for treated eye versus the contralateral
eye (paired t-test).
Contralateral Difference
Treated Eye (Caatralateral Eye % Reduction
Eye - Treated Eye) value
Day 5 9.1 0.46 8.0 0.27 1.1 0.51 11.5
0.05 0.095
(after 6 h)
Overnight 5 13.7 0.90 10.3 0.75 3.4
0.38 24.7 0.02 0.0009
(After 12h)
=
=
=
61
=
CA 02640986 2010-02-08
OTHER EMBODIMENTS
[195] While the invention has been described in conjunction with the detailed
description
thereof, the foregoing description is intended to illustrate and not limit the
scope of the
invention, which is defined by the scope of the appended claims. Other
aspects, advantages, and
modifications are within the scope of the following claims.
[196] The patent and scientific literature referred to herein establishes the
knowledge that is
available to those with skill in the art.
[197] While this invention has been particularly shown and described with
references to
preferred embodiments thereof, it will be understood by those skilled in the
art that various
changes in form and details may be made therein without departing from the
scope of the
invention encompassed.by the appended claims.
62
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