U.S. patent application number 14/813885 was filed with the patent office on 2016-01-28 for methods for administering hypoglycemic agents.
This patent application is currently assigned to GlaxoSmithKline LLC. The applicant listed for this patent is GlaxoSmithKline LLC. Invention is credited to Mark A. Bush, Mary Colleen O'Neill.
Application Number | 20160022781 14/813885 |
Document ID | / |
Family ID | 38024051 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160022781 |
Kind Code |
A1 |
Bush; Mark A. ; et
al. |
January 28, 2016 |
METHODS FOR ADMINISTERING HYPOGLYCEMIC AGENTS
Abstract
The present invention relates to methods of administering
hypoglycemic agents and/or GLP-1 agonists.
Inventors: |
Bush; Mark A.; (Research
Triangle Park, NC) ; O'Neill; Mary Colleen; (San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GlaxoSmithKline LLC |
Wilmington |
DE |
US |
|
|
Assignee: |
GlaxoSmithKline LLC
|
Family ID: |
38024051 |
Appl. No.: |
14/813885 |
Filed: |
July 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13464045 |
May 4, 2012 |
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14813885 |
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12092433 |
May 2, 2008 |
8202837 |
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PCT/US2006/060508 |
Nov 3, 2006 |
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13464045 |
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60742600 |
Dec 6, 2005 |
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60733920 |
Nov 4, 2005 |
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Current U.S.
Class: |
514/5.2 ;
514/6.8; 514/7.2 |
Current CPC
Class: |
A61P 3/10 20180101; A61K
38/26 20130101; A61P 3/04 20180101; A61K 38/28 20130101; A61K
31/426 20130101; A61K 31/64 20130101; A61K 38/385 20130101; A61K
45/06 20130101; A61K 9/0019 20130101; A61K 31/155 20130101 |
International
Class: |
A61K 38/26 20060101
A61K038/26; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method for treating a disease selected from the group
consisting of Type 1 diabetes, Type II diabetes, obesity and
hyperglycemia in a human in need thereof comprising administering a
composition comprising a polypeptide having GLP-1 activity as a
subcutaneous injection once weekly to said human via an injection
device comprising a tube having a gauge of 28, 29 or, 30 wherein
the composition comprises about 0.01 mg to about 104 mg of the
polypeptide having GLP-1 activity and wherein the polypeptide
having GLP-1 activity comprises two fragment and variant
polypeptides of human GLP-1, wherein said fragment and variant
polypeptides of human GLP-1 are tandemly oriented and covalently
bound by a chemical linker.
2. The method of claim 1 wherein the disease is Type II
diabetes.
3. The method of claim 1 wherein the needle has a gauge selected
from is 29.
4. The method of claim 1, wherein said two fragment and variant
polypeptides of human GLP-1 are separated by other amino acids
sequences.
5. The method of claim 1 wherein the chemical linker is a
disulphide bond.
6. The method of claim 1, wherein the fragment and variant
polypeptides of human GLP-1 comprise a substitution of the alanine
residue analogous to alanine 8 of wild type GLP-1.
7. The method of claim 7 wherein the alanine at position 8 is
substituted with glycine.
8. The method of claim 1 wherein the polypeptide is administered to
said human at a dose selected from about 0.5 mg/week, 0.8 mg/week,
1.0 mg/week and 2 mg/week.
9. The method of claim 1 wherein the GLP-1 activity is selected
from stimulating glucose-dependent insulin secretion, suppressing
glucagon secretion, delaying gastric emptying, and promoting beta
cell competence and neogenesis.
Description
[0001] This Application is a continuation of U.S. Ser. No. 13/464,
045 which is a divisional application of U.S. Ser. No. 12/092,433,
now U.S. Pat. No. 8,202,837, which is a .sctn.371 of International
Application No. PCT/US2006/060508, filed 3 Nov. 2006, which claims
priority of U.S. Provisional Application No. 60/733,920, filed 4
Nov. 2005 and U.S. Provisional Application No. 60/742,600, filed 6
Dec. 2005.
BACKGROUND
[0002] Hypoglycemic agents may be used in the treatment of both
Type I and Type II diabetes to lower glucose concentration in
blood. Insulinotropic peptides have been implicated as possible
therapeutic agents for the treatment of diabetes. Insulinotropic
peptides include incretin hormones such as, but are not limited to,
gastric inhibitory peptide (GIP) and glucagon like peptide-1
(GLP-1) as well as fragments, variants, and conjugates thereof.
Insulinotropic peptides also include exendin 3 and exendin 4. GLP-1
is a 30 amino acid long incretin hormone secreted by the L-cells in
the intestine. GLP-1 has been shown to stimulate insulin secretion
in a physiological and glucose-dependent manner, decrease glucagon
secretion, inhibit gastric emptying, decrease appetite, and
stimulate proliferation of .beta.-cells.
[0003] Insulin and insulinotrpoic peptides may be administered via
subcutaneous injection, such as with a needle containing device,
for example, a pen injector, and/or syringe. Patients may need to
inject several times a day to control blood glucose, which can be
burdensome as well as painful. Thus, there is a need for methods of
administering hypoglycemic agents less frequently and by methods
that will minimize such burdensome regimens as well as site
injection pain.
SUMMARY OF THE INVENTION
[0004] In one embodiment of the present invention, methods are
provided for administering a GLP-1 agonist composition comprising
at least one polypeptide to a patient in need thereof, comprising
the step of injecting the GLP-1 agonist composition via an
injection device comprising a tube having a gauge of about 28 or
greater, wherein said polypeptide is administered no more than once
daily. The polypeptide may be, but is not limited to, GLP-1 or a
fragment, variant, and/or conjugate thereof. Certain embodiments of
a GLP-1 or a fragment, variant, or conjugate thereof comprise human
serum albumin. Human serum albumin, variants and/or fragments
thereof, may be conjugated to a GLP-1 or fragment or variant
thereof Human serum albumin may be conjugated through a chemical
linker, including but not limited to naturally occurring or
engineered disulfide bonds, or by genetic fusion to GLP-1, or a
fragment or variant thereof
[0005] In another aspect of the present invention, methods are
provided for administering a hypoglycemic agent comprising at least
one polypeptide to a patient in need thereof, comprising the step
of injecting the hypoglycemic agent via an injection device
comprising a tube having a gauge of about 28 or greater, wherein
said polypeptide is administered no more than once daily.
[0006] In another aspect of the present invention, methods are
provided for treating or preventing a disease in a mammal in need
thereof comprising administering a composition comprising at least
one polypeptide having GLP-1 agonist activity once weekly, wherein
the composition comprises about 0.25 mg to about 104 mg of at least
one polypeptide having GLP-1 activity. The mammal may suffer from
one or more of the following diseases: Type 1 diabetes, Type II
diabetes, obesity and hyperglycemia.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1: A device for delivery of lyophilized hypoglycemic
agent and/or GLP-1 agonist
[0008] FIG. 2: SEQ ID NO:1 which comprises two tandemly oriented
GLP-1(7-36)(A8G) fused to the N-Terminus of human serum
albumin.
DEFINITIONS
[0009] "GLP-1 agonist composition" as used herein means any
composition capable of stimulating the secretion of insulin,
including, but not limited to an incretin hormone.
[0010] "Incretin hormone" as used herein means any hormone that
potentiates insulin secretion. One example of an incretin hormone
is GLP-1. GLP-1 is an incretin secreted by intestinal L cells in
response to ingestion of food. In a healthy individual, GLP-1 plays
an important role regulating post-prandial blood glucose levels by
stimulating glucose-dependent insulin secretion by the pancreas
resulting in increased glucose absorption in the periphery. GLP-1
also suppresses glucagon secretion, leading to reduced hepatic
glucose output. In addition, GLP-1 delays gastric emptying time and
slows small bowel motility delaying food absorption. GLP-1 promotes
continued beta cell competence by stimulating transcription of
genes involved in glucose dependent insulin secretion and by
promoting beta-cell neogenesis (Meier, et al. "Glucagon-Like
Peptide 1 and Gastric Inhibitory Polypeptide Potential Applications
in Type 2 Diabetes Mellitus" Biodrugs 2003; 17 (2): 93-102). "GLP-1
activity" as used herein means one or more of the activities of
naturally occurring human GLP-1, including but not limited to,
stimulating glucose-dependent insulin secretion, suppressing
glucagon secretion, delaying gastric emptying, and promoting beta
cell competence and neogenesis.
[0011] An "incretin mimetic" as used herein is a compound capable
of potentiating insulin secretion. An incretin mimetic may be
capable of stimulating insulin secretion, increasing beta cell
neogenesis, inhibiting beta cell apoptosis, inhibiting glucagon
secretion, delaying gastric emptying and inducing satiety in a
mammal. An incretin mimetic may include, but is not limited to, any
polypeptide which has GLP-1 activity, including but not limited to,
exendin 3 and exendin 4, including any fragments and/or variants
and/or conjugates thereof
[0012] "Hypoglycemic agent" as used herein means any compound or
composition comprising a compound capable of reducing blood
glucose. A hypoglycemic agent may include, but is not limited to,
any GLP-1 agonist including incretin hormones or incretin mimetics,
GLP-1 or fragment, variant and/or conjugate thereof. Other
hypoglycemic agents include, but are not limited to, drugs that
increase insulin secretion (e.g., sulfonylureas (SU) and
meglitinides), increase glucose utilization (e.g., glitazones),
reduce hepatic glucose production (e.g., metformin), and delay
glucose absorption (e.g., a-glucosidase inhibitors).
[0013] "Polypeptide" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to both short chains, commonly referred to as peptides,
oligopeptides or oligomers, and to longer chains, generally
referred to as proteins. Polypeptides may contain amino acids other
than the 20 gene-encoded amino acids. "Polypeptides" include amino
acid sequences modified either by natural processes, such as
posttranslational processing, or by chemical modification
techniques that are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature. Modifications can
occur anywhere in a polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched as a result of
ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched and branched cyclic polypeptides may result from
posttranslation natural processes or may be made by synthetic
methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination. See,
for instance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and
Wold, F., Posttranslational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983;
Seifter, et al., "Analysis for protein modifications and nonprotein
cofactors", Meth. Enzymol. (1990) 182:626-646 and Rattan, et al.,
"Protein Synthesis: Posttranslational Modifications and Aging", Ann
NY Acad Sci (1992) 663:48-62.
[0014] "Variant" as the term is used herein, is a polynucleotide or
polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptide
encoded by the reference sequence, as discussed below. A typical
variant of a polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequences of the reference polypeptide and the variant
are closely similar overall and, in many regions, identical. A
variant and reference polypeptide may differ in amino acid sequence
by one or more substitutions, additions, deletions in any
combination. A substituted or inserted amino acid residue may or
may not be one encoded by the genetic code. A variant of a
polynucleotide or polypeptide may be a naturally occurring such as
an allelic variant, or it may be a variant that is not known to
occur naturally. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by mutagenesis
techniques or by direct synthesis.
[0015] As used herein "conjugate" or "conjugated" refers to two
molecules that are bound to each other. For example, a first
polypeptide may be covalently or non-covalently bounded to a second
polypeptide. The first polypeptide may be covalently bound by a
chemical linker or may be fused genetically to the second
polypeptide, wherein the first and second polypeptide share a
common polypeptide backbone.
[0016] As used herein "tandemly oriented" refers to two or more
polypeptides that are adjacent to one another as part of the same
molecule. They may be linked either covalently or non-covalently.
Two or more tandemly oriented polypeptides may form part of the
same polypeptide backbone. Tandemly oriented polypeptides may have
direct or inverted orientation and/or may be separated by other
amino acid sequences.
[0017] As used herein "fragment," when used in reference to a
polypeptide, is a polypeptide having an amino acid sequence that is
the same as part but not all of the amino acid sequence of the
entire naturally occurring polypeptide. Fragments may be
"free-standing" or comprised within a larger polypeptide of which
they form a part or region as a single continuous region in a
single larger polypeptide. By way of example, a fragment of
naturally occurring GLP-1 would include amino acids 7 to 36 of
naturally occurring amino acids 1 to 36. Furthermore, fragments of
a polypeptide may also be variants of the naturally occurring
partial sequence. For instance, a fragment of GLP-1 comprising
amino acids 7-30 of naturally occurring GLP-1 may also be a variant
having amino acid substitutions within its partial sequence.
[0018] As used herein, "reduce" or "reducing" blood glucose refers
to a decrease in the amount of blood glucose observed in the blood
of a patient after administration a hypoglycemic agent.
[0019] As used herein "disease associated with elevated blood
glucose" include, but are not limited to, Type I and Type II
diabetes and hyperglycemia.
[0020] As used herein "co-administration" or "co-administering" as
used herein refers to administration of two or more compounds to
the same patient. Co-administration of such compounds may be at
about the same time (e.g., within the same hour) or it may be
within several hours or days of one another. For example, a first
compound may be administered once weekly while a second compound is
co-administered daily.
DETAILED DESCRIPTION OF THE INVENTION
[0021] In one embodiment of the present invention, methods are
provided for administering a GLP-1 agonist composition comprising
at least one polypeptide to a patient in need thereof, comprising
the step of injecting the GLP-1 agonist composition via an
injection device comprising a tube having a gauge of about 28 or
greater, wherein said polypeptide is administered no more than once
daily. As is understood in the art, examples of needles with gauges
of 28 or greater include, but are not limited to, 28, 29 and 30
gauge needles. As is also understood in the art, the higher the
needle gauge the smaller the needle aperture. An embodiment of the
invention comprises a polypeptide that may be, but is not limited
to, GLP-1 or a fragment, variant, or conjugate thereof. A GLP-1 or
a fragment, variant, or conjugate thereof may comprise human serum
albumin. Human serum albumin may be conjugated to the GLP-1 or
fragment or variant thereof. Human serum albumin may be conjugated
through a chemical linker or genetically fused to the GLP-1
fragment or variant. Examples of GLP-1, fragments or variants,
fused with human serum albumin are provided in the following PCT
applications: WO 2003/060071, WO 2003/59934, WO 2005/003296, WO
2005/077042.
[0022] A further embodiment of the invention comprises one, two,
three, four, five, or more tandemly oriented molecules of GLP-1
fused to the N- or C-terminus of human serum albumin or variant
thereof. GLP-1 fragments may include, but are not limited to,
molecules of GLP-1 comprising, or alternatively consisting of,
amino acids 7 to 36 of GLP-1 (hereinafter designated as
"GLP-1(7-36)"). Variants of GLP-1 or fragments of GLP-1 may
include, but are not limited to, substitutions of an alanine
residue analogous to alanine 8 of wild type GLP-1, such alanine
being mutated to a glycine (hereinafter designated as "A8G") (See
for Example, the mutants disclosed in U.S. Pat. No. 5,545,618,
herein incorporated by reference in its entirety). Other
embodiments have such A8G polypetides fused to the N- or C-terminus
of albumin or variant thereof. An example of two tandemly oriented
GLP-1(7-36)(A8G) fragments/variants fused to the N-terminus of
human serum albumin comprises SEQ ID NO:1, which is presented in
FIG. 2.
[0023] In another aspect of the present invention, the GLP-1
agonist composition further comprises one or more compounds
selected from the group of: peroxisome proliferating activated
receptor (PPAR) ligand, thiazolidinedione, metformin, insulin, and
sulfonylurea. In another aspect, methods are provided comprising
the step of co-administering at least one GLP-1 agonist with one or
more compounds selected from the group of: peroxisome proliferating
activated receptor (PPAR) ligand, thiazolidinedione, metformin,
insulin, and sulfonylurea. The GLP-1 agonist composition may have
one or more of these compounds in addition to at least one
polypeptide.
[0024] In another aspect, the GLP-1 agonist composition is
lyophilized. In another aspect of this invention, methods are
provided that further comprise admixing said GLP-1 agonist
composition with a liquid prior to administration of said GLP-1
agonist composition. In yet another aspect, the GLP-1 agonist
composition is in solution form, and may be an aqueous
solution.
[0025] In yet another aspect of the present invention, at least one
polypeptide of a GLP-1 agonist composition may be administered to
said patient once weekly, twice weekly, once every two weeks,
and/or once monthly. In another aspect, the patient suffers from
Type II diabetes. An injection device of the invention may be
reusable and/or disposable. In one aspect, an injection device
comprises a needle. In another aspect the injection a delivery
device of the invention comprises a catheter.
[0026] In yet another aspect, GLP-1 agonist composition is
administered via subcutaneous injection. In another aspect, the
injection may be intramuscular or intravenous, intraperitoneal,
intranasal, transmucosal or topical. In another aspect, GLP-1
agonist composition is self-administered, meaning a patient
receiving an injection administers a GLP-1 agonist composition to
himself or herself. Subcutaneous, injections may be administered,
for example, at the abdomen, upper arm, and/or thigh.
[0027] In another aspect of the present invention, methods are
provided for administering a hypoglycemic agent comprising at least
one polypeptide to a patient in need thereof, comprising the step
of injecting a hypoglycemic agent via an injection device
comprising a tube having a gauge of about 28 or greater, wherein
said polypeptide is administered no more than once daily. The
needle may have a 29 or 30 gauge. In another aspect, the
polypeptide of the invention is not insulin. P In another aspect of
the present invention methods are provided for treating or
preventing a disease in a mammal in need thereof comprising
administering a composition comprising at least one polypeptide
having GLP-1 agonist activity once weekly, wherein the composition
comprises about 0.010 mg to about 104 mg of at least one
polypeptide having GLP-1 activity. The disease may be selected from
the group consisting of Type 1 diabetes, Type II diabetes, obesity
and hyperglycemia. In one aspect, the disease is Type II
diabetes.
[0028] In another aspect of the present invention, the polypeptide
having GLP-1 activity comprises at least one fragment or variant of
human GLP-1 genetically fused with human serum albumin. This
fragment or variant of GLP-1 may comprise GLP-1(7-36(A8G)). At
least one fragment or variant of GLP-1 may be genetically fused to
human serum albumin. In another aspect, the polypeptide having
GLP-1 activity comprises at least two GLP-1(7-36(A8G)) tandemly and
genetically fused to the human serum albumin. In another aspect,
the two GLP-1(7-36(A8G)) are genetically fused at the N-terminus of
the human serum albumin. In another aspect, at least one
polypeptide having GLP-1 activity comprises SEQ ID No.: 1. At least
one polypeptide having GLP-1 activity may be administered at a dose
of about 0.25 mg to about 32 mg weekly. Some examples of doses for
a once weekly administration of a polypeptide having GLP-1 activity
include, but are not limited to, 0.010 mg/week, 0.25 mg/week, 0.5
mg/week, 0.8 mg/week, 1.0 mg/week, 2 mg/week, 3.2 mg/week, 8
mg/week, 12.8 mg/week, 32 mg/week, 51.2 mg/week, and/or 104
mg/week.
EXAMPLES
[0029] The following examples illustrate various aspects of this
invention. These examples do not limit the scope of this invention
which is defined by the appended claims.
Example 1
[0030] The following devices may be used to deliver a hypoglycemic
and/or a GLP-1 agonist composition: [0031] 1. STATdose.RTM., GSK,
Liquid Drug Product Reusable Auto-injector; [0032] 2. Autoject
mini.RTM., Owen Mumford, Liquid Drug Product Reusable
Auto-injector; [0033] 3. Penlet.RTM., Becton Dickinson, Liquid Drug
Product Disposable Auto-injector; [0034] 4.
Tigerlily/Snapdragon.RTM., Owen Mumford, Liquid Drug Product
Disposable Auto-injector; [0035] 5. AutoSafety Injector.RTM., The
Medical House, Liquid Drug Product Disposable Auto-injector; [0036]
6. Liquid Dry Injector.RTM. Becton Dickinson, Lyophilised Drug
Product Disposable Pen Injector; and/or [0037] 7. Pre-filled
syringes.
Example 2
[0038] A lyophilized hypoglycemic agent and/or GLP-1 agonist
composition may be delivered by a device as shown in FIG. 1. This
device system can contain a powder comprising a lyophilized
hypoglycemic agent and/or GLP-1 agonist and a liquid. This device
system can be held upright to mix a contained powder and liquid.
The two halves of an injection pen can be pressed together firmly
until the powder completely dissolves. Once dissolved the
hypoglycemic agent and/or GLP-1 agonist composition can be
administered to the patient.
Example 3
[0039] A hypoglycemic agent and/or GLP-1 agonist composition can
comprise a polypeptide having GLP-1 activity. A hypoglycemic agent
and/or GLP-1 agonist composition comprising a polypeptide having
GLP-1 activity can be delivered by subcutaneous injection to a
person in need thereof, wherein the agent or composition comprises
polypeptide having GLP-1 activity at a dose in the range of about
0.010 mg to about 104 mg once weekly. Some examples of doses for a
once weekly administration of a GLP-1 agonist composition
comprising a polypeptide having GLP-1 activity include, but are not
limited to, 010 mg/week, 0.25 mg/week, 0.5 mg/week, 0.8 mg/week,
1.0 mg/week, 2 mg/week, 3.2 mg/week, 8 mg/week, 12.8 mg/week, 32
mg/week, 51.2 mg/week, and/or 104 mg/week.
Example 4
[0040] A lyophilized hypoglycemic agent and/or GLP-1 agonist
composition can be reconstituted with water for injection. Examples
of excipients that can be included in the composition include, but
are not limited to, trehalose dehydrate, mannitol, sodium phosphate
(such as dibasic, anhydrous and monobasic, monohydrate),
polysorbate 80, sodium hydroxide, phosphoric acid, and water for
injection.
[0041] All patent applications to which this application claims
priority are incorporated by reference herein in their entirety as
if each application is specifically and individually being fully
set forth.
Sequence CWU 1
1
11645PRTHomo sapiens 1His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Arg His Gly 20 25 30 Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly Gln Ala 35 40 45 Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg Asp Ala His Lys 50 55 60 Ser Glu Val
Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys65 70 75 80 Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe 85 90
95 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr
100 105 110 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu
His Thr 115 120 125 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
Arg Glu Thr Tyr 130 135 140 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln
Glu Pro Glu Arg Asn Glu145 150 155 160 Cys Phe Leu Gln His Lys Asp
Asp Asn Pro Asn Leu Pro Arg Leu Val 165 170 175 Arg Pro Glu Val Asp
Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 180 185 190 Thr Phe Leu
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 195 200 205 Phe
Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 210 215
220 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu
Pro225 230 235 240 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
Ser Ala Lys Gln 245 250 255 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
Gly Glu Arg Ala Phe Lys 260 265 270 Ala Trp Ala Val Ala Arg Leu Ser
Gln Arg Phe Pro Lys Ala Glu Phe 275 280 285 Ala Glu Val Ser Lys Leu
Val Thr Asp Leu Thr Lys Val His Thr Glu 290 295 300 Cys Cys His Gly
Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu305 310 315 320 Ala
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 325 330
335 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu
340 345 350 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala
Ala Asp 355 360 365 Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
Glu Ala Lys Asp 370 375 380 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr
Ala Arg Arg His Pro Asp385 390 395 400 Tyr Ser Val Val Leu Leu Leu
Arg Leu Ala Lys Thr Tyr Glu Thr Thr 405 410 415 Leu Glu Lys Cys Cys
Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 420 425 430 Val Phe Asp
Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 435 440 445 Lys
Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln 450 455
460 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser
Thr465 470 475 480 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
Val Gly Ser Lys 485 490 495 Cys Cys Lys His Pro Glu Ala Lys Arg Met
Pro Cys Ala Glu Asp Tyr 500 505 510 Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His Glu Lys Thr Pro 515 520 525 Val Ser Asp Arg Val Thr
Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 530 535 540 Arg Pro Cys Phe
Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys545 550 555 560 Glu
Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu 565 570
575 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu
580 585 590 Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala
Val Met 595 600 605 Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys
Ala Asp Asp Lys 610 615 620 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys
Leu Val Ala Ala Ser Gln625 630 635 640 Ala Ala Leu Gly Leu 645
* * * * *