U.S. patent application number 12/799204 was filed with the patent office on 2010-10-07 for method for treating symptoms of diabetes.
This patent application is currently assigned to THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK. Invention is credited to Ann Marie Schmidt, David M. Stern.
Application Number | 20100255081 12/799204 |
Document ID | / |
Family ID | 25038266 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100255081 |
Kind Code |
A1 |
Stern; David M. ; et
al. |
October 7, 2010 |
Method for treating symptoms of diabetes
Abstract
The present invention provides a method for treating symptoms of
diabetes in a diabetic subject which comprises administering to the
subject a therapeutically effective amount of an agent which
inhibits binding of advanced glycation endproducts to any receptor
for advanced glycation endproducts so as to treat chronic symptoms
of diabetes in the subject.
Inventors: |
Stern; David M.; (Augusta,
GA) ; Schmidt; Ann Marie; (Franklin Lakes,
NJ) |
Correspondence
Address: |
John P. White, Esq.;Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
THE TRUSTEES OF COLUMBIA UNIVERSITY
IN THE CITY OF NEW YORK
|
Family ID: |
25038266 |
Appl. No.: |
12/799204 |
Filed: |
April 20, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10850861 |
May 20, 2004 |
7700085 |
|
|
12799204 |
|
|
|
|
08755235 |
Nov 22, 1996 |
6790443 |
|
|
10850861 |
|
|
|
|
Current U.S.
Class: |
424/450 ;
424/130.1; 514/23; 514/44R; 514/6.9 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61P 3/12 20180101; A61K 38/00 20130101; A61P 3/10 20180101; A61P
9/02 20180101; C07K 14/70503 20130101; A61P 17/02 20180101; A61P
15/10 20180101; A61P 29/00 20180101; C07K 16/2803 20130101; A61P
25/00 20180101; A61P 27/02 20180101 |
Class at
Publication: |
424/450 ;
514/6.9; 514/23; 514/44.R; 424/130.1 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 38/02 20060101 A61K038/02; A61K 31/70 20060101
A61K031/70; A61K 31/7088 20060101 A61K031/7088; A61K 39/395
20060101 A61K039/395; A61P 3/10 20060101 A61P003/10 |
Goverment Interests
[0001] The invention disclosed herein was made with Government
support under Grant Nos. HL21006 and AG00603 from the National
Institutes of Health, U.S. Department of Health and Human Services.
Accordingly, the U.S. Government has certain rights in this
invention.
Claims
1. A method for treating symptoms of diabetes in a diabetic subject
which comprises administering to the subject a therapeutically
effective amount of sRAGE so as to treat symptoms of diabetes in
the subject.
2. The method of claim 1, wherein the symptoms comprise abnormal
wound healing, symptoms of a heart attach, symptoms of a strake,
symptoms of peripheral vascular disease, amputation, symptoms of
kidney disease, kidney failure, blindness, neuropathy, inflammation
or impotence.
3. A method for treating symptoms of diabetes in a diabetic subject
which comprises administering to the subject a therapeutically
effective amount of an agent which inhibits binding of advanced
glycation endproducts to any receptor for advanced glycation
endproducts so as to treat symptoms of diabetes in the subject.
4. The method of claim 3, wherein the agent comprises a
polypeptide, a peptidomimetic, an organic molecule, a carbohydrate,
a lipid, an antibody or a nucleic acid.
5. The method of claim 4, wherein the polypeptide comprises an
advanced glycation endproduct polypeptide or a portion thereof, a
receptor for advanced glycation endproduct polypeptide or a portion
thereof, a soluble receptor for advanced glycation endproduct
polypeptide or a portion thereof.
6. The method of claim 4, wherein the antibody comprises an
anti-RAGE antibody or an anti-RAGE F(ab').sub.2 fragment.
7. The method of claim 3, wherein the symptoms comprise abnormal
wound healing, symptoms of a heart attack, symptoms of a stroke,
symptoms of peripheral vascular disease, amputation, symptoms of
kidney disease, kidney failure, blindness, neuropathy, inflammation
or impotence.
8. The method of claim 3, wherein the subject is a mammal.
9. The method of claim 8, wherein the mammal is a human.
10. The method of claim 3, wherein the administration comprises
intralesional, intraperitoneal, intramuscular or intravenous
injection; infusion; liposome-mediated delivery, topical, nasal,
oral, anal, ocular or otic delivery.
11. The method of claim 3, wherein the agent is administered
daily.
12. The method of claim 3, wherein the therapeutically effective
amount comprises a dose of from about 200 ng/day/kg body weight to
about 200,000 ng/day/kg body weight.
13. A method for improving wound healing in a diabetic subject
which comprises administering to the subject a therapeutically
effective amount of an agent which inhibits binding of advanced
glycation endproducts to a receptor for advanced glycation
endproducts, over a sufficient period of time in a sufficient
amount so as to improve would healing in the subject.
14. The method of claim 13, wherein the agent comprises a
polypeptide, a peptidomimetic, an organic molecule, a carbohydrate,
a lipid, an antibody or a nucleic acid.
15. The method of claim 14, wherein the polypeptide comprises an
advanced glycation endproduct polypeptide or a portion thereof, a
receptor for advanced glycation endproduct polypeptide or a portion
thereof, a soluble receptor for advanced glycation endproduct
polypeptide or a portion thereof.
16. The method of claim 15, wherein the antibody comprises an
anti-RAGE antibody or an anti-RAGE F(ab').sub.2 fragment.
17. The method of claim 13, wherein the subject is a mammal.
18. The method of claim 17, wherein the mammal is a human.
19. The method of claim 13, wherein the intraperitoneal,
intramuscular or intravenous injection; infusion; liposome-mediated
delivery; topical, nasal, oral, anal, ocular or otic delivery.
20. The method of claim 13, wherein the administration comprises
daily administration from about the day of wounding to about ten
days after wounding.
21-22. (canceled)
Description
BACKGROUND OF THE INVENTION
[0002] Throughout this application, various publications are
referenced by author and date. Full citations for these
publications may be found listed alphabetically at the end of the
specification immediately the claims. The disclosures of these
publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the
state of the art as known to those skilled therein.
[0003] Ineffective healing of wounds is a serious problem in
diabetes, contributing to increased morbidity (Reynolds, 1985;
Galloway and Shuman, 1963; and Pearl and Kanat, 1988). The
reparative response in wound healing is orchestrated by multiple
cellular elements which work together in many ways, including
infiltration of the lesion by inflammatory effector cells.
Subsequent to this, fibroblastic elements together with
inflammatory effector cells provide antibacterial mechanisms and
promote removal of necrotic tissue, as well as laying down of new
connective tissue. A fundamental disorder of glucose metabolism
might perturb these complex and interactive protective processes.
Previous work has suggested that cellular dysfunction in diabetic
wound healing involves defective neutrophil function (Bagdade et
al., 1978; Nolan et al., 1978; and Mowat and Baum, 1971), delayed
infiltration of the wound with inflammatory cells (Greenhalgh et
al., 1990 and Fahey et al., 1991), decreased production of collagen
(Goodson and Hunt, 1977 and Goodson and Hunt, 1986), and diminished
activity of endogenous growth factors, such as basic fibroblast
growth factor (Giardino et al., 1994), which could provide a basis
for the slower formation of granulation tissue and wound
closure.
SUMMARY OF THE INVENTION
[0004] The present invention provides a method for treating
symptoms of diabetes in a diabetic subject which comprises
administering to the subject a therapeutic amount of an agent which
inhibits binding of advanced glycation endproducts to any receptor
for advanced glycation endproducts so as to treat symptoms of
diabetes in the subject.
BRIEF DESCRIPTION OF THE FIGURES
[0005] FIG. 1. Effect of sRAGE on wound healing in the
genetically-diabetic db+/db+ mouse. A full-thickness 1.5.times.1.5
cm wound was created on the backs of db+/db+ mice or control,
heterozygote db+/m+ mice and covered with TEGADERM.RTM.. Diabetic
wounds were treated with either phosphate-buffered saline (PBS)
directly under the TEGADERM.RTM. daily for 7 days commencing on day
3 following surgery or with sRAGE (200 ng). Wound area was measured
at baseline through day 21 by placing a glass slide over the wound
area, tracing the wound area, and placing this information into a
computer in order to calculate the percentage of wound closure as a
function of time. Left axis represents percent wound closure.
[0006] FIG. 2. Administration of sRAGE to the genetically-diabetic
db+/db+ mouse improves wound healing: dose-response studies. Wounds
were created as above and treated from days 3 through 9 with sRAGE
(either 2,000, 200, or 20 ng/day) or with phosphate-buffered
saline. At day 10, wound area was measured and compared with
initial wound area as above. Results are presented as fold increase
in percent wound healing compared with mice treated with phosphate
buffered saline (defined as one in figure). All statistical
analyses are shown comparing wound healing in the presence of
different doses of sRAGE vs. treatment of diabetic wounds with
phosphate-buffered saline.
[0007] FIGS. 3A and 3B. AGE-immunoreactive epitopes in the wounds
of diabetic (db+/db+) mice. 1.5.times.1.5 cm full-thickness wounds
created in the backs of diabetic mice (db+/db+ mice; FIG. 3A) and
non-diabetic mice (db+/m+; FIG. 3B) were excised, fixed and
sections stained with affinity-purified anti-AGE IgG.
Magnification: 200.times..
DETAILED DESCRIPTION OF THE INVENTION
[0008] The present invention provides a method for treating
symptoms of diabetes in a diabetic subject which comprises
administering to the subject a therapeutically effective amount of
sRAGE so as to treat symptoms of diabetes in the subject. The
symptoms may comprise abnormal wound healing, symptoms of a heart
attack, symptoms of a stroke, symptoms of peripheral vascular
disease, amputation, symptoms of kidney disease, kidney failure,
blindness, neuropathy, inflammation or impotence.
[0009] The present invention also provides a method for treating
symptoms of diabetes in a diabetic subject which comprises
administering to the subject a therapeutically effective amount of
an agent which inhibits binding of advanced glycation endproducts
to any receptor for advanced glycation endproducts so as to treat
symptoms of diabetes in the subject. In accordance with the method
of this invention, the agent may comprise a polypeptide, a
peptidomimetic, an organic molecule, a carbohydrate, a lipid, an
antibody or a nucleic acid. In accordance with the method of this
invention, the polypeptide may comprise an advanced glycation
endproduct polypeptide or a portion thereof, a receptor for an
advanced glycation endproduct polypeptide or a portion thereof, a
soluble receptor for advanced glycation endproduct polypeptide or a
portion thereof. In accordance with the method of this invention,
the antibody may comprise an anti-RAGE antibody or an anti-RAGE
F(ab').sub.2 fragment. In accordance with the method of this
invention, the therapeutically effective amount may comprise a dose
of from about 200 ng/day/kg body weight to about 200,000 ng/day/kg
body weight or from about 50 ng/day/kg to about 500,000 ng/day/kg
body weight.
[0010] The present invention provides a method for improving wound
healing in a diabetic subject which comprises administering to the
subject a therapeutically effective amount of an agent which
inhibits binding of advanced glycation endproducts to a receptor
for advanced glycation endproducts, over a sufficient period of
time in a sufficient amount so as to improve wound healing in the
subject.
[0011] In accordance with the method of this invention, the agent
may comprise a polypeptide, a peptidomimetic, an organic molecule,
a carbohydrate, a lipid, an antibody or a nucleic acid. The
polypeptide of this invention may comprise an advanced glycation
endproduct polypeptide or a portion thereof, a receptor for
advanced glycation endproduct polypeptide or a portion thereof, a
soluble receptor for advanced glycation endproduct polypeptide or a
portion thereof.
[0012] In one embodiment of this invention, the administration may
comprise daily administration from about the day of wounding to
about ten days after wounding. The present invention provides that
the sufficient amount comprises a dose of from about 10 ng/day/kg
body weight to about 500,000 ng/day/kg body weight or a dose of
from about 150 ng/day/kg body weight to about 200,000 ng/day/kg
body weight.
[0013] The present invention provides a method for treating
symptoms of diabetes in a diabetic subject which comprises
administering to the subject a therapeutically effective amount of
an agent which agent inhibits binding of advanced glycation
endproducts to any receptor for advanced glycation endproducts so
as to treat symptoms of diabetes in the subject.
[0014] In accordance with the method of this invention, the agent
may be a polypeptide, a peptidomimetic, an organic molecule, a
carbohydrate, a lipid, an antibody or a nucleic acid. In the case
of polypeptides, the polypeptide may be an advanced glycation
endproduct (AGE) polypeptide or a portion thereof, a receptor for
advanced glycation endproduct polypeptide or a portion thereof, a
soluble receptor for advanced glycation endproduct polypeptide or a
portion thereof, e.g., soluble RAGE, or a recombinant polypeptide.
The polypeptide may be synthesized chemically or produced by
standard recombinant DNA methods. In the case of antibodies, the
antibody may be an anti-RAGE antibody or an anti-RAGE F(ab').sub.2
fragment.
[0015] In accordance with the method of the present invention, the
symptoms which may be treated include abnormal wound healing,
symptoms related to having a heart attack, such as chest pain,
symptoms related to having a stroke, peripheral vascular disease,
amputation, kidney disease, kidney failure, blindness, neuropathy,
inflammation and impotence.
[0016] The subject on which the method is employed may be any
mammal, e.g. a human, mouse, cow, pig, dog, cat, or monkey.
[0017] The administration of the agent may be effected by
intralesional, intraperitoneal, intramuscular or intravenous
injection; by infusion; or may involve liposome-mediated delivery;
or topical, nasal, oral, anal, ocular or otic delivery.
[0018] In the practice of the method administration may comprise
daily, weekly, monthly or hourly administration, the precise
frequency being subject to various variables such as age and
condition of the subject, amount to be administered, half-life of
the agent in the subject, area of the subject to which
administration is desired and the like.
[0019] In connection with the method of this invention, a
therapeutically effective amount of may include dosages which take
into account the size and weight of the subject, the age of the
subject, the severity of the symptom, the surface area of the
wound, the efficacy of the agent, the method of delivery of the
agent and the history of the symptoms in the subject. One of
ordinary skill in the art would be readily able to determine the
exact dosages and exact times of administration based upon such
factors. For example, a therapeutically effective amount may a dose
of from about 200 ng/day/kg body weight to about 200,000 ng/day/kg
body weight. In this regard, it has been shown that 24 micrograms
administered intraperitoneally daily (on days 3-9) to wounded
diabetic mice resulted in greatly improved wound healing. In this
regard, the dose may also be administered as a single dose or as a
series of doses over a period of time.
[0020] The present invention also provides a method for improving
wound healing in a diabetic subject which comprises administering
to the subject a therapeutically effective amount of an agent which
inhibits binding of advanced glycation endproducts so as to improve
wound healing in the subject.
[0021] The present invention provides a method for alleviating
inflammation in a subject which comprises administering a
therapeutically effective amount of an agent which inhibits binding
of advanced glycation endproducts to any receptor for advanced
glycation endproducts so as to treat symptoms of inflammation in
the subject.
[0022] In accordance with the method of the invention, the agent
may be a polypeptide, a peptidomimetic, an organic molecule, a
carbohydrate, a lipid, an antibody or a nucleic acid. In the case
of polypeptides, the polypeptide may be an advanced glycation
endproduct polypeptide or a portion thereof, a receptor for
advanced glycation endproduct polypeptide or a portion thereof, a
soluble receptor for advanced glycation endproduct polypeptide or a
portion thereof, or a recombinant polypeptide. The polypeptide may
be synthesized chemically or produced by standard recombinant DNA
methods. In the case of antibodies, the antibody may be an
anti-RAGE antibody or an anti-RAGE F(ab').sub.2 fragment.
[0023] There may be other mechanisms by which soluble RAGE may
improve diabetic wound healing. Soluble RAGE may have other
effects, such as anti-inflammatory effects that are at least in
part, independent of binding up AGE's and interfering with their
ability to activate cellular RAGE.
[0024] The administration of the agent may be effected by
intralesional, intraperitoneal, intramuscular or intravenous
injection; by infusion; by liposome-mediated delivery or by
topical, nasal, oral, anal, ocular or otic delivery.
[0025] In one embodiment of the claimed invention, the
administration may include daily administration from about the day
of wounding to about ten days after wounding.
[0026] In another embodiment of the invention, the sufficient
amount may include a dose of from about 200 ng/day/mouse body
weight to about 200,000 ng/day/mouse body weight.
[0027] The present invention also provides a method for improving
wound healing in a diabetic subject which comprises administering
to the subject a therapeutic amount of an agent so as to improve
wound healing in the subject. The mechanism of improving wound
healing may be biochemical in nature or competitive in nature.
[0028] As used herein "AGE" means an advanced glycation endproduct;
"RAGE" means a receptor for an advanced glycation endproduct;
"sRAGE" means a soluble form of a receptor for an advanced
glycation endproducts, such as the extracellular two-thirds of the
RAGE polypeptide.
[0029] In the practice of the methods of the invention a
"therapeutically effective amount" is an amount which is capable of
inhibiting the binding of AGE to any receptor for advanced
glycation endproduct. Accordingly, the effective amount will vary
with the subject being treated, as well as the condition to be
treated. For the purposes of this invention, the methods of
administration are to include, but are not limited to,
administration cutaneously, subcutaneously, intravenously,
parenterally, orally, topically, or by aerosol.
[0030] Portions of the agent of the invention may be "labeled" by
association with a detectable marker substance (e.g., radiolabeled
with .sup.125I or biotinylated) to provide reagents useful in
detection and quantification of such agent or its receptor bearing
cells or its derivatives in solid tissue and fluid samples such as
blood, cerebral spinal fluid or urine.
[0031] The administration of compounds and pharmaceuticals to
subjects to improve wound healing is known in the art because the
need for improving the symptoms associated with diabetes has been a
long-felt need. The following publications are hereby incorporated
by reference: U.S. Pat. No. 5,561,116, Solid product containing
propolis components, and preparation and uses thereof; U.S. Pat.
No. 4,971,954, Collagen-based matrices ribose cross-linked; U.S.
Pat. No. 5,567,417, Method for inhibiting angiogenesis using
heparinase; U.S. Pat. No. 5,565,428, Method of administration of
IGF-1. Administering insulin-like growth factor-I to a mammal so as
to sustain its biological activity in the mammal comprising
administering a therapeutically effective amount of IGF-I to the
mammal for a period of time that stimulates the maximum biological
response in the mammal is disclosed. The '428 patent also discloses
administration over a period of time and repeated administration
and discontinuance of administration for a period as long as
necessary to achieve or maintain the desired biological response in
the mammal. Thus, methods of administration of therapeutic amounts
of a peptide or protein are known to one of skill in the art. U.S.
Pat. No. 5,561,137, Thio-heterocyclic macrolactam immunomodulators;
U.S. Pat. No. 5,561,110, Method for the treatment of the
complications and pathology of diabetes; U.S. Pat. No. 5,547,672,
Accelerated wound healing; U.S. Pat. No. 5,532,227, Tetracyclines
including non-antimicrobial chemically-modified tetracyclines
inhibit excessive glycosylation of different types of collagen and
other proteins during diabetes; U.S. Pat. No. 5,527,772 Regulation
of cell proliferation and differentiation using peptides; U.S. Pat.
No. 5,468,737, Wound healing accelerated by systemic administration
of polysaccharide from aloe; U.S. Pat. No. 5,395,398, Microelectric
apparatus for the antisepsis, promulgation of healing and analgesia
of wound and chronic skin ulcers; U.S. Pat. No. 5,378,475,
Sustained release drug delivery devices; U.S. Pat. No. 5,246,708,
Methods for promoting wound healing with deoxyribonucleosides; U.S.
Pat. No. 5,532,227, Tetracycline, including non-antimicrobial
chemically-modified tetracycline inhibit excessive glycosylation of
different types of collagen and other proteins during diabetes. The
disclosures of the publications referred to herein, in their
entireties, are hereby incorporated by reference into this
application in order to more fully describe the state of the art as
known to those skilled therein.
[0032] When administered, compounds are often cleared rapidly from
the circulation and may therefore elicit relatively short-lived
pharmacological activity. Consequently, frequent injections of
relatively large doses of bioactive compounds may by required to
sustain therapeutic efficacy. Compounds modified by the covalent
attachment of water-soluble polymers such as polyethylene glycol,
copolymers of polyethylene glycol and polypropylene glycol,
carboxymethyl cellulose, dextran, polyvinyl alcohol,
polyvinylpyrrolidone or polyproline are known to exhibit
substantially longer half-lives in blood following intravenous
injection than do the corresponding unmodified compounds
(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al.,
1987). Such modifications may also increase the compound's
solubility in aqueous solution, eliminate aggregation, enhance the
physical and chemical stability of the compound, and greatly reduce
the immunogenicity and reactivity of the compound. As a result, the
desired in vivo biological activity may be achieved by the
administration of such polymer-compound adducts less frequently or
in lower doses than with the unmodified compound.
[0033] Attachment of polyethylene glycol (PEG) to compounds is
particularly useful because PEG has very low toxicity in mammals
(Carpenter et al., 1971). For example, a PEG adduct of adenosine
deaminase was approved in the United States for use in humans for
the treatment of severe combined immunodeficiency syndrome. A
second advantage afforded by the conjugation of PEG is that of
effectively reducing the immunogenicity and antigenicity of
heterologous compounds. For example, a PEG adduct of a human
protein might be useful for the treatment of disease in other
mammalian species without the risk of triggering a severe immune
response. The compound of the present invention capable of
improving wound healing in a subject may be delivered in a
microencapsulation device so as to reduce or prevent a host immune
response against the compound or against cells which may produce
the compound. The compound of the present invention may also be
delivered microencapsulated in a membrane, such as a liposome.
[0034] Polymers such as PEG may be conveniently attached to one or
more reactive amino acid residues in a protein such as the
alpha-amino group of the amino terminal amino acid, the epsilon
amino groups of lysine side chains, the sulfhydryl groups of
cysteine side chains, the carboxyl groups of aspartyl and glutamyl
side chains, the alpha-carboxyl group of the carboxy-terminal amino
acid, tyrosine side chains, or to activated derivatives of glycosyl
chains attached to certain asparagine, serine or threonine
residues.
[0035] Numerous activated forms of PEG suitable for direct reaction
with proteins have been described. Useful PEG reagents for reaction
with protein amino groups include active esters of carboxylic acid
or carbonate derivatives, particularly those in which the leaving
groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or
1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing
maleimido or haloacetyl groups are useful reagents for the
modification of protein free sulfhydryl groups. Likewise, PEG
reagents containing amino hydrazine or hydrazide groups are useful
for reaction with aldehydes generated by periodate oxidation of
carbohydrate groups in proteins.
[0036] The invention also provides a kit which comprises a
therapeutic amount of an agent, which agent is capable of
inhibiting binding of advanced glycation endproducts to a receptor
for advanced glycation endproducts, over a sufficient period of
time in a sufficient amount so as to treat chronic symptoms of
diabetes in the subject. A kit may include a composition which
includes sRAGE or a portion thereof in a form which is previously
dose regulated and time regulated so that a subject may easily take
such therapeutic at home or away from a clinical setting.
[0037] This invention is illustrated in the Experimental Details
section which follows. These sections are set forth to aid in an
understanding of the invention but are not intended to, and should
not be construed to, limit in any way the invention as set forth in
the claims which follow thereafter.
EXPERIMENTAL DETAILS
Example 1
Improved Wound Healing in Diabetic Mice by Treatment with the
Soluble Receptor for Advanced Glycation Endproducts (sRAGE)
[0038] Defective wound healing in diabetes continues to be an
important cause of morbidity in the postoperative period, following
trauma, and in the repair of cutaneous lesions. Advanced Glycation
Endproducts (AGEs) are the result of nonenzymatic
glycation/oxidation of proteins/lipids. Accelerated formation and
accumulation of AGEs in tissues of patients with diabetes has been
linked, in certain situations, to the development of secondary
complications. An important means by which AGEs perturb homeostatic
processes is through their interaction with cellular binding sites;
the best characterized of these is Receptor for AGE or RAGE, an
immunoglobulin superfamily molecule expressed by endothelium,
monocytes, and smooth muscle cells, as well as mesangial cells and
neurons. AGE engagement of RAGE leads to endothelial activation,
with expression of adhesion molecules, enhanced procoagulant
properties, and diminished barrier function; and perturbation of
monocytes, with changes in cell motility and activation, resulting
in expression of proinflammatory cytokines. The interaction of AGES
with RAGE-bearing cells, especially endothelium and mononuclear
phagocytes, may promote chronic cellular activation thereby
preventing optimal wound healing as reflected by formation of
granulation tissue and new connective tissue. The data herein are
consistent with this concept: using a secondary intention wound
model in diabetic mice, wound closure is enhanced following
administration of soluble(s) RAGE, the extracellular domain of the
receptor. These experiments contribute to a long-term goal and
long-felt need, understanding the contribution of cellular
interactions of AGEs in the pathogenesis of diabetic
complications.
[0039] Poor wound healing in diabetes is likely to be a
manifestation of a basic defect in the host inflammatory-reparative
response, in addition to possible underlying vascular
insufficiency. Exposure of macromolecules to aldose sugars results
in nonenzymatic glycation and oxidation (Baynes, 1991; Sell and
Monnier, 1989; Ruderman et al., 1992; and Vlassara et al., 1994),
initially the reversible early glycation adducts, Schiff bases and
Amadori products, form. Following further complex molecular
rearrangements, the irreversible AGEs come about. The latter
comprise a heterogenous group of structures characterized by
fluorescence, propensity to form cross-links, generation of
reactive oxygen intermediates (ROIs) and interaction with cellular
receptors, the best characterized of which is Receptor for AGE, or
RAGE (Schmidt et al., 1992; Neeper et al., 1992; and Schmidt et
al., 1994a). AGEs accumulated in the tissues in diabetes influence
end-organ function by two general mechanisms: directly, via effects
on tissue architecture, consequent to the formation of cross-links
and trapping of plasma proteins, and indirectly, by interaction
with cellular elements, such as endothelial cells (Ecs),
mononuclear phagocytes (Mps), central to homeostasis as well as the
host response to pathophysiologically relevant stimuli.
[0040] Studies have suggested that the extracellular two-thirds of
the molecule, soluble or sRAGE, appeared to be able to inhibit the
interaction of circulating AGEs with cellular surfaces (Schmidt et
al., 1994b). For example, binding of radiolabelled AGE albumin, a
prototypic ligand developed in the laboratory, to cultured
endothelial cells or peripheral blood-derived mononuclear
phagocytes, was inhibited in the presence of increasing doses of
sRAGE. In vivo, clearance of radiolabelled AGE albumin from the
circulation of a normal mouse after intravenous injection, was
delayed upon treatment with sRAGE. Extrapolation of these findings
was attempted to the setting of wound healing. The goal in these
studies was to assess the role of AGE-RAGE interaction in the
setting of the host response to wounding.
[0041] In order to assess the contribution of AGE-RAGE interaction
to defective wound healing in diabetes, the wound healing response
in diabetic was compared to normal animals, and to determine if
blockade of RAGE would ameliorate wound closure in diabetes. In
these studies, it was found that administration of soluble RAGE
improved wound healing in genetically-diabetic mice. These data
support the hypothesis that RAGE blockade may represent a feasible
target for intervention in diabetic wound healing as well as other
complications of diabetes, such as renal, retinal, neurological,
cardiovascular, cerebrovascular and peripheral vascular diseases.
Diabetic subjects experience increased restenosis and local
problems after angioplasty which suggests that soluble RAGE may be
beneficial in reducing restenosis after balloon/stent injury.
Materials and Methods
[0042] Murine model of diabetes. A genetic model of
insulin-resistant/hyperglycemic diabetes (db+/db+mice) due to an
autosomal recessive trait (chromosome 4) which results in
abnormalities of glucose metabolism and obesity in homozygote mice
was employed. Heterozygote mice (db+/+m) do not develop these
abnormalities, and are employed as controls (Coleman, 1982 and Wyse
and Dulin, 1970). Diabetic animals are hyperglycemic
(glucose>400 mg/dl by age 3 months), and develop abnormalities
similar to human complications, including a defective wound repair.
Life expectancy of homozygote mice is 6-8 months. Wounding studies
began when mice reached 8 weeks of age, as AGEs are present by that
time.
[0043] Model of wound healing. For analysis of wound healing in
diabetes, a secondary intention wound model was employed
(Greenhalgh et al., 1990), as it stimulates, in part, the clinical
situation following breakdown of skin in an ulcerated area. A
full-thickness 1.5.times.1.5 cm wound was created on the back of
the mouse which was subsequently covered by TEGADERM (clear,
plastic closure). The initial area of the wound was measured by
placing a sterile glass slide over the area, and tracing the edges
of the wound. The area was then determined by using a computer
program (NIH Image 157). Serial measurement of the wound dimensions
were made on days 3, 5, 7, 10, 14, and 17. This data, consistent
with those of previous studies (Greenhalgh et al., 1990), showed
significant delay of wound repair in the diabetic mouse especially
within the first 2-3 weeks after creation of the wound. Animals in
each group were sacrificed at days 17 for analysis. Studies began
when mice reached 8-10 weeks of age. In certain experiments, mice
were treated with soluble RAGE (the extracellular two-thirds of the
molecule) under the TEGADERM on days 3 through 9 after the initial
wounding procedure.
Immunohistochemistry for Detection of Advanced Glycation
Endproducts.
[0044] At the time of the wounding procedure, 1.5.times.1.5 cm
wounds were excised, fixed in formalin (10%) and then processed for
immunohistochemistry Using affinity-purified anti-AGE IgG (Miyata
et al., 1996).
Results
[0045] In order to understand the role of RAGE in diabetic wound
healing, 1.5.times.1.5 cm wounds were created on the backs of
db+/db+ or db+/m+mice. It was first determined that there was no
statistically-significant difference in original wound area among
the groups of mice receiving the various treatment regimens. When
sRAGE (200 ng/day) was administered under the TEGADERM daily from
days 3 through 9, the wound healing observed in diabetic mice was
significantly enhanced compared with diabetic mice treated with
vehicle (phosphate buffered saline; p<0.05; FIG. 1).
Furthermore, the healing observed in diabetic mice treated with
sRAGE approximated that observed in control, db+/m+ mice treated
with vehicle (differences were not statistically significant).
(FIG. 1).
[0046] Consistent with the hypothesis that these findings were due
to receptor-mediated mechanisms, dose-response studies revealed
that there was no enhancement of diabetic wound healing upon
administration of sRAGE, 2,000 ng/day, compared with a daily dose
of 200 ng/day (differences were not significant; FIG. 2). However,
consistent with the studies described herein in diabetic mice,
treatment with either 200 or 2,000 ng/day sRAGE (administered on
days 3 through 9) was significantly superior to treatment of these
mice with phosphate buffered saline when the final wound area was
measured on day ten after creation of the wound (FIG. 2). However,
at a daily dose of sRAGE of 20 ng/day, there was no significant
difference in wound healing in the diabetic mice receiving sRAGE
versus those diabetic mice receiving vehicle. (FIG. 2).
[0047] In order to determine if diabetic wounds were enriched in
AGE-immunoreactive material, immunohistochemistry was performed of
diabetic versus control mice wounds using affinity-purified
anti-AGE IgG. These studies demonstrated that there was a
significant increase in AGE-reactive material in the wound tissue
of the diabetic mice (FIG. 3A) compared with the nondiabetic
control animals (FIG. 3B).
Discussion
[0048] The results of these studies indicate that in diabetic
tissue such as wounds, there is increased deposition/formation of
AGEs. Such AGEs, upon interaction with their cellular receptor
RAGE, result in the generation of a sustained inflammatory
environment in which healing and quiescence of the potent effector
cells and mediators is markedly delayed. It was hypothesized that
interference with AGE-RAGE interaction might result in accelerated
healing. In these studies, it was demonstrated that local
administration of soluble RAGE improved diabetic wound healing in a
dose-dependent manner. The specific mechanisms which underlie the
efficacy of administration of sRAGE is important. It is possible
that administration of sRAGE improves any one of a number of
important steps in physiologic wound healing such as inflammation,
angiogenesis and/or formation and deposition of new granulation
tissue, specifically collagen.
[0049] Taken together, these data suggest that in an AGE-enriched
environment such as that observed in diabetes, interference with
AGE-cellular RAGE interaction might result in amelioration of the
chronic complications of diabetes. Given that RAGE is expressed in
the endothelium and smooth muscle of the vasculature, in mesangial
cells, in certain neural and vascular cells of the retina, and in
certain neurons of both the central and peripheral nervous systems
as well as other cells, it is likely that blockade of cellular RAGE
might result in improved diabetic complications that might
otherwise lead to heart attacks, stroke, peripheral vascular
disease, amputation of the extremities, kidney disease/failure,
blindness, impotence and neuropathy. RAGE is found in monocytes and
macrophages and may be present in other cell types wherein
therapeutic intervention may also be possible. The present studies
support the concept that administration of sRAGE (or other forms of
RAGE blockade; such as recombinant sRAGE, RAGE-based peptides,
anti-RAGE IgG or anti-RAGE F(ab').sub.2) might present a novel form
of therapeutic intervention in this chronic, debilitating
disorder.
REFERENCES
[0050] Bagdade, J. et al. (1978) Impaired granulocyte adherence. A
reversible defect in host defense in patients with poorly
controlled diabetes. Diabetes 27:677-681. [0051] Baynes, J. (1991)
Role of oxidative stress in development of complications in
diabetes. Diabetes 40:405-412. [0052] Coleman, D. (1982)
Diabetes-obesity syndromes in mice. Diabetes 31 (Suppl.):1-6.
[0053] Fahey, T. et al. (1991) Diabetes impairs the late
inflammatory response to wound healing. Surg. Res. 50:308-313.
[0054] Galloway, J. and Shuman, D. (1963) Diabetes and Surgery. Am.
J. Med. 34:177-191. [0055] Giardino, I. et al. (1994) Nonenzymatic
glycosylation in vitro and in bovine endothelial cells after basic
fibroblast growth factor activity. J. Clin. Invest. 94:110-117.
[0056] Goodson, W. and Hunt T. (1977) Studies of wound healing in
experimental diabetes mellitus. J. Surg. Res. 22:221-227. [0057]
Goodson, W. and Hunt T. (1986) Wound collagen accumulation in obese
hyperglycemic mice. Diabetes 35:491-495. [0058] Greenhalgh, D. et
al. (1990) PDGF and FGF stimulate wound healing in the genetically
diabetic mouse. Am. J. Pathol. 136:1235-1246. [0059] Mowat, A. and
Baum, J. (1971) Chemotaxis of polymorphonuclear leukocytes from
patients with diabetes mellitus. NEJM 284:621-627. [0060] Neeper,
M. et al. (1992) Cloning and expression of RAGE: a cell surface
receptor for AGEs. J. Biol. Chem. 267:14998-15004. [0061] Nolan, C.
et al. (1978) Further characterization of the impaired bactericidal
function of granulocytes in patients with poorly controlled
diabetes. Diabetes 27:889-894. [0062] Pearl, S. and Kanat, I.
(1988) Diabetes and healing: a review of the literature. J. Foot
Surg. 27:268-273. [0063] Reynolds, C. (1985) Management of the
diabetic surgical patient. A systematic but flexible plan is the
key. Postgrad. Med. 77:265-279. [0064] Ruderman, N. et al. (1992)
Glucose and diabetic vascular disease. FASEB J. 6:2905-2914. [0065]
Schmidt, A-M et al. (1994a) Cellular receptors for AGEs.
Arterioscler. Thromb. 14:1521-1528. [0066] Schmidt, A-M. et al.
(1994b) RAGE has a central role in vessel wall interactions and
gene activation in response to AGESs. PNAS, USA 91:8807-8811.
[0067] Schmidt, A-M et al. (1992) Isolation and characterization of
binding proteins for AGEs from lung tissue which are present on the
endothelial surface. J. Biol. Chem. 267:14987-14997. [0068] Sell,
D. and Monnier, V. (1989) Structure elucidation of senescence
cross-link from human extracellular matrix. J. Biol. Chem.
264:21597-21602. [0069] Vlassara, H. et al. (1994) Pathogenic
effects of AGEs: biochemical, biologic, and clinical implications
for diabetes and aging. Lab. Invest. 70:138-151. [0070] Wyse, B.
and Dulin, W. (1970) The influence of age and dietary conditions on
diabetes in the Db mouse. Diabetologia 6:268-273.
Sequence CWU 1
1
411438DNABovine 1cggagaagga tggcagcagg ggcagtggtc ggagcctgga
tgctagtcct cagtctgggg 60gggacagtca cgggggacca aaacatcaca gcccggatcg
ggaagccact ggtgctgaac 120tgcaagggag cccccaagaa accaccccag
cagctggaat ggaaactgaa cacaggccgg 180acagaagctt ggaaagtcct
gtctccccag ggagacccct gggatagcgt ggctcgggtc 240ctccccaacg
gctccctcct cctgccggct gttgggatcc aggatgaggg gactttccgg
300tgccgggcaa cgagccggag cggaaaggag accaagtcta actaccgagt
ccgagtctat 360cagattcctg ggaagccaga aattgttgat cctgcctctg
aactcatggc tggtgtcccc 420aataaggtgg ggacatgtgt gtccgagggg
ggctaccctg cagggactct taactggctc 480ttggatggga aactctgatt
cctgatggca aaggagtgtc agtgaaggaa gagaccaaga 540gacacccaaa
gacagggctt ttcactctcc attcggagct gatggtgacc ccagctcggg
600gaggagctct ccaccccacc ttctcctgta gcttcacccc tggccttccc
cggcgccgag 660ccctgcacac ggcccccatc cagctcaggg tctggagtga
gcaccgaggt ggggagggcc 720ccaacgtgga cgctgtgcca ctgaaggaag
tccagttgtg gtagagccag aagggggagc 780agtagctcct ggtggtactg
tgaccttgac ctgtgaagcc cccgcccagc ccccacctca 840aatccactgg
atcaaggatg gcaggcccct gccccttccc cctggcccca tgctgctcct
900cccagaggta gggcctgagg accagggaac ctacagttgt gtggccaccc
atcccagcca 960tgggggggag gagagccgtg ctgtcagcgt cacgatcatc
gaaacaggcg aggaggggac 1020gactgcaggc tctgtggaag ggccggggct
ggaaacctag ccctgaccct ggggatcctg 1080ggaggcctgg ggacagtcgc
cctgctcatt ggggtcatcg tgtggcatcg aaggccggca 1140acgcaaagga
caggagagga aggtcccgga aaaccaggag gaggaagagg aggagagagc
1200ggaactgaac cagccagagg agcccgaggc ggcagagagc agcacaggag
ggccttgagg 1260agcccacggc cagacccgat ccatcagccc cttttctttt
cccacactct gttctggccc 1320cagaccagtt ctcctctgta taatctccag
cccacatctc ccaaactttc ttccacaacc 1380agagcctccc acaaaaagtg
atgagtaaac acctgccaca tttaaaaaaa aaaaaaaa 14382416PRTBovine 2Met
Ala Ala Gly Ala Val Val Gly Ala Trp Met Leu Val Leu Ser Leu1 5 10
15Gly Gly Thr Val Thr Gly Asp Gln Asn Ile Thr Ala Arg Ile Gly Lys
20 25 30Pro Leu Val Leu Asn Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln
Gln 35 40 45Leu Glu Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys
Val Leu 50 55 60Ser Pro Gln Gly Asp Pro Trp Asp Ser Val Ala Arg Val
Leu Pro Asn65 70 75 80Gly Ser Leu Leu Leu Pro Ala Val Gly Ile Gln
Asp Glu Gly Thr Phe 85 90 95Arg Cys Arg Ala Thr Ser Arg Ser Gly Lys
Glu Thr Lys Ser Asn Tyr 100 105 110Arg Val Arg Val Tyr Gln Ile Pro
Gly Lys Pro Glu Ile Val Asp Pro 115 120 125Ala Ser Glu Leu Met Ala
Gly Val Pro Asn Lys Val Gly Thr Cys Val 130 135 140Ser Glu Gly Gly
Tyr Pro Ala Gly Thr Leu Asn Trp Leu Leu Asp Gly145 150 155 160Lys
Thr Leu Ile Pro Asp Gly Lys Gly Val Ser Val Lys Glu Glu Thr 165 170
175Lys Arg His Pro Lys Thr Gly Leu Phe Thr Leu His Ser Glu Leu Met
180 185 190Val Thr Pro Ala Arg Gly Gly Ala Leu His Pro Thr Phe Ser
Cys Ser 195 200 205Phe Thr Pro Gly Leu Pro Arg Arg Arg Ala Leu His
Thr Ala Pro Ile 210 215 220Gln Leu Arg Val Trp Ser Glu His Arg Gly
Gly Glu Gly Pro Asn Val225 230 235 240Asp Ala Val Pro Leu Lys Glu
Val Gln Leu Val Val Glu Pro Glu Gly 245 250 255Gly Ala Val Ala Pro
Gly Gly Thr Val Thr Leu Thr Cys Glu Ala Pro 260 265 270Ala Gln Pro
Pro Pro Gln Ile His Trp Ile Lys Asp Gly Arg Pro Leu 275 280 285Pro
Leu Pro Pro Gly Pro Met Leu Leu Leu Pro Glu Val Gly Pro Glu 290 295
300Asp Gln Gly Thr Tyr Ser Cys Val Ala Thr His Pro Ser His Gly
Pro305 310 315 320Gln Glu Ser Arg Ala Val Ser Val Thr Ile Ile Glu
Thr Gly Glu Glu 325 330 335Gly Thr Thr Ala Gly Ser Val Glu Gly Pro
Gly Leu Glu Thr Leu Ala 340 345 350Leu Thr Leu Gly Ile Leu Gly Gly
Leu Gly Thr Val Ala Leu Leu Ile 355 360 365Gly Val Ile Val Trp His
Arg Arg Arg Gln Arg Lys Gly Gln Glu Arg 370 375 380Lys Val Pro Glu
Asn Gln Glu Glu Glu Glu Glu Glu Arg Ala Glu Leu385 390 395 400Asn
Gln Pro Glu Glu Pro Glu Ala Ala Glu Ser Ser Thr Gly Gly Pro 405 410
41531405DNAHuman 3ggggcagccg gaacagcagt tggagcctgg gtgctggtcc
tcagtctgtg gggggcagta 60gtaggtgctc aaaacatcac agcccggatt ggcgagccac
tggtgctgaa gtgtaagggg 120gcccccaaga aaccacccca gcggctggaa
tggaaactga acacaggccg gacagaagct 180tggaaggtcc tgtctcccca
gggaggaggc ccctgggaca gtgtggctcg tgtccttccc 240aacggctccc
tcttccttcc ggctgtcggg atccaggatg aggggatttt ccggtgcagg
300gcaatgaaca ggaatggaaa ggagaccaag tccaactacc gagtccgtgt
ctaccagatt 360cctgggaagc cagaaattgt agattctgcc tctgaactca
cggctggtgt tcccaataag 420gtggggacat gtgtgtcaga gggaagctac
cctgcaggga ctcttagctg gcacttggat 480gggaagcccc tggtgcctaa
tgagaaggga gtatctgtga aggaacagac caggagacac 540cctgagacag
ggctcttcac actgcagtcg gagctaatgg tgaccccagc ccggggagga
600gatccccgtc ccaccttctc ctgtagcttc agcccaggcc ttccccgaca
ccgggccttg 660cgcacagccc ccatccagcc ccgtgtctgg gagcctgtgc
ctctggagga ggtccaattg 720ggtggtggag ccagaaggtg gagcagtagc
tcctggtgga accgtaaccc tgacctgtga 780agtccctgcc cagccctctc
ctcaaatcca ctggatgaag gatggtgtgc cctgcccctt 840ccccccagcc
ctgtgctgat cctccctgag atagggcctc aggaccaggg aacctacagc
900tgtgtggcca cccattccag ccacgggccc caggaaagcc gtgctgtcag
catcagcatc 960atcgaaccag gcgaggaggg gccaactgca ggctctgtgg
gaggatcagg gctgggaact 1020ctagccctgg ccctggggat cctgggaggc
ctggggacag ccgccctgct cattggggtc 1080atcttgtggc aaaggcggca
acgccgagga gaggagagga ggccccagaa aaccaggagg 1140aagaggagga
gcgtgcagaa ctgaatcagt cggaggaacc tgaggcaggc gagagtacta
1200ctggagggcc ttgaggggcc cacagacaga tcccatccat cagctccctt
ttctttttcc 1260cttgaactgt tctggcctca gaccaactct ctcctgtata
atctctctcc tgtataaccc 1320caccttgcca agctttcttc tacaaccaga
gccccccaca atgatgatta aacacctgac 1380acatctttgc aaaaaaaaaa aaaaa
14054405PRTHuman 4Gly Ala Ala Gly Thr Ala Val Gly Ala Trp Val Leu
Val Leu Ser Leu1 5 10 15Trp Gly Ala Val Val Gly Ala Gln Asn Ile Thr
Ala Arg Ile Gly Glu 20 25 30Pro Leu Val Leu Lys Cys Lys Gly Ala Pro
Lys Lys Pro Pro Gln Arg 35 40 45Leu Glu Trp Lys Leu Asn Thr Gly Arg
Thr Glu Ala Trp Lys Val Leu 50 55 60Ser Pro Gln Gly Gly Gly Pro Trp
Asp Ser Val Ala Arg Val Leu Pro65 70 75 80Asn Gly Ser Leu Phe Leu
Pro Ala Val Gly Ile Gln Asp Glu Gly Ile 85 90 95Phe Arg Cys Arg Ala
Met Asn Arg Asn Gly Lys Glu Thr Lys Ser Asn 100 105 110Tyr Arg Val
Arg Val Tyr Gln Ile Pro Gly Lys Pro Glu Ile Val Asp 115 120 125Ser
Ala Ser Glu Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr Cys 130 135
140Val Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser Trp His Leu
Asp145 150 155 160Gly Lys Pro Leu Val Pro Asn Glu Lys Gly Val Ser
Val Lys Glu Gln 165 170 175Thr Arg Arg His Pro Glu Thr Gly Leu Phe
Thr Leu Gln Ser Glu Leu 180 185 190Met Val Thr Pro Ala Arg Gly Gly
Asp Pro Arg Pro Thr Phe Ser Cys 195 200 205Ser Phe Ser Pro Gly Leu
Pro Arg His Arg Ala Leu Arg Thr Ala Pro 210 215 220Ile Gln Pro Arg
Val Trp Glu Pro Val Pro Leu Glu Glu Val Gln Leu225 230 235 240Val
Val Glu Pro Glu Gly Gly Ala Val Ala Pro Gly Gly Thr Val Thr 245 250
255Leu Thr Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile His Trp Met
260 265 270Lys Asp Gly Val Pro Leu Pro Leu Pro Pro Ser Pro Val Leu
Ile Leu 275 280 285Pro Glu Ile Gly Pro Gln Asp Gln Gly Thr Tyr Ser
Cys Val Ala Thr 290 295 300His Ser Ser His Gly Pro Gln Glu Ser Arg
Ala Val Val Ser Ile Ser305 310 315 320Ile Ile Glu Pro Gly Glu Glu
Gly Pro Thr Ala Gly Ser Val Gly Gly 325 330 335Ser Gly Leu Gly Thr
Leu Ala Leu Ala Leu Gly Ile Leu Gly Gly Leu 340 345 350Gly Thr Ala
Ala Leu Leu Ile Gly Val Ile Leu Trp Gln Arg Arg Gln 355 360 365Arg
Arg Gly Glu Glu Arg Lys Ala Pro Glu Asn Gln Glu Glu Glu Glu 370 375
380Glu Arg Ala Glu Leu Asn Gln Ser Glu Glu Pro Glu Ala Gly Glu
Ser385 390 395 400Ser Thr Gly Gly Pro 405
* * * * *