U.S. patent application number 11/992152 was filed with the patent office on 2010-08-19 for polyphenol conjugates as rgd-binding compounds and methods of use.
This patent application is currently assigned to ORDWAY RESEARCH INSTITUTE, INC.. Invention is credited to Maria Alexander-Bridges, Paul J. Davis, Shaker A. Mousa.
Application Number | 20100209382 11/992152 |
Document ID | / |
Family ID | 37667164 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100209382 |
Kind Code |
A1 |
Alexander-Bridges; Maria ;
et al. |
August 19, 2010 |
Polyphenol Conjugates as RGD-Binding Compounds and Methods of
Use
Abstract
Provided herein are compositions and methods for preventing and
treating diseases and risk factors associated with metabolic
syndrome by targeting the RGD-binding site of selected intra- and
extracellular proteins. Exemplary compositions include
RGD-polyphenol conjugates via an ester linkage; polyphenol polymer
conjugated to RGD analogs or mimetics; and RGD polymer conjugates
linked to polyphenol.
Inventors: |
Alexander-Bridges; Maria;
(Boston, CA) ; Mousa; Shaker A.; (Wynantskill,
NY) ; Davis; Paul J.; (West Sand Lake, NY) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Assignee: |
ORDWAY RESEARCH INSTITUTE,
INC.
Albany
NY
|
Family ID: |
37667164 |
Appl. No.: |
11/992152 |
Filed: |
September 18, 2006 |
PCT Filed: |
September 18, 2006 |
PCT NO: |
PCT/US2006/036243 |
371 Date: |
November 3, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60718105 |
Sep 16, 2005 |
|
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Current U.S.
Class: |
424/78.31 ;
424/78.37; 435/6.16; 435/7.21; 436/86; 506/9; 514/456; 514/54;
514/685; 514/723 |
Current CPC
Class: |
A61K 47/60 20170801;
A61K 47/593 20170801; G01N 33/5023 20130101; A61K 47/58 20170801;
A61K 47/61 20170801; A61P 3/00 20180101; A61P 3/10 20180101; A61P
5/48 20180101; C12N 15/1086 20130101 |
Class at
Publication: |
424/78.31 ;
424/78.37; 514/54; 514/723; 514/456; 514/685; 436/86; 435/6; 506/9;
435/7.21 |
International
Class: |
A61K 31/74 20060101
A61K031/74; A61K 31/765 20060101 A61K031/765; A61K 31/785 20060101
A61K031/785; A61K 31/08 20060101 A61K031/08; A61K 31/352 20060101
A61K031/352; A61K 31/12 20060101 A61K031/12; G01N 33/50 20060101
G01N033/50; C12Q 1/68 20060101 C12Q001/68; C40B 30/04 20060101
C40B030/04; G01N 33/567 20060101 G01N033/567; A61K 31/715 20060101
A61K031/715; A61P 3/10 20060101 A61P003/10 |
Claims
1. A pharmaceutical composition for treating conditions associated
with metabolic syndrome in a subject in need thereof, comprising a
therapeutically effective dose of an RGD-binding compound, or
mimetics thereof, comprising a polyphenol conjugated to a
polymer.
2. The pharmaceutical composition of claim 1, wherein said
polyphenol is selected from the group consisting of resveratrol,
fisetin, butein, piceatannol, quercetin, and mimetics and analogs
thereof.
3. The pharmaceutical composition of claim 2, wherein said
polyphenol is resveratrol, or mimetics or analogs thereof.
4. The pharmaceutical composition of claim 1, wherein said polymer
is selected from the group consisting of polyvinyl alcohol,
polyacrylic acid, polyethylene glycol, polylactic acid, hyaluronic
acid, polyamidoamine, and combinations thereof.
5. A pharmaceutical composition for preventing fat cell
differentiation or fat cell accumulation in a subject in need
thereof, comprising a therapeutically effective dose of an
RGD-binding compound, or mimetics thereof.
6. The pharmaceutical composition of claim 5, wherein the compound
is polymer conjugated resveratrol, or mimetics or analogs
thereof.
7. A method of treating conditions associated with metabolic
syndrome comprising administering to a subject in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising a compound that binds to one or more intra-cellular
lipogenic proteins encoding the amino acid sequence
Arg-Gly-Asp.
8. The method of claim 7, wherein said intra-cellular protein is
selected from the group consisting of sirtuins, PI3 kinase, and
sterol regulated binding protein-1.
9. The method of claim 7, wherein said compound is polymer
conjugated resveratrol or mimetics or analogs thereof.
10. The method of claim 7, wherein the compound inhibits
insulin/integrin signaling, thereby treating conditions associated
with metabolic syndrome.
11. A method of treating conditions associated with metabolic
syndrome comprising administering to a subject in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising a compound that binds to one or more extra-cellular
proteins encoding the amino acid sequence Arg-Gly-Asp.
12. The method of claim 11, wherein said extra-cellular protein is
selected from the group consisting of insulin-like binding
protein-1 (igfbp-1), vegf, and osteopontin.
13. The method of claim 11, wherein the compound inhibits
insulin/integrin signaling, thereby treating conditions associated
with metabolic syndrome.
14. A method for identifying target peptides encoding the amino
acid sequence Arg-Gly-Asp, the method comprising: (a) providing an
affinity column containing at least one compound that binds to the
amino acid sequence Arg-Gly-Asp; (b) contacting said target
peptides with said at least one compound in said affinity column;
and (c) collecting the target peptides that bind to the compounds
in the affinity column, wherein the target peptides that bind to
the compounds in the affinity column contain a functional
RGD-binding site.
15. A method for identifying genes encoding the amino acid sequence
Arg-Gly-Asp that are regulated in opposing directions by insulin
and at least one RGD-binding compound, the method comprising: (a)
selectively stimulating expression of an unknown gene product that
encodes the amino acid sequence Arg-Gly-Asp; (b) isolating said
gene product from control, resveratrol- and insulin-stimulated
cells alone and in combination; (c) pooling said gene products; and
(c) analyzing the pools of gene product to identify genes regulated
in opposing directions by insulin and at least one RGD-binding
compound.
16. The method of claim 15, wherein said analyzing step includes
performing a gene expression microarray.
17. A method of screening for indicators of insulin sensitivity
that encode the amino acid sequence Arg-Gly-Asp, the method
comprising: (a) providing an affinity column containing at least
one compound that binds to the amino acid sequence Arg-Gly-Asp; (b)
introducing serum collected from a subject exposed to nutritional
manipulations, or from a subject afflicted with a condition
associated with metabolic syndrome to said column, said serum
containing at least one indicator of insulin sensitivity; and (c)
collecting at least one indicator that binds to the compounds in
the affinity column, wherein an indicator that binds to the
compounds in the affinity column encode the amino acid sequence
Arg-Gly-Asp and represents an indicator of insulin sensitivity.
18. The method of claim 17, wherein said indicators are
extra-cellular secretory proteins.
19. A method of screening a test compound for modulating insulin
sensitivity, the method comprising: (a) providing a cell culture
comprising a first cell line overexpressing a recombinant
expression construct containing at least one intra-cellular protein
encoding the amino acid sequence Arg-Gly-Asp, and a second cell
line overexpressing a recombinant expression construct containing
at least one mutant derivative of the protein expressed in said
first cell line; (b) adding the test compound to the cell culture;
(c) assaying the cell culture to determine whether the test
compound is taken up by the first and second cell lines; (d)
assaying the first and second cell lines to determine whether the
test compound binds the protein expressed by the first and second
cell line, wherein binding of the test compound to the protein
expressed by the first cell line, but not the protein expressed by
the second cell line, indicates that the test compound modulates
insulin activity.
20. The method of claim 19, wherein said mutant derivative encodes
the amino acid sequence Arg-Gly-Glu.
21. The method of claim 19, wherein the first cell line in step (a)
alternatively contains an extra-cellular protein encoding the amino
acid sequence Arg-Gly-Asp.
22. The method of claim 19, wherein the first and second cell line
contains preadipocytes.
23. The method of claim 19, wherein the test compound modulates
insulin activity by preventing insulin stimulated fat cell
differentiation or insulin stimulated fat accumulation.
24. The method of claim 19, wherein the test compound modulates
insulin activity by potentiating insulin stimulated fat cell
differentiation or insulin stimulated fat accumulation.
25. The method of claim 19, wherein the first cell line in step (a)
alternatively comprises a reporter gene driven by the promoter of a
gene product encoding the amino acid sequence Arg-Gly-Asp, and
wherein the second cell line in step (a) alternatively comprises a
reporter gene driven by the promoter of a gene product encoding the
amino acid sequence Arg-Gly-Glu.
26. The method of claim 19, further comprising the step of adding
insulin to the cell culture prior to performing step (b).
27. The method of claim 26, wherein the assaying step of step (d)
includes determining whether the test compound binds the promoter
expressed by the first and second cell line, wherein binding of the
test compound to the promoter expressed by the first cell line, but
not the promoter expressed by the second cell line, indicates that
the test compound modulates insulin activity.
28. The method of claim 19, wherein the method further comprises
the step of assaying libraries for a hit.
29. A method for inhibiting the activity or expression of a protein
encoding the amino acid sequence Arg-Gly-Asp, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising an
RGD-binding compound.
30. The method of claim 29, wherein the RGD-binding compound is
polymer conjugated resveratrol, or mimetics or analogs thereof.
31. The method of claim 29, wherein the RGD-binding compound
inhibits the synthesis of new proteins encoding the amino acid
sequence Arg-Gly-Asp.
32. The method of claim 29, wherein the protein is involved in the
insulin-signaling pathway.
33. The method of claim 29, wherein the RGD-binding compound
regulates fat cell differentiation and/or fat accumulation by
inhibiting a regulatory component involved in the activity or
synthesis of the protein encoding the amino acid sequence
Arg-Gly-Asp.
34. The method of claim 33, wherein said regulatory component is
any one, or a combination, of a member selected from the group
consisting of: insulin regulation, the effect of nutritional
manipulation or inflammatory processes, hormone/nuclear receptor
regulation, and coregulatory proteins that alter activity or
synthesis of transcription factors that regulate fat cell
differentiation and fat accumulation.
Description
FIELD OF THE INVENTION
[0001] The invention described herein pertains to compounds and
methods for the prevention or treatment of conditions associated
with metabolic syndrome, and more particularly to the use of
resveratrol-like compounds in the treatment of such conditions.
BACKGROUND
[0002] Metabolic syndrome (or Syndrome X) is characterized by a
group of metabolic risk factors in one person that include, for
example, central obesity (i.e., excessive fat tissue in and around
the abdomen), atherogenic dyslipidemia (i.e., blood fat
disorders--mainly high triglycerides and low HDL cholesterol--that
foster plaque buildups in artery walls), raised blood pressure
(e.g., 130/85 mmHg or higher), insulin resistance or glucose
intolerance (the body can't properly use insulin or blood sugar),
prothrombotic state (e.g., high fibrinogen or plasminogen activator
inhibitor in the blood), and proinflammatory state (e.g., elevated
high-sensitivity C-reactive protein in the blood).
[0003] The underlying causes of this syndrome are
overweight/obesity, physical inactivity and genetic factors. People
with the metabolic syndrome are at increased risk of coronary heart
disease, other diseases related to plaque buildups in artery walls
(e.g., stroke and peripheral vascular disease) and type 2
diabetes.
[0004] Metabolic syndrome has become increasingly common in the
United States. It's estimated that about 47 million U.S. adults
have it. The syndrome is closely associated with a generalized
metabolic disorder called insulin resistance, in which the body
cannot use insulin efficiently. This is why the metabolic syndrome
is also called the insulin resistance syndrome. Some people are
genetically predisposed to insulin resistance. Acquired factors,
such as excess body fat and physical inactivity, can elicit insulin
resistance and the metabolic syndrome in these people. Most people
with insulin resistance have central obesity. The biologic
mechanisms at the molecular level between insulin resistance and
metabolic risk factors are not fully understood and appear to be
complex.
[0005] Conventional treatment typically includes dietary changes to
limit fat and calories, increased exercise, and changes in habits
or patterns of eating. Medications commonly prescribed for weight
loss include numerous metabolic stimulants, perhaps in combination
with cholesterol lowering drugs and/or high blood pressure
medications. However, such compounds may not be effective in all
subjects, or may be of limited efficacy. Accordingly, new
treatments for metabolic syndrome and its associated factors are
needed.
[0006] Caloric restriction is known to extend lifespan in mammals.
Caloric restriction also reduces the incidence of age-associated
conditions such as obesity, insulin resistance, dyslipidemia, and
cancer. Resveratrol, a polyphenol derived from red wine, is a
"phytoestrogen" that mimics caloric restriction by activating Sir2
and extending lifespan in several species. Resveratrol is also
known to have anti-inflammatory and anti-angiogenic effects, as
well as preventive effects to atherosclerosis.
[0007] Resveratrol has been shown to reduce fat accumulation in C.
elegans and in several insulin-sensitive mammalian cell lines.
Inhibition of AMP-activated kinase gene expression is also known to
prevent the effect of resveratrol on fat accumulation. Accordingly,
the effect of resveratrol on fat accumulation depends on Sir2.1.
Thus, resveratrol-like compounds will prove useful in the
prevention and treatment of diseases and risk factors associated
with metabolic syndrome.
[0008] Consequently, it would be desirable to provide resveratrol
analogs, resveratrol-like compounds, and polymers thereof, as well
as methods of using such compounds in preventing and treating
diseases and risk factors associated with metabolic syndrome.
SUMMARY
[0009] In view of the foregoing, it is an object of the present
invention to provide resveratrol-like compounds and methods for
preventing and/or treating diseases and risk factors associated
with metabolic syndrome.
[0010] It is a further object of the invention to screen for
resveratrol-like compounds
[0011] It is yet a further object of the present invention to
provide methods of preventing and/or treating such disorders by
targeting one or more RGD sites in selected intracellular or
extracellular proteins.
[0012] Accordingly, these and other objects are accomplished by
providing compounds and methods for preventing and/or treating
diseases and risk factors associated with metabolic syndrome. In
one aspect, the invention includes a compound that binds an RGD
sequence is provided for the treatment of conditions associated
with metabolic syndrome. The compound can include, but is not
limited to, a polyphenol such as resveratrol, fisetin, butein,
piceatannol, or quercetin, conjugated to a polymer.
[0013] In certain embodiments, the polymer can include, for
example, polyvinyl alcohol, polyacrylic acid, polylactic acid, and
other polymers with different molecular weight ranging from
2,000-20,000 Dalton.
[0014] In another aspect, the invention includes a compound that
binds an RGD sequence and inhibits insulin/integrin-signaling to
fat cell differentiation and fat cell accumulation. The compound
can include, but is not limited to, resveratrol conjugated to a
polymer, encapsulated inside or immobilized on nanoparticles, or
mimetics and analogs or polymers thereof.
[0015] In another aspect, the invention includes methods of
treating conditions associated with metabolic syndrome by
administering a therapeutically effective amount of a compound that
inhibits insulin-integrin-signaling via one or more intracellular
lipogenic proteins, or one or more extracellular proteins,
containing the RGD sequence. In particular embodiments, the
insulin/integrin-signaling intracellular protein can include, for
example, sirtuins, PI3 kinase, or SREBP. In other embodiments, the
insulin-integrin-signaling extracellular protein can include, for
example, IGFBP-1, VEGF, or osteopontin.
[0016] The invention also provides methods for identifying target
peptides possessing RGD-containing binding sites by providing an
affinity column containing at least one resveratrol-like compound
or anti-angiogenic compound, contacting the target peptide with the
affinity column, and collecting the target peptides that bind to
the resveratrol-like compound or anti-angiogenic compound.
[0017] In still another aspect, the invention provides methods for
identifying genes that are regulated in opposing directions by
insulin and an RGD-binding compound by selectively stimulating
expression of an unknown gene product that encodes the RGD
sequence, isolating the gene product from control cells,
resveratrol-stimulated cells and insulin-stimulated cells, alone
and in combination, and analyzing the pools of gene product to
identify genes regulated in opposing directions by insulin and
RGD-binding compounds. In particular embodiments, the gene products
can include, for example, mRNA or protein. In further embodiments,
the analyzing step can include performing a gene expression
microarray or mass spectroscopy to identify RGD containing gene
products.
[0018] The invention further provides methods of screening a test
compound for modulating insulin sensitivity via an intracellular
protein containing an RGD sequence, via an extracellular protein
containing an RGD sequence, or by using the promoter of a gene
product that encodes the RGD sequence. In screening a test compound
for modulating insulin sensitivity via an intracellular protein
containing an RGD sequence, the method includes providing a
transformed cultured cell line overexpressing a recombinant
expression construct containing at least one RGD protein, and
another transformed cultured cell line overexpressing a mutant
derivative bearing RGE, adding a test compound to each of the cell
cultures, assaying the cell cultures to determine whether the test
compound is imported into the cell lines, assaying the cell
cultures to determine whether the desired phenotypic change has
occurred in wild-type but not mutant cell lines, and binding the
test compound to the wild-type protein, but not the mutant protein,
in vitro, wherein binding of the test compound to the wild-type
protein indicates that the protein modulates insulin activity.
[0019] In screening a test compound for modulating insulin
sensitivity via an extracellular protein containing an RGD
sequence, the method includes providing a cultured preadipocyte
cell line, adding the protein containing an RGD sequence and a
mutant protein containing an RGE sequence to the preadipocyte cell
culture, assaying the transformed cells to determine whether the
desired phenotypic change has occurred in wild-type but not mutant
proteins, adding the test compound in the presence and absence of
the RGD and RGE containing proteins, assaying the transformed cell
line to determine whether the test compound prevents fat cell
differentiation and/or fact accumulation, and determining whether
the test compound binds to the wild-type but not the mutant
protein, wherein binding of the test compound to the wild-type but
not the mutant protein indicates that the test compound modulates
insulin sensitivity by reducing adipogenesis.
[0020] In screening a test compound for modulating insulin
sensitivity by using the promoter of a gene containing an RGD
sequence, the method includes providing a cultured cell line with a
reporter gene driven by the promoter of a gene product regulated at
the transcription level by insulin or an RGD-binding compound,
adding insulin to the cell culture, adding an RGD-binding compound
to the cell culture, assaying the transformed cell to determine
whether the effect of insulin on the promoter is inhibited by the
RGD-binding compound, assaying libraries for a hit, and determining
whether the test compound binds a wild-type but not a mutant
protein, wherein binding indicates that the test compound modulates
insulin sensitivity.
[0021] In still a further aspect, the invention provides methods
for inhibiting the activity or expression of a protein containing
an RGD sequence involved in the insulin-signaling pathway by
administering an effective amount of an RGD-binding compound to a
subject suffering from an insulin-signaling disorder and/or a
disorder associated with metabolic syndrome. In certain
embodiments, the insulin-signaling disorder includes, but is not
limited to, obesity, insulin resistance, diabetes, and complication
thereof such as hyperlipidemia, cardiovascular disease,
neurological disease, and renal disease. In other embodiments, the
protein containing the RGD sequence is currently expressed, and the
RGD-binding compound inhibits the active protein as well as the
syntheses of new proteins containing the RGD sequence. In further
embodiments the RGD-binding compound inhibits the activities and/or
processes that regulate fat cell differentiation and fat
accumulation.
[0022] Other features and advantages of the invention will be
apparent from the detailed description and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 Representative polyphenols.
[0024] FIG. 2 Representative RGD analogs.
[0025] FIG. 3 Additional examples of RGD analogs.
[0026] FIG. 4 Polymer conjugates of polyphenolic compounds.
[0027] FIG. 5 Synthesis of polyphenolic polymer conjugates.
[0028] FIG. 6 Additional examples of polymer conjugates
[0029] FIG. 7 Polymer or protein bound catechin/epicatechin.
DETAILED DESCRIPTION
[0030] The details of one or more embodiments of the invention have
been set forth in the accompanying description below. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
Other features, objects, and advantages of the invention will be
apparent from the description and from the claims. In the
specification and the appended claims, the singular forms include
plural references unless the context clearly dictates otherwise.
All patents and publications cited in this specification are
incorporated by reference in their entirety.
[0031] For convenience, certain terms used in the specification,
examples and claims are collected here. Unless otherwise defined,
all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art
to which this invention pertains.
[0032] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule (such as a nucleic acid, an antibody, a protein or
portion thereof, e.g., a peptide), or an extract made from
biological materials such as bacteria, plants, fungi, or animal
(particularly mammalian) cells or tissues. The activity of such
agents may render it suitable as a "therapeutic agent" which is a
biologically, physiologically, or pharmacologically active
substance (or substances) that acts locally or systemically in a
subject.
[0033] The term "RGD" as used herein refers to the single letter
amino acid code and references the tripeptide amino acid sequence
arginine-glycine-aspartic acid (Arg-Gly-Asp).
[0034] The term "RGD-binding compound" is used herein to mean a
compound that modulates at least one activity of a protein encoding
the amino acid sequence Arg-Gly-Asp.
[0035] The terms "peptide mimetic", "mimetic", or "peptidomimetic"
as used herein refer to a compound that mimics at least one
activity of a peptide or compound. For example, an RGD-binding
compound mimetic refers to a compound that mimics a compound that
modulates at least one activity of a protein encoding the amino
acid sequence Arg-Gly-Asp.
[0036] A "form that is naturally occurring" when referring to a
compound means a compound that is in a form, e.g., a composition,
in which it can be found naturally. For example, since resveratrol
can be found in red wine, it is present in red wine in a form that
is naturally occurring. A compound is not in a form that is
naturally occurring if, e.g., the compound has been purified and
separated from at least some of the other molecules that are found
with the compound in nature. A "naturally occurring compound"
refers to a compound that can be found in nature, i.e., a compound
that has not been designed by man. A naturally occurring compound
may have been made by man or by nature.
[0037] "Sirtuin protein" refers to a member of the sirtuin
deacetylase protein family or preferably to the Sir2 family, which
include yeast Sir2 (GenBank Accession No. P53685), C. elegans
Sir-2.1 (GenBank Accession No. NP.sub.--501912), and human SIRT1
(GenBank Accession No. NM.sub.--012238 and NP.sub.--036370 (or
AF083106), and SIRT2 (GenBank Accession No. NM.sub.--030593 and
AF083107) proteins. Other family members include the four
additional yeast Sir2-like genes termed "HST genes" (homologues of
Sir two) HST1, HST2, HST3 and HST4, and the five other human
homologues hSIRT3, hSIRT4, hSIRT5, hSIRT6 and hSIRT7 (Brachmann et
al. (1995) Genes Dev. 9:2888 and Frye et al. (1999) BBRC 260:273).
Preferred sirtuins are those that share more similarities with
SIRT1, i.e., hSIRT1, and/or Sir2 than with SIRT2, such as those
members having at least part of the N-terminal sequence present in
SIRT1 and absent in SIRT2 such as SIRT3 has.
[0038] "Diabetes" refers to high blood sugar or ketoacidosis, as
well as chronic, general metabolic abnormalities arising from a
prolonged high blood sugar status or a decrease in glucose
tolerance. "Diabetes" encompasses both the type I and type II (Non
Insulin Dependent Diabetes Mellitus or NIDDM) forms of the disease.
The risk factors for diabetes include the following factors:
waistline of more than 40 inches for men or 35 inches for women,
blood pressure of 130/85 mmHg or higher, triglycerides above 150
mg/dl, fasting blood glucose greater than 100 mg/dl or high-density
lipoprotein of less than 40 mg/dl in men or 50 mg/dl in women.
[0039] The term "hyperinsulinemia" refers to a state in an
individual in which the level of insulin in the blood is higher
than normal.
[0040] The term "insulin resistance" refers to a state in which a
normal amount of insulin produces a subnormal biologic response
relative to the biological response in a subject that does not have
insulin resistance.
[0041] The term "metabolic syndrome" or "disorders or conditions
associated with metabolic syndrome," as discussed herein, refers to
any disease or condition that is caused by or contributed to by
insulin resistance. Examples include: diabetes, obesity,
insulin-resistance syndromes, syndrome X, insulin resistance, high
blood pressure, hypertension, high blood cholesterol, dyslipidemia,
hyperlipidemia, dyslipidemia, atherosclerotic disease including
stroke, coronary artery disease or myocardial infarction,
hyperglycemia, hyperinsulinemia and/or hyperproinsulinemia,
impaired glucose tolerance, delayed insulin release, diabetic
complications, including coronary heart disease, angina pectoris,
congestive heart failure, stroke, cognitive functions in dementia,
retinopathy, peripheral neuropathy, nephropathy,
glomerulonephritis, glomerulosclerosis, nephrotic syndrome,
hypertensive enephrosclerosis, some types of cancer (such as
endometrial, breast, prostate, and colon), complications of
pregnancy, poor female reproductive health (such as menstrual
irregularities, infertility, irregular ovulation, polycystic
ovarian syndrome (PCOS)), lipodystrophy, cholesterol related
disorders, such as gallstones, cholescystitis and cholelithiasis,
gout, obstructive sleep apnea and respiratory problems,
osteoarthritis, and prevention and treatment of bone loss, e.g.
osteoporosis.
[0042] "Obese" individuals or individuals suffering from obesity
are generally individuals having a body mass index (BMI) of at
least 25 or greater. Obesity may or may not be associated with
insulin resistance.
[0043] A "patient," "individual," "subject" or "host" refers to
either a human or a non-human animal.
[0044] The term "modulation" is art-recognized and refers to up
regulation (i.e., activation or stimulation), down regulation
(i.e., inhibition or suppression) of a response, or the two in
combination or apart.
[0045] The term "prophylactic" or "therapeutic" treatment is
art-recognized and refers to administration of a drug to a host. If
it is administered prior to clinical manifestation of the unwanted
condition (e.g., disease or other unwanted state of the host
animal) then the treatment is prophylactic, i.e., it protects the
host against developing the unwanted condition, whereas if
administered after manifestation of the unwanted condition, the
treatment is therapeutic (i.e., it is intended to diminish,
ameliorate or maintain the existing unwanted condition or side
effects therefrom).
[0046] The term "mammal" is known in the art, and exemplary mammals
include humans, primates, bovines, porcines, canines, felines, and
rodents (e.g., mice and rats).
[0047] The term "pharmaceutically-acceptable salt" is
art-recognized and refers to the relatively non-toxic, inorganic
and organic acid addition salts of compounds, including, for
example, those contained in compositions described herein.
[0048] The term "pharmaceutically acceptable carrier" is
art-recognized and refers to a pharmaceutically-acceptable
material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in
carrying or transporting any subject composition or component
thereof from one organ, or portion of the body, to another organ,
or portion of the body. Each carrier must be "acceptable" in the
sense of being compatible with the subject composition and its
components and not injurious to the patient. Some examples of
materials which may serve as pharmaceutically acceptable carriers
include: (1) sugars, such as lactose, glucose and sucrose; (2)
starches, such as corn starch and potato starch; (3) cellulose, and
its derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt;
(6) gelatin; (7) talc; (8) excipients, such as cocoa butter and
suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
(10) glycols, such as propylene glycol; (11) polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,
such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering
agents, such as magnesium hydroxide and aluminum hydroxide; (15)
alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)
Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer
solutions; and (21) other non-toxic compatible substances employed
in pharmaceutical formulations.
[0049] The terms "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" are art-recognized and refer to the administration of
a subject composition, therapeutic or other material other than
directly into the central nervous system, such that it enters the
patient's system and, thus, is subject to metabolism and other like
processes.
[0050] The terms "parenteral administration" and "administered
parenterally" are art-recognized and refer to modes of
administration other than enteral and topical administration,
usually by injection, and includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intra-articulare, subcapsular, subarachnoid, intraspinal, and
intrasternal injection and infusion.
[0051] "Treating" a condition or disease refers to curing as well
as ameliorating at least one symptom of the condition or
disease.
[0052] The term "therapeutic agent" is art-recognized and refers to
any chemical moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or
systemically in a subject. The term also means any substance
intended for use in the diagnosis, cure, mitigation, treatment or
prevention of disease or in the enhancement of desirable physical
or mental development and/or conditions in an animal or human.
[0053] The term "therapeutic effect" is art-recognized and refers
to a local or systemic effect in animals, particularly mammals, and
more particularly humans caused by a pharmacologically active
substance. The phrase "therapeutically-effective amount" means that
amount of such a substance that produces some desired local or
systemic effect at a reasonable benefit/risk ratio applicable to
any treatment. The therapeutically effective amount of such
substance will vary depending upon the subject and disease or
condition being treated, the weight and age of the subject, the
severity of the disease or condition, the manner of administration
and the like, which can readily be determined by one of ordinary
skill in the art. For example, certain compositions described
herein may be administered in a sufficient amount to produce a
desired effect at a reasonable benefit/risk ratio applicable to
such treatment.
[0054] The term "synthetic" is art-recognized and refers to
production by in vitro chemical or enzymatic synthesis.
Compositions
[0055] RGD is the single letter amino acid code for
arginine-glycine-aspartate. This tripeptide motif can be found in
proteins of the extracellular matrix. Integrins link the
intracellular cytoskeleton of cells with the extracellular matrix
by recognizing this RGD motif. Without attachment to the
extracellular matrix, cells normally undergo apoptosis. Soluble RGD
peptides induce apoptosis and might be used as drugs against
angiogenesis, inflammation and cancer mestastasis since small
soluble peptides containing the RGD motif inhibit cell attachment
and consequently induce apoptosis.
[0056] Provided herein are RGD-binding compounds for treating a
variety of conditions associated, directly or indirectly, with
metabolic syndrome. Exemplary compounds include, but are not
limited to, various polyphenols conjugated to a polymer.
Polyphenols can include, for example, resveratrol, fisetin, butein,
piceatannol, quercetin, and analogs thereof. In a preferred
embodiment, the polyphenol is resveratrol or a resveratrol-like
compound (i.e., a resveratrol analog).
[0057] Exemplary polymers that the polyphenols can be conjugated
with include, but are not limited to polyvinyl alcohol, polyacrylic
acid, or polylactic acid with different molecular weight ranging
from 2,000-20,000 Dalton.
[0058] The RGD-binding compounds disclosed herein can also be used
for preventing fat cell differentiation and/or fat accumulation. In
addition to polymer conjugated resveratrol, or analogs thereof,
RGD-binding compounds can include, for example, RGD, and analogs or
polymers thereof. In one embodiment, a polymer conjugated RGD
analog can include an integrin antagonist that inhibits
angiogenesis.
[0059] Structure models for RGD-binding compounds contemplated by
the present invention are more fully described in FIGS. 1-7.
The Role of Resveratrol or Resveratrol-Like Compounds, Analogs, and
Polymeric Conjugations in Affecting Insulin-Sensitivity
[0060] Also provided herein are methods for treating a variety of
conditions, directly or indirectly, associated with metabolic
syndrome. In one embodiment, the method includes administering to a
subject in need thereof a therapeutically effective amount of a
compound that binds to one or more intra-cellular or extra-cellular
lipogenic proteins encoding the amino acid sequence Arg-Gly-Asp
(i.e., an RGD-binding compound). Such RGD-binding compounds inhibit
insulin/integrin signaling, thereby treating conditions associated
with metabolic syndrome.
[0061] Intra-cellular proteins contemplated by the present
invention include, but are not limited to sirtuins, PI3 kinase, and
sterol regulated binding protein-1 ("SREBP-1"). Extra-cellular
proteins contemplated by the present invention include, but are not
limited to, insulin-like binding protein-1 ("igfbp-1"), vascular
endothelial growth factor ("vegf"), and osteopontin
[0062] As before, the compounds that bind the RGD sequence encoded
by the intra-cellular or extra-cellular proteins include, for
example, polymer conjugated resveratrol or analogs thereof, and RGD
analogs and polymers thereof.
Screens for Identifying Peptides Encoding the Amino Acid Sequence
Arg-Gly-Asp which Mediate the Adverse Effects of Conditions
Associated with Metabolic Syndrome
[0063] Also provided are methods for identifying target peptides
encoding the amino acid sequence Arg-Gly-Asp. Such peptides often
mediate the adverse effects of conditions associated with metabolic
syndrome via signaling pathways, e.g., the insulin-signaling
pathway or the integrin-signaling pathway. In one embodiment, the
method can include providing an affinity column containing at least
one compound that binds to the amino acid sequence Arg-Gly-Asp,
contacting the target peptides with the at least one compound in
the affinity column, and collecting the target peptides that bind
to the compounds in the affinity column. Target peptides that bind
to the compounds in the affinity column contain a functional RGD
binding site.
[0064] In another aspect, methods for identifying genes encoding
the amino acid sequence Arg-Gly-Asp that are regulated in opposing
directions by insulin and at least one RGD-binding compound are
provided. One method features selectively stimulating expression of
an unknown gene product that encodes the amino acid sequence
Arg-Gly-Asp, isolating the gene product from control, resveratrol-
and insulin-stimulated cells alone and in combination, pooling the
gene products, and analyzing the pools of gene product to identify
genes regulated in opposing directions by insulin and at least one
RGD-binding compound. In a preferred embodiment of the method, the
analyzing step includes performing a gene expression microarray. In
another embodiment, the analyzing step includes performing mass
spectroscopy. The gene product can be mRNA or a protein.
[0065] Also provided are methods of screening for indicators of
insulin sensitivity that encode the amino acid sequence
Arg-Gly-Asp. One method includes providing an affinity column
containing at least one compound that binds to the amino acid
sequence Arg-Gly-Asp, introducing serum collected from a subject
exposed to nutritional manipulations, or from a subject afflicted
with a condition associated with metabolic syndrome to the column,
wherein the serum contains at least one indicator of insulin
sensitivity, and collecting at least one indicator that binds to
the compounds in the affinity column. An indicator that binds to
the compounds in the affinity column encodes the amino acid
sequence Arg-Gly-Asp and represents an indicator of insulin
sensitivity. In a preferred embodiment, the indicators are
extra-cellular secretory proteins.
Screens for Identifying Resveratrol-Like Compounds which Bind to
Peptides Encoding the Amino Acid Sequence Arg-Gly-Asp.
[0066] In a further aspect, the invention also provides methods of
screening a test compound for modulating insulin sensitivity. The
target compound can modulate insulin activity by preventing insulin
stimulated fat cell differentiation or insulin stimulated fat
accumulation, or by potentiating insulin stimulated fat cell
differentiation or insulin stimulated fat accumulation. One method
includes providing a cell culture comprising a first cell line
overexpressing a recombinant expression construct containing at
least one intra-cellular protein encoding the amino acid sequence
Arg-Gly-Asp, and a second cell line overexpressing a recombinant
expression construct containing at least one mutant derivative of
the protein expressed in said first cell line. The test compound
can then be added to the Cell culture and the cell culture assayed
to determine whether the test compound is taken up by the first and
second cell lines. The first and second cell lines can also be
assayed to determine whether the test compound binds the protein
expressed by the first and second cell line. Binding of the test
compound to the protein expressed by the first cell line, but not
the protein expressed by the second cell line, indicates that the
test compound modulates insulin activity.
[0067] In a preferred embodiment of the method, the mutant
derivative encodes the amino acid sequence Arg-Gly-Glu, which is
different at only one amino acid location from the wild-type
protein. In another embodiment, the first cell line in step (a) can
alternatively contains an extra-cellular protein encoding the amino
acid sequence Arg-Gly-Asp. The first and second cell line can
contain, for example, preadipocytes.
[0068] In a further embodiment, the first cell line in step (a) can
alternatively contain a reporter gene driven by the promoter of a
gene product encoding the amino acid sequence Arg-Gly-Asp, whereas
the second cell line in step (a) can alternatively contain a
reporter gene driven by the promoter of a gene product encoding the
amino acid sequence Arg-Gly-Glu (i.e., a mutant to the gene product
expressed by the first cell line). This embodiment can further
feature the step of adding insulin to the cell culture prior to
adding the test compound to the cell culture. In a further
embodiment, the assaying step can include determining whether the
test compound binds the promoter expressed by the first and second
cell line. Binding of the test compound to the promoter expressed
by the first cell line, but not the promoter expressed by the
second cell line, indicates that the test compound modulates
insulin activity. Using this method, libraries of compounds can be
screened to determine if the library contains compounds capable of
binding to the amino acid sequence Arg-Gly-Asp, which is an
indicator of a potential pharmaceutical that binds to proteins
encoding the Arg-Gly-Asp amino acid sequence that mediates various
signaling pathways in conditions associated with metabolic
syndrome.
Methods for Inhibiting Peptides Encoding the Amino Acid Sequence
Arg-Gly-Asp which Affect Insulin Sensitivity
[0069] The present invention also provides methods for inhibiting
the activity or expression of a protein encoding the amino acid
sequence Arg-Gly-Asp. The method can include administering to a
subject in need thereof an effective amount of an RGD-binding
compound. In a preferred embodiment, the RGD-binding compound is
polymer conjugated resveratrol, or analogs thereof.
[0070] A typical subject in need of administration of an
RGD-binding compound as contemplated herein can include those
suffering from a variety of conditions associated with metabolic
syndrome. For example, the subject may be afflicted with any one,
or a combination, of the conditions selected from the group
consisting of insulin resistance, diabetes, obesity,
insulin-resistance syndromes, syndrome X, high blood pressure,
hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia,
atherosclerotic disease including stroke, coronary artery disease
or myocardial infarction, hyperglycemia, hyperinsulinemia and/or
hyperproinsulinemia, impaired glucose tolerance, delayed insulin
release, diabetic complications, including coronary heart disease,
angina pectoris, congestive heart failure, stroke, cognitive
functions in dementia, retinopathy, peripheral neuropathy,
nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic
syndrome, hypertensive enephrosclerosis, some types of cancer (such
as endometrial, breast, prostate, and colon), complications of
pregnancy, poor female reproductive health (such as menstrual
irregularities, infertility, irregular ovulation, polycystic
ovarian syndrome (PCOS)), lipodystrophy, cholesterol related
disorders, such as gallstones, cholescystitis and cholelithiasis,
gout, obstructive sleep apnea and respiratory problems,
osteoarthritis, and prevention and treatment of bone loss, e.g.
osteoporosis.
[0071] In one embodiment, the method is useful for inhibiting
synthesis of new proteins encoding the amino acid sequence
Arg-Gly-Asp, and/or inhibiting proteins that are currently
expressed. Such proteins can be involved in the insulin-signaling
and/or integrin-signaling pathways.
[0072] In another embodiment of the method, the RGD-binding
compound regulates fat cell differentiation and/or fat accumulation
by inhibiting a regulatory component involved in the activity or
synthesis of the protein encoding the amino acid sequence
Arg-Gly-Asp. The regulatory component can include, for example, any
one, or a combination, of a member selected from the group
consisting of: insulin regulation, the effect of nutritional
manipulation or inflammatory processes, hormone/nuclear receptor
regulation, and coregulatory proteins that alter activity or
synthesis of transcription factors that regulate fat cell
differentiation and fat accumulation.
Formulations
[0073] The RGD-binding compounds described above are preferably
administered in a formulation including analogs or polymeric forms,
and derivatives together with an acceptable carrier for the mode of
administration. Any formulation or drug delivery system containing
the active ingredients, which is suitable for the intended use, as
are generally known to those of skill in the art, can be used.
Suitable pharmaceutically acceptable carriers for oral, rectal,
topical or parenteral (including subcutaneous, intraperitoneal,
intramuscular and intravenous) administration are known to those of
skill in the art. The carrier must be pharmaceutically acceptable
in the sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0074] Formulations suitable for parenteral administration
conveniently include sterileaqueous preparation of the active
compound, which is preferably isotonic with the blood of the
recipient. Thus, such formulations may conveniently contain
distilled water, 5% dextrose in distilled water or saline. Useful
formulations also include concentrated solutions or solids
containing the compound of formula (I), which upon dilution with an
appropriate solvent give a solution suitable for parental
administration above.
[0075] For enteral administration, a compound can be incorporated
into an inert carrier in discrete units such as capsules, cachets,
tablets or lozenges, each containing a predetermined amount of the
active compound; as a powder or granules; or a suspension or
solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup,
an elixir, an emulsion or a draught. Suitable carriers may be
starches or sugars and include lubricants, flavorings, binders, and
other materials of the same nature.
[0076] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active compound
in a free-flowing form, e.g., a powder or granules, optionally
mixed with accessory ingredients, e.g., binders, lubricants, inert
diluents, surface active or dispersing agents. Molded tablets may
be made by molding in a suitable machine, a mixture of the powdered
active compound with any suitable carrier.
[0077] A syrup or suspension may be made by adding the active
compound to a concentrated, aqueous solution of a sugar, e.g.,
sucrose, to which may also be added any accessory ingredients. Such
accessory ingredients may include flavoring, an agent to retard
crystallization of the sugar or an agent to increase the solubility
of any other ingredient, e.g., as a polyhydric alcohol, for
example, glycerol or sorbitol.
[0078] Formulations for rectal administration may be presented as a
suppository with a conventional carrier, e.g., cocoa butter or
Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany), for a
suppository base.
[0079] Alternatively, the compound may be administered in liposomes
or microspheres (or microparticles). Methods for preparing
liposomes and microspheres for administration to a patient are well
known to those of skill in the art. U.S. Pat. No. 4,789,734, the
contents of which are hereby incorporated by reference, describes
methods for encapsulating biological materials in liposomes.
Essentially, the material is dissolved in an aqueous solution, the
appropriate phospholipids and lipids added, along with surfactants
if required, and the material dialyzed or sonicated, as necessary.
A review of known methods is provided by G. Gregoriadis, Chapter
14, "Liposomes," Drug Carriers in Biology and Medicine, pp. 287-341
(Academic Press, 1979).
[0080] Microspheres formed of polymers or proteins are well known
to those skilled in the art, and can be tailored for passage
through the gastrointestinal tract directly into the blood stream.
Alternatively, the compound can be incorporated and the
microspheres, or composite of microspheres, implanted for slow
release over a period of time ranging from days to months. See, for
example, U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214, and
Jein, TIPS 19:155-157 (1998), the contents of which are hereby
incorporated by reference.
[0081] In one embodiment, the RGD-binding compounds or its
polymeric forms can be formulated into nanoparticles.
[0082] Preferred nanoparticles are those prepared from
biodegradable polymers, such as polyethylene glycols,
polyglycolide, polylactide and copolymers thereof. Those of skill
in the art can readily determine an appropriate carrier system
depending on various factors, including the desired rate of drug
release and the desired dosage.
[0083] In one embodiment, the formulations are administered via
catheter directly to the inside of blood vessels. The
administration can occur, for example, through holes in the
catheter. In those embodiments wherein the active compounds have a
relatively long half life (on the order of 1 day to a week or
more), the formulations can be included in biodegradable polymeric
hydrogels, such as those disclosed in U.S. Pat. No. 5,410,016 to
Hubbell et al. These polymeric hydrogels can be delivered to the
inside of a tissue lumen and the active compounds released over
time as the polymer degrades. If desirable, the polymeric hydrogels
can have microparticles or liposomes which include the active
compound dispersed therein, providing another mechanism for the
controlled release of the active compounds.
[0084] The formulations may conveniently be presented in unit
dosage form and may be prepared by any of the methods well known in
the art of pharmacy. All methods include the step of bringing the
active compound into association with a carrier, which constitutes
one or more accessory ingredients. In general, the formulations are
prepared by uniformly and intimately bringing the active compound
into association with a liquid carrier or a finely divided solid
carrier and then, if necessary, shaping the product into desired
unit dosage form.
[0085] The formulations can optionally include additional
components, such as various biologically active substances such as
growth factors (including TGF-.beta., basic fibroblast growth
factor (FGF2), epithelial growth factor (EGF), transforming growth
factors .alpha. and .beta. (TGF alpha. and beta.), nerve growth
factor (NGF), platelet-derived growth factor (PDGF), and vascular
endothelial growth factor/vascular permeability factor (VEGF/VPF)),
antiviral, antibacterial, anti-inflammatory, immuno-suppressant,
analgesic, vascularizing agent, and cell adhesion molecule.
[0086] In addition to the aforementioned ingredients, the
formulations may further include one or more optional accessory
ingredient(s) utilized in the art of pharmaceutical formulations,
e.g., diluents, buffers, flavoring agents, binders, surface active
agents, thickeners, lubricants, suspending agents, preservatives
(including antioxidants) and the like.
[0087] The invention will be further illustrated in the following
non-limiting examples.
EXAMPLES
Example 1
Size Measurement of Resveratrol Doped Poly(Lactic-Co-Glycolic Acid)
(PLGA) or Poly(Lactic-Co-Glycolic Acid)/Polyvinyl Alcohol
(PLGA/PVA) Nanoparticles by Dynamic Light Scattering (DLS)
[0088] Resveratrol nanoparticles were prepared. The size of the
nanoparticles was determined by DLS (single emulsion/solvent
evaporation method), and ranged from 100-300 nm. The surface of
PLGA nanoparticles was coated with chitosan and/or PVA.
Example 2
Polymer Conjugated Polyphenols Activate AMPK and Stimulate Fat
Mobilization
[0089] Polyphenols, such as resveratrol, have beneficial effects on
dyslipidemia, which accelerates atherosclerosis in diabetes. Using
HEPG2 cell models or 3T3 adipocyte models it the polymer conjugated
polyphenols described herein were observed to activate AMPK and
stimulate fat mobilization. Specifically, the polymer conjugated
polyphenols described herein increased phosphorylation of AMPK and
its downstream target, acetyl-CoA carboxylase (ACC), and they
increased activity of AMPK. The polymer conjugated polyphenols
described herein also prevented the lipid accumulation that
occurred in HepG2 cells exposed to high glucose. These studies
demonstrate that the polymer conjugated polyphenols described and
claimed herein are useful as a new therapeutic avenue to treat
hyperlipidemia and atherosclerosis specifically in diabetes via
activating AMPK.
Other Embodiments
[0090] 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.
* * * * *