U.S. patent application number 11/827135 was filed with the patent office on 2009-03-05 for compositions and methods for treating obesity, obesity related disorders and for inhibiting the infectivity of human immunodeficiency virus.
Invention is credited to Ju Bao, Young Tae Chang, Paul L. Huang, Philip Lin Huang, Sylvia Lee-Huang, Jae Wook Lee, Yongtao Sun, Dawei Zhang, John Z.H. Zhang.
Application Number | 20090061031 11/827135 |
Document ID | / |
Family ID | 40407913 |
Filed Date | 2009-03-05 |
United States Patent
Application |
20090061031 |
Kind Code |
A1 |
Lee-Huang; Sylvia ; et
al. |
March 5, 2009 |
Compositions and methods for treating obesity, obesity related
disorders and for inhibiting the infectivity of human
immunodeficiency virus
Abstract
The present invention relates to methods and pharmaceutical
compositions for treating obesity or obesity-related disorders in a
subject suffering from or predisposed to developing obesity or an
obesity-related disorder, or for inhibiting the infectivity of HIV,
by administering oleuropein, an analogue or derivative thereof, or
the major metabolites of oleuropein including oleuropein aglycone,
hydroxytyrosol, and elenolic acid or their analogues, or
derivatives thereof, an iridoid glycoside, or a secoiridoid
glycoside or analogues or derivatives thereof, or any combination
of the foregoing including olive leave extract. The invention also
relates to methods for screening/diagnosing a subject having, or
predisposed to having obesity or a related disorder by measuring
the expression profiles of an adipogenic gene selected from
PPAR.gamma.2, LPL and .alpha.P2 gene and gene product, or other
adipogenic, lipogenic, or lipolytic genes and gene products in an
individual. The invention further provides for screening for novel
oleuropein analogues.
Inventors: |
Lee-Huang; Sylvia; (New
York, NY) ; Huang; Paul L.; (Boston, MA) ;
Huang; Philip Lin; (Maple Glen, PA) ; Zhang;
Dawei; (Elmhurst, NY) ; Zhang; John Z.H.; (New
York, NY) ; Chang; Young Tae; (New York, NY) ;
Lee; Jae Wook; (San Diego, CA) ; Bao; Ju;
(Brooklyn, NY) ; Sun; Yongtao; (Chaanxi Province,
CN) |
Correspondence
Address: |
KLAUBER & JACKSON
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
US
|
Family ID: |
40407913 |
Appl. No.: |
11/827135 |
Filed: |
July 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60819172 |
Jul 7, 2006 |
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60897702 |
Jan 26, 2007 |
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Current U.S.
Class: |
424/774 ; 514/27;
514/277; 514/331; 514/342; 514/369; 514/419; 514/42; 514/423;
514/460; 514/592; 514/6.5; 514/634; 514/635; 514/636; 514/734 |
Current CPC
Class: |
A61K 31/155 20130101;
A61K 31/445 20130101; A61K 31/40 20130101; A61K 31/435 20130101;
A61P 3/00 20180101; A61K 36/00 20130101; A61K 31/175 20130101; A61K
45/06 20130101; A61K 31/70 20130101; A61K 31/366 20130101; A61K
31/4439 20130101; A61K 31/351 20130101; A61K 31/427 20130101; A61K
31/05 20130101; A61K 31/7036 20130101; A61K 38/28 20130101; A61P
31/12 20180101; A61K 31/404 20130101; A61K 31/7048 20130101 |
Class at
Publication: |
424/774 ;
514/460; 514/734; 514/27; 514/636; 514/4; 514/369; 514/342;
514/423; 514/277; 514/419; 514/635; 514/634; 514/331; 514/42;
514/592 |
International
Class: |
A61K 36/00 20060101
A61K036/00; A61K 31/351 20060101 A61K031/351; A61K 31/70 20060101
A61K031/70; A61K 38/28 20060101 A61K038/28; A61K 31/4439 20060101
A61K031/4439; A61K 31/435 20060101 A61K031/435; A61K 31/366
20060101 A61K031/366; A61K 31/7036 20060101 A61K031/7036; A61P 3/00
20060101 A61P003/00; A61P 31/12 20060101 A61P031/12; A61K 31/175
20060101 A61K031/175; A61K 31/445 20060101 A61K031/445; A61K 31/404
20060101 A61K031/404; A61K 31/40 20060101 A61K031/40; A61K 31/427
20060101 A61K031/427; A61K 31/155 20060101 A61K031/155; A61K 31/05
20060101 A61K031/05 |
Claims
1. A method of modulating adipocyte differentiation or adipogenic
gene or gene product expression or lipolytic gene or gene product
expression, comprising administering a therapeutically effective
amount of oleuropein or an analogue, derivative, or oleuropein
aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogue, derivative, or any combination thereof
including but not limited to olive leave extract to a mammalian
subject in need thereof.
2. A method of treating, controlling or preventing obesity, or of
reducing body weight, or of inhibiting fat accumulation in vivo, or
of promoting fat burning in vivo, comprising administering a
therapeutically effective amount of oleuropein or an analogue,
derivative or oleuropein aglycone or their analogues, or
derivatives thereof, or hydrotyrosol, or dihydroxy phenol or their
analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside or their analogue, derivative, or any
combination thereof including but not limited to olive leave
extract to a mammalian subject in need thereof.
3. A method of treating, controlling or preventing the onset of one
or more obesity related disorders or conditions selected from the
group consisting of diabetes mellitus, hyperglycemia,
hyperlipidemia, hypercholesteremia, atherosclerosis, hypertension,
hypertriglyceridemia, insulin resistance, or hyperinsulinemia, in a
mammalian subject in need of treatment, comprising administering a
therapeutically effective amount of oleuropein or an analogue,
derivative or oleuropein aglycone or their analogues, or
derivatives thereof, or hydrotyrosol, or dihydroxy phenol or their
analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside or their analogue, derivative, or any
combination thereof including but not limited to olive leave
extract.
4. A method of treating, controlling or preventing the onset of one
or more obesity related disorders or conditions selected from the
group consisting of Asthma and related diseases including but not
limited to, Allergy, Atopic Dermatitis (Eczema), Gastroesophageal
Reflux Disease, Pneumothorax, Airway, Pulmonary and Lung disorders,
Churg-Strauss Syndrome in a mammalian subject in need of treatment,
comprising administering a therapeutically effective amount of
oleuropein or an analogue, derivative or oleuropein aglycone or
their analogues, or derivatives thereof, or hydrotyrosol, or
dihydroxy phenol or their analogues, or derivatives thereof, or
elenolic acid or their analogues, or derivatives thereof, or an
iridoid glycoside, or a secoiridoid glycoside or their analogue,
derivative, or any combination thereof including but not limited to
olive leave extract.
5. A method of treating, controlling or preventing the onset of one
or more obesity related disorders or conditions selected from the
group consisting of AIDS and HAART related diseases including but
not limited to lipodystrophy in a mammalian subject in need of
treatment, comprising administering a therapeutically effective
amount of oleuropein or an analogue, derivative or oleuropein
aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogue, derivative, or any combination thereof
including but not limited to olive leave extract.
6. A method of treating, controlling or preventing the onset of one
or more viral infection related disorders or conditions selected
from the group consisting of viral infection related diseases
including but not limited to the AIDS virus HIV-1, and other
viruses with type I transmembrane envelope glycoprotein, such as
simian immunodeficiency viruses (SIV), Sendai virus, feline
immunodeficiency virus (FIV), respiratory syncytial virus (RSV),
measles virus, Ebola virus, Nipah and Hendra viruses, the severe
acute respiratory syndrome associated coronavirus (SARS-CoV), and
the avain flu virus in mammalian, avian, poultry, non human
primates subjects in need of treatment, comprising administering a
therapeutically effective amount of oleuropein or an analogue,
derivative or oleuropein aglycone or their analogues, or
derivatives thereof, or hydrotyrosol, or dihydroxy phenol or their
analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside or their analogue, derivative, or any
combination thereof including but not limited to olive leave
extract.
7. The method of claim 1, wherein the adipogenic genes and gene
products whose expression is modulated includes, but is not limited
to, PPAR.gamma., PPAR.gamma.2, LPL and the .alpha.P2 genes and gene
products.
8. The method of claim 1, wherein the lipolytic genes and gene
products whose expression is modulated including but not limited to
PPAR .delta. and its modulated genes and gene products.
9. The method of claim 1, wherein the lipogenic or lipolytic genes
whose expression is modulated including but not limited to PPAR
.alpha. and its modulated genes and gene products.
10. A method of de-differentiation and transdifferentiating
adipocytes into osteoblasts (bone cells), myoblasts (muscle cells),
and chondrocytes (cartilage cells), the method comprising
administering to a mammal in need thereof a therapeutically
effective amount of oleuropein or an analogue, derivative or
oleuropein aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogue, derivative, or any combination thereof
including but not limited to olive leave extract.
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18. The method of claim 2 further comprising administering a second
agent in combination with oleuropein, or an analogue or oleuropein
aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogue, derivative, or any combination thereof
including but not limited to olive leave extract.
19. The method of claim 18, wherein the second agent is selected
from the group consisting of a different PPAR modulating agent, a
cholesterol or lipid lowering agent, a biguanide, insulin, an
antihyperglycemic agent and any agent useful for treating metabolic
syndrome or type 2 diabetes.
20. The method of claim 19, wherein the different PPAR modulating
agent is selected from the group consisting of troglitazone,
pioglitazone and rosiglitazone.
21. The method of claim 19, wherein the cholesterol or lipid
lowering agent is a HMG-CoA reductase inhibitor, and wherein the
HMG-CoA reductase inhibitor is a statin selected from the group
consisting of atorvastatin, bervastatin, cerivastatin, dalvastatin,
fluvastatin, itavastatin, lovastatin, mevastatin, nicostatin,
nivastatin, pravastatin and simvastatin.
22. The method of claim 19, wherein the biguanide is selected from
the group consisting of metformin, phenformin, and buformin.
23. The method of claim 19, wherein the antihyperglycemic is a
prandial glucose regulator or an alpha-glucosidase inhibitor.
24. The method of claim 23, wherein the prandial glucose regulator
is repaglinide or nateglinide.
25. The method of claim 23, wherein the alpha-glucosidase inhibitor
is selected from the group consisting of acarbose, voglibose and
miglitol.
26. The method of claim 19, wherein the agent useful for treating
metabolic syndrome or type 2 diabetes is a sulfonylurea selected
from the group consisting of glimepiride, glibenclamide
(glyburide), gliclazide, glipizide, gliquidone, chloropropamide,
tolbutamide, acetohexamide, glycopyramide, carbutamide,
glibonuride, glisoxepid, glybuthiazole, glibuzole, glyhexamide,
glymidine, glypinamide, phenbutamide, tolcylamide and
tolazamide.
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Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to methods and
compositions for modulating the body weight of mammals including
animals and humans, and more particularly to materials identified
herein as modulators of weight, and the use of these materials for
treating obesity and disorders related to obesity and to the
diagnostic and therapeutic uses to which such modulators may be
put. The present invention also relates to methods and compositions
for inhibiting the infectivity of human immunodeficiency virus
(HIV).
BACKGROUND OF THE INVENTION
[0002] Obesity, which is defined in general terms as an excess of
body fat relative to lean body mass, is now a world wide epidemic,
and is one of the most serious contributors to increased morbidity
and mortality. Obesity is prevalent in the United States, affecting
more than 61% of the total population (Flegal, et al., Overweight
and Obesity in the United States: Prevalence and Trends, 1960-1994.
Int J Obes 22:39-47, 1998). Obesity is defined more specifically by
the United States Centers for Disease Control and Prevention (CDC)
as an excessively high amount of body fat or adipose tissue in
relation to lean body mass and overweight is defined as an
increased body weight in relation to height, when compared to some
standard of acceptable or desirable weight. The CDC alternatively
defines overweight as a person with a body mass index (BMI) between
25.0 and 29.9 and obesity is defined as a BMI greater than or equal
to 30.0. Obesity is often associated with psychological and medical
morbidities, the latter of which includes increased joint problems,
vascular diseases such as coronary artery disease, hypertension,
stroke, and peripheral vascular disease. Obesity also causes
metabolic abnormalities such as insulin resistance and Type II
diabetes (non-insulin-dependent diabetes mellitus (NIDDM)),
hyperlipidemia, and endothelial dysfunction. These abnormalities
predispose the vasculature to injury, cellular proliferation and
lipid oxidation, with resulting atherosclerosis leading to heart
attack, stroke, and peripheral vascular diseases. In 1998,
consumers spent $33 billion in the United States for weight-loss
products and services with a less than positive outcome (Serdula,
et al., Prevalence of Attempting Weight Loss and Strategies for
Controlling Weight, JAMA 282:1353-1358, 1999). Thus, obesity and
its associated complications continue to be a major problem
throughout the worldwide health care system.
[0003] Obesity is clearly an important clinical problem with very
broad reaching implications. There is a pressing need for more
research on the molecular mechanisms that underlie obesity and its
medical consequences, as well as new approaches for its treatment.
To date, these approaches have been limited to diet and exercise
(therapeutic lifestyle changes), surgical procedures such as
gastric bypass, and pharmacologic agents. Drug treatment for
obesity has been disappointing since almost all drug treatments for
obesity are associated with undesirable side effects that
contributed to their termination. A number of monoamines and
neuropeptides are known to reduce food intake (Bray, et al.,
Pharmacological Treatment of Obesity, Am J Clin Nutr 55:151S-319S,
1992). Available pharmacotherapies have included Sibutramine (an
appetite suppressant), Orlistat (a lipase inhibitor), fenfluramine
and dexfenfluramine. Although body weight loss is effective, these
sympathomimetic drugs cause side effects including pulmonary
hypertension, neuroanatomic changes, and a typical valvular heart
diseases. For example, fenfluramine and dexfenfluramine were
withdrawn from the market in 1997 because of associated cardiac
valvulopathy (Connolly, et al., Valvular Heart Disease Associated
With Fenfluramine-Phentermine, New Engl J Med 337-581-588, 1997).
Thus, nutrition and dietary restriction are most desirable for
weight loss. However, long-term success of dietary regulation is
low because of noncompliance. The loss of motivation to change
dietary habits necessary to consume less fat and fewer calories
results in regaining weight.
[0004] Thus, there are no real treatments based on the biology of
the primary metabolic abnormalities found in obesity and its
related conditions, such as metabolic syndrome or atherosclerosis.
Accordingly, there is still a need for new compositions and methods
that address treating individuals suffering from obesity and
obesity-related disorders. There is also a need for new agents and
compositions for treating individuals infected with human
immunodeficiency virus (HIV). It is toward the development of new
compositions and methods for treating obesity and obesity-related
disorders and for treating individuals infected with HIV that the
present invention is directed.
[0005] The citation of any reference herein should not be construed
as an admission that such reference is prior art to the instant
invention.
SUMMARY OF THE INVENTION
[0006] In accordance with the broadest aspect of the invention,
methods and compositions comprising oleuropein, or an analogue or
derivative thereof, or the major metabolites of oleuropein
including oleuropein aglycone, hydroxytyrosol, and elenolic acid or
their analogues, or derivatives thereof, or an iridoid glycoside,
or a secoiridoid glycoside or their analogues or derivatives
thereof, or any combination of the foregoing including but not
limited to olive leave extract, are disclosed for treating obesity
and obesity-related conditions or disorders, as well as for
inhibiting the infectivity of HIV. In particular, these
compositions modulate adipogenesis, lipodystrophy, reduce fat
accumulation and weight gain. These agents also prevent HIV viral
fusion/entry into a host cell and bind the catalytic site of the
HIV integrase. Thus, these agents provide an advantage over other
anti-viral therapies in that both viral entry and integration are
inhibited. They exert their effect by modulating adipocyte
differentiation (adipogenesis), de-differentiation,
transdifferentiation, and by decreasing the number of adipocytes
(fat cells), or by modulating adipocyte metabolism (lipid
synthesis, storage, accumulation, and utilization) so as to
decrease fat accumulation and decrease the size of the fat cell
(decrease fat mass), increase fat burning and expenditure. These
compositions also have an effect on adipogenic, lipogenic and
lipolytic gene/gene product expression, perturbation of
pre-adipocyte to adipocyte balance by promoting de-differentiation
or transdifferentiation of adipocytes, the end result being a
reduction in fat accumulation (adipose mass) and a reduction in
weight gain. Accordingly, these compositions are useful for
treating obesity and obesity-related disorders. In addition, these
compositions have been shown to reduce diet-induced atherogenesis,
thus allowing for a means of treating one of the major conditions
or disorders associated with, or resulting from, obesity. Moreover,
the compositions disclosed may also be utilized for treating other
obesity-related disorders, including but not limited to, coronary
artery disease, hypertension, stroke, peripheral vascular disease,
insulin resistance, glucose intolerance, diabetes mellitus,
hyperlipidemia, atherosclerosis, cellular proliferation and
endothelial dysfunction, diabetic dyslipidemia, HIV-related
lipodystrophy, e.g. Highly Active Anti-Retroviral Therapy
(HAART)-induced lipodystrophy, and metabolic syndrome, type II
diabetes, hyperinsulinemia, diabetic complications including
diabetic neuropathy, nephropathy, retinopathy or cataracts, heart
failure, hypercholesterolemia, inflammation, thrombosis, congestive
heart failure, and any other cardiovascular disease related to
obesity or an overweight condition, or obesity induced asthma,
airway dysfunction and pulmonary disorders. The compositions may
also be contemplated for use in the production of lean meat from
meat animals, e.g., beef cattle, lambs, hogs, chickens and
turkeys.
[0007] Accordingly, a first aspect of the invention provides a
method of modulating adipocyte differentiation or adipogenic gene
expression, comprising administering a therapeutically effective
amount of oleuropein or an analogue, or derivative thereof, or
oleuropein aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues, or derivatives thereof, or any
combination of any of the foregoing thereof including but not
limited to olive leave extract, to a mammalian subject in need
thereof.
[0008] In one embodiment, oleuropein or its analogues, or
derivatives thereof, or oleuropein aglycone or their analogues, or
derivatives thereof, or hydrotyrosol, or dihydroxy phenol or their
analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside or their analogues, or derivatives thereof,
or any combinations of any of the foregoing including but not
limited to olive leave extract, up-regulates fat utilization,
energy uncoupling, related regulators, factors and enzymes
including but not limited to lipolytic genes/gene products in these
pathways.
[0009] In another embodiment, oleuropein or an analogue, or
derivative thereof, or oleuropein aglycone or their analogues, or
derivatives thereof, or hydrotyrosol, or dihydroxy phenol or their
analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside or their analogues, or derivatives thereof,
or any combinations of any of the foregoing including but not
limited to olive leave extract, blocks or down-regulates adipocyte
differentiation, regulators, factors, and enzymes involved in the
adipogenic pathway. In yet another embodiment, oleuropein or an
analogue, or derivative thereof or oleuropein aglycone or their
analogues, or derivatives thereof, or hydrotyrosol, or dihydroxy
phenol or their analogues, or derivatives thereof, or elenolic acid
or their analogues, or derivatives thereof, or an iridoid
glycoside, or a secoiridoid glycoside or their analogues, or
derivatives thereof, or any combinations of any of the foregoing
thereof including but not limited to olive leave extract, enhances
de-differentiation of adipocytes and allows for
transdifferentiation into osteoblasts, muscle cells, cartilage, and
bone as well as regulators and enzymes involved in these
pathways.
[0010] In yet another embodiment, the adipogenic gene whose
expression is modulated by oleuropein or an analogue, or derivative
thereof, or oleuropein aglycone or their analogues, or derivatives
thereof, or hydrotyrosol, or dihydroxy phenol or their analogues,
or derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues, or derivatives thereof, or any
combinations of any of the foregoing including but not limited to
olive leave extract, is selected from the group consisting of
Peroxisome Proliferator-Activated Receptor .gamma. (PPAR.gamma.),
lipoprotein lipase (LPL) and the .alpha.P2 gene or gene product.
Oleuropein or its analogues, or derivatives, or oleuropein aglycone
or their analogues, or derivatives thereof, or hydrotyrosol, or
dihydroxy phenol or their analogues, or derivatives thereof, or
elenolic acid or their analogues, or derivatives thereof, or an
iridoid glycoside, or a secoiridoid glycoside or their analogues,
or derivatives thereof, or any combinations of any of the foregoing
thereof including but not limited to olive leave extract, also
modulate the expression of all of the three types of PPARs .alpha.,
.delta., and .gamma. resulting in a coordinated regulation of
adipocyte differentiation, de-differentiation,
transdifferentiation, adipocyte metabolism and energy homeostasis.
In yet another embodiment, the lipogenic, lipolytic and energy
uncoupling genes and gene products whose expression is modulated
includes, but is not limited to, PPAR .delta. and its modulated
genes and gene products. In yet another embodiment, the lipogenic,
lipolytic and energy uncoupling genes whose expression is modulated
includes, but is not limited to, PPAR .alpha. and its modulated
genes and gene products.
[0011] A second aspect of the invention provides a method of
treating, controlling or preventing obesity, or of reducing body
weight, or of inhibiting fat accumulation, or of promoting fat
burning and energy uncoupling in vivo, or of treating, controlling
or preventing the onset of one or more obesity-related disorders or
conditions, comprising administering a therapeutically effective
amount of oleuropein or an analogue, or derivative thereof, or
oleuropein aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues, or derivatives, or any combinations
of any of the foregoing thereof including but not limited to olive
leave extract, to a mammalian subject in need thereof.
[0012] In one embodiment, the one or more obesity-related disorders
or conditions are selected from the group consisting of coronary
artery disease, hypertension, stroke, peripheral vascular disease,
insulin resistance, glucose intolerance, diabetes mellitus,
hyperglycemia, hyperlipidemia, hypercholesteremia,
hypertriglyceridemia, hyperinsulinemia, atherosclerosis, cellular
proliferation and endothelial dysfunction, diabetic dyslipidemia,
HIV-related lipodystrophy and metabolic syndrome, type II diabetes,
diabetic complications including diabetic neuropathy, nephropathy,
retinopathy or cataracts, heart failure, inflammation, thrombosis,
congestive heart failure, any other asthmatic or pulmonary disease
related to obesity and any other viral infection infection related
diseases and any other cardiovascular disease related to obesity or
an overweight condition.
[0013] A third aspect provides a method of treating, controlling or
preventing the onset of one or more obesity related disorders or
conditions selected from the group consisting of Asthma and related
diseases including but not limited to, Allergy, Atopic Dermatitis
(Eczema), Gastroesophageal Reflux Disease, Airway, Pulmonary and
Lung disorders, in a mammalian subject in need of treatment,
comprising administering a therapeutically effective amount of
oleuropein or an analogue, derivative or oleuropein aglycone or
their analogues, or derivatives thereof, or hydrotyrosol, or
dihydroxy phenol or their analogues, or derivatives thereof, or
elenolic acid or their analogues, or derivatives thereof, or an
iridoid glycoside, or a secoiridoid glycoside or their analogues,
or derivatives, or any component or any combinations of any of the
foregoing thereof including but not limited to olive leave
extract.
[0014] A fourth aspect provides a method of treating, controlling
or preventing the onset of one or more obesity related disorders or
conditions selected from the group consisting of AIDS and HAART
related diseases including but not limited to lipodystrophy in a
human and or mammalian subject in need of treatment, comprising
administering a therapeutically effective amount of oleuropein or
an analogue, derivative or oleuropein aglycone or their analogues,
or derivatives thereof, or hydrotyrosol, or dihydroxy phenol or
their analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside or their analogues, or derivatives, or any
component or any combinations of any of the foregoing thereof
including but not limited to olive leave extract.
[0015] A fifth aspect provides a method of treating, controlling or
preventing the onset of one or more viral infection related
disorders or conditions selected from the group consisting of viral
infection related diseases including but not limited to the AIDS
virus HIV-1, and other viruses including but not limited to simian
immunodeficiency viruses (SIV), Sendai virus, feline
immunodeficiency virus (FIV), respiratory syncytial virus (RSV),
measles virus, Ebola virus, Nipah and Hendra viruses, the severe
acute respiratory syndrome associated coronavirus (SARS-CoV), and
the avain flu virus in a humans, mammalian, avian, poultry, non
human primates subjects in need of treatment, comprising
administering a therapeutically effective amount of oleuropein or
an analogue, or derivative, or oleuropein aglycone or their
analogues, or derivatives thereof, or hydrotyrosol, or dihydroxy
phenol or their analogues, or derivatives thereof, or elenolic acid
or their analogues, or derivatives thereof, or an iridoid
glycoside, or a secoiridoid glycoside or their analogues, or
derivatives, or any component or any combinations of any of the
foregoing thereof including but not limited to olive leave
extract.
[0016] A sixth aspect of the invention provides a method for
modulating Peroxisome Proliferator Activation Receptor (PPAR)
activity, comprising administering to a mammal in need thereof a
therapeutically effective amount of oleuropein or an analogue, or
derivative thereof, or oleuropein aglycone or their analogues, or
derivatives thereof, or hydrotyrosol, or dihydroxy phenol or their
analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside or their analogues, or derivatives, or any
component or any combinations of any of the foregoing thereof. or
an iridoid glycoside, or a secoiridoid glycoside or their
analogues, or derivatives, or any components or any combinations
thereof including but not limited to olive leave extract.
[0017] A seventh aspect provides a method of treating a subject
suffering from, or at risk for developing a disease or condition
for which PPAR modulation provides a therapeutic benefit,
comprising administering to said subject a therapeutically
effective amount of oleuropein, or an analogue or derivative
thereof or oleuropein aglycone or their analogues, or derivatives
thereof, or hydrotyrosol, or dihydroxy phenol or their analogues,
or derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues, or derivatives, or any component or
any combinations of any of the foregoing thereof including but not
limited to olive leave extract.
[0018] In one embodiment, the subject is a human or a non-human
mammal. In a preferred embodiment, the subject is a mammal. In
another preferred embodiment, the subject is a non-human mammal
selected from the group consisting of cows, horses, pigs, sheep,
goats, birds, rodents, dogs, cats and other domestic animals or
farm animals.
[0019] In yet another particular embodiment, the condition for
which PPAR modulation provides a therapeutic benefit is selected
from the group consisting of obesity, obesity-related disorders and
the sequelae thereof. In yet another embodiment, accordingly, the
present invention provides for a method of treating inflammatory
diseases or conditions comprising administering a PPAR gamma
modulator to a subject in need of such therapy. In one particular
embodiment, the method provides for treating neurological or
neurodegenerative diseases or conditions caused in part by the
presence or influx of inflammatory cells, such as for example,
multiple sclerosis, stroke or Alzheimer's disease. The use of a
PPAR modulator for treating a nervous system injury is also
contemplated, for example, a spinal cord injury or traumatic brain
injury. The use of a PPAR modulator for wound healing is also
contemplated. In one particular embodiment, the PPAR modulator is a
PPAR gamma, alpha, or delta agonist, or a combined agonist.
[0020] In yet another particular embodiment, the PPAR is selected
from PPAR .delta., .gamma. or .alpha., or a dual, or pan PPAR
.delta., .gamma. and .alpha. modulators.
[0021] In yet another particular embodiment, the obesity-related
disorder or sequelae is selected from the group consisting of
coronary artery disease, hypertension, stroke, peripheral vascular
disease, insulin resistance, diabetes mellitus, hyperlipidermia,
atherosclerosis, cellular proliferation and endothelial
dysfunction, diabetic dyslipidemia, type II diabetes,
hyperinsulinemia, diabetic complications including diabetic
neuropathy, nephropathy, retinopathy or cataracts, heart failure,
hypercholesterolemia, inflammation, thrombosis, congestive heart
failure, and any other cardiovascular disease related to obesity or
an overweight condition.
[0022] An eighth aspect of the invention provides a method of
reducing or preventing formation of atherosclerotic lesions or
preventing diet-induced atherogenesis, comprising administering to
a mammal in need thereof a therapeutically effective amount of
oleuropein or an analogue or derivative thereof, or oleuropein
aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues, or derivatives, or any combinations
of any of any of the foregoing thereof.
[0023] A ninth aspect of the invention provides a method of
modulating endothelial dysfunction comprising administering to a
mammal in need thereof a therapeutically effective amount of
oleuropein or an analogue, or derivative, or oleuropein aglycone or
their analogues, or derivatives thereof, or hydrotyrosol, or
dihydroxy phenol or their analogues, or derivatives thereof, or
elenolic acid or their analogues, or derivatives thereof, or an
iridoid glycoside, or a secoiridoid glycoside or their analogues,
derivatives, or any combinations of any of the foregoing thereof
including but not limited to olive leave extract.
[0024] A tenth aspect of the invention provides a method for
treating obesity or obesity related disorders or other disorders
for which PPAR modulation provides a therapeutic benefit comprising
administering a second agent in combination with oleuropein, or an
analogue or derivative thereof, or oleuropein aglycone or their
analogues, or derivatives thereof, or hydrotyrosol, or dihydroxy
phenol or their analogues, or derivatives thereof, or elenolic acid
or their analogues, or derivatives thereof, or an iridoid
glycoside, or a secoiridoid glycoside or their analogues,
derivatives, or any combination of any of the foregoing thereof
including but not limited to olive leave extract.
[0025] In one particular embodiment, the second agent is selected
from the group consisting of a different PPAR modulating agent, a
cholesterol or lipid lowering agent, a biguanide, insulin, an
antihyperglycemic agent, GLP-1 or analogues thereof, DPP4
inhibitors, a weight loss agent, and any agent useful for treating
metabolic syndrome or type 2 diabetes.
[0026] In another particular embodiment, the different PPAR
modulating agent is a thiazolidinedione selected from the group
consisting of troglitazone, pioglitazone and rosiglitazone.
[0027] In yet another particular embodiment, the cholesterol or
lipid lowering agent is a HMG-CoA reductase inhibitor, and wherein
the HMG-CoA reductase inhibitor is a statin selected from the group
consisting of atorvastatin, bervastatin, cerivastatin, dalvastatin,
fluvastatin, itavastatin, lovastatin, mevastatin, nicostatin,
nivastatin, pravastatin and simvastatin. In yet another particular
embodiment, the cholesterol or lipid lowering agent is niacin or a
fibrate.
[0028] In yet another particular embodiment, the biguanide is
selected from the group consisting of metformin, phenformin, and
buformin.
[0029] In yet another particular embodiment, the antihyperglycemic
is a prandial glucose regulator or an alpha-glucosidase
inhibitor.
[0030] In yet another particular embodiment, the prandial glucose
regulator is repaglinide or nateglinide.
[0031] In yet another particular embodiment, the alpha-glucosidase
inhibitor is selected from the group consisting of acarbose,
voglibose and miglitol.
[0032] In yet another particular embodiment, the agent useful for
treating metabolic syndrome or type 2 diabetes is a sulfonylurea
selected from the group consisting of glimepiride, glibenclamide
(glyburide), gliclazide, glipizide, gliquidone, chloropropamide,
tolbutamide, acetohexamide, glycopyramide, carbutamide,
glibonuride, glisoxepid, glybuthiazole, glibuzole, glyhexamide,
glymidine, glypinamide, phenbutamide, tolcylamide and
tolazamide.
[0033] In yet another particular embodiment, the agent useful for
treating AIDS and Highly Active Anti-Retroviral Therapy
(HAART)-induced lipodystrophy, metabolic syndrome or type 2
diabetes is a thiazolidinedione (TZD) and a fibrate.
[0034] In yet another particular embodiment, the agent useful for
treating HIV-1 infection and AIDS is a component or the full
composition of the Highly Active Anti-Retroviral Therapy (HAART),
including a protease inhibitor (PI), a nucleotide reverse
transcriptase inhibitor (NRTI), and a non-nucleotide reverse
transcriptase inhibitor (NNRTI).
[0035] In yet another particular embodiment, the agent useful for
treating obesity induced asthma and related disorders is selected
from the group consisting of a glucocorticoid, an antileukotriene
and an antihistamine.
[0036] An eleventh aspect of the invention provides a
pharmaceutical composition comprising a therapeutically effective
amount of oleuropein, or an analogue or derivative thereof or
oleuropein aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues, derivatives, or any combinations
thereof including but not limited to olive leave extract, and a
pharmaceutically acceptable carrier for delivery to a mammal in
need of such therapy.
[0037] In one particular embodiment, the pharmaceutical composition
may be administered orally, nasally, transdermally, intravenously,
intramuscularly, or subcutaneously.
[0038] In another particular embodiment, the subject has or is at
risk for unwanted weight gain, obesity or an obesity related
disorder, e.g., diabetes or glucose intolerance, insulin resistant
states, hypertension, HIV-1 infection, or any of the other
disorders disclosed herein. In preferred embodiments, the method
includes identifying a subject as being in need of treatment or
prevention of unwanted weight gain, obesity or an obesity related
disorder. In another particular embodiment, the pharmaceutical
composition is formulated for delivery to a human or non-human
mammal. In a preferred embodiment, the mammal is a human. In
another preferred embodiment, the subject is a non-human mammal
selected from the group consisting of cows, horses, pigs, sheep,
goats, birds, rodents (including rats, mice and gerbils), dogs,
cats and other domestic animals or farm animals.
[0039] A twelfth aspect of the invention provides a pharmaceutical
composition directed to combination therapy, whereby oleuropein or
oleuropein aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues or derivatives, or any combination
thereof including but not limited to olive leave extract, is
combined with one or more other therapeutic agents that are useful
in the treatment of disorders associated with the development and
progression of obesity and obesity related disorders, such as
atherosclerosis, hypertension, hyperlipidemias, dyslipidemias,
diabetes and other related disorders as described herein.
[0040] In one particular embodiment, the composition for
combination therapy comprises oleuropein and at least one other
agent. In another particular embodiment, the composition for
combination therapy comprises oleuropein and at least two or more
other agents.
[0041] In one particular embodiment, the at least one other
therapeutic agent is a different PPAR modulating agent other than
oleuropein.
[0042] In a more particular embodiment, the different PPAR
modulating agent is a thiazolidinedione selected from the group
consisting of troglitazone, pioglitazone and rosiglitazone.
[0043] In yet another particular embodiment, the at least one other
therapeutic agent is a cholesterol-lowering agent.
[0044] In yet another particular embodiment, the cholesterol
lowering agent is a HMG-CoA reductase inhibitor.
[0045] In yet another particular embodiment, the HMG-CoA reductase
inhibitor is a statin selected from the group consisting of
atorvastatin, bervastatin, cerivastatin, dalvastatin, fluvastatin,
itavastatin, lovastatin, mevastatin, nicostatin, nivastatin,
pravastatin and simvastatin.
[0046] In yet another particular embodiment, oleuropein is combined
with a therapy useful for the treatment of metabolic syndrome or
type 2 diabetes.
[0047] In yet another particular embodiment, the therapy useful for
the treatment of metabolic syndrome or type 2 diabetes and its
associated complications is selected from the group consisting of a
biguanide drug, (including metformin, phenformin and buformin),
insulin (synthetic insulin analogues, amylin) and oral
antihyperglycemics.
[0048] In yet another particular embodiment, oleuropein is combined
with an oral antihyperglycemic agent, which is a prandial glucose
regulator or an alpha-glucosidase inhibitor. In yet another
particular embodiment, the prandial glucose regulator is
repaglinide or nateglinide. In yet another particular embodiment,
the alpha-glucosidase inhibitor is acarbose, voglibose or
miglitol.
[0049] In yet another particular embodiment, oleuropein is combined
with a sulfonylurea.
[0050] In yet another particular embodiment, the sulfonylurea is
selected from the group consisting of glimepiride, glibenclamide
(glyburide), gliclazide, glipizide, gliquidone, chloropropamide,
tolbutamide, acetohexamide, glycopyramide, carbutamide,
glibonuride, glisoxepid, glybuthiazole, glibuzole, glyhexamide,
glymidine, glypinamide, phenbutamide, tolcylamide and
tolazamide.
[0051] In one particular embodiment, the composition comprises a
mixture of oleuropein or oleuropein aglycone or their analogues, or
derivatives thereof, or hydrotyrosol, or dihydroxy phenol or their
analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside or their analogues or derivatives, or any
combination thereof including but not limited to olive leave
extract, and the at least one or two other agents, wherein the at
least one or two other agents may be administered prior to,
concurrent with, or subsequent to, oleuropein or oleuropein
aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues or derivatives, or any combination
thereof including but not limited to olive leave extract.
[0052] In another particular embodiment, the composition comprising
oleuropein or an analogue or derivative thereof or oleuropein
aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues or derivatives, or any combination
thereof including but not limited to olive leave extract, interacts
with, e.g., binds to, PPAR.delta., PPAR.gamma. and PPAR.alpha..
[0053] In yet another particular embodiment, oleuropein or
oleuropein aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogues or derivatives, or any combination
thereof including but not limited to olive leave extract, is
combined with a therapy useful for the treatment of the AIDS virus
HIV-1, and other viruses with a type I transmembrane envelope
glycoprotein, such as simian immunodeficiency viruses (SIV), Sendai
virus, feline immunodeficiency virus (FIV), respiratory syncytial
virus (RSV), measles virus, Ebola virus, Nipah and Hendra viruses,
the severe acute respiratory syndrome associated coronavirus
(SARS-CoV), and the avain flu virus H5N1 including but not limited
to PI, NRTI, NNRTI, HAART, tamiflu, ribavirin, steroid, recombinant
nematode anticoagulant protein c2 (rNAPC2).
[0054] In yet another particular embodiment, oleuropein is combined
with a therapy useful for the treatment of obesity induced asthma
and related disorders, including but not limited to the PPARs,
glucocorticoids, antileukotrienes and antihistamines.
[0055] In another particular embodiment, the oleuropein or an
analogue or derivative thereof, or oleuropein aglycone or their
analogues, or derivatives thereof, or hydrotyrosol, or dihydroxy
phenol or their analogues, or derivatives thereof, or elenolic acid
or their analogues, or derivatives thereof, an iridoid glycoside,
or a secoiridoid glycoside, or their analogues, or derivatives
thereof, or any combination thereof including but not limited to
olive leave extract, is targeted to adipose tissue in a subject.
The oleuropein or an analogue or derivative thereof or oleuropein
aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or an analogue, or derivative thereof, or any combination
thereof including but not limited to olive leave extract, may be
targeted to adipose tissue by virtue of an inherent characteristic,
e.g., lipid solubility. In other embodiments, the agent may include
(e.g., the agent can be linked, fused or conjugated to, or
enveloped in) a targeting reagent that targets the agent to an
adipose tissue. The targeting reagent can be a nucleic acid, a
protein (e.g., a hormone, e.g., leptin, conjugate or an antibody to
an adipocyte-specific antigen), a lipid (e.g., a liposome), a
carbohydrate, or other molecule that is targeted to an adipose
tissue.
[0056] In yet another particular embodiment, the oleuropein or an
analogue or derivative thereof, or oleuropein aglycone or their
analogues, or derivatives thereof, or hydrotyrosol, or dihydroxy
phenol or their analogues, or derivatives thereof, or elenolic acid
or their analogues, or derivatives thereof, or an iridoid
glycoside, or a secoiridoid glycoside, or their analogues or
derivatives thereof, or any combination of the foregoing including
but not limited to olive leave extract, is targeted to adipose
tissue via Resistin (resistance to insulin) or Resistin-Like
Molecules (RELMs or FIZZ1-3) (Degawa-Yamauchi M, Serum Resistin
(FIZZ3) Protein Is Increased in Obese Humans, J. Clin. Endocrinol.
Metab., 88: 5452-5455, (2003); (Diabetologia. (2006) May 10; Anal
Chem. (2006) May 15; 78(10):3271-6; Hum Reprod. (2006) May 12) or
Adipocyte-Specific Secretory Factor (ADSF) Proteins (Endocrine.
(2006) February; 29(1):81-90 Proc Natl Acad Sci USA. (2004) April
27; 101(17):6780-5. Epub (2004) April 16) or antibodies to such
proteins, as well as Leptin (Int J Obes (Lond). (2006) May 16) or
Leptin Receptor antibodies, Acrp30/Adiponectin (Endocrinology.
(2006) June; 147(6):2690-5. Epub (2006) March 2; (Hum Reprod.
(2006) May 12; [Epub ahead of print]; Biochem Biophys Res Commun.
(2006) June 23; 345(1):332-9. Epub (2006) April 27. J Endocrinol
Invest. (2006) March; 29(3):231-6;) (Hum Reprod. 2006 May 12; [Epub
ahead of print]; Biochem Biophys Res Commun. 2006 Jun. 23;
345(1):332-9. Epub 2006 Apr. 27. J Endocrinol Invest. 2006 March;
29(3):231-6;) or Adipsin antibodies, Orexins (Br J Nutr. 2004
August; 92 Suppl 1:S47-57) or Orexins Receptor antibodies, a
Glucose Transporter (Glut1-Glut14) (Clin Exp Pharmacol Physiol.
2006 April; 33(4):395-9; Am J Med. 2006 May; 119(5 Suppl 1):S10-6)
antibody, or an antibody to a Hypoxia Induced Factor (HIF-alpha,
beta) (HIF-alpha, beta, Diabetologia. 2006 May; 49(5):1049-63. Epub
2006 Feb. 28; Biochem Biophys Res Commun. 2006 Mar. 10;
341(2):549-56.) Am J Physiol Endocrinol Metab. 2006 March;
290(3):E591-7. Epub 2005 Oct. 18.
[0057] In yet another particular embodiment, the oleuropein or an
analogue or derivative thereof, or oleuropein aglycone or their
analogues, or derivatives thereof, or hydrotyrosol, or dihydroxy
phenol or their analogues, or derivatives thereof, or elenolic acid
or their analogues, or derivatives thereof, or an iridoid
glycoside, or a secoiridoid glycoside or their analogue or
derivative, or any combination thereof including but not limited to
olive leave extract, is targeted to a preadipocyte and its specific
proteins such as pref-1 and C1q to modulate adipocyte
differentiation and maturation.
[0058] In yet another particular embodiment, the oleuropein or an
analogue or derivative thereof, or oleuropein aglycone or their
analogues, or derivatives thereof, or hydrotyrosol, or dihydroxy
phenol or their analogues, or derivatives thereof, or elenolic acid
or their analogues, or derivatives thereof, or an iridoid
glycoside, or a secoiridoid glycoside, or their analogues, or
derivatives thereof, or any combination of the foregoing including
but not limited to olive leave extract, is targeted to a
mesenchymal stem cell (MSC) or an embryonic stem cell. MSCs
differentiate into adipocytes, chondrocytes, osteoblasts, and
myoblasts. Thus, these stem cells are promising candidates for
adipogenesis management by oleuropein and derivatives or oleuropein
aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogue or derivative, or any combination
thereof including but not limited to olive leave extract, targeting
and/or ex-vivo treatment so as to modulate adipocyte
differentiation, de-differentiation and trans-differentiation. The
application of oleuropein and derivatives in this system is not
only effective in treating obesity and related metabolic syndromes
by modulating adipocyte differentiation and development but also
provide tools to manipulate adult stem cells for cell-based
approaches in regenerative medicine. For example, aged and
osteoporotic patients have a high fat to bone ratio in their bones
compared with young and healthy counterparts, an outcome possibly
due to the conversion of bone to fat cells. Application of
oleuropein or an analogue or derivative thereof, or oleuropein
aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside or their analogue or derivative, or any combination
thereof including but not limited to olive leave extract to mediate
transdifferentiation between osteoblasts and adipocytes should be
of relevance to the development of therapeutic control of bone loss
in osteoporosis. For specific targeting of MSC, these cells express
leukemia inhibitory factor, macrophage colony-stimulating factor,
and stem cell factor specifically. Thus, these proteins and
antibodies specific for or that bind these proteins can be used for
targeting.
[0059] In another particular embodiment, the targeting reagent is
lipid soluble.
[0060] In another particular embodiment, the administration of the
compositions of the invention can be initiated, e.g., (a) when the
subject begins to show signs of unwanted weight gain, obesity or an
obesity-related disease; (b) when obesity or an obesity-related
disease is diagnosed; (c) before, during or after a treatment for
obesity or an obesity-related disease is begun or begins to exert
its effects; or (d) generally, as is needed to maintain health,
e.g., normal weight. The period over which the agent is
administered (or the period over which clinically effective levels
are maintained in the subject) can be long term, e.g., for six
months or more or a year or more, or short term, e.g., for less
than a year, six months, one month, two weeks or less.
[0061] In another particular embodiment, the pharmaceutical
compositions described herein, including oleuropein, or an analogue
or derivative thereof, or oleuropein aglycone or their analogues,
or derivatives thereof, or hydrotyrosol, or dihydroxy phenol or
their analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside or their analogues, or derivatives thereof,
or any combination thereof including but not limited to olive leave
extract and any second agent to be administered with oleuropein or
its analogues or derivatives thereof, or oleuropein aglycone or
their analogues, or derivatives thereof, or hydrotyrosol, or
dihydroxy phenol or their analogues, or derivatives thereof, or
elenolic acid or their analogues, or derivatives thereof, or an
iridoid glycoside, or a secoiridoid glycoside or their analogues or
derivatives thereof, or any combination thereof including but not
limited to olive leave extract as described above, are administered
in a therapeutically effective dose.
[0062] In yet another particular embodiment, oleuropein or an
analogue, or derivative thereof, or oleuropein aglycone or their
analogues, or derivatives thereof, or hydrotyrosol, or dihydroxy
phenol or their analogues, or derivatives thereof, or elenolic acid
or their analogues, or derivatives thereof, or an iridoid
glycoside, or a secoiridoid glycoside, or their analogues, or
derivatives thereof, or any combination thereof including but not
limited to olive leave extract may have an effect on white or brown
adipose tissue or a combination thereof. For example, since PPAR
delta is involved in thermogenesis and energy uncoupling (the main
function of brown fat) and oleuropein has high affinity for PPAR
delta, it may also have an effect on brown adipose tissue.
Accordingly, oleuropein may be involved in the dissipation of
stored fat as heat in brown adipose tissue. Brown fat plays an
important role in the control of body weight, and mitochondrial
uncoupling proteins may be one of many factors involved in the
development of obesity. An interesting demonstration of this is
found in a report in which transgenic mice with genetic ablation of
brown fat developed obesity in the absense of overeating (Bachman E
S, Dhillon H, Zhang C--Y, et al. BetaAR signaling required for
diet-induced thermogenesis and obesity resistance. Science 297:843,
2002).
[0063] A thirteenth aspect of the invention provides a method for
identifying a candidate compound or an analogue or derivative of
oleuropein that modulates adipocyte differentiation,
de-differentiation, trans-differentiation, fat accumulation and
adipogenic gene expression or for treating obesity or
obesity-related disorders, comprising: [0064] a. treating a cell
with an inducing agent in the presence or absence of a candidate
compound; [0065] b. determining whether the candidate compound
inhibits differentiation of the pre-adipocyte cell to an adipocyte,
or whether the candidate compound down-regulates adipogenic and
lipogenic gene or gene product expression and/or up-regulates
lipolytic and/or an energy uncoupling gene or gene product
expression; [0066] c. comparing the results obtained with the
candidate compound in vitro with the results obtained using
oleuropein; [0067] d. testing the candidate compound and oleuropein
in an animal model of obesity to determine the in vivo effects of
both the candidate compound and oleuropein; [0068] e. determining
whether the candidate compound decreases the amount of adipose
tissue in vivo or whether the test compound prevents further fat
accumulation in vivo, and [0069] f. selecting a candidate compound
that has equivalent or better activity than oleuropein.
[0070] In one particular embodiment, the invention relates to a
method for identifying a therapeutic agent having analogous
activity to oleuropein comprising treating a type of cell that
expresses a type of PPAR with a candidate compound/therapeutic; and
determining the level of expression of at least one gene selected
from the group consisting of PPAR.gamma.2, lipoprotein lipase
(LPL), and .alpha.P2 lipid binding protein, wherein a change in the
profile of expression of at least one of these genes in the cell
treated with the candidate compound/therapeutic relative to a cell
that was not treated with the candidate compound/therapeutic
indicates that the candidate compound/therapeutic is a therapeutic
for treating a disease associated with a PPAR. The "profile",
"profiling" or "profile of expression" refers to both the level of
expression of one or more genes and also the activity or function
of one or more of these genes. Accordingly, the candidate compound
may have an effect not only on the expression of one or more genes,
but on the function or activity of one or more genes. In one
particular embodiment, an increase in the profile of expression of
at least one of these genes following treatment with a candidate
compound/therapeutic indicates that the candidate
compound/therapeutic is a therapeutic for treating a disease
associated with a PPAR. The profile in expression of one or more of
these genes relates to the differentiation or maturation of the
adipocyte. In another particular embodiment, the inducing agent
that is used for in vitro analysis is any compound or molecule that
induces differentiation of a pre-adipocyte or a mesenchymal stem
cell into a mature adipocyte, eg. dexamethasone, insulin and methyl
xanthine. In another particular embodiment, following
contacting/treating a cell containing PPAR with a candidate
compound/therapeutic, one may determine the profile of expression
of other genes selected from the group consisting of leptin,
TNF.alpha., IL-6, PAI-1, adipsin, complement factor C3 and
angiotensinogen. In yet another particular embodiment, in addition
to classic markers of adipogenesis such as hormone-sensitive lipase
(HSL), lipoprotein lipase (LPL), adiponectin, fatty acid-binding
protein 4, perilipin and CCAAT enhancer binding protein, and
enzymes of energy metabolism such as glycerol-3-phosphate
dehydrogenase 1, acetyl-coenzyme A carboxykinase,
phosphoenolpyruvate carboxykinase and pyruvate dehydrogenase
kinase, profiles on the expression of other related genes including
PPAR.alpha., PPAR.delta., PPAR.gamma., AP2, MARPK3, SREBP-1,
leptin, and GLUT4 are envisioned following treatment of a type of
cell with a candidate compound/therapeutic. These are important
because they are not only adipocyte "markers" but also important in
adipocyte function and pathogenesis. Any agent that affects
adipogenesis would modulate the expression of these genes. In one
particular embodiment, an adipogenic agent would up-regulate while
an anti-adipogenic agent would down regulate the expression of one
or more of these genes. However, physiologically it is not a simple
up or down regulation. It involves the regulation of other related
genes upstream and downstream as including PPAR.delta.,
PPAR.alpha., enzymes involve in lipid metabolism, fatty acid
oxidation, energy uncoupling as well as genes modulated by the
PPARs. In addition to monitoring the up or down regulation of these
genes, another particular embodiment provides for measuring the
differential expression as well as polymorphisms of these genes. In
yet another particular embodiment, the PPAR is selected from the
group consisting of .alpha., .gamma., or .gamma..
[0071] In another embodiment of the invention, the candidate
therapeutic is selected from the group consisting of proteins,
peptides, peptidomimetics, antibodies, nucleic acids, including RNA
(eg. siRNA), DNA, derivatives of fatty acids, and small molecules.
The small molecules may be synthetic or may be derived from a
natural source, such as a plant, animal, microbe, or soil.
[0072] In another embodiment, the disease is obesity or an
obesity-related disorder, such as, but not limited to, Type II
diabetes.
[0073] In another embodiment, the expression level of at least one
of the genes noted above is detected. In another particular
embodiment, the expression level of at least two or more of the
genes noted above is detected.
[0074] In another embodiment, the composition is an oral capsule or
tablet, a liquid suspension, or a chip or wafer for oral delivery.
In another embodiment, the composition is formulated for
intravenous use, for intramuscular use, for subcutaneous delivery
or for intraperitoneal injection.
[0075] In another embodiment, the composition comprising oleuropein
or an analogue, derivative or oleuropein aglycone or their
analogues, or derivatives thereof, or hydrotyrosol, or dihydroxy
phenol or their analogues, or derivatives thereof, or elenolic acid
or their analogues, or derivatives thereof, or an iridoid
glycoside, or a secoiridoid glycoside, or their analogues, or
derivatives thereof, or any combination thereof including but not
limited to olive leave extract may be targeted to (e.g., the
oleuropein or analogue or derivative thereof can be linked, fused
or conjugated to, or enveloped in) the adipocyte by attachment of
the oleuropein or analogue or derivative thereof to adipose tissue
via Resistin or Resistin-Like Molecules (RELMs or FIZZ1-3) or
Adipocyte-Specific Secretory Factor (ADSF) Proteins or antibodies
to such proteins, as well as Leptin or Leptin Receptor antibodies,
Acrp30/Adiponectin or Adipsin antibodies, Orexins or Orexins
Receptor antibodies, a Glucose Transporter (Glut1-Glut14) antibody,
or an antibody to a Hypoxia Induced Factor (HIF-alpha, beta).
[0076] In yet another particular embodiment, the composition
comprising the oleuropein or an analogue or derivative thereof, or
oleuropein aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside, or their analogues, or derivatives thereof, or any
combination thereof including but not limited to olive leave
extract, is targeted to a preadipocyte and its specific proteins
such as Preadipocyte factor-1 (Pref-1), which is an epidermal
growth factor-like domain-containing transmembrane protein and is
implicated in inhibiting preadipocytes differentiation and
component of complement (C1) or C1q, which is a serum glycoprotein
involved in immune complexes to modulate adipocyte differentiation
and maturation.
[0077] In yet another particular embodiment, the composition
comprising the oleuropein or an analogue or derivative thereof, or
oleuropein aglycone or their analogues, or derivatives thereof, or
hydrotyrosol, or dihydroxy phenol or their analogues, or
derivatives thereof, or elenolic acid or their analogues, or
derivatives thereof, or an iridoid glycoside, or a secoiridoid
glycoside, or their analogues, or derivatives thereof, or any
combination thereof including but not limited to olive leave
extract, is targeted to a mesenchymal stem cell (MSC). MSCs
differentiate into adipocytes, chondrocytes, osteoblasts, and
myoblasts. Accordingly, in yet another embodiment, the invention
provides a method of transdifferentiating adipocytes into
osteoblasts, myoblasts and chondrocytes, wherein the method
comprises administering to a mammal in need thereof a
therapeutically effective amount of oleuropein or an analogue, or
derivative thereof, or oleuropein aglycone or their analogues, or
derivatives thereof, or hydrotyrosol, or dihydroxy phenol or their
analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside, or their analogues, or derivatives thereof,
or any combination thereof including but not limited to olive leave
extract. Thus, these stem cells are promising candidates for
adipogenesis management by oleuropein and derivatives (as mentioned
above) targeting and/or ex-vivo treatment so as to modulate
adipocyte differentiation, trans-differentiation and
de-differentiation. The application of oleuropein and derivatives
(as mentioned above) in this system is not only effective in
treating obesity and related metabolic syndromes by modulating
adipocyte differentiation and development but also provide tools to
manipulate adult stem cells for cell-based approaches in
regenerative medicine. For example, aged and osteoporotic patients
have a high fat to bone ratio in their bones compared with young
and healthy counterparts, an outcome possibly due to the conversion
of bone to fat cells. Application of oleuropein or an analogue, or
derivative thereof, or oleuropein aglycone or their analogues, or
derivatives thereof, or hydrotyrosol, or dihydroxy phenol or their
analogues, or derivatives thereof, or elenolic acid or their
analogues, or derivatives thereof, or an iridoid glycoside, or a
secoiridoid glycoside, or their analogues, or derivatives thereof,
or any combination thereof including but not limited to olive leave
extract to mediate transdifferentiation between osteoblasts and
adipocytes should be of relevance to the development of therapeutic
control of bone loss in osteoporosis. For specific targeting of
MSC, these cells express leukaemia inhibitory factor, macrophage
colony-stimulating factor, and stem cell factor specifically. Thus
these proteins and their antibodies can be used for targeting.
[0078] In another particular embodiment, the targeting reagent is
lipid soluble.
[0079] In a fourteenth aspect, the invention provides a method of
determining whether a subject is responsive to treatment with a
therapeutic such as oleuropein or a therapeutic having analogous
activity to oleuropein, comprising determining the level of
expression of one or more genes or gene products selected from the
group consisting of PPAR.gamma.2, LPL, or .alpha.P2 in a cell or a
bodily fluid sample of the subject, and/or determining the
differential expression of the adipogenic, lipogenic and lipolytic
genes or gene products, wherein a change in level of expression of
any one or more of these genes or gene products in a cell or bodily
fluid sample of the subject relative to that in a cell or bodily
fluid sample of a subject that was not treated with oleuropein or a
therapeutic having analogous activity to oleuropein, or a change of
the profiles and or functions of adipogenic, lipogenic and
lipolytic genes, indicates that the subject is responsive to
treatment with oleuropein or an oleuropein analogue or
derivative.
[0080] A fifteenth aspect of the invention provides a method for
determining whether a subject is responsive to treatment with a
therapeutic having analogous activity to oleuropein, comprising
determining the profile of expression of adipogenic, lipogeneic and
lipolytic, genes or gene products selected from the group
consisting of PPAR.delta., PPAR.gamma. and PPAR.alpha. in cells of
the subject, wherein a higher or lower level of expression or
function/activity of any one of these genes in the cells of the
subject relative to that in cells of a subject that was not treated
with a PPAR ligand indicates that the subject is responsive to
treatment with the PPAR ligand.
[0081] In one particular embodiment, the cells are obtained from
whole blood, e.g. peripheral blood mononuclear cells (PBMC). In
another particular embodiment, the cells are adipocytes. In yet
another particular embodiment, the cells are pre-adipocytes. In yet
another embodiment, the cells are mesenchymal stem cells. In yet
another particular embodiment, the bodily fluid sample is plasma or
serum.
[0082] In a sixteenth aspect, the invention provides a method for
predicting whether a subject would be responsive to treatment with
a compound having analogous activity to oleuropein, comprising
treating the subject with the candidate or test compound followed
by collecting preadipocytes or whole blood of the subject and
determining the profile of expression of at least one of the genes
selected from the group consisting of PPAR.gamma.2, LPL, .alpha.P2,
leptin, TNF.alpha., IL-6, PAI-1, adipsin, complement factor C3,
angiotensinogen, hormone-sensitive lipase (HSL), lipoprotein lipase
(LPL), adiponectin, fatty acid-binding protein 4, perilipin and
CCAAT enhancer binding protein, and enzymes of energy metabolism
such as glycerol-3-phosphate dehydrogenase 1, acetyl-coenzyme A
carboxykinase, phosphoenolpyruvate carboxykinase and pyruvate
dehydrogenase kinase, wherein a change in the profile of expression
(the level of expression and/or the change in function or activity)
of at least one of these genes relative to expression in cells or
in a whole blood sample of subjects not treated with oleuropein,
indicates that the subject would be responsive to treatment with
the compound having analogous activity to oleuropein. In another
particular embodiment, a method for predicting whether a subject
would be responsive to treatment with a compound having analogous
activity to oleuropein, comprises incubating cells of the subject
with oleuropein and determining the level of expression of at least
one of the genes selected from the group consisting of PPAR.alpha.
and PPAR.delta. as well as their responsive genes in lipid
metabolism, lipid utilization and energy uncoupling. The expression
of these genes can be profiled in a cellular sample or in a blood
sample, for example, whole blood, or plasma or serum. In addition
to PPAR.alpha., PPAR.delta. and PPAR.gamma. and their responsive
genes, the levels of glucose, fasting insulin, insulin AUC, total
cholesterol, LDL cholesterol, HDL cholesterol, triglyceride,
adiponectin, free fatty acid and TNF.alpha., are also indications
of an effect of oleuropein or an analogue or derivative
thereof.
[0083] In one embodiment, the PPAR is selected from the group
consisting of .alpha., .gamma., or .gamma..
[0084] A seventeenth aspect of the invention provides a method for
purifying biologically active oleuropein, an oleuropein derivative,
or an oleuropein metabolite from olive leave extract, the method
comprising the steps: [0085] a) extracting the oleuropein, or a
derivative or metabolite thereof by heating olive leaves to at
least 80.degree. C. but not above 85.degree. C. for about 10-12
hours in water, saline or phosphate buffer; [0086] b) collecting
the liquid from step a) and repeating the extracting procedure of
step a) at least one additional time; [0087] c) combining the
liquid from steps a) and b) and centrifuging at least
20,000.times.g for at least 30 minutes to remove small particulates
or insoluble material; [0088] d) concentrating the liquid from step
c) by lyophilization until dried; [0089] e) dissolving the dried
extract from step d) into sterile water to a concentration of about
10-20 mg/ml and filter sterilizing; [0090] f) distributing the
material from step e) into sterile cryotubes under aseptic
conditions and storing at a temperature of at least -80.degree. C.;
[0091] g) fractionation, characterization and analyzing the extract
by a method selected from the group consisting of high pressure
liquid chromatography (HPLC), thin layer chromatography (TLC) and
liquid chromatography-mass spectrometry (LC-MS); and [0092] h)
comparing the material obtained in step g) with a known
standard.
[0093] In one embodiment, the ratio of dried leaves to water,
saline or phosphate buffer in step c) above is about 1 gram of
dried leaves to about 40 ml of water, saline or phosphate
buffer.
[0094] In another embodiment, the filter sterilizing is
accomplished using a filter of about 0.45 microns.
[0095] An eighteenth aspect of the invention provides a method for
synthesizing biologically active hydroxytyrosol, or a
hydroxytyrosol analogue or derivative, comprising: [0096] a)
providing 3,4-dihydroxylphenylacetic acid; [0097] b) reacting
3,4-dihydroxylphenylacetic acid with acetyl chloride and methanol
by stirring at room temperature overnight to yield
3,4-dihydroxylphenylacetic ester [0098] c) purifying the
3,4-dihydroxylphenylacetic ester by column chromatography. [0099]
d) dissolving the 3,4-dihydroxylphenylacetic ester of step c) in
tetrahydrofuran; [0100] e) adding 1 molar LiAlH4 into the reaction
mixture from step d); [0101] stirring at 0.degree. C. for about 2
hours; [0102] f) purifying the hydroxytyrosol, or a hydroxytyrosol
analogue or derivative by column chromatography; and [0103] g)
characterizing the hydroxytyrosol, or a hydroxytyrosol analogue or
derivative by liquid chromatography-mass spectrometry.
[0104] A nineteenth aspect of the invention provides for the
preparation of biologically active hydroxytyrosol, comprising:
[0105] a) providing oleuropein; [0106] b) treating the oleuropein
of step a) with beta-glycosidase to yield oleuropein aglycone;
[0107] c) hydrolyzing the oleuropein aglycone to yield
hydroxytyrosol and elenolic acid.
[0108] In one embodiment, the hydrolyzing of step c) above is
accomplished by treating the oleuropein aglycone of step b) with an
esterase to yield hydroxytyrosol and elenolic acid.
[0109] A twentieth aspect of the invention provides for a method of
inhibiting human immunodeficiency virus (HIV) infectivity,
comprising administering a therapeutically effective amount of
oleuropein or hydroxytyrosol, or a derivative or analogue thereof.
In one embodiment, the administering may be in vitro or in
vivo.
[0110] In another embodiment, the oleuropein or hydroxytyrosol, or
a derivative or analogue thereof, prevents the fusion of the virus
to the host cell, and/or prevents cell to cell transmission of the
virus, and/or prevents viral replication by binding to the
active/catalytic site of the HIV integrase.
[0111] In yet another embodiment, the inhibiting of HIV infectivity
is the result of the binding of oleuropein or hydroxytyrosol, or a
derivative or analogue thereof, to a conserved hydrophobic pocket
on the surface of the central trimeric coiled-coil of the HIV gp41
fusion domain.
[0112] In yet another embodiment, the oleuropein or hydroxytyrosol,
or a derivative or analogue thereof, interacts with the N-terminal
heptad repeat (NHR) coiled-coil trimer N36 helices and interferes
with the formation of 6HB with the C-terminal heptad repeat (CHR),
C34.
[0113] In yet another embodiment, the oleuropein or hydroxytyrosol,
or a derivative or analogue thereof, inhibits 6HB formation.
[0114] In yet another embodiment, the oleuropein or hydroxytyrosol,
or a derivative or analogue thereof, binds to both regions I and II
of said integrase.
[0115] In yet another embodiment, the oleuropein or hydroxytyrosol,
or a derivative or analogue thereof, inhibits one or more of the
following activities of the integrase: [0116] a) inhibition of 3'
processing activity of the HIV integrase; [0117] b) inhibition of
strand-transfer activity of the HIV integrase; or [0118] c)
inhibition of the disintegration activity of the HIV integrase.
[0119] In yet another embodiment, the human immunodeficiency virus
is HIV-1 or HIV-2.
[0120] In yet another embodiment, the oleuropein or hydroxytyrosol,
or a derivative or analogue thereof, inhibits the fusion or
replication of any one of an M tropic or a T tropic strain from
different HIV clades.
[0121] Other objects and advantages will become apparent from a
review of the ensuing detailed description and attendant claims
taken in conjunction with the following illustrative drawings. All
references cited in the present application are incorporated herein
in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0122] FIG. 1 Demonstrates that oleuropein modulates adipocyte
differentiation
[0123] FIG. 2 Demonstrates that oleuropein de-differentiates
adipocytes and allows transdifferentiation
[0124] FIG. 3 Represents aortas from apoE knock out mice fed a
Western diet for 4 months, and then stained with Oil Red 0
[0125] FIG. 4 Shows aortic lesions in apoE knock out mice fed a
Western diet for 4 months
[0126] FIG. 5 Shows the predicted PPAR.delta.-oleuropein binding
structure
[0127] FIG. 6 Shows the predicted PPAR.gamma.-oleuropein binding
structure
[0128] FIG. 7 Shows the predicted PPAR.alpha.-oleuropein binding
structure
[0129] FIG. 8 Shows the chemical structure of oleuropein
[0130] FIG. 9 Shows the HPLC elution profile of Olive Leaf Extract
(OLE). The numbers represent the identified peaks. The identities
of the peaks are shown in Table 5. Peak 6 is oleuropein, and
contains the bulk of the biological activity.
[0131] FIG. 10 Shows the chemical structure of oleuropein and its
major metabolites.
[0132] FIG. 11 Shows the results of the standardization of olive
leaf extract by liquid chromatography-mass spectrometry (LC-MS)
based on oleuropein content.
[0133] FIG. 12 Shows the results of the quantitation of oleuropein
and metabolites in serum and urine of mice fed oleuropein.
[0134] FIG. 13. LC-MS analysis of Ole and HT, metabolism of Ole and
chemical synthesis of HT
A. LC elution profile of Ole. B. MS analysis of Ole, showing one
major component with a molecular mass of 539. C. LC elution profile
of HT. D. MS analysis of HT, showing one major component with a
molecular mass of 153. E. Metabolism of Ole, showing major
reactions in the production of HT from Ole. F. Chemical synthesis
of HT.
[0135] FIG. 14. HIV-1 gp41 and the formation of fusion core
6HB.
A. Structure map of HIV-1 gp41, FP (fusion peptide), NHR
(N-terminal heptad repeat), CHR(C-terminal heptad repeat), TR
(tryptophan-rich), TM (transmembrane), and CP (cytoplasmic)
domains. Residue numbers correspond to positions in gp160 of
HIV-1HXB2. B. Formation of 6HB by N36 and C34 helices, showing the
trimeric coiled-coil core of N36 and the surrounding three C34s. C.
and D. Effect of Ole and HT on fusion core, 6HB formation, showing
the binding of Ole or HT to the N36 trimeric coiled-coil core thus
inhibiting 6HB formation.
[0136] FIG. 15. Molecular docking of Ole and HT with HIV-1 gp41
A. Structures of the 5-helical gp41 bundle. B. Structure of Ole.
The 9 freely rotating bonds are shown. C. and D. The predicted
binding structures of Ole (C) and HT (D) inside the HIV-1 gp41
hydrophobic site. gp41 is shown as a surface model and Ole and HT
are shown as stick models. Both molecules for m stable hydrogen
bonds with Q577 (green) on N36 peptide. E. and F. Ribbon
representation showing hydrogen binding of gp41 5HB with Ole (E)
and HT (F). The trimeric coiled-coil core of N36 peptides are pink
and C34 peptides green. The Ole and HT molecules are shown as stick
formation and hydrogen bonds with Q577 are in green dashed
lines.
[0137] FIG. 16. The effect of Ole and HT on the formation of 6HB
between HIV-1 peptides N36 and C34
A. Native PAGE was carried out in Tris-glycine 18% gels, at 120 V
constant voltages at room temperature for 2 h. The gel was then
stained with Coomassie Blue and analyzed by densitometry. Lane 1,
molecular weight markers; lane 2, N36; lane 3, C34; lane 4 fusion
complex, (N36+C34); lanes 5-8 and 9-12, in the presence of Ole and
HT at 25, 50, 75 and 100 nM. B. CD analysis, CD spectra for N36
(Green), C34 (Red), (N36+C34) 6HB (Blue), and N36+C34 in the
presence of 25 and 50 nM of Ole or HT.
[0138] FIG. 17. Molecular docking of Ole and HT with HIV-1
integrase
The predicted binding structures of Ole (A) and HT (B) inside the
HIV-1 integrase catalytic site. Integrase is shown as a surface
model, while Ole and HT are shown as van der Waals models and the
purple sphere represents Mg.sup.2+. Hydrogen bonds formed by Ole
(C) and HT (D) with integrase are indicated as green dotted lines,
and the integrase backbone is represented by the cyan ribbon.
[0139] FIG. 18. The effect of Ole and HT on HIV-1 integrase
3'-processing, strand transfer and disintegration activities
A. The Effect of Ole and HT on 141V-1 Integrase 3'-Processing
Activity
[0140] Left: Schematic representation of the 3'-processing activity
of HIV-1 integrase. A 5'-.sup.32P-labeled 21-mer of HIV-1 LTR U3
double-stranded (ds) DNA was used as the substrate. Specific
cleavage of the dinucleotide GT from the 3' end of the substrate
results in the formation of 19-mer 3'-recessed U3-GT. Right:
Inhibition of HIV-1 integrase 3'-processing activity by Ole and HT.
Inhibition was monitored by the formation of labeled 19-mer
product. Lane 1, the 21-mer substrate, 5'-.sup.32P-labeled U3. Lane
2, cleavage of the 3' GT of the 21-mer substrate by HIV-1 integrase
results in the formation of the 19-mer 3'-recessed U3-GT. Lanes 3-7
or 8-12, in the presence of 25, 50, 75, 100, and 200 nM Ole or
HT.
B. The Effect of Ole and HT on HIV-1 Integrase Strand Transfer
Activity
[0141] Left: Schematic representation of strand transfer
(integration). Pre-cleaved 5'-.sup.32P-labeled U3-GT 19-mer was
used as the viral substrate and, unlabeled pUC18 DNA (2.69 kb) was
used as the heterologous target substrate. Right: Inhibition of
strand-transfer activity of HIV-1 integrase by Ole and HT.
Integration was monitored by the conversion of the unlabeled
plasmid into labeled DNA. Lanes 1,5'-.sup.32P-labeled size marker,
HindIII fragments of .lamda. phage DNA. Lanes 2, target substrate,
pUC18; because it is unlabeled, it is not seen in the
autoradiogram. Lanes 3, the product of integration (ST) by HIV-1
integrase. The integration of the 5'.sup.32P-labeled U3-GT into
pUC18 results in the appearance of labeled band at 2.69 kb,
corresponding to the size of pUC18. Lanes 4-8, in the presence of
25, 50, 75, 100 and 200 .mu.M Ole or HT. C. The effect of Ole and
HT on HIV-1 Integrase Disintegration Activity Left: Schematic
representation of the disintegration activity of HIV-1 integrase.
The 5' .sup.32P-labeled 38-mer dumbbell was used as the substrate
and shown with the predicted secondary structure. Disintegration
yields a .sup.32P-labeled 14-mer consisting of the viral sequences
in the hairpin stem and a 24-mer unlabeled target sequence that has
been repaired. Right: Inhibition of disintegration activity of
HIV-1 integrase by Ole and HT. Lane 1, the 5' .sup.32P-labeled
38-mer dumbbell substrate. Lane 2, treatment with HIV-1 integrase
results in the formation of the 5' .sup.32P-labeled 14-mer
disintegration product. Lanes 3, 4, 5, 6, 7 disintegration assays
in the presence of 25, 50, 75, 100, and 200 nM Ole. Lanes 8, 9, 10,
11, 12, in the presence of 25, 50, 75, 100, and 200 nM HT.
DETAILED DESCRIPTION OF THE INVENTION
[0142] Before the present methods and treatment methodology are
described, it is to be understood that this invention is not
limited to particular methods, and experimental conditions
described, as such methods and conditions may vary. It is also to
be understood that the terminology used herein is for purposes of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only in the appended claims.
[0143] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0144] Unless defined otherwise, 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 belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention, the
preferred methods and materials are now described. All publications
mentioned herein are incorporated herein by reference.
DEFINITIONS
[0145] The terms used herein have the meanings recognized and known
to those of skill in the art, however, for convenience and
completeness, particular terms and their meanings are set forth
below.
[0146] The term "adipocyte" refers to a cell existing in or derived
from fat tissue which is terminally differentiated. In their
differentiated state, adipocytes assume a rounded morphology
associated with cytoskeletal changes and loss of mobility. They
further accumulate lipid as multiple small vesicles that later
coalesce into a single, large lipid droplet displacing the nucleus.
The term "human adipocyte" refers to an adipocyte existing in or
isolated from human fat tissue. Adipocytes play a critical role in
energy homeostasis. They synthesize and store lipids when nutrients
are plentiful, and release fatty acids into the circulation when
nutrients are required. Numerous adipogenic genes are expressed in
functional adipocytes, whereas they are not expressed in
preadipocytes in which lipid are not accumulated either. Adipocyte
development has been extensively studied in cell culture as well as
in animal models. There are several lines of evidence supporting
that adipose tissue dysfunction plays an important role in the
pathogenesis of type II diabetes mellitus, i.e. failure of
adipocyte differentiation is a predisposition to developing
diabetes, (see, e.g., Danforth (2000) Nature Genetics 26: 13).
[0147] The term "adipogenic gene expression" refers to the
expression of several genes known as "adipogenesis marker genes"
and more particularly refers to one or more genes specifically
associated with a specific adipogenesis/differentiation stage. Such
marker genes are well-known in the art. For example, Peroxisome
Proliferator-Activated Receptor .gamma.2 (PPAR.gamma.2),
lipoprotein lipase (LPL), and the adipocyte-selective fatty acid
binding protein (the .alpha.P2 gene). In addition, other
differentiated adipocyte marker genes include glycerophosphate
dehydrogenase (GPDH), fatty acid synthase, acetyl CoA carboxylase,
malic enzyme, Glut 4, and the insulin receptor (see Spiegelman et
al. J. Biol. Chem. 268: 6823-6826, 1993, incorporated herein by
reference). Preadipocytes also have characteristic marker genes,
such as the cell surface antigen recognized by the monoclonal
antibody AD-3. Expression level changes of the various isoforms of
the C/EBP (CCAAT/enhancer-binding proteins) family of transcription
factors may also indicate different stages of adipogenesis (see Yu
and Hausman, Exp Cell Res Dec. 15, 1998; 245(2): 343-9). Other
genes include the lipolytic genes involved in the mobilization and
1-oxidation of stored fat as well as those in the mitochrondia
involved in thermogenesis (the uncoupling enzymes, factors and
modulators) such as PPAR .alpha., PPAR.delta., and their related
genes, hormone-sensitive lipase (HSL), lipoprotein lipase (LPL),
and enzymes of energy metabolism such as glycerol-3-phosphate
dehydrogenase 1, acetyl-coenzyme A carboxykinase,
phosphoenolpyruvate carboxykinase and pyruvate dehydrogenase
kinase.
[0148] The phrase "essentially pure" refers to a cell population,
e.g., a human adipocyte population, that has been isolated from its
natural source (e.g., has been isolated or purified from fat
tissue, for example, from human fat tissue) and, through a
purification step or series of purification steps, has been
separated from other cells (e.g., non-adipocyte cells) and cellular
debris. An essentially pure cell population, as defined according
to the instant invention, is at least 90% pure, i.e., at least 90%
of the cells are of the desired cell type (e.g., human adipocytes)
and less then 10% are contaminating (e.g. non-adipocyte) cells. In
a preferred embodiment, an essentially pure cell population (e.g.,
an essentially pure adipocyte population) is at least 95% pure. In
a more preferred embodiment, an essentially pure cell population
(e.g., an essentially pure adipocyte population) is at least 96%,
97%, 98%, 99% or 100% pure).
[0149] The phrase "differentiation-inducing agent" refers to a
compound or agent that initiates or stimulates the differentiation
of preadipocytes into adipocytes. Preferred
"differentiation-inducing agents" include but are not limited to
insulin, insulin-sensitizing agents, substrates for lipid
synthesis, PPAR ligands (e.g., natural ligands, for example,
prostaglandin J.sub.2, and synthetic ligands, for example,
thiazolidinediones, and the like). The phrase
"differentiation-promoting agent" refers to a compound or an agent
that enhances or accelerates the differentiation of preadipocytes
into adipocytes. Preferred "differentiation-inducing agents"
include but are not limited to insulin, insulin-sensitizing agents,
substrates for lipid synthesis, PPAR ligands, and the like.
"Differentiation-inducing agents" or "differentiation-promoting
agents" vary considerably in effectiveness but share common effects
on several cellular signaling pathways including, but not limited
to: (1) tyrosine kinase pathways (e.g., IGF-1-mediated tyrosine
kinase pathway); (2) adenylyl cyclase/phosphodiesterase signaling
pathways; (3) steroid/thyroid/peroxisome proliferator activated
(PPAR)/retinoid nuclear receptors signaling pathways; and (4)
protein kinase signaling pathways (MacDougald, O. A. et al. (1995)
Annu. Rev. Biochem 64:345-73; Smas, C. M. et al. (1995) Biochem J.
309, 697-710; Cornelius, P. et al. (1994) Annu. Rev. Nutr.
14:99-129). Following induction of differentiation through these
signal transduction pathways, coordinated changes in the expression
of over 600 genes occurs leading to the acquisition and maintenance
of the fat cell phenotype (MacDougald, O. A. et al. (1995) Annu.
Rev. Biochem 64:345-73; Smas, C. M. et al. (1995) Biochem J. 309,
697-710; Cornelius, P. et al. (1994) Annu. Rev. Nutr. 14:99-129).
These changes in differentiation-dependent gene expression are
orchestrated by several transcription factors including CCAAT
enhancer binding proteins (C/EBP.alpha., .beta., and .gamma.),
PPAR.gamma., and others (reviewed in MacDougald, O. A. et al.
(1995) Annu. Rev. Biochem 64:345-73; Smas, C. M. et al. (1995)
Biochem J. 309, 697-710; Kirkland, J. L., et al. (1997) J. Amer.
Geriatr. Soc. 45:959-67). Overexpression of some of these
transcription factors, including C/EBP.alpha. and PPAR .gamma., is
sufficient to induce the differentiation of preadipocytes (Lin, F.
T., et al (1994) Proc. Natl. Acad: Sci. USA 91:8757-8761; Hu, E. et
al. (1995) Proc. Natl. Acad. Sci. USA 92:8956-60; Wu, Z., et al.
(1995) Genes Defer 9:2350-63; Yeh, W. C., et al. (1995) Genes
Devel. 9:168-81).
[0150] Markers of differentiation include (presented in order of
detectable changes in expression): (1) cytoskeletal genes; (2)
lipoprotein lipase (LPL) and collagen isoforms; (3) adipocyte fatty
acyl binding protein (aP2) and glycerol-3-phosphate dehydrogenase
(G3PD); and/or (4) the insulin sensitive glucose transporter
(GLUT4), angiotensinogen (ang), apolipoprotein E (apoE), leptin,
adipsin (complement factor D), protein C3, factor B, and other
genes occur that contribute to the endocrine/paracrine function of
adipose tissue. Increased fat cell mass, which is dependent on the
balance between rates of adipogenesis, lipogenesis and
lipolysis.
[0151] "Modulation" or "modulates" or "modulating" 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. As used herein, a fat cell, preadipocyte or
adipocyte "modulator" or "modulating" compound or agent is a
compound or agent that modulates at least one biological marker or
biological activity characteristic of fat cells and/or fat tissue.
The term "modulating" as related to adipocyte differentiation or
adipogenic gene expression, refers to the ability of a compound or
agent to exert an effect on adipocyte differentiation,
de-differentiation or transdifferentiation or to alter the
expression of at least one gene (as noted above) related to
adipogenesis. In addition, compounds or agents of the invention
modulate at least one of (1) differentiation-specific gene
expression, (2) lipid metabolism (e.g., lipogenesis and/or
lipolysis), (3) fatty acid uptake, (4) fat accumulation and/or (5)
accumulation of cytoplasmic lipid and (6) modulation of PPAR
activity.
[0152] "Differentiate" or "differentiation" as used herein,
generally refers to the process by which precursor or progenitor
cells differentiate into specific cell types. In the matter of the
present invention, the term refers to the process by which
pre-adipocytes become adipocytes. Differentiated cells can be
identified by their patterns of gene expression and cell surface
protein expression. The genes associated with adipocyte
differentiation are noted above. As an example, cells of adipocyte
lineage typically express the following genes: ob, Ucp, PPAR.gamma.
and C/EBPs (see, e.g., Kozak and Kozak, Endocrinology 134(2):906-13
(1994)) and Lee et al., J. Clin. Invest. 111 (4): 453-461 (2003).
As used herein, the term "differentiate" refers to having a
different character or function from the original type of tissues
or cells. Thus, "differentiation" is the process or act of
differentiating.
[0153] "Dedifferentiate" or "dedifferentiation" as used herein,
refers to the process by which lineage committed cells reverse
their lineage commitment and become precursor or progenitor cells.
Dedifferentiated cells can be identified by loss of patterns of
gene expression and cell surface protein expression associated with
the lineage committed cells. A loss of expression or decrease in
expression levels of one or more of the adipocyte genes noted above
indicates that an adipocyte has undergone dedifferentiation.
[0154] "Transdifferentiation" refers to the process by which
precursor or progenitor cells pre-committed to cell types of one
lineage differentiate into specific cell types of another lineage,
e.g., pre-adipocytes can transdifferentiate into osteoblasts and
vice versa. Transdifferentiated cells can be identified by their
patterns of gene expression and cell surface protein expression.
Typically, cells of an osteoblast lineage express genes such as,
for example, alkaline phosphatase, collagen type I, osteocalcin,
and osteoponin; and bone specific transcription factors such as,
for example, Cbfa1/Runx2, Osx, gsc, Dlx1, Dlx5, Msx1, Cart1, Hoxa1,
Hoxa2, Hoxa3, Hoxb1, rae28, Twist, AP-2, Mf1, Pax1, Pax3, Pax9,
TBX3, TBX4, TBX5, and Brachyury (see, e.g., Olsen et al, 2000 supra
and Nakashima et al., Cell 108(1):17-29 (2002). The cells can be
transdifferentiated within the same progenitors. For example,
mesenchymal stem cells or marrow stromal cells (MSC), are stem
cells that can differentiate into osteoblasts, chondrocytes,
myocytes, adipocytes, neuronal cells, and, as described lately,
into beta-pancreatic islets cells. Thus these cells can be cross
transdifferentiated under optimal culture conditions and/or growth
factors. MSCs cultured in the presence of transformation growth
factor (TGF), specifically bone morphogenetic protein (BMP), will
differentiate into chondrocytes, whereas MSCs cultured in serum
with ascorbic acid, inorganic phosphate and dexamethasone will
differentiate into osteoblasts. On the other hand, MSCs cultured
under adipogenic conditions in the presence of dexamethasone,
insulin and isobutyl-methylxanthine will differentiate into
adipocytes.
[0155] Adipocyte differentiation is a multistep process controlled
by the action of a complex interactive network of transcription
factors in mammals. In the fruit fly the serpent gene is critical
for the genesis of the fruit fly fat body, which corresponds to
mammalian liver and adipose tissue. The GATA family of
transcription factors in mice are the mammalian homologues of the
Drosophila serpent gene. Both GATA-2 and GATA-3 directly bind to
specific sites in the proximal promoter of the adipogenic
transcription factor, peroxisome proliferaror activated
receptor-gamma (PPAR-gamma), and negatively regulate its activity.
Both GATA-2 and GATA-3 expression are severely defective in the
white adipose tissue of several different models of obesity. These
results indicate that GATA-2 and GATA-3 are preadipocyte markers
and play an important role in adipogenesis.
[0156] As used herein, the term "candidate compound" or "candidate
therapeutic" or "test compound" or "agent" or "test agent" refers
to any compound or molecule that is to be tested. As used herein,
the terms, which are used interchangeably, refer to biological or
chemical compounds such as simple or complex organic or inorganic
molecules, peptides, proteins, antibodies, oligonucleotides,
polynucleotides, carbohydrates, or lipoproteins. A vast array of
compounds can be synthesized, for example oligomers, such as
oligopeptides and oligonucleotides, and synthetic organic compounds
based on various core structures, and these are also included in
the terms noted above. In addition, various natural sources can
provide compounds for screening, such as plant or animal extracts,
and the like. Compounds can be tested singly or in combination with
one another. Agents or candidate compounds can be randomly selected
or rationally selected or designed. As used herein, an agent or
candidate compound is said to be "randomly selected" when the agent
is chosen randomly without considering the specific interaction
between the agent and the target compound or site. As used herein,
an agent is said to be "rationally selected or designed", when the
agent is chosen on a nonrandom basis which takes into account the
specific interaction between the agent and the target site and/or
the conformation in connection with the agent's action. Moreover,
the agent may be selected by its effect on the gene expression
profile obtained from screening in vitro or in vivo. For example,
the gene expression data for primary human preadipocytes and
adipocytes can be accessed online through databases including Pub
Med, Human Genome Project (HGP), Gene Bank and PDB (Protein Data
Bank).
[0157] Biochemically, "adipogenesis" is referred to as the process
of fat cell formation. It involves commitment, differentiation and
maturation of adipocytes. Adipogenic genes, gene products, enzymes,
factors, and pathways are the genes, gene products, enzymes,
factors, and pathways for adipocyte differentiation (adipogenesis
or fat cell formation), and these genes are described throughout
the present application. "Lipogenesis" is referred to as the
process of lipid biosynthesis (fat formation), the conversion of
carbohydrate or protein to fat and the synthesis of triglycerides
from 2-monogylcerol and free fatty acids as well as from
glycerol-3-phosphate and fatty acyl CoA. In addition, the synthesis
of long chain fatty acids, using acetyl CoA as the primer and
malonyl CoA as the addition unit also refers to as lipogenesis.
Lipogenic genes, gene products, enzymes, factors, and pathways are
the genes, gene products, enzymes, factors, and pathways for lipid
biosynthesis (fat formation), and these genes are also described
throughout the present application. Thus, adipogenesis is different
from lipogenesis and the two terms are not synonymous. In addition,
"adipogenesis" refers to the process whereby adipose tissue, a
mesodermal derivative, develops from preadipocytes. "Adipogenesis"
refers to the process by which an undifferentiated precursor cell
differentiates into an adipocyte (a fat cell, which is a cell
characterized by the cellular function of fat storage e.g., in
cytoplasmic lipid droplets). Precursor cells that are involved in
the process of adipogensis include pre-adipocytes, mesenchymal stem
cells and progenitor cells. Generally, diseases associated with
adipogenesis include body weight disorders such as obesity and
cachexia, and nonshivering and shivering thermogenesis.
Accordingly, in one aspect of the invention, the agents identified
as modulators of adipogenesis are potentially useful for modulating
body weight-related processes, including, for example, treatment of
body weight disorders such as obesity and cachexia, and
thermogenesis. Treatment of other obesity related disorders or
conditions are also contemplated. Obesity related disorders or
conditions may be selected from the group consisting of coronary
artery disease/cardiovascular disease, hypertension,
cerebrovascular disease, stroke, peripheral vascular disease,
insulin resistance, glucose intolerance, diabetes mellitus,
hyperglycemia, hyperlipidemia, dyslipidemia, hypercholesteremia,
hypertriglyceridemia, hyperinsulinemia, atherosclerosis, cellular
proliferation and endothelial dysfunction, diabetic dyslipidemia,
HIV-related lipodystrophy, peripheral vessel disease, cholesterol
gallstones, cancer, menstrual abnormalities, infertility,
polycystic ovaries, osteoarthritis, sleep apnea, metabolic syndrome
(Syndrome X), type II diabetes, diabetic complications including
diabetic neuropathy, nephropathy, retinopathy, cataracts, heart
failure, inflammation, thrombosis, congestive heart failure, and
any other cardiovascular disease related to obesity or an
overweight condition. Obesity induced or related asthma, airway
dysfunction and pulmonary disorders are also important diseases
linked to obesity. "Lipolysis" or "lipolytic" refers to the process
of lipid degradation/oxidation (fat burning, utilization, energy
production). Lipolytic genes, gene products, enzymes, factors, and
pathways are the genes, gene products, enzymes, factors, and
pathways for lipid oxidation (fat burning, .beta.-oxidation).
"Thermogenesis" refers to the process of heat and energy
generation, also known as energy uncoupling. Thermogenic genes,
gene products, enzymes, factors, and pathways are the genes, gene
products, enzymes, factors, and pathways for body heat generation
and energy uncoupling. This process also increases metabolic
rate.
[0158] The PPARs play a central role in adipocyte differentiation.
PPAR gamma responsive genes in human adipocyte differentiation are
briefly discussed below: Affymetrix profiling of gene expression in
human adipocytes identified about 1000 genes that were
significantly up-regulated subsequent to induction of
differentiation and 278 statistically significantly down-regulated
genes [Gene. 2006 Mar. 15; 369:90-9]. In addition, it is also
necessary to consider PPAR alpha and delta responsive genes. A
recent report (Diabetologia. 2005 September; 48(9):1776-83. Epub
2005 Jul. 30) on microarray gene profiling of isolated abdominal
subcutaneous adipocytes from 20 non-obese (BMI 25.+-.3 kg/m.sup.2)
and 19 obese (BMI 55.+-.8 kg/m.sup.2) non-diabetic Pima Indians
using Affymetrix HG-U95 GeneChip arrays showed that the most
differentially expressed genes in adipocytes of obese individuals
consisted of 433 upregulated and 244 downregulated genes. Of these,
410 genes could be classified into 20 functional Gene Ontology
categories. The analyses indicated that the inflammation/immune
response category was over-represented, and that most
inflammation-related genes were upregulated in adipocytes of obese
subjects. This study provides evidence supporting the active role
of mature adipocytes in obesity-related inflammation. It also
provides potential candidate genes for susceptibility to
obesity.
[0159] Depending on the desired result, an agent identified to
induce adipogenesis is potentially useful for increasing body
weight and an agent identified to prevent adipogenesis is
potentially useful for decreasing body weight. Adipogenesis in vivo
and in vitro is subject to hormonal and transcriptional control, in
part mediated by a cascade of transcription factors including
members of the CCAAT/enhancer binding protein family, basic
helix-loop-helix leucine zipper (bBLH-LZ) family, e.g., ADD1/SREBP1
and peroxisome proliferator activated receptor gamma (PPARgamma)
(See, e.g., Wu et al. (1999) Transcriptional activation of
adipogenesis Current Opin. Cell Biol 11:689-694, Rosen and
Spiegelman (2000) Molecular regulation of adipogenesis Annu Rev
Cell Dev Biol 16:145-171, for recent reviews, as well as Kim and
Spiegelman (1996) ADD1/SREBP1 promotes adipocyte differentiation
and gene expression linkedfatty acid metabolism Genes Devel
10:1096-1107). However details regarding cellular targets of such
transcription factors remain largely undetermined, as do the
mechanisms underlying their action in physiological and
pathological processes.
[0160] "Subject" or "patient" refers to a mammal, preferably a
human, in need of treatment for a condition, disorder or
disease.
[0161] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water or aqueous solution
saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly for injectable
solutions. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin.
[0162] A "therapeutically effective amount" or "an effective
amount", which are used interchangeably, is an amount sufficient to
decrease or prevent the symptoms associated with the conditions
disclosed herein, including diseases associated with obesity, or an
amount to inhibit the infectivity of human immunodeficiency virus
(HIV-1 and HIV-2) and other related conditions contemplated for
therapy with the compositions of the present invention. The effect
on HIV infectivity may be measured by any of the commonly used
methods known to those skilled in the art, for example, cell fusion
may be measured by assessing the level of syncitia formation. Viral
replication may be measured by using PCR procedures to monitor the
level of viral nucleic acid in a host cell.
[0163] "Metabolic Syndrome" or otherwise known as "Syndrome X"
means a disease characterized by spontaneous hypertension,
dyslipidemia, insulin resistance, hyperinsulinemia, increased
abdominal fat and increased risk of coronary heart disease. As used
herein, the terms "Syndrome X", "Metabolic Syndrome" and "Metabolic
Syndrome X" shall mean a disorder that presents risk factors for
the development of Type II diabetes mellitus and cardiovascular
disease and is characterized by insulin resistance and
hyperinsulinemia and may be accompanied by one or more of the
following: (a) glucose intolerance, (b) Type II diabetes, (c)
dyslipidemia, (d) hypertension and (e) obesity.
[0164] "Obesity" refers to a condition in which the body weight of
a mammal exceeds medically recommended limits by at least about
20%, based upon age and skeletal size. "Obesity" is characterized
by fat cell hypertrophy and hyperplasia. "Obesity" may be
characterized by the presence of one or more obesity-related
phenotypes, including, for example, increased body mass (as
measured, for example, by body mass index, or "BMI"), altered
anthropometry, basal metabolic rates, or total energy expenditure,
chronic disruption of the energy balance, increased Fat Mass as
determined, for example, by DEXA (Dexa Fat Mass percent), altered
maximum oxygen use (VO2), high fat oxidation, high relative resting
rate, glucose resistance, hyperlipidemia, insulin resistance, and
hyperglycemia. See also, for example, Hopkinson et al. (1997) An J
Clin Nutr 65(2): 432-8 and Butte et al. (1999) Am J Clin Nutr
69(2): 299-307. "Overweight" individuals are generally having a
body mass index (BMI) between 25 and 30. "Obese" individuals or
individuals suffering from "obesity" are generally individuals
having a BMI of 30 or greater. Obesity may or may not be associated
with insulin resistance.
[0165] An "obesity-related disease" or "obesity related disorder"
or "obesity related condition", which are all used interchangeably,
refers to a disease, disorder, or condition, which is associated
with, related to, and/or directly or indirectly caused by obesity.
The "obesity-related diseases", or the "obesity-related disorders"
or the "obesity related conditions" include but are not limited to,
coronary artery disease/cardiovascular disease, hypertension,
cerebrovascular disease, stroke, peripheral vascular disease,
insulin resistance, glucose intolerance, diabetes mellitus,
hyperglycemia, hyperlipidemia, dyslipidemia, hypercholesteremia,
hypertriglyceridemia, hyperinsulinemia, atherosclerosis, cellular
proliferation and endothelial dysfunction, diabetic dyslipidemia,
HIV-related lipodystrophy, peripheral vessel disease, cholesterol
gallstones, cancer, menstrual abnormalities, infertility,
polycystic ovaries, osteoarthritis, sleep apnea, metabolic syndrome
(Syndrome X), type II diabetes, diabetic complications including
diabetic neuropathy, nephropathy, retinopathy, cataracts, heart
failure, inflammation, thrombosis, congestive heart failure, and
any other cardiovascular disease related to obesity or an
overweight condition and/or obesity related asthma, airway and
pulmonary disorders.
[0166] An individual "at risk" may or may not have detectable
disease, and may or may not have displayed detectable disease prior
to the treatment methods described herein. "At risk" denotes that
an individual who is determined to be more likely to develop a
symptom based on conventional risk assessment methods or has one or
more risk factors that correlate with development of obesity or an
obesity-related disease or a disease for which PPAR modulation
provides a therapeutic benefit, or an individual who is more likely
to acquire HIV through use of shared needles or those individuals
who may practice unprotected sexual activity. An individual having
one or more of these risk factors has a higher probability of
developing obesity or an obesity-related disease, or HIV than an
individual without these risk factors. Examples (i.e., categories)
of risk groups are well known in the art and discussed herein.
[0167] "Development" or "progression" of obesity herein means
initial manifestations and/or ensuing progression of the disorder.
Development of obesity can be detectable and assessed using
standard clinical techniques, such as measurement of increased body
mass (as measured, for example, by body mass index, or "BMI"),
altered anthropometry, basal metabolic rates, or total energy
expenditure, chronic disruption of the energy balance, increased
Fat Mass as determined, for example, by DEXA (Dexa Fat Mass
percent), altered maximum oxygen use (VO2), high fat oxidation,
high relative resting rate, glucose resistance, hyperlipidemia,
insulin resistance, and hyperglycemia. See also, for example,
Hopkinson et al. (1997) Am J Clin Nutr 65(2): 432-8 and Butte et
al. (1999) Am J Clin Nutr 69(2): 299-307. However, development also
refers to disease progression that may be undetectable. For
purposes of this invention, development or progression refers to
the biological course of the disease state. "Development" includes
occurrence, recurrence, and onset. As used herein "onset" or
"occurrence" of obesity includes initial onset and/or
recurrence.
[0168] As used herein, "delaying development" of obesity, or an
obesity-related disease, means to defer, hinder, slow, retard,
stabilize, and/or postpone development of the disease. This delay
can be of varying lengths of time, depending on the history of the
disorder and/or the medical profile of the individual being
treated. As is evident to one skilled in the art, a sufficient or
significant delay can, in effect, encompass prevention, in that the
individual does not develop detectable disease. A method that
"delays" development of disease is a method that reduces the extent
of the disease in a given time frame, when compared to not using
the method. Such comparisons are typically based on animal or
clinical studies, using a statistically significant number of
subjects, although this knowledge can be based upon anecdotal
evidence. "Delaying development" can mean that the extent and/or
undesirable clinical manifestations are lessened and/or time course
of the progression is slowed or lengthened, as compared to not
administering the agent. Thus the term also includes, but is not
limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease, delay
or slowing of disease progression, and remission (whether partial
or total) whether detectable or undetectable.
[0169] "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.
[0170] The term "hyperinsulinemia" refers to a state in an
individual in which the level of insulin in the blood is higher
than normal.
[0171] 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.
[0172] An "insulin resistance disorder" as discussed herein, refers
to any disease or condition that is caused by or contributed to by
insulin resistance. Examples include: diabetes, obesity, metabolic
syndrome, 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 nephrosclerosis 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.
[0173] "Alpha-glucosidase inhibitors" act to inhibit
alpha-glucosidase, which is an enzyme that converts fructose to
glucose, thus these inhibitors delay the digestion of
carbohydrates. The undigested carbohydrates are subsequently broken
down in the gut, thereby reducing the post-prandial glucose peak.
Suitable examples include, but are not limited to, acarbose,
voglibose and miglitol.
[0174] "Sulfonylureas" increase insulin production by stimulating
pancreatic beta cells, and therefore act as insulin secretagogues.
The primary mechanism of action of sulfonylureas is to close
ATP-sensitive potassium channels in the beta-cell plasma membrane,
initiating a chain of events that result in insulin release.
Suitable examples of sulfonylureas include, but are not limited to
chlorpropamide, tolazamide, tolbutamide, glyburide, glipizide,
glimepiride, and like. Meglitinides, another class of insulin
secretagogues, that have a mechanism of action distinct from that
of the sulfonylureas. Suitable examples of meglitinides include,
but are not limited to repaglinide. Agents which modify insulin
secretion such as Glucagon-like Peptide-1 (GLP-1) and it's
mimetics, Glucose-insulinotropic peptide (GIP) and it's mimetics,
Exendin and it's mimetics, and Dipeptyl Protease Inhibitors (DPPIV)
are also contemplated for use with the invention.
[0175] "Biguanides" are compounds that decrease liver glucose
production and increase the uptake of glucose. Suitable examples
include, but are not limited to metformin, phenformin and
buformin.
[0176] A "peroxisome proliferator activated receptor" or "PPAR" is
a member of a family of nuclear receptors, distinguished in alpha
(.alpha.), delta (6), and gamma (.gamma.) subtypes as described
herein. As used herein the term "PPAR" refers to a peroxisome
proliferator-activated receptor as recognized in the art. As
indicated above, the PPAR family includes PPAR .alpha. (also
referred to as PPARa or PPARalpha), PPAR .delta. (also referred to
as PPAR beta, PPARd or PPARdelta), and PPAR .gamma. (also referred
to as PPARg or PPARgamma). The individual PPARs can be identified
by their sequences, where exemplary reference sequence accession
numbers are: NM.sub.--005036 (cDNA sequence for hPPARa (SEQ ID
NO:1)), NP.sub.--005027 (protein sequence for hPPARa (SEQ ID NO:
2)), NM.sub.--015869 (cDNA sequence for hPPARg isoform 2 (SEQ ID
NO: 3)), NP.sub.--056953 (protein sequence for hPPARg isoform 2
(SEQ ID NO: 4)), NM.sub.--006238 (cDNA sequence for hPPARd (SEQ ID
NO: 5)), and NP.sub.--006229 (protein sequence for hPPARd (SEQ ID
NO: 6)). One of ordinary skill in the art will recognize that
sequence differences will exist due to allelic variation, and will
also recognize that other animals, particularly other mammals have
corresponding PPARs, which have been identified or can be readily
identified using sequence alignment and confirmation of activity,
can also be used. One of ordinary skill in the art will also
recognize that modifications can be introduced in a PPAR sequence
without destroying PPAR activity. Such modified PPARs can also be
used in the present invention, e.g., if the modifications do not
alter the binding site conformation to the extent that the modified
PPAR lacks substantially normal ligand binding.
[0177] A "PPAR modulating agent" or "PPAR modulator" refers to any
agent that binds to any of the PPAR receptors and acts to either
enhance the activity or function of that particular receptor
(agonist) or acts to inhibit or depress the activity or function of
that particular receptor (antagonist). The PPAR receptors include
the .alpha., .gamma., and .delta. receptors. It is also possible to
have the following types of PPAR modulators: [0178] Selective PPAR
modulators that serve as selective or partial agonist/antagonist
and uncouple therapeutic effects from adverse side effects. For
example, a selective/partial PPAR .gamma. agonist/antagonist that
uncouples the therapeutic effect of insulin sensitizing activity
from adipogenic effect (side effect for weight gain during
treatment) [0179] Dual PPAR modulators that act on two PPARs
collectively, selectively or partially either as agonists or
agonist-antagonists combined. For example, the dual
PPAR.gamma./PPAR.alpha. agonists have two separate therapeutic
targets in metabolic pathways in adipose tissue and liver. They may
improve both hyperglycemia and atherogenic dyslipidaemia and may
further reduce the inflammatory component of atherogenesis. [0180]
Pan PPAR modulators that interact with all of the three PPARs
collectively, selectively or partially either as agonists or
agonist-antagonists combined. For example, the pan
PPAR.gamma./PPAR.alpha./PPAR .delta. agonists have three separate
therapeutic targets--metabolic pathways in adipose tissue, liver
and muscle (and other tissue). They may improve hyperglycemia,
atherogenic dyslipidemia, inflammatory component of atherogenesis,
energy uncoupling and weight reduction.
[0181] "Thiazolidinediones" are insulin sensitizing drugs, which
decrease peripheral insulin resistance by enhancing the effects of
insulin at target organs and tissues. These drugs bind and activate
the nuclear receptor, peroxisome proliferator-activated
receptor-gamma (PPAR-gamma) which increases transcription of
specific insulin-responsive genes. Suitable examples of PPAR-gamma
agonists are the thiazolidinediones which include, but are not
limited to rosiglitazone, pioglitazone, troglitazone, isaglitazone
(known as MCC-555),
2-[2-[(2R)-4-hexyl-3,4-dihydro-3-oxo-2H-1,4-benzoxazin-2-yl]ethoxy]-benze-
ne acetic acid, and the like. Additionally, the
non-thiazolidinediones also act as insulin sensitizing drugs, and
include, but are not limited to GW2570, and the like.
[0182] The concept of "combination therapy" is well exploited in
current medical practice. Treatment of a pathology by combining two
or more agents that target the same pathogen or biochemical pathway
sometimes results in greater efficacy and diminished side effects
relative to the use of the therapeutically relevant dose of each
agent alone. In some cases, the efficacy of the drug combination is
additive (the efficacy of the combination is approximately equal to
the sum of the effects of each drug alone), but in other cases the
effect can be synergistic (the efficacy of the combination is
greater than the sum of the effects of each drug given alone). As
used herein, the term "combination therapy" means the two compounds
can be delivered in a simultaneous manner, e.g. concurrently, or
wherein one of the compounds is administered first, followed by the
second agent, e.g sequentially. The desired result can be either a
subjective relief of one or more symptoms or an objectively
identifiable improvement in the recipient of the dosage.
[0183] "Atherogenesis" is a process of forming atheromas, that is,
plaques in the inner lining (the intima) of arteries. It is a
process in which the immune system appears to take an active part,
and refers to the build-up of plaque in the blood vessels.
Accordingly, activated lymphocytes have been detected in human and
murine plaques, sometimes even preceding the infiltrating
lipid-laden macrophages. The atherosclerotic process entails a
proliferative phenotype, involving, apart from lymphocytes and
macrophages, also smooth muscle cells, which occupy lesions that
are relatively more advanced. Furthermore, atherosclerosis is the
accumulation of lipid, inflammatory cells, and fibrous tissue in
the intima, which causes intimal thickening of large and mid-sized
arteries. The clinical manifestations differ depending on the
circulatory bed affected. The coronary arteries are particularly
susceptible to atherogenesis; atherosclerosis of the coronary
arteries may lead to angina pectoris and myocardial infarction.
Dyslipidemia is a primary, major risk factor for coronary artery
disease (CAD) and may even be a prerequisite for CAD, occurring
before other major risk factors come into play. "Diet-induced
atherogenesis" refers to the induction or initiation of the
atherogenic process by consumption of food that contains a high fat
content, which ultimately results in build-up of plaque in the
blood vessels.
[0184] "Endothelial dysfunction" is a physiological dysfunction of
normal biochemical processes carried out by endothelial cells, the
cells that line the inner surface of all blood vessels, arteries
and veins. Compromise of normal function of endothelial cells is
characteristic of endothelial dysfunction. Normal functions of
endothelial cells include mediation of coagulation, platelet
adhesion, immune function, control of volume and electrolyte
content of the intravascular and extravascular spaces. An important
consequence of endothelial dysfunction is the inability of a vessel
to dilate in response to physiological stimuli, such as increases
in blood flow, reflecting impaired flow-dependent,
endothelium-mediated vasodilation (FDD). Accumulating evidence
suggests that endothelial dysfunction contributes to exercise
intolerance, impaired myocardial perfusion, and left ventricular
remodelling in congestive heart failure. Moreover, impaired
endothelial function is associated with a number of disease states,
including cardiovascular disease (CVD) and its major risk factors.
Endothelial dysfunction precedes overt vascular disease by years
and may itself be a potentially modifiable CVD risk factor.
Although no gold standard for the measurement of endothelial
function exists, the measurement of flow-mediated dilation (FMD) in
the brachial artery, assessed with Doppler ultrasonography, is the
most studied method and shows the most promise for clinical
application. It is a well-tolerated, noninvasive, and low-risk
procedure. Brachial artery FMD is an attractive screening tool for
assessing endothelial dysfunction.
[0185] "Treatment" or "treating" refers to therapy, prevention and
prophylaxis and particularly refers to the administration of
medicine or the performance of medical procedures with respect to a
patient, for either prophylaxis (prevention) or to cure (if
possible) or reduce the extent of or likelihood of occurrence of
the infirmity or malady or condition or event in the instance where
the patient is afflicted. More particularly, as related to the
present invention, "treatment" or "treating" is defined as the
application or administration of a therapeutic agent to a patient,
or application or administration of a therapeutic agent to an
isolated tissue or cell line from a patient, who has a disease, a
symptom of disease or a predisposition toward development of a
disease. Treatment can slow, cure, heal, alleviate, relieve, alter,
remedy, ameliorate, improve or affect the disease, a symptom of the
disease or the predisposition toward disease, e.g., by at least
10%. In the present invention, the treatments using the agents
described may be provided to slow or halt weight gain, or to aid in
weight reduction, or to prevent fat accumulation or obesity, or to
inhibit adipocyte differentiation or adipogenic gene expression, or
to increase the amount or quality of lean muscle mass present in
the mammal, or to prevent the infectivity of HIV, or to slow viral
spread, or to alleviate one or more symptoms associated with the
presence of the viral disease. More preferably, the goal is the
treatment of obesity or obesity-related diseases, disorders or
conditions and the morbidity associated with such conditions and
the treatment of human immunodeficiency virus infections and the
symptoms and sequelae associated with the presence of the viral
infection.
[0186] "Diagnosis" or "screening" refers to diagnosis, prognosis,
monitoring, characterizing, selecting patients, including
participants in clinical trials, and identifying patients at risk
for or having a particular disorder or clinical event or those most
likely to respond to a particular therapeutic treatment, or for
assessing or monitoring a patient's response to a particular
therapeutic treatment.
[0187] A "small molecule" refers to a composition that has a
molecular weight of less than 3 kilodaltons (kDa), and preferably
less than 1.5 kilodaltons, and more preferably less than about 1
kilodalton. Small molecules may be nucleic acids, peptides,
polypeptides, peptidomimetics, carbohydrates, lipids or other
organic (carbon-containing) or inorganic molecules. As those
skilled in the art will appreciate, based on the present
description, extensive libraries of chemical and/or biological
mixtures, often fungal, bacterial, or algal extracts, may be
screened with any of the assays of the invention to identify
compounds that modulate a bioactivity. A "small organic molecule"
is an organic compound (or organic compound complexed with an
inorganic compound (e.g., metal)) that has a molecular weight of
less than 3 kilodaltons, and preferably less than 1.5 kilodaltons,
and more preferably less than about 1 kilodalton.
[0188] "Disease associated with PPAR .gamma., .delta., or .alpha."
or "a disease associated with a PPAR .gamma., .delta., or .alpha.
receptor" includes diseases treatable with a ligand of any of the
above-noted PPAR receptors, such as but not limited to Type II
diabetes and obesity, or other obesity related disorders. Diseases
related to PPAR expression and/or activity would also be considered
as associated with the PPAR receptor, such as obesity or other
disorders expected to be affected by alterations in the PPAR's role
in adipocyte differentiation, de-differentiation and
transdifferentiation as well as adipocyte functions including fat
synthesis, storage, utilization and energy uncoupling. The diseases
associated with the modulation of the different PPAR receptors are
known to those skilled in the art. For example, possible diseases
and/or risk factors associated with PPARs are: the metabolic
syndrome and its associated risk factors for atherosclerotic
cardiovascular disease (ASCVD) and diabetes, atherogenic
dyslipidemia, elevated blood pressure, elevated plasma glucose, a
prothrombotic state and a pro-inflammatory state. In addition,
other conditions, including fatty liver, polycystic ovary disease,
sleep apnea, cholesterol gallstones, asthma and cancer.
[0189] "Prophylactic" or "therapeutic" treatment refers to
administration to the host of one or more of the subject
compositions. 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).
[0190] "Therapeutic agent" or "therapeutic" refers to an agent
capable of having a desired biological effect on a host.
Chemotherapeutic and genotoxic agents are examples of therapeutic
agents that are generally known to be chemical in origin, as
opposed to biological, or cause a therapeutic effect by a
particular mechanism of action, respectively. Examples of
therapeutic agents of biological origin include growth factors,
hormones, and cytokines. A variety of therapeutic agents are known
in the art and may be identified by their effects. Certain
therapeutic agents are capable of regulating cell proliferation and
differentiation. Examples include chemotherapeutic nucleotides,
drugs, hormones, non-specific (non-antibody) proteins,
oligonucleotides (e.g., antisense oligonucleotides that bind to a
target nucleic acid sequence (e.g., mRNA sequence)), peptides, and
peptidomimetics. Examples of other therapeutic agents that modulate
PPAR gamma, alpha and delta are the thiazolidinediones, firbrates,
fatty acids and eicosanoids.
[0191] "Therapeutic effect" refers to a local or systemic effect in
animals, particularly mammals, and more particularly humans caused
by a pharmacologically active substance. The term thus 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 conditions in an
animal or human.
[0192] "Agonist" refers to an agent that mimics or up-regulates
(e.g., potentiates or supplements) the bioactivity of a protein. An
agonist may be a wild-type protein or derivative thereof having at
least one bioactivity of the wild-type protein. An agonist may also
be a compound that up-regulates expression of a gene or which
increases at least one bioactivity of a protein. An agonist may
also be a compound which increases the interaction of a polypeptide
with another molecule, e.g., a target peptide or nucleic acid. An
agonist may also be a compound that increase or up-regulate the
activity and/or function of a protein, peptide, an enzyme or
biofactor.
[0193] "Antagonist" refers to an agent that down-regulates (e.g.,
suppresses or inhibits) at least one bioactivity of a protein. An
antagonist may be a compound which inhibits or decreases the
interaction between a protein and another molecule, e.g., a target
peptide or enzyme substrate. An antagonist may also be a compound
that down-regulates expression of a gene or which reduces the
amount of expressed protein present. An antagonist may also be a
compound that decrease or down-regulate the activity and/or
function of a protein, peptide, an enzyme or biofactor.
[0194] "Analog" or "analogue" as used herein, refers to a chemical
compound, a nucleotide, a protein, or a polypeptide that possesses
similar or identical activity or function(s) as the chemical
compounds, nucleotides, proteins or polypeptides having the desired
activity and therapeutic effect of the present invention (eg. to
inhibit fat accumulation, or to modulate adipocyte differentiation
or adipogenic gene expression or to treat obesity or
obesity-related diseases, disorders or conditions), but need not
necessarily comprise a compound that is similar or identical to
those compounds of the preferred embodiment, or possess a structure
that is similar or identical to the agents of the present
invention. An agent having activity "analogous to oleuropein" is an
agent that possesses similar functions and activities as
oleuropein, including the effects on the adipogenic and lipolytic
genes described herein, and on adipocyte metabolism,
differentiation, de-differentiation, or transdifferentiation or
effects on the PPAR receptors described herein.
[0195] "Derivative" refers to the chemical modification of
molecules, either synthetic organic molecules or proteins, nucleic
acids, or any class of small molecules such as fatty acids, or
other small molecules that are prepared either synthetically or
isolated from a natural source, such as a plant, that retain at
least one function of the active parent molecule, but may be
structurally different. Chemical modifications may include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group.
It may also refer to chemically similar compounds which have been
chemically altered to increase bioavailability, absorption, or to
decrease toxicity. A derivative polypeptide is one modified by
glycosylation, pegylation, or any similar process that retains at
least one biological or immunological function of the polypeptide
from which it was derived.
[0196] As used herein in connection with the design or development
of PPAR ligands, the term "bind" and "binding" and like terms refer
to a non-convalent energetically favorable association between the
specified molecules (i.e., the bound state has a lower free energy
than the separated state, which can be measured calorimetrically).
For binding to a target, the binding is at least selective, that
is, the compound binds preferentially to a particular target or to
members of a target family at a binding site, as compared to
non-specific binding to unrelated proteins not having a similar
binding site. For example, BSA is often used for evaluating or
controlling for non-specific binding. In addition, for an
association to be regarded as binding, the decrease in free energy
going from a separated state to the bound state must be sufficient
so that the association is detectable in a biochemical assay
suitable for the molecules involved.
[0197] By "binding pocket" is meant a specific volume within a
binding site. A binding pocket is a particular space within a
binding site at least partially bounded by target molecule atoms.
Thus a binding pocket is a particular shape, indentation, or cavity
in the binding site. Binding pockets can contain particular
chemical groups or structures that are important in the
non-covalent binding of another molecule such as, for example,
groups that contribute to ionic, hydrogen bonding, van der Waals,
or hydrophobic interactions between the molecules.
[0198] In the context of target molecules in the present invention,
the term "crystal" refers to an ordered complex of target molecule,
such that the complex produces an X-ray diffraction pattern when
placed in an X-ray beam. Thus, a "crystal" is distinguished from a
disordered or partially ordered complex or aggregate of molecules
that do not produce such a diffraction pattern. Preferably a
crystal is of sufficient order and size to be useful for X-ray
crystallography. A crystal may be formed only of target molecule
(with solvent and ions) or may be a co-crystal of more than one
molecule, for example, as a co-crystal of target molecule and
binding compound, and/or of a complex of proteins (such as a
holoenzyme).
[0199] By "designing a ligand", "preparing a ligand", "discovering
a ligand", and like phrases is meant the process of considering
relevant data (especially, but not limited to, any individual or
combination of binding data, X-ray co-crystallography data,
molecular weight, clogP, and the number of hydrogen bond donors and
acceptors) and making decisions about advantages that can be
achieved with resort to specific structural modifications to a
molecule, and implementing those decisions. This process of
gathering data and making decisions about structural modifications
that can be advantageous, implementing those decisions, and
determining the result can be repeated as many times as necessary
to obtain a ligand with desired properties.
[0200] By "docking" is meant the process of attempting to fit a
three-dimensional configuration of a binding pair member into a
three-dimensional configuration of the binding site or binding
pocket of the partner binding pair member, which can be a protein,
and determining the extent to which a fit is obtained. The extent
to which a fit is obtained can depend on the amount of void volume
in the resulting binding pair complex (or target molecule-ligand
complex). The configuration can be physical or a representative
configuration of the binding pair member, e.g., an in silico
representation or other model.
[0201] In the context of development of modulators using molecular
scaffolds, by "ligand" is meant a molecular scaffold that has been
chemically modified at one or more chemically tractable structures
to bind to the target molecule with altered or changed binding
affinity or binding specificity relative to the molecular scaffold.
The ligand can bind with a greater specificity or affinity for a
member of the molecular family relative to the molecular scaffold.
A ligand binds non-covalently to a target molecule, which can
preferably be a protein or enzyme.
[0202] By "orientation", in reference to a binding compound bound
to a target molecule is meant the spatial relationship of the
binding compound and at least some of its consitituent atoms to the
binding pocket and/or atoms of the target molecule at least
partially defining the binding pocket.
[0203] Binding compounds can be characterized by their affinity for
the target molecule as measured by determining the dissociation
constant or by measuring their effect on the activity of the target
molecule. For example, the IC50 (or EC50) is defined as the
concentration of compound at which 50% of the activity of the
target molecule (e.g., enzyme or other protein) activity being
measured is lost (or gained) relative to activity when no compound
is present. Activity can be measured using methods known to those
of ordinary skill in the art, e.g., by measuring any detectable
product or signal produced by occurrence of an enzymatic reaction,
or other activity by a protein being measured. For PPAR agonists,
activities can be determined as described in the Examples, or using
other such assay methods known in the art.
[0204] In addition, the interaction of a ligand with its target
structure can be assessed by measurement of the free energy in
kcal/mol using approaches that involve AutoDock, Molecular Dynamics
(MD) and Molecular Mechanics/Poisson-Boltzmann Solvent Accessible
surface area (MM-PBSA) calculations. This is done using the crystal
structures of the ligand binding domains of the PPAR receptors.
Molecular docking is used to generate several distinct binding
orientations. Molecular dynamics simulation is used to further
relax the complex. MM-PBSA is then used to estimate the affinity
for each binding mode. The binding modes with the lowest free
energy are expected to be the most favorable. The energetically
most favorable binding mode would provide for a free energy of
<0 kcal/mol (negative value), and larger the free energy, the
less favorable the interaction. The binding between a ligand and
its target is unique in each system and can not be compared to
other systems. However, it can be stated that the smaller the free
energy the more favorable the binding. For favorable interactions,
the free energy is negative. The more negative, the more favorable
the interaction is.
[0205] The MM/PBSA approach uses a set of structures collected with
molecular dynamics to evaluate free energies of binding. This
method combines the molecular mechanical energies with the
continuum solvent approaches. The molecular mechanical energies are
determined based on AMBER force field and represent the internal
energy (bond, angle and dihedral), van der Waals and electrostatic
interactions. The electrostatic contribution to the solvation free
energy is calculated with the Poisson-Boltzmann (PB) method. The
hydrophobic contribution to the solvation free energy is determined
with solvent-accessible-surface-area dependent terms. And estimates
of conformational entropies can be made with the mode module from
AMBER. Briefly, MM/PBSA methodology involves the following
steps:
[0206] 1. Perform short molecular dynamics simulation.
[0207] 2. Calculate average molecular mechanical and solvation
energies from a set of simulation snapshots.
[0208] 3. Use the quasi-harmonic approximation to/approximate/the
entropy of the system.
[0209] By putting energy terms together, free energy of binding can
be estimated with the formula
.DELTA.G.apprxeq.U(EE)+.DELTA.G(solv)-T.DELTA.S
Where .DELTA.G=standard free energy (kcal/mol), U (EE)=MM energy
(kcal/mol), .DELTA.G (solv)=free energy of solvent (kcal/mol),
T=temperature (Kelvin) and .DELTA.S=entropy (kcal/mol K).
[0210] "Resistin" (resistance to insulin) or "Resistin-Like
Molecules" "RELMs" or "FIZZ1-3" are found in the inflammatory zone
involved in allegory and inflammation. Resistin is an adipokine
associated with obesity and type 2 diabetes. Serum resistin level
is related to insulin resistance. (Degawa-Yamauchi M, Serum
Resistin (FIZZ3) Protein Is Increased in Obese Humans, J. Clin.
Endocrinol. Metab., 88: 5452-5455, (2003); Anal Chem. (2006) May
15; 78(10):3271-6)
[0211] "Adipocyte-Specific Secretory Factor" (ADSF) is a small
cysteine-rich protein secreted from adipose tissue
(adipocyte-derived hormones, adipokines) that belongs to a gene
family found in inflammatory zone (FIZZ) or found in resistin-like
molecule (RELM). ADSF has been implicated in modulating
adipogenesis and insulin resistance. It inhibits adipogenesis,
decreases plasma triglyceride and free fatty acid, improves glucose
tolerance and insulin sensitivity. (Endocrine. (2006) February;
29(1):81-90; Proc Natl Acad Sci USA. (2004) April 27;
101(17):6780-5. Epub (2004) April 16).
[0212] "Leptin" is a 16 kDa adipokine (protein hormone), encoded by
the obese (ob) gene, expressed predominantly by adipocytes. It is
important in regulating body weight, metabolism and reproductive
function. Smaller amounts of leptin are also secreted by cells in
the epithelium of the stomach and in the placenta. Leptin receptors
are highly expressed in areas of the hypothalamus known to be
important in regulating body weight, as well as in T lymphocytes
and vascular endothelial cells (Int J Obes (Lond). (2006) May
16)
[0213] "Acrp30/Adiponectin" (adipocyte complement-related protein
of 30 kDa) also known as AdipoQ, APM1, Adiponectin, Gelatin binding
protein 28 kDa/GBP28 or adipocyte most abundant gene transcript is
identified as a novel adipocyte-specific synthesized and secreted
protein with structural resemblance to complement factor C1q. Like
adipsin, Acrp30 secretion is induced .about.10-fold during
adipocyte differentiation. Plasma levels are reduced in obese
humans, and low levels are associated with insulin-resistance.
(Endocrinology. (2006) June; 147(6):2690-5. Epub (2006) March 2).
Adiponectin is an adipokine with insulin-sensitizing,
anti-inflammatory, and anti-atherogenic properties. Plasma levels
of adiponectin are reduced in insulin resistant states such as
obesity, type 2 diabetes and cardiovascular disease. (Hum Reprod.
(2006) May 12; [Epub ahead of print]; Biochem Biophys Res Commun.
(2006) June 23; 345(1):332-9. Epub (2006) April 27. J Endocrinol
Invest. (2006) March; 29(3):231-6).
[0214] "Orexins" is a neuropeptide that stimulates appetite (Br J
Nutr. (2004) August; 92 Suppl 1:S47-57)
[0215] "Glucose Transporter" (Glut1-Glut14) "Glut 4" is a glucose
transporter expressed uniquely in muscle and fat tissues. It moves
from intracellular sites to the plasma membrane following insulin
stimulation and thus increases the rate of glucose transport into
these tissues. GLUT4 expression is absent or low in most cultured
cell models, which is a disadvantage for studies of glucose
metabolism. However, in conditionally immortalized muscle there is
a clear rise in GLUT4 levels following differentiation. (Clin Exp
Pharmacol Physiol. 2006 April; 33(4):395-9; Am J Med. 2006 May;
119(5 Suppl 1):S10-6).
[0216] "Hypoxia-inducible factor-1" (HIF-1) is a transcription
factor. It plays a critical role in the transduction of the
metabolic response to hypoxia. HIF-1 is composed of .alpha. and
.beta. subunits, the .beta.-subunit is expressed constitutively
whereas the .alpha.-subunit is induced by hypoxia. HIF-1 is
activated in hypoxia by the stimulation of the expression of the
HIF-1.alpha. subunit to form the functional transcription factor.
HIF-1 are expressed in adipocytes and hypoxia cell culture leads to
increased levels of adipokines. Insulin activates hypoxia-inducible
factor-1 alpha in human vascular smooth muscle cells via
phosphatidylinositol-3 kinase and mitogen-activated protein kinase
pathways (HIF-alpha, beta, Diabetologia. (2006) May; 49(5):1049-63.
Epub (2006) February 28; Biochem Biophys Res Commun. (2006) March
10; 341(2):549-56.) Am J Physiol Endocrinol Metab. (2006) March;
290(3):E591-7. Epub 2005 Oct. 18
[0217] "Pref-1" or Preadipocyte factor-1 is a transmembrane protein
with epidermal growth factor-like domain. It is highly expressed in
preadipocytes. Pref-1 expression is, however, completely abolished
in adipocytes.
[0218] "Adipsin" is a serine protease that is secreted by
adipocytes. It is deficient in several animal model of obesity.
Adipsin has now been identified as the same protein as complement
factor D or C3 convertase activator or properdin factor D. Its
expression is induced upon differentiation of preadipocytes.
GENERAL DESCRIPTION
[0219] Olive leaf has a number of constituents, including
oleuropein and several types of flavinoids, including rutin,
apigenin, luteolin. In the studies presented herein, "ole" refers
to oleuropein, and "OLE" refers to Olive Leaves Extract. Recent
studies have centered on oleuropein (ole), which is a non-toxic
glucoside isolated from olive leaves, which has been shown to have
a number of beneficial medicinal properties. For example, early
studies by Fleming et al. demonstrated the anti-microbial effects
of oleuropein (Fleming, H. P. et al. (1973), Applied Microbiol. 26:
777-782). This work was further supported by the studies of
Zanichelli, et al. (Zanichelli, D. et al. (2005) J. Food Prot.
68(7):1492-1496) and Micol et al. (Micol, V. et al. (2005),
Antiviral Res. 66(2-3): 129-136). Carluccio et al. have shown in
vitro that oleuropein inhibits endothelial adhesion molecule
expression (Carluccio, M. A. et al. (2003) Arterioscler Thromb Vasc
Biol. 23: 622-629). Hamdi et al. have shown that oleuropein has
anti-tumor properties and acts to disrupt actin filaments in cells
in culture (Hamdi, H. K. et al. (2005) Biochem Biophys. Res.
Commun. July 14 Epub). Miles et al. demonstrated that oleuropein
inhibits certain inflammatory cytokines (Miles, E. A. et al.
(2005), Nutrition, 21(3): 389-394). Further studies by Manna, et
al. demonstrated that oleuropein prevents ischemia and reperfusion
induced myocardial injury (Manna C. et al. (2004) J. Nutr. Biochem.
15(8): 461-466). Studies by Puel et al. demonstrated that
oleuropein prevented inflammation-induced osteopenia in
ovariectomised rats (Puel, C. et al. (2004) 92(1): 119-127).
Earlier studies by Visioli et al. demonstrated that oleuropein
protects low density lipoprotein from CuSO4-induced oxidation. Coni
et al (2000) have subsequently demonstrated that oleuropein can
increase the ability of low density lipoprotein to resist oxidation
in vivo (Coni E. et al. (2000), Lipids, 35(1): 45-54), while Caruso
et al. have also shown that oleuropein can prevent cholesterol
oxide formation in vitro (Caruso, D. (1999) Nutr. Metab.
Cardiovasc. Dis. 9(3):102-107).
[0220] Oleuropein is reported herein to be a novel modulator of
adipocyte differentiation, de-differentiation,
trans-differentiation, and adipogenic and lipolytic genes and gene
products expression and also decreases in fat accumulation and
increases in fat burning. The oleuropein was prepared from olive
leave extract as reported in Lee-Huang et al. (Lee-Huang S; Zhang
L; Huang P L; Chang Y T; Huang P L. "Anti-HIV activity of olive
leaf extract (OLE) and modulation of host cell gene expression by
HIV-1 infection and OLE treatment". Biochemical & biophysical
research communications. 2003; 307:1029); 307:1029. Commercial
oleuropein was used as a standard for the comparison with the
oleuropein preparations used in the present studies. In addition,
oleuropein has been found to be effective in modulation of
endothelial dysfunction and to reduce diet induced atherosclerosis.
Moreover, oleuropein and derivatives or analogues thereof reduce
the number of fat cells by modulating adipocyte differentiation,
de-differentiation and trans-differentiation. Likewise, oleuropein
and its derivatives or analogues thereof reduce fat accumulation,
decrease the size of the fat cells, increase fat burning and
expenditure by modulating adipocyte metabolism in terms of
down-regulation of lipogenesis (fat formation), up-regulation of
lipolysis (fat burning) and thermogenesis via modulating the
lipogenic, lipolytic and thermogenic genes, gene products, enzymes,
factors, and pathways. Accordingly, based on the findings presented
here, oleuropein is proposed to be useful for treating obesity or
obesity related disorders, such as, but not limited to, coronary
artery disease/cardiovascular disease, hypertension,
cerebrovascular disease, stroke, peripheral vascular disease,
insulin resistance, glucose intolerance, diabetes mellitus,
hyperglycemia, hyperlipidemia, dyslipidemia, hypercholesteremia,
hypertriglyceridemia, hyperinsulinemia, atherosclerosis, cellular
proliferation and endothelial dysfunction, diabetic dyslipidemia,
HIV-related lipodystrophy, peripheral vessel disease, cholesterol
gallstones, cancer, menstrual abnormalities, infertility,
polycystic ovaries, osteoarthritis, sleep apnea, metabolic syndrome
(Syndrome X), type II diabetes, diabetic complications including
diabetic neuropathy, nephropathy, retinopathy, cataracts, heart
failure, inflammation, thrombosis, congestive heart failure, and
any other cardiovascular disease related to obesity or an
overweight condition. Moreover, while not wishing to be bound by
theory it has also been determined that oleuropein binds to all of
the PPAR receptors .alpha., .delta., and .gamma. and modulate their
actions coordinately. Whether or not this is the mechanism by which
oleuropein acts remains to be determined. Furthermore, with the
discovery that oleuropein selectively modulates all of the PPAR
receptors, the potential for using high throughput assays to
identify other similar/analogous PAN-PPAR modulators
pharmacologically useful oleuropein-like compounds which modulate
these receptors stimulatanously is feasible. Such compounds will be
useful in the treatment of PPAR-mediated diseases and conditions as
well as any for which oleuropein was previously considered to be
useful. In addition, oleuropein may be used in diagnostic assays to
monitor the progression of disease or to monitor the effectiveness
of therapy with agents that are administered to patients suffering
from obesity or an obesity related condition, or who are prone to
development of such conditions.
Proliferator-Activated Receptors (PPARs)
[0221] The Proliferator-Activated Receptors (PPARs) are members of
the nuclear receptor superfamily, which upon binding to specific
DNA response elements and in response to ligand binding, result in
the activation of several genes. The PPARs contain a DNA-binding
domain, a ligand-binding domain, and a flexible hinge connecting
the two. The PPARs function as ligand-regulated transcription
factors that control the expression of target genes by binding to
their responsive DNA sequence (DNA response elements or PPREs) as
heterodimers with the retinoid X receptor (RXR). The target genes
encode enzymes involved in lipid metabolism and differentiation of
adipocytes. The PPAR receptor experiences a conformational change
upon ligand binding, which results in activation of gene
transcription. The term "nuclear receptor" mainly refers to factors
which control transcription of a target gene by binding upstream
from the target gene promoter in a nuclear receptor
ligand-dependent fashion. However, some nuclear receptors lack
nuclear receptor ligands. Consequently, even nuclear receptors
which lack nuclear receptor ligands are called "nuclear receptors"
if their structural and functional homology places them in the
nuclear receptor gene superfamily. Nuclear receptors include
estrogen receptors (ER), vitamin D receptors (VDR), peroxisome
proliferator-activated receptors (PPAR), liver X receptors (LXR),
retinoic acid receptors (RAR), retinoid X receptors (RXR), androgen
receptors (AR), glucocorticoid receptors (GR), farnesoid x
receptors (FXR), mineralcorticoid receptors (MR) and the like for
example, but are not limited by these.
[0222] There are three known subtypes of PPARs. They include the
subtypes PPAR alpha, PPAR gamma, and PPAR delta. Natural agonists
of the three types of PPARs include fatty acids, which implicates
them as critical regulators in metabolic pathways involving energy
storage and utilization. Furthermore, this also suggests that the
PPARs may be potential targets for development of therapeutics
against disorders such as obesity, and obesity-related disorders,
including diabetes and dyslipidemia (Kliewer, et al., Recent
Progress in Hormone Research, (2001), 56: 239-63).
[0223] PPAR.alpha. is expressed predominantly in the liver and, to
a lesser extent, in cardiac and skeletal muscle. It plays a crucial
role in fatty acid oxidation in response to fasting, providing
ketone bodies that serve as an energy source for peripheral
tissues. PPAR.alpha. knockout mice cannot meet energy demands
during fasting, and develop hypoglycemia, hyperlipidemia, and fatty
liver (Kersten S, Seydoux J, Peters J M, Gonzalez F J, Desvergne B,
and Wahli W, Peroxisome proliferator-activated receptor alpha
mediates the adaptive response to fasting. J Clin Invest, 1999.
103: p. 1489-98.). PPAR.alpha. agonists include the fibrates, which
are used clinically to treat hypertriglyceridemia.
[0224] PPAR.gamma. has several critical roles. First, PPAR.gamma.
is essential for fat cell differentiation. PPAR.gamma. knockout
mice lack adipose tissue (Barak Y, Nelson M C, Ong E S, Jones Y Z,
Ruiz-Lozano P, Chien K R, Koder A, and Evans R M, PPAR gamma is
required for placental, cardiac, and adipose tissue development.
Mol Cell, 1999.4: p. 585-95.; Kubota N, Terauchi Y, Miki H,
Tamemoto H, Yamauchi T, Komeda K, Satoh S, Nakano R, Ishii C,
Sugiyama T, Eto K, Tsubamoto Y, Okuno A, Murakami K, Sekihara H,
Hasegawa G, Naito M, Toyoshima Y, Tanaka S, Shiota K, Kitamura T,
Fujita T, Ezaki O, Aizawa S, Kadowaki T, and et al., PPAR gamma
mediates high-fat diet-induced adipocyte hypertrophy and insulin
resistance. Mol Cell, 1999. 4: p. 597-609.; Rosen E D, Sarraf P,
Troy A E, Bradwin G, Moore K, Milstone D S, Spiegelman B M, and
Mortensen R M, PPAR gamma is required for the differentiation of
adipose tissue in vivo and in vitro. Mol Cell, 1999. 4: p. 611-7.),
and overexpression of PPAR.gamma. converts non-adipocytes into fat
cells (Tontonoz P, Hu E, and Spiegelman B M, Stimulation of
adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated
transcription factor. Cell, 1994. 79: p. 1147-56.). Second,
PPAR.gamma. acts as a fatty acid sensor that regulates whole-body
glucose homeostasis. PPAR.gamma. activates genes involved in
lipogenesis and lipid storage. It also modulates adipokine
expression, increasing production of adiponectin, while it blocks
expression of TNF-.alpha. and resistin. The net result of these
changes increases insulin sensitivity. PPAR.gamma. is the molecular
target for the thiazolidinedione class of drugs that improve
insulin sensitivity.
[0225] PPAR.delta., also known as PPAR.beta., is expressed
ubiquitously, and regulates expression of genes involved in fatty
acid catabolism and adaptive thermogenesis. Transgenic expression
of PPAR.delta. results in lean mice that are resistant to obesity
and hyperlipidemia, while PPAR.delta. knockout mice show reduced
energy uncoupling, and are prone to obesity (Wang Y X, Lee C H,
Tiep S, Yu R T, Ham J, Kang H, and Evans R M,
Peroxisome-proliferator-activated receptor delta activates fat
metabolism to prevent obesity. Cell, 2003. 113: p. 159-70.).
[0226] PPARs may modulate vascular effects by affecting insulin
resistance, and by expression of adipokines. In addition, PPARs may
have direct effects on gene transcription in vascular tissues and
endothelial cells.
[0227] The significance of these receptors in physiology and
disease is evidenced by the fact that PPAR-.gamma. and PPAR.alpha.
are respective molecular targets for the insulin-sensitizing
thiazolidinedione (TZD) and lipid-lowering fibrate drugs that total
more than $5 billion in annual sales.
[0228] PPAR.delta. agonist GW501516
(2-methyl-4-(((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-met-
hyl)sulfanyl)phenoxy)acetic acid) and PPAR.delta., .alpha.,
.gamma., pan agonist GW2433
(2-(4-(3-(1-(2-(2-chloro-6-fluoro-phenyl)-ethyl)-3-(2,3-dichloro-phenyl)--
ureido)-propyl)-phenoxy)-2-methyl-propionic acid) were shown to
lower plasma triglyceride levels in obese monkeys while raising
high-density lipoprotein levels, prompting the initiation of
clinical trials to assess efficacy in hyperlipidemic patients. The
medical potential of PPAR .delta. agonist and PPAR.delta., .alpha.,
.gamma., pan agonist is believed to exceed that of both PPAR.alpha.
and PPAR.gamma. single agonist or dual agonists.
[0229] The findings presented herein demonstrate that oleuropein
interacts with PPAR.alpha., PPAR.delta. and PPAR.gamma., and as
such, offers a novel application for therapeutic pan modulation of
these important transcription factors in the treatment of obesity
and related disorders.
[0230] PPAR gamma: Out of the three subtypes, PPAR gamma has been
most extensively studied. It is known to play an important role in
the regulation of glucose and lipid homeostasis as well as in
adipocyte differentiation (Willson, et al., Journal of Medicinal
Chemistry, (2000), 43: 527-550). The PPAR gamma protein is
conserved across several species including mice and humans. One of
the first synthetic ligands of PPAR gamma identified as agonists
was a class of antidiabetic compounds known as thiazolidinediones
(TZDs). The efficacy of individual TZDs in anti-diabetic therapy
appears to correlate with their ability to bind and activate the
PPAR gamma receptor (Auwerx, J., Diabetologia, (1999), 42:
1033-1049). This class of compounds has also been shown to induce
gene expression in adipocytes, which correlates with lowered
glucose levels (Willson, et al.). TZDs have also been shown to
reduce lipid and insulin levels. Known thiazolidinedionies include
Rosiglitazone, Troglitazone, Pioglitazone, and MCC-555. Each of
these compounds binds preferentially to PPAR gamma over the other
PPAR subtypes. Pioglitazone and rosiglitazone are Food and Drug
Administration approved drugs that are currently sold for the
treatment of Type II diabetes. Troglitazone was also FDA approved
for Type II diabetes, but has been withdrawn from commercial use
due to the occurrence of undesirable side effects.
[0231] Although the advances made with the thiazolidinedione class
of antidiabetes agents is significant, there are unacceptable side
effects associated with this class of drugs, which have limited
their clinical use. Accordingly, there remains a need for potent,
selective modulators of PPAR gamma, of which the activators of PPAR
gamma will be useful for the treatment of NIDDM and other disorders
related to lipid metabolism and energy homeostasis. Still further,
compounds that block PPAR gamma activity would be useful for
interfering with the maturation of preadipocytes into adipocytes
and thus would be useful for the treatment of obesity and
obesity-related disorders associated with undesirable adipocyte
maturation. In addition, the response of patients to particular
TZDs is variable, with about 20-30% of patients using these
compounds being classified as non-responders. Accordingly, it is
highly desirable to identify compounds for treating diabetes, as
well as for treating obesity and other obesity-related disorders
that are more therapeutically effective with fewer side effects.
There is also a need to develop more accurate methods for
predicting whether a subject is likely to respond to a particular
treatment as well as methods that determine the extent to which a
patient has responded to the treatment.
[0232] PPAR gamma has been shown to be expressed in an adipose
tissue-specific manner. Furthermore, it is induced early during the
course of differentiation of preadipocytes. Further studies have
now demonstrated that PPAR gamma not only plays a pivotal role in
the adipogenic signaling cascade, but also regulates the ob/leptin
gene which is involved in regulating energy homeostasis. PPAR gamma
also plays a role in adipocyte differentiation, which has been
shown to be a critical step for targeting therapeutics for treating
obesity and obesity-related conditions, such as diabetes.
[0233] PPAR gamma ligands also have in vitro anticancer activity
against a wide variety of neoplastic cells and in vivo anticancer
effects and chemotherapeutic or chemopreventive effects have been
seen in animal studies. PPAR gamma ligands may slow the growth of
cancer cells and may induce the partial differentiation of some
cancer cells. Overall, much literature indicates that PPAR gamma
ligands have antiproliferative activity and may be useful in the
treatment of cancer, including particularly several common cancers,
including those of the colon, prostate, and breast. (See, Koeffler
H P Clin Cancer Res. 9(1):1-9 (2003)).
[0234] Furthermore, there is evidence to suggest that PPAR (e.g.,
PPAR-gamma) acts by a number of mechanisms to influence the
permeability of skin, inhibit the growth of epidermal cells,
promote the terminal differentiation of epidermis, and regulate the
inflammatory response of skin. Accordingly, PPAR ligands may be
useful in the modulation of skin conditions characterized by
hyperproliferation, inflammatory infiltrates and abnormal
differentiation (e.g., psoriasis), including inflammatory skin
diseases (e.g. atopic dermatitis), proliferative skin diseases,
acne vulgaris, protease inhibitor associated lipodystrophia and
wound healing. (See, Kuenzli S, et al., Br J. Dermatol.
149(2):229-36 (2003)).
[0235] Additional studies suggest that PPAR gamma plays a role in
the pathophysiology of senile osteoporosis. For example,
adipogenesis in bone marrow increases with aging. Mesenchymal stem
cells expressing a subtype of this receptor (PPAR gamma 2)
differentiate into adipocytes. Appropriate modulation of this
receptor may promote mesenchymal stem cell differentiation into
osteoblasts. Furthermore, activators of PPAR alpha, delta, and
gamma have been reported to induce alkaline phosphatase activity
and bone matrix calcification. Accordingly, pharmacological use of
PPAR activators should promote bone mineral density by modulating
osteoblastic maturation. (See, Duque G, Drug News Perspect.
16(6):341-6 (2003); Jackson S M, et al., FEBS Lett. 471(1):119-24
(2000). Based on the studies presented herein, another aspect of
the present invention is the use of oleuropein, or an analogue or
derivative thereof for the treatment of osteoporosis, or other
diseases or conditions associated with bone loss.
[0236] The anti-proliferative and anti-inflammatory effects of PPAR
gamma are observed in glial cells and lymphocytes. It has been
postulated that activation of such cells may be involved in the
pathophysiology of neurological diseases associated with brain
inflammation (e.g, Alzheimer's disease and multiple sclerosis).
Studies indicate that PPAR gamma modulators would be
therapeutically useful in such diseases. (See, Feinstein D L,
Diabetes Technol Ther. 5(1):67-73 (2003)). Accordingly, the present
invention provides for a method of treating inflammatory diseases
or conditions comprising administering a PPAR gamma modulator to a
subject in need of such therapy. In one particular embodiment, the
method provides for treating neurological or neurodegenerative
diseases or conditions caused in part by the presence or influx of
inflammatory cells, such as for example, multiple sclerosis, stroke
or Alzheimer's disease. The use of a PPAR modulator for treating a
nervous system injury is also contemplated, for example, a spinal
cord injury or traumatic brain injury. In one particular
embodiment, the PPAR modulator is a PPAR gamma agonist.
[0237] Based on the studies done with the PPAR family of receptors
in various disease conditions, it is possible that compounds that
interact with the various PPAR receptors may be useful in treating
these various diseases. Depending on the biological environment
(e.g., cell type, pathological condition of the host, etc.), these
compounds can activate or block the actions of PPARgamma. By
activating the PPAR gamma receptor, the compounds find use as
therapeutic agents capable of modulating conditions mediated by the
PPAR gamma receptor. As noted above, an example of such a condition
is non-insulin dependent diabetes mellitus (NIDDM). Additionally,
the compounds may be useful for the prevention and treatment of
complications of diabetes (e.g., neuropathy, retinopathy,
glomerulosclerosis, and cardiovascular disorders), and treating
hyperlipidemia. Still further, the compounds may be useful for the
modulation of inflammatory conditions which most recently have been
found to be controlled by PPAR gamma (see, Ricote, et al., Nature,
391:79-82 (1998) and Jiang, et al., Nature, 391:82-86 (1998).
Examples of inflammatory conditions include asthma, rheumatoid
arthritis and atherosclerosis. Such compounds may also be useful in
the treatment of other skin diseases such as acne, atopic
dermatitis, psoriasis, photodermatitis, eczema, and seborrhea.
[0238] It has also been described in WO 96/33724 that compounds
selective for PPAR gamma receptors, such as prostaglandin-J2 or
-D2, may be potentially active agents for the treatment of obesity
and diabetes. Compounds that act via antagonism of PPAR gamma may
be useful for treating obesity, hypertension, hyperlipidemia,
hypercholesterolemia, hyperlipoproteinemia, and metabolic
disorders.
[0239] Compounds which are PPAR gamma agonists or activators by
virtue of their anti-inflammatory effects can have neuroprotective
effects and find use in the treatment of brain inflammatory
conditions such as Alzheimer's disease and multiple sclerosis. PPAR
alpha, PPAR delta and their ligands are important for lipid
oxidation and energy uncoupling. They increase fat consumption and
decrease fat accumulation, thus they can have weight reduction and
anti-obesity effects. A ligand such as oleuropein with the
capability to bind all of the three PPARs has the potential to act
as a PAN modulator. These are discussed below.
PPAR Gamma
[0240] Owing to their ability to induce gene expression in
adipocytes and to enhance adipocyte differentiation, TZDs induce
weight gain in often already obese patients. For this reason,
efforts are being made to identify new partial agonists or
antagonists for PPAR.gamma. in an attempt to combine their
anti-diabetic and anti-obesity effects.
Combined PPAR.alpha./PPAR.gamma. Agonists
[0241] The effects of TZDs on the lipid profile in diabetic
patients are not optimal. Given the favorable effect of PPAR
activators on plasma lipoprotein metabolism, combined activation of
PPAR.alpha. and PPAR.gamma. could lead to a complementary and
synergistic action on lipid metabolism, insulin sensitivity and
inflammation control. Dual activation of PPAR.alpha. and .gamma.
could, in theory, also limit the occurrence of side effects
associated with TZD therapy, such as edema and body-weight gain,
although this has not been observed so far in clinical trials with
coagonists. Thus, combined PPAR.alpha./.gamma. activation has
recently emerged as an intriguing concept and spawned the
development of co-agonists.
PPAR-Delta and PPAR.alpha..delta..gamma. Pan Agonist or
Modulator
[0242] Because of its ubiquitous expression and the paucity of
selective ligands, PPAR-delta is the least understood PPAR subtype.
Nevertheless, early PPAR-6-selective agonists were found to elevate
HDL-C levels in diabetic mice, an observation that indicated that
PPAR.delta. ligands might have beneficial effects on dyslipidemia
(Eur J Pharmacol. 2006 Apr. 24; 536(1-2):182-91. Epub 2006 Feb.
28). Subsequently, the potent PPAR.delta. agonist GW501516 was
shown to increase HDL-C while decreasing elevated TG and insulin
levels in obese rhesus monkeys. GW501516 also attenuates weight
gain and insulin resistance in mice fed high-fat diets by
increasing the expression in skeletal muscle of genes that promote
lipid catabolism and mitochondrial uncoupling, thereby increasing
.beta.-oxidation of fatty acids in skeletal muscle (Curr Opin
Investig Drugs. 2006 April; 7(4):360-70).
Expected Results of PPAR Pan Modulator Such as
OLE/Oleuropein/Hydroxytyrosol) on Metabolic Endpoints
[0243] Our discovery that Oleuropein is able to act as a PPAR pan
agonist that targets all of the three isoforms of PPARs,
PPAR.alpha., PPAR.delta. and PPAR.gamma. offers a new class of
orally available potential novel drug. A compound targeting all of
these PPAR subtypes could provide a combination of triglyceride,
LDL and glucose lowering activities, coupled with increases in
insulin sensitivity, HDL and reverse cholesterol transport (see
Table 1). Its antihyperglycemic, lipid-modulating,
insulin-sensitizing activities could be used in the treatment of a
variety of metabolic and cardiovascular diseases, including Type II
diabetes, impaired glucose tolerance, dyslipidemia, hypertension,
metabolic syndrome X, asthma and HIV/HAART associated
lipodystrophys. Furthermore, synergies of such a combination may
enable lower dosing and consequently mitigate side effects and
toxicities observed with current therapies. Although treatment
options for Type II diabetes are available, their usefulness is
significantly limited due to their failure to ameliorate concurrent
hyperglycemia and hyperlipidemia (e.g. triglycerides,
LDL-cholesterol) or to raise HDL, in addition to their side
effects.
TABLE-US-00001 TABLE 1 Cardiovascular Disease PPAR Pan-Agonist Fact
Sheet and Expected Results of OLE/Oleuropein/Hydroxytyrosol on
Metabolic Endpoints Target Glucose Insulin TG FAA LDL HDL
Limitations PPAR.alpha. No effect No effect .dwnarw. .dwnarw.
.dwnarw. .uparw. Ineffective on glucose and insulin sensitivity
PPAR.delta. No effect .uparw. .dwnarw..dwnarw. .dwnarw. .dwnarw.
.uparw. Less validation for PPAR.delta. PPAR.gamma.*
.dwnarw..dwnarw. .uparw. .dwnarw. .dwnarw. .uparw..uparw. No effect
Edema, weight gain, anemia, LDL/cholesterol.uparw., need to monitor
liver function PPAR.alpha..gamma.* .dwnarw. .uparw. .dwnarw.
.dwnarw. .dwnarw. .uparw. Edema, weight gain, anemia,
LDL/cholesterol.uparw., need to monitor liver function
PPAR.alpha..delta..gamma.** .dwnarw..dwnarw. .uparw..uparw.
.dwnarw..dwnarw..dwnarw. .dwnarw..dwnarw. .dwnarw..dwnarw.
.uparw..uparw..uparw. Less validation for PPAR.delta.
Abbreviations: Insulin S, insulin sensitivity; TG, triglycerides;
FFA, free fatty acids; LDL, low-density lipoprotein cholesterol;
HDL, high-density lipoprotein *Reported clinical effects in obese,
rhesus monkeys and in patients Edema, weight gain, anemia, .uparw.
LDL cholesterol, requirement to monitor liver **Predicted effect of
oleuropein and derivatives
Use of Oleuroein and/or Hydroxytyrosol for Inhibiting Viral
Infectivity
[0244] The work presented herein also demonstrates that oleuropein
(Ole) and hydroxytyrosol (HT) are a unique class of HIV-1
inhibitors from olive leaf extracts, which are effective against
viral fusion and integration. Molecular docking simulation was used
to study the interactions of Ole and HT with viral targets. It was
determined that Ole and HT bind to the conserved hydrophobic pocket
on the surface of the HIV-gp41 fusion domain by hydrogen bonds with
Q577 and hydrophobic interactions with I573, G 572, and L 568 on
gp41 N-terminal heptad repeat peptide N36, interfering with
formation of the gp41 fusion-active core. To test and confirm
modeling predications, the effect of Ole and HT on HIV-1 fusion
complex formation was studied using native polyacrylamide gel
electrophoresis and circular dichroism spectroscopy. Ole and HT
exhibit dose dependent inhibition on HIV-1 fusion core formation
with EC.sub.50s of 66-58 .mu.M, with no detectable toxicity.
[0245] At present, 29 drugs are licensed by the FDA for the
treatment of HIV-1 infection in the United States [S. M. Hammer,
Clinical practice. Management of newly diagnosed HIV infection, N
Engl J Med 353 (2005) 1702-1710]. These agents can be classified
according to their mechanism of action: reverse transcriptase
inhibitors (RTIs) (nucleoside, NRTIs and non-nucleoside, NNRTIs),
protease inhibitors (PIs), fusion inhibitors, and multi-class
combination products (MCCP). The combination of RTIs and P is,
commonly known as Highly Active Antiretroviral Therapy (HAART) [S.
M. Hammer, Clinical practice. Management of newly diagnosed HIV
infection, N Engl J Med 353 (2005) 1702-1710, J. Cohen, Therapies.
Confronting the limits of success, Science 296 (2002) 2320-2324],
has significantly reduced morbidity and mortality, transforming
HIV/AIDS into a manageable chronic illness. Although HAART can
favorably influence disease progression, it does not cure HIV
infection. Antiviral therapy must be maintained long-term, and
serious chronic toxicity, therapy fatigue, and drug resistance have
become major issues.
[0246] New therapeutic approaches include the fusion inhibitor
Fuzeon (T-20, enfuvirtide), the non-peptidic PI Tipranavir, the new
PI darunavir and the recently approved MCCP Atripla. However,
existing experience with HIV-1 highlights the need to use multiple
effective agents in combination for maximal and durable effect.
Thus, the search for novel anti-HIV agents continues to be of great
significance, especially those capable of affecting multiple stages
of the viral life cycle.
[0247] We previously reported that olive leaf extract is potent
against HIV-1 [S. Lee-Huang, L. Zhang, P. L. Huang, and Y. T.
Chang, Anti-HIV activity of olive leaf extract (OLE) and modulation
of host cell gene expression by HIV-1 infection and OLE treatment,
Biochem Biophys Res Commun 307 (2003) 1029-1037]. Subsequent
studies as described herein demonstrate that the anti-HIV
properties of oleuropein (Ole) and its main metabolite,
hydroxytyrosol (HT) are the key anti-HIV components of Olive Leaf
Extract. They are active against multiple stages of the HIV-1 life
cycle, inhibiting cell-to-cell HIV-1 transmission and viral core
antigen p24 production. Molecular docking simulations indicate that
Ole and HT interact with the conserved hydrophobic pocket on the
surface of the central trimeric coiled-coil of HIV-gp41 fusion
complex, the six helical bundle (6HB), and the catalytic core
domain (CCD) of HIV-1 integrase active site.
[0248] Molecular modeling and functional confirmation of Ole and HT
binding to HIV-1 integrase was also studied and the results
summarized herein. Docking simulations identified two binding
regions for Ole within the integrase active site. Region I
encompasses the conserved D64-D 116-E 152 motif, while region II
involves the flexible loop region formed by amino acid residues
140-149. HT, on the other hand, binds to region II. Both Ole and HT
exhibit favorable interactions with important amino acid residues
through strong H-bonding and van der Waals contacts, predicting
integrase inhibition. To test and confirm modeling predictions, we
examined the effect of Ole and HT on HIV-1 integrase activities
including 3'-processing, strand transfer and disintegration. Ole
and HT exhibit dose-dependent inhibition on all three activities,
with EC.sub.50s in the nM range. These studies demonstrate that
molecular modeling of target-ligand interaction coupled with
structural-activity analysis should facilitate the design and
identification of innovative integrase inhibitors and other
therapeutics.
[0249] HIV-1 integrase is one of three viral enzymes required for
viral replication, along with RT and protease [P. O. Brown,
Retroviruses, in Coffin, J., Hughes, S., and Varmus, H., (Eds.)
Cold Spring Harbor Press, Cold Spring Harbor, 1998, 161-203; T. K.
Chiu, and D. R. Davies, Structure and function of HIV-1 integrase,
Curr Top Med Chem 4 (2004) 965-977; K. Zhu, C. Dobard, and S. A.
Chow, Requirement for integrase during reverse transcription of
human immunodeficiency virus type I and the effect of cysteine
mutations of integrase on its interactions with reverse
transcriptase, J Virol 78 (2004) 5045-5055]. Integration of HIV-1
cDNA into the host chromosome is essential for stable maintenance
of the viral genome, efficient viral gene expression and productive
infection. Thus, viral integrase is a critical target for anti-HIV
therapy [Y. Pommier, A. A. Johnson, and C. Marchand, Integrase
inhibitors to treat HIV/AIDS, Nat Rev Drug Discov 4 (2005) 236-248;
P. A. Sherman, and J. A. Fyfe, Human immunodeficiency virus
integration protein expressed in Escherichia coli possesses
selective DNA cleaving activity, Proc Natl Acad Sci USA 87 (1990)
5119-5123]. The first step leading to viral DNA integration is the
binding of viral integrase to HIV long terminal repeat (LTR)
sequences. This is followed by three sequential reactions: 1)
3'-processing, the removal of two nucleotides, GT, from the 3'-end
of HIV-LTR, 2) strand transfer, a concerted cleavage-ligation
reaction, in which the integrase makes a staggered cut in the
target DNA and ligates the recessed 3' ends of the viral DNA to the
5' ends of the target DNA, and 3) gap repair, removal of the two
unpaired nucleotides at the 5' end of the viral DNA and repair of
the gap between the viral and the target DNA. In the presence of a
DNA substrate that mimics the product of viral integration,
integrase can catalyze the reversal of strand transfer, known as
"disintegration" [S. A. Chow, K. A. Vincent, V. Ellison, and P. O.
Brown, Reversal of integration and DNA splicing mediated by
integrase of human immunodeficiency virus, Science 255 (1992)
723-726 S. A. Chow, K. A. Vincent, V. Ellison, and P. O. Brown,
Reversal of integration and DNA splicing mediated by integrase of
human immunodeficiency virus, Science 255 (1992) 723-726], in which
viral DNA is released and the target DNA is sealed.
Therapeutic and Prophylactic Compositions and their Use
[0250] Candidates for therapy with the agents identified by the
methods described herein are patients either suffering from, or at
risk for development of obesity, or an obesity related disease,
disorder or condition, including diabetes, dyslipidemia,
hypertension and cardiovascular diseases, asthma to name a few.
Other obesity related disorders have been described previously. In
addition, patients who are obese or are prone to developing obesity
or an obesity related disorder based on a genetic predisposition
are also considered to be candidates for therapy using oleuropein
or a compound having activity analogous to oleuropein. Furthermore,
patients who have limited mobility or patients who are bed-ridden
due to a surgical procedure or illness and are not capable of
exercise and are thus prone to accumulation of body fat may be
candidates for therapy with the agents identified by the methods
described. In addition, the present invention contemplates the use
of oleuropein, hydroxytyrosol, and their derivatives, analogues or
mimics thereof, for inhibiting the growth and/or infectivity of
HIV-1 by virtue of the effects of oleuroein and hydroxytyrosol on
inhibition of viral fusion and/or by their effects on the viral
integrase.
[0251] The invention provides methods of treatment comprising
administering to a subject an effective amount of an agent of the
invention. In a preferred aspect, the compound is substantially
purified (e.g., substantially free from substances that limit its
effect or produce undesired side-effects). The subject is
preferably an animal, including but not limited to animals such as
monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is
preferably a mammal, and most preferably human. In one specific
embodiment, a non-human mammal is the subject. In another specific
embodiment, a human mammal is the subject. Accordingly, the agents
identified by the methods described herein may be formulated as
pharmaceutical compositions to be used for prophylaxis or
therapeutic use to treat these patients. Moreover, the agents of
the invention may be useful for administering to non-human mammals
to aid in the build-up of lean muscle which may then prove highly
beneficial for the meat industry.
[0252] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, or microcapsules. Methods of
introduction can be enteral or parenteral and include but are not
limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, topical and oral
routes. The compounds may be administered by any convenient route,
for example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compositions of the invention into the central
nervous system by any suitable route, including intraventricular
and intrathecal injection; intraventricular injection may be
facilitated by an intraventricular catheter, for example, attached
to a reservoir, such as an Ommaya reservoir. Pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent. In a
specific embodiment, it may be desirable to administer the
pharmaceutical compositions of the invention locally to the area in
need of treatment.
[0253] Such compositions comprise a therapeutically effective
amount of an agent, and a pharmaceutically acceptable carrier. In a
particular embodiment, the term "pharmaceutically acceptable" means
approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans. The term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the therapeutic is administered.
Such pharmaceutical carriers can be sterile liquids, such as water
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. The composition, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions,
emulsion, tablets, pills, capsules, powders, sustained-release
formulations and the like. The composition can be formulated as a
suppository, with traditional binders and carriers such as
triglycerides. Oral formulation can include standard carriers such
as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin. Such
compositions will contain a therapeutically effective amount of the
compound, preferably in purified form, together with a suitable
amount of carrier so as to provide the form for proper
administration to the subject. The formulation should suit the mode
of administration.
[0254] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lidocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0255] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects (a) approval by the agency of manufacture,
use or sale for human administration, (b) directions for use, or
both.
[0256] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved, for example, and
not by way of limitation, by local infusion during surgery, by
topical application, by injection, by means of a catheter, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers or co-polymers such as Elvax (see Ruan et al,
(1992), Proc Natl Acad Sci USA, 89:10872-10876). In one embodiment,
administration can be by direct injection by aerosol inhaler.
[0257] In another embodiment, the compound can be delivered in a
vesicle, in particular a liposome (see Langer (1990) Science
249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.)
[0258] In yet another embodiment, the compound can be delivered in
a controlled release system. In one embodiment, a pump may be used
(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng.
14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989)
N. Engl. J. Med. 321:574). In another embodiment, polymeric
materials can be used (see Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
(1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger
and Peppas, J. (1983) Macromol. Sci. Rev. Macromol. Chem. 23:61;
see also Levy et al. (1985) Science 228:190; During et al. (1989)
Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In
yet another embodiment, a controlled release system can be placed
in proximity of the therapeutic target, i.e., the airways, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release (1984) supra, vol. 2,
pp. 115-138). Other suitable controlled release systems are
discussed in the review by Langer (1990) Science 249:1527-1533.
Effective Doses
[0259] Toxicity and therapeutic efficacy of compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
that exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects can be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to unaffected cells and, thereby, reduce side effects.
[0260] The data obtained from cell culture assays and animal
studies can be used in formulating a dose range for use in humans.
The dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage can vary within this range depending
upon the dosage form employed and the route of administration
utilized. For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (i.e., the concentration of the test compound which
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to optimize efficacious
doses for administration to humans. Plasma levels can be measured
by any technique known in the art, for example, by high performance
liquid chromatography.
[0261] In addition, in vitro assays may optionally be employed to
help identify optimal dosage ranges. The precise dose to be
employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each subject's circumstances. Normal dose ranges used for
particular therapeutic agents employed for specific diseases can be
found in the Physicians' Desk Reference, 54.sup.th Edition
(2000).
[0262] While the subject is being treated, the health of the
patient may be monitored by measuring one or more relevant indices
at predetermined times during a 24-hour period. For example, lipid
levels may be monitored during the course of therapy using standard
procedures known to those skilled in the art, in order to monitor
the effectiveness of therapy. Alternatively, for patients suffering
from HIV, blood samples may be obtained during the course of
therapy to monitor levels of viral nucleic acid using standard PCR
procedures known to those skilled in the art. Treatment, including
supplement, amounts, times of administration and formulation, may
be optimized according to the results of such monitoring. The
patient may be periodically reevaluated to determine the extent of
improvement by measuring the same parameters, the first such
reevaluation typically occurring at the end of four weeks from the
onset of therapy, and subsequent reevaluations occurring every four
to eight weeks during therapy and then every three months
thereafter. Therapy may continue for several months or even years,
with a minimum of one month being a typical length of therapy for
humans. Adjustments to the amount(s) of agent administered and
possibly to the time of administration may be made based on these
reevaluations.
[0263] Treatment may be initiated with smaller dosages which are
less than the optimum dose of the compound. Thereafter, the dosage
may be increased by small increments until the optimum therapeutic
effect is attained.
[0264] The combined use of several compounds of the present
invention, or alternatively other therapeutic agents, may reduce
the required dosage for any individual component because the onset
and duration of effect of the different components may be
complimentary. In such combined therapy, the different active
agents may be delivered together or separately, and simultaneously
or at different times within the day.
[0265] The invention includes use of any modifications or
equivalents of the above agents which do not exhibit a
significantly reduced activity. The statements of effect and use
contained herein are therefore to be construed accordingly, with
such uses and effects employing modified or equivalent gene
products being part of the invention.
[0266] Oleuropein is a phenolic secoiridoid glycoside with the
structure as shown in the figure below. It is an organic molecule
that consists of a dihydroxy phenol moiety, a secoiridoid moiety
and a glucose moiety. The glucose moiety can be removed by
.beta.-glycosidase to yield oleuropein aglycone. Oleuropein
aglycone can than be hydrolyzed into hydroxytyrosol and elenolic
acid. These are the metabolites of oleuropein and they are all
physiological active (See FIG. 10).
[0267] The present agents that prevent fat accumulation, or that
modulate differentiation, de-differentiation and
trans-differentiation of adipocytes or that have an effect on the
expression of adipogenic, lipogenic and lipolytic genes and gene
products can be used as the sole active agents, or can be used in
combination with other active ingredients
Combination Therapy
[0268] The compounds of the invention may be combined with another
therapeutic agent that is useful in the treatment of obesity or
disorders associated with the development and progression of
obesity and obesity related disorders, such as, diabetes,
atherosclerosis, hypertension, hyperlipidaemias, dyslipidaemias,
and cardiovascular disease. The compounds of the invention may be
combined with another therapeutic agent that decreases the ratio of
LDL:HDL or an agent that causes a decrease in circulating levels of
LDL-cholesterol. In patients with diabetes mellitus the compounds
of the invention may also be combined with therapeutic agents used
to lower blood sugar or to treat complications associated with
diabetes, including nephropathy, neuropathy, retinopathy,
cardiovascular disease, stroke, or any other complication
associated with diabetes.
[0269] Accordingly, the compounds of the invention may be used
alongside other therapies for the treatment of metabolic syndrome
or type 2 diabetes and its associated complications, these include
biguanide drugs, for example metformin, phenformin and buformin,
insulin (synthetic insulin analogues, amylin) and oral
antihyperglycemics (these are divided into prandial glucose
regulators and alpha-glucosidase inhibitors). An example of an
alpha-glucosidase inhibitor is acarbose or voglibose or miglitol.
An example of a prandial glucose regulator is repaglinide or
nateglinide. Alternatively, oleuropein and/or hydroxytyrosol may be
used in combination with other anti-virals known in the art to be
effective against HIV.
[0270] In another aspect of the invention, the agents of the
invention, e.g. oleuropein or an analogue or derivative thereof, or
a pharmaceutically acceptable salt, solvate, solvate of such a salt
or a prodrug thereof, may be administered in association with
another PPAR modulating agent. PPAR modulating agents include but
are not limited to a PPAR alpha, delta and/or gamma agonist, or
pharmaceutically acceptable salts, solvates, solvates of such salts
or prodrugs thereof. Suitable PPAR alpha, delta and/or gamma
agonists, pharmaceutically acceptable salts, solvates, solvates of
such salts or prodrugs thereof are well known in the art. These
include the compounds described in WO 01/12187, WO 01/12612, WO
99/62870, WO 99/62872, WO 99/62871, WO 98/57941, WO 01/40170, J Med
Chem, 1996, 39, 665, Expert Opinion on Therapeutic Patents, 10 (5),
623-634 and J Med Chem, 2000, 43, 527, which are all incorporated
herein by reference in their entireties. Particularly a PPAR alpha
and/or gamma agonist refers to NN622/Ragaglitazar, BMS 298585,
WY-14643, clofibrate, fenofibrate, bezafibrate, gemfibrozil and
ciprofibrate; GW 9578, ciglitazone, troglitazone, pioglitazone,
rosiglitazone, eglitazone, proglitazone, BRL-49634, KRP-297,
JIT-501, SB 213068, GW 1929, GW 7845, GW 0207, L-796449, L-165041,
GW 2433 and a PPAR alpha and/or gamma agonist such as
(S)-2-ethoxy-3-[4-(2-{4-methanesulphonyloxyphenyl}ethox-y)-phenyl]propano-
ic acid and pharmaceutically acceptable salts thereof.
[0271] In addition the combination of the invention may be used in
conjunction with a sulfonylurea for example: glimepiride,
glibenclamide (glyburide), gliclazide, glipizide, gliquidone,
chloropropamide, tolbutamide, acetohexamide, glycopyramide,
carbutamide, glibonuride, glisoxepid, glybuthiazole, glibuzole,
glyhexamide, glymidine, glypinamide, phenbutamide, tolcylamide and
tolazamide. Preferably the sulfonylurea is glimepiride or
glibenclamide (glyburide). More preferably the sulfonylurea is
glimepiride. Therefore the present invention includes
administration of a compound of the present invention in
conjunction with one, two or more existing therapies described in
this paragraph. The doses of the other existing therapies for the
treatment of obesity or an obesity related disorder, such as, type
2 diabetes and its associated complications will be those known in
the art and approved for use by regulatory bodies for example the
FDA and may be found in the Orange Book published by the FDA.
Alternatively smaller doses may be used as a result of the benefits
derived from the combination. The present invention also includes a
compound of the present invention in combination with a
cholesterol-lowering agent. The cholesterol-lowering agents
referred to in this application include but are not limited to
inhibitors of HMG-CoA reductase (3-hydroxy-3-methylglutaryl
coenzyme A reductase). Suitably the HMG-CoA reductase inhibitor is
a statin selected from the group consisting of atorvastatin,
bervastatin, cerivastatin, dalvastatin, fluvastatin, itavastatin,
lovastatin, mevastatin, nicostatin, nivastatin, pravastatin and
simvastatin, or a pharmaceutically acceptable salt, especially
sodium or calcium, or a solvate thereof, or a solvate of such a
salt. A particular statin is atorvastatin, or a pharmaceutically
acceptable salt, solvate, solvate of such a salt or a prodrug
thereof. More particular statins are rosuvastatin and atorvastatin
calcium salts.
[0272] In the present application, the term "cholesterol-lowering
agent" also includes chemical modifications of the HMG-CoA
reductase inhibitors, such as esters, prodrugs and metabolites,
whether active or inactive.
[0273] Diabetes mellitus is a syndrome resulting from the
interaction of hereditary and environmental factors; it is
characterized by disturbances in insulin secretion and other
metabolic and vascular abnormalities, i.e. an elevated
concentration of glucose in the blood, non-specific accelerated
arteriosclerosis, neuropathy and thickening of the capillary basal
lamina caused by a degeneration of the kidney and the retina.
[0274] According to a modern classification, the diabetes is
divided into two main categories:
[0275] Insulin-dependent diabetes mellitus (also known as Type I
diabetes); patients with this type of diabetes literally depend on
insulin to prevent ketoacidosis and death. As far as the endogenous
insulin secretion is concerned, patients with Type I diabetes
mellitus exhibit insulinopenia.
[0276] Noninsulin-dependent diabetes mellitus (also known as Type 2
diabetes); patients with this type of diabetes do not need insulin
to live: they can decide whether using it or not to control the
symptoms of the diabetes. As far as the endogenous insulin
secretion is concerned, patients with Type 2 diabetes can be
further classified into two groups. In the first group, insulin
levels are either normal or lower than normal; in the second group,
insulin values are higher than normal and patients exhibit insulin
resistance.
[0277] Accordingly, given the data presented herein, oleuropein may
be useful as a stand-alone agent for the treatment of obesity, or
for preventing fat accumulation in individuals prone to obesity.
However, it is also to be understood that oleuropein may be used as
adjunct therapy with other known agents used to treat obesity or
obesity related disorders.
Monitoring a Patient's Response to the Therapeutic
[0278] Monitoring a patient's response to the therapeutic is
usually done by measuring metabolic endpoints including plasma
levels of glucose, insulin, TG, FAA, LDL, HDL in addition to
adipogenic, lipogenic and lipolytic genes and gene products
(proteins and/or enzymes). These are usually evaluated from whole
blood, blood cells, plasma or serum of the patient's blood by
specific bioassays. These assays are routinely used in clinical
analysis of patient's blood samples. The modulation of the PPARs
(genes or proteins), adipogenic, lipogenic and lipolytic genes and
gene products, cytokines, hormones (insulin etc), adipokines as
well as other genes and gene products involved in the targeted
regulatory pathways can be determined from peripheral blood
mononuclear cells (PBMC) purified from the blood sample using
RT-PCR, cDNA microarray blots for specific genes and ELISA or
proteomics for gene products (proteins). Specific enzyme assays can
be used for monitoring enzymatic activities. An example of the
metabolic variables to be measured in a proposed clinical trial is
presented in Table 2 below. However, in addition to the variables
described below, other measurements may include, but not be limited
to, determination of the specific levels of cytokine and adipokines
such as IL-6, resistin, MCP-1, PAI-1, adiponection or leptin.
TABLE-US-00002 TABLE 2 Metabolic Variables at Baseline and the
Changes Observed after, for example, 3 months, 6 Months and 12
Months of Treatment with Oleuropein, and derivatives (including but
not limit to its metabolites) for diabetics/obesity/cardiovascular
disease Variable Mean Change after 3, 6, or 12 Months or Baseline
other defined times Placebo OLE Placebo OLE Group Group Group Group
P Metabolic characteristics (n = xx) (n = xx) (n = xx) (n = xx)
Value.dagger. Ratio of glucose disposal rate to mean serum insulin
level at steady state Fasting glucose level, mmol/L (mg/dL) 2-h
glucose level, mmol/L (mg/dL) Fasting insulin level, pmol/L Insulin
AUC level, pmol/L .times. 10.sup.4 (120 min) Total cholesterol
level, mmol/L (mg/dL) LDL cholesterol level, mmol/L (mg/dL) HDL
cholesterol level, mmol/L (mg/dL) Triglyceride level, mmol/L
(mg/dL) Adiponectin level, .mu.g/mL Free fatty acid level, mmol/L
TNF.alpha., IL-6, adipokines PPAR.alpha., PPAR.delta., PPAR.gamma.
Adipogenic genes and gene products Lipogenic genes and gene
products Lipolytic genes and gene products Safety variables
Hemoglobin level, g/L ALT level, U/L * All values are means .+-.
SD. ALT = alanine aminotransferase; AUC = area under the curve; HDL
= high-density lipoprotein; LDL = low-density lipoprotein.
.dagger.P values for mean change from baseline represent
between-group effect from analysis of covariance with baseline
value as covariate. For treating AIDS related lipodystrophy one
also measure CD.sup.+ counts and Log HIV RNA level, copies/mL
[0279] The present invention further provides for methods of
diagnosing a patient's response to treatment with oleuropein or an
analogue or derivative thereof. Furthermore, the present invention
provides prognostic methods for evaluating the progression of
treatment with oleuropein or an analogue or derivative thereof. The
invention provides a array of genes and gene products identified as
being modulated following treatment with oleuropein or an analogue
or derivative thereof and which may be used to monitor a
therapeutic response to treatment with oleuropein or an analogue or
derivative thereof. The genes and gene products, which are up- or
down-regulated in response to treatment with oleuropein may be used
diagnostically and prognostically for treatment of a PPAR
associated disease, such as obesity or an obesity related disorder
or disease such as Type II diabetes, with a PPAR ligand. These
genes or gene products (proteins or enzymes) can be monitored from
the patient's blood (whole blood cells, serum or plasma) by enzyme
assay, ELISA (antibody), RT-PCR, microarray, and/or proteomics
techniques. Exemplary diagnostic tools and assays are set forth
below, under followed by exemplary methods for conducting these
assays.
Diagnostic Tools and Assays and Methods for Identifying Novel
Therapeutics for Treating a Disease Associated with a PPAR
Receptor
[0280] One aspect of the invention provides a means of determining
whether a subject is responsive to treatment with oleuropein or an
analogue or derivative thereof or a combination of oleuropein and a
second agent used to treat obesity or an obesity related disorder,
or of assessing the final outcome of therapy with oleuropein or an
analogue or derivative thereof or a combination of oleuropein and a
second agent used to treat obesity or an obesity related disorder
in a subject in need of such therapy. Another aspect of the
invention provides for the use of methods for screening for novel
therapeutics for treating a disease associated with a PPAR
receptor. The methods described herein are merely exemplary and are
not meant to be limiting, and as such, it is to be recognized that
one of skill in the art would be cognizant of the various other
methodologies that may be used to determine effectiveness of
therapy with oleuropein or analogues or derivatives thereof, or to
identify novel analogues or derivatives or metabolites of
oleuropein for use in treating obesity or obesity related
disorders.
[0281] In one particular embodiment, the method comprises measuring
any one or more of the following clinical parameters: lipid
profile, glucose levels, insulin levels, or any one or more
cytokines or adipokines present in the serum or plasma of a subject
undergoing treatment with oleuropein, or an analogue or derivative
or metabolite of oleuropein. Any one or more of these sensitive and
reliable end-points may be effective at determining whether
oleuropein, or an analogue, or a derivative, or a metabolite
thereof, or a combination of oleuropein or an analogue or
derivative or metabolite thereof together with one or more agents
useful for treating obesity or an obesity related disorder has
proven to be effective in treating these patients suffering from
these conditions.
[0282] In another particular embodiment, the effectiveness of
therapy with oleuropein or hydroxytyrosol or an analogue or
derivative thereof in patients infected with HIV may be measured by
standard procedures known to those skilled in the art. For example,
in the early stages of infection, HIV often causes no symptoms and
the infection can be diagnosed only by testing a person's blood.
Two tests are available to diagnose HIV infection--one that looks
for the presence of antibodies produced by the body in response to
HIV and the other that looks for the virus itself. The first test
is an (Enzyme Linked Immunosorbent Assay). an ELISA test and it is
used to measure antibodies produced by the patient against the
virus. When the body is infected with HIV, one looks for such
antibodies in blood.
[0283] If antibodies are present, the test gives a positive result,
but this must be confirmed by another test called Western Blot or
Immunofluoroscent Assay (IFA). All positive tests by ELISA may not
be accurate and hence Western Blot and repeated tests are necessary
to confirm a person's HIV status. A person infected with HIV is
termed HIV--positive or seropositive.
[0284] Antibodies to HIV generally do not reach detectable levels
in the blood till about three months after infection. This period,
from the time of infection till the blood is tested positive for
antibodies, is called the Window Period. Some times, the antibodies
might take even six months to show up. Even if the tests are
negative, during the Window Period, the amount of virus is very
high in an infected person. Hence, if a person is newly infected,
the risk of transmission is higher.
[0285] If a person is highly likely to be infected with HIV and yet
both the tests are negative, a doctor may suggest a repetition of
the tests after three months or six months when the antibodies are
more likely to have developed.
[0286] The second test is called PCR (Polymerase Chain Reaction),
which looks for HIV itself in the blood. This test, which
recognizes the presence of the virus' genetic material in the
blood, can detect the virus within a few days of infection.
[0287] There are also tests like Radio Immuno Precipitation Assay
(RIPA), a confirmatory blood test that may be used when antibody
levels are difficult to detect or when Western Blot test results
are uncertain. Other available tests are Rapid Latex Agglutination
Assay, a simplified, inexpensive blood test that may prove useful
in medically disadvantaged areas where there is a high prevalence
of HIV infection, and p24 Antigen Capture Assay.
[0288] In another particular embodiment, the responsiveness of a
subject to treatment with oleuropein or an analogue or derivative
or metabolite thereof can be assessed by measuring body mass index
(BMI) or by monitoring the weight of the subject. In a particular
embodiment, one would expect to see an improvement in the BMI or a
decrease in body weight or at least a stabilization in body mass
(eg. no additional weight gain over a given period of time).
[0289] In another particular embodiment, such a method comprises
determining the levels of expression of one or more genes or gene
products (proteins) which are modulated in a cell of the subject
undergoing treatment with oleuropein (or an oleuropein analogue,
derivative, or metabolite thereof) and comparing these levels of
expression with the levels of expression of the genes and gene
products in a cell of a subject not treated with oleuropein (or an
oleuropein analogue, derivative, or metabolite thereof), or of the
same subject before treatment with oleuropein (or an oleuropein
analogue, derivative, or metabolite thereof), such that the
modulation (either up or down-regulation of the gene or gene
product) of one or more genes is indicative that the subject is
responsive to treatment with oleuropein (or with an analogue,
derivative or metabolite thereof). In one embodiment, the cell is
obtained from a sample of whole blood, for example, white blood
cells, including lymphocytes, monocytes, neutrophils and the like,
although other cells expressing these genes are also contemplated
for analysis. In one embodiment, the genes may be any of the
adipogenic genes or gene products selected from the group
consisting of Peroxisome Proliferator-Activated Receptor .gamma.2
(PPAR.gamma.2), lipoprotein lipase (LPL), and the
adipocyte-selective fatty acid binding protein (the .alpha.P2
gene). In addition, other differentiated adipocyte marker genes
include glycerophosphate dehydrogenase (GPDH), fatty acid synthase,
acetyl CoA carboxylase, malic enzyme, Glut 4, and the insulin
receptor (see Spiegelman et al. J. Biol. Chem. 268: 6823-6826,
1993, incorporated herein by reference). Preadipocytes also have
characteristic marker genes, such as the cell surface antigen
recognized by the monoclonal antibody AD-3. Expression level
changes of the various isoforms of the C/EBP
(CCAAT/enhancer-binding proteins) family of transcription factors
may also indicate different stages of adipogenesis (see Yu and
Hausman, Exp Cell Res Dec. 15, 1998; 245(2): 343-9). A person of
skill in the art will recognize that in certain diagnostic and
prognostic assays, it will be sufficient to assess the level of
expression of a single adipogenic, lipogenic or lipolytic gene as
noted above and that in others, the expression of two or more is
preferred. For example, the level of expression of a gene or gene
product (protein) may be determined by a method selected from, but
not limited to, cDNA microarray, reverse transcription-polymerase
chain reaction (RT-PCR), real time PCR and proteomics analysis.
Other means such as electrophoretic gel analysis, enzyme
immunoassays (ELISA assays), Western blots, dotblot analysis,
Northern blot analysis and in situ hybridization may also be
contemplated for use, although it is to be understood that the
former assays that are noted (eg. microarrays, RT-PCR, real time
PCR and proteomics analysis) provide a more sensitive, quantitative
and reliable measurement of genes or gene products that are
modulated by oleuropein or analogues, derivatives or metabolites
thereof. Sequences of the genes or cDNA from which probes are made
(if needed) for analysis may be obtained, e.g., from GenBank, other
public databases or publications, and are shown here for certain of
the exemplary markers shown in Tables 3 and 4. Magnetic resonance
imaging may also be used for assessing the effect of oleuropein or
analogues or derivatives or metabolites thereof on protein
expression.
[0290] In another particular embodiment, novel candidate
therapeutics (eg. oleuropein analogues or derivatives or
metabolites thereof) may be tested for activity by measuring their
effect on adipocyte differentiation, de-differentiation or
trans-differentiation, as described herein. The candidate
therapeutics may be selected from the following classes of
compounds: proteins, peptides, peptidomimetics, antibodies,
derivatives of fatty acids, nucleic acids, including DNA or RNA,
antisense molecules or siRNA molecules, or other small organic
molecules, either synthetic or naturally derived. In some
embodiments, the candidate therapeutics are selected from a library
of compounds. These libraries may be generated using combinatorial
synthetic methods.
Use of Microarrays for Determining Gene Expression Levels
[0291] Microarrays may also be used for determining gene expression
levels and may be prepared by methods known in the art, or they may
be custom made by companies, e.g., Affymetrix (Santa Clara, Calif.)
(see www.affymetrix.com). Numerous articles describe the different
microarray technologies, (e.g., Shena, et al., Tibtech, (1998), 16:
301; Duggan, et al., Nat. Genet., (1999), 21:10; Bowtell, et al.,
Nat. Genet., (1999), 21:25; Hughes, et al., Nat. Biotechn., (2001),
19:342). While many of the microarrays utilize nucleic acids and
relevant probes for the analysis of gene expression profiles,
protein arrays, in particular, antibody arrays or glycosylation
arrays also hold promise for studies related to protein or
glycoprotein expression from biological samples (see for example,
RayBiotech, Inc. at www.raybiotech.com/product.htm, Panomics at
www.panomics.com, Clontech Laboratories, inc. at www.clontech.com,
Procognia in Maidenhead, UK and Qiagen at www.qiagen.com.
Samples for Analysis
[0292] While the efficacy of oleuropein or an analogue, derivative,
or metabolite thereof may be tested in a subject for its effect on,
for example, glucose levels, insulin levels, lipid profile, body
mass index or weight loss, it may also be of interest to assess its
effects on the modulation of the genes or gene products listed in
Table 4. While it may be possible to look at the level of a
particular gene in certain cellular samples (whole blood cells or
peripheral blood mononuclear cells), a more particular method would
involve the analysis of the protein expression in these cell types
or in the plasma or serum form the subjects exposed or treated with
oleuropein or an analogue or derivative thereof. For example,
protein and nucleic acid prepared from specimens may be obtained
from an individual to be tested using either "invasive" or
"non-invasive" sampling means. A sampling means is said to be
"invasive" if it involves the collection of the biosamples from
within the skin or organs of an animal (including, especially, a
human, a murine, an ovine, an equine, a bovine, a porcine, a
canine, or a feline animal). Examples of invasive methods include
needle biopsy, pleural aspiration, etc. Examples of such methods
are discussed by Kim, C. H. et al., J. Virol., (1992),
66:3879-3882; Biswas, B. et al., Annals NY Acad. Sci., (1990),
590:582-583; Biswas, B., et al., J. Clin. Microbiol., (1991),
29:2228-2233. Extraction of adipose tissue from individuals used in
some embodiments of this invention is well known to those skilled
in the art, for example as described by Lonnroth, et al., Diabetes,
(1983), 32980: 748-54.
[0293] In one embodiment the assays of the present invention will
be performed on cells including but not limited to whole blood
cells, or isolated white blood cells from a mammal, or from
adipocyte cultures propagated for laboratory purposes, 3T3-L1
adipocytes, cells of skeletal muscle derived from a mammal,
skeletal muscle cells propagated for laboratory purposes, C2C12
myotube cells, or mesenchymal stem cells, etc. Primary cultures or
cell lines can be used. Alternatively, embroyonic stem (ES) cells
differentiated into adipocytes can be used, for example, as
described in Poliard, et al., Journal of Cell Biology, (1995), 130:
1461-72. Appropriate cell lines that can be obtained for screening
purposes are commercially available from the ATCC. In yet another
embodiment, a sample of whole blood, blood plasma or serum is
obtained for further analysis.
Other Methods for Determining Gene Expression Levels
[0294] In certain embodiments, it is sufficient to determine the
expression of one or only a few genes, as opposed to hundreds or
thousands of genes. Although microarrays may be used in these
embodiments, various other methods of detection of gene expression
are available.
[0295] For example, the modulation of gene expression can be
performed using a RT-PCR or Real Time-PCR assay. Total RNA is
extracted using procedures known to those skilled in the art and
subjected to reverse transcription using an RNA-directed DNA
polymerase, such as reverse transcriptase isolated from AMV, MoMuLV
or recombinantly produced. The cDNAs produced can be amplified in
the presence of Taq polymerase and the amplification monitored in
an appropriate apparatus in real time as a function of PCR cycle
number under the appropriate conditions that yield measurable
signals, for example, in the presence of dyes that yield a
particular absorbance reading when bound to duplex DNA. The
relative concentrations of the mRNAs corresponding to chosen genes
can be calculated from the cycle midpoints of their respective Real
Time-PCR amplification curves and compared between cells exposed to
a candidate therapeutic relative to a control cell in order to
determine the increase or decrease in mRNA levels in a quantitative
fashion.
[0296] In addition, a method for high throughput analysis of gene
expression is the serial analysis of gene expression (SAGE)
technique, first described in Velculescu, et al., Science, (1995),
270, 484-487. Among the advantages of SAGE is that it has the
potential to provide detection of all genes expressed in a given
cell type, provides quantitative information about the relative
expression of such genes, permits ready comparison of gene
expression of genes in two cells, and yields sequence information
that may be used to identify the detected genes. Thus far, SAGE
methodology has proved itself to reliably detect expression of
regulated and nonregulated genes in a variety of cell types
(Velculescu, et al., (1997), Cell, 88, 243-251; Zhang, et al.,
Science, (1997), 276, 1268-1272 and Velculescu, et al., Nat Genet,
(1999), 23, 387-388. Techniques for producing and probing nucleic
acids are further described, for example, in Sambrook, et al.,
Molecular Cloning: A Laboratory Manual (New York, Cold Spring
Harbor Laboratory, 1989).
[0297] In other methods, the level of expression of a gene is
detected by measuring the level of protein encoded by the gene. In
the case of polypeptides which are secreted from cells, the level
of expression of these polypeptides may be measured in biological
fluids. While methods such as immunoprecipitation, ELISA, Western
blot analysis, or immunohistochemistry using an agent, e.g., an
antibody, that specifically detects the protein encoded by the gene
may be contemplated, other more sensitive and quantitative methods
are preferred, as described below. The invention is not limited to
a particular assay procedure, and therefore is intended to include
both homogeneous and heterogeneous procedures. General techniques
to be used in performing the various immunoassays noted above are
known to those of ordinary skill in the art.
[0298] Proteomics: Rationale for Use
[0299] While genomic profiling provides information about
susceptibility to disease, proteomic profiling reflects snapshots
of metabolic dynamics, reveals heterogeneous gene expression,
identifies biologically relevant phenotypes and generates
information on protein structure-function relationships in the
severity and prognosis of a disease. Thus, results from proteomic
studies should offer insight into the pathology of obesity and
effects by oleuropein therapy. Many forms of protein alterations
can be associated with pathophysiological changes and therapeutic
treatments. In addition to expression levels and patterns, these
include alternative splicing, post-translational modifications,
proteolytic processes, co-secretion and protein-protein
interactions. Thus, the identification and quantification of
proteins alone is not sufficient to understand functional
interactions. Changes as small as the addition of a single
phosphate, cleavage of a leader peptide, amidation, or oxidation,
can drastically alter the biological function of a protein. Thus,
it is important to detect these minute changes using sensitive and
accurate proteomic technology.
Sample Preparation
[0300] Serum or plasma proteins may be differentially expressed in
response to pathophysiological changes in obesity and related
diseases and to therapeutic treatments of these disorders.
Proteomic study of plasma proteins in normal and obese patients
demonstrated significant differences in protein patterns. Plasma
will be prepared from blood samples and used in proteomic
analyses.
Proteomics: The Use of 2DE, MS, MALDI-TOF, MS/MS, LC/MS, and
SELDI-TOF
[0301] 2-dimensional polyacrylamide gel electrophoresis (2DE)
coupled to mass spectrometry (MS) is currently the standard
analysis in proteomics. Plasma samples may be subjected to 2DE
(first dimension isoelectic focusing, second dimension SDS-PAGE).
Selected spots from 2DE may be extracted from the gels, digested
with trypsin and subjected to MS analysis to determine their
identities (Aebersold, R. & Mann, M. Mass spectrometry-based
proteomics. Nature 422, 198-207 (2003)). MALDI-TOF (matrix-assisted
laser desorption/ionization coupled with time of flight (TOF)) is a
method of choice to be used for proteins (Tanaka, K. The origin of
macromolecule ionization by laser irradiation (Nobel lecture).
Angew Chem Int Ed Engl 42, 3860-70 (2003).). Tandem MS (MS/MS) may
be used for selective isolation of peptide fragments to read out
the (partial) amino acid sequence, and LC/MS (liquid chromatography
coupled to MS) may be used for the identification of small
peptides. However, the 2DE/MS detection is restricted to pI between
4 and 10 and proteins within an MW range of 10-200 kDa. Thus,
peptides or small proteins (0.5-10 kDa), such as hormones,
adipokines and growth factors, which are related to obesity
pathogenesis may not be detected by 2DE/MS. Thus, additional
initial separation systems such as C/MS using HPLC coupled
MALDI-TOF for differential small peptide display is contemplated
for use (America, A. H., Cordewener, J. H., van Geffen, M. H.,
Lommen, A., Vissers, J. P., Bino, R. J. & Hall, R. D. Alignment
and statistical difference analysis of complex peptide data sets
generated by multidimensional LC-MS. Proteomics 6, 641-53 (2006).
Surface enhanced laser desorption ionization and time of flight
(SELDI-TOF) using chromatographic chip surfaces based on amino acid
sequence, protein structure, charge or hydrophobicity is also
contemplated for use (Weinberger, S. R., Dalmasso, E. A. &
Fung, E. T. Current achievements using ProteinChip Array
technology. Curr Opin Chem Biol 6, 86-91 (2002)), as well as
antibody proteomics based on immunoaffinity (Ingvarsson, J.,
Lindstedt, M., Borrebaeck, C. A. & Wingren, C. One-step
fractionation of complex proteomes enables detection of low
abundant analytes using antibody-based microarrays. J Proteome Res
5, 170-6 (2006).).
TABLE-US-00003 TABLE 3 PCR primers for differentiation specific
genes Gel Product lane Gene Primer sequence (sense/antisense) size
(bp) Gen ID # 1 Marker 1 kb plus DNA ladder of 200, 300, 200-1,000
400, 500, 650, 850, and 1000 bp 2 PPAR.gamma.2
5'-GGATGTCGTGTCTGTGGAGA-3' (SEQ ID NO:1 630 BC006811 Adipogenic
5'-TGAGGAGAGTTACTTGGTCG-3' (SEQ ID NO:2) 3 LPL
5'-GAGATTTCTCTGTATGGCACC-3' (SEQ ID NO:3) 276 BC011353 Adipogenic
3'-CTGCAAATGAGACACTTTCTC-3' (SEQ ID NO:4) 4 .alpha.P2
5'-GTACCTGGAAACTTGTCTCC-3' (SEQ ID NO:5) 418 BC007538 Adipogenic
5'-GTTCAATGCGAACTTCAGTC-3' (SEQ ID NO:6) 5 PPAR.delta.
5'-GGTGAATGGCCTGCCTCCCTACAA-3' (SEQ ID NO:7) 380 BC007578 Anti-
5'-CACAGAATGATGGCC GCAATGAAT-3' (SEQ ID NO:8) adipogenic 6 ALP
5'-TGGAGCTTCAGAAGCTCAACACCA-3' (SEQ ID NO:9) 452 BC014139
Osteogenic 5'-ATCTCGTTGTCTGAGTACCAGTCC-3' (SEQ ID NO:10) 7 OC
5'-CATGAGAGCCCTCACA-3' (SEQ ID NO:11) 310 NM199173 Osteogenic
5'-AGAGCGACACCTAGAC-3' (SEQ ID NO:12) 8 28S rRNA
5'-GTGCAGATCTTGGTGGTAGTAGC-3' (SEQ ID NO:13) 589 BC000380 Internal
5'-AGAGCCAATCCTTATCCCGAAGTT-3' (SEQ ID NO:14) Control
TABLE-US-00004 TABLE 4 Sequence Identifiers for Relevant Proteins
Whose Genes may be Modulated by Exposure to Oleuropein or Analogues
or Derivatives thereof. GenBank Name of Accession Protein Number
DNA/Protein SEQ. I.D. NO. PPAR-gamma isoform 2 NP_056953 Protein 15
Leptin DD247154 DNA 16 P2 Adipocyte Protein XM_939801 DNA 17
Adipsin M84526 DNA 18 Angiotensinogen Protein AAD14288 Protein 19
Angiotensinogen DNA BC011519 DNA 20 Complement Factor H AAH58009
Protein 21 Complement Factor D BC057807 DNA 22 Lipoprotein Lipase
M15856 DNA 23 BC011353 DNA 24 NM_000237 DNA 29 Perilipin NM_002666
DNA 25 Glucose Transporter 4 NP_001033 Protein 26 (GLUT4) SREBF1
NM_004176 DNA 27 CCAAT NM_005194 DNA 28 Plasminogen Activator
NP_000593 Protein 30 Inhibitor-1 Adiponectin NM_004797 DNA 31
Interleukin-6 NM_000600 DNA 32 Interleukin-6 NP_000591 Protein 33
TNF-alpha NP_000585 Protein 34 PPAR-alpha NM_005036 DNA 35
PPAR-alpha NP_005027 Protein 36 PPAR-gamma NM_015869 DNA 37
PPAR-gamma NP_056953 Protein 38 PPAR-delta NM_006238 DNA 39
PPAR-delta NP_006229 Protein 40
EXAMPLES
[0302] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the methods and compositions of
the invention, and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers used (e.g., amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
Example 1
Modulation of Adipogenesis and Lipodystrophy by Oleuropein
Oleuropein Modulates Adipocyte Differentiation
Methods
[0303] Human mesenchymal stem cells (hMSCs) at 10.sup.4
cells/cm.sup.2 were cultured in MSC growth medium first to
confluence. Adipogenesis was induced two days post confluence by
culturing the cells in adipogenic medium (AIM) containing 10 .mu.M
dexamethasone, 1 .mu.g/ml insulin, and 0.5 mM
3-isobutyl-1-methylxanthine, in the absence and presence of
oleuropein at 1, 10 and 100 .mu.M. Fresh culture medium was changed
every 3 days. Full differentiation of hMSC to adipocytes was
detected by Oil Red 0 staining at day 12. Gene expression was
monitored by RT-PCR, using primers shown in Table 3.
Results
[0304] It was determined that oleuropein down-regulates adipocyte
differentiation, fat accumulation and adipogenic gene expression in
human mesenchymal stem cells (hMSCs). As seen in FIG. 1, hMSCs
grown in control medium do not stain with Oil Red O (counterstained
with hematoxylin), while hMSCs grown in AIM contain lipid droplets
that stain with Oil Red O. Oleuropein shows dose dependent
inhibition of adipocyte differentiation. We are confining these
results using flow cytometry. Oleuropein down-regulates the
expression of adipogenic genes PPAR.gamma.2, LPL (lipoprotein
lipase), and .alpha.P2 (lipid binding protein). Oleuropein
up-regulates PPAR.delta. expression while the expression of the
internal control 28S rRNA remains relatively constant.
Oleuropein De-Differentiates Adipocytes and Allows
Transdifferentiation into Osteoblasts
Methods
[0305] For transdifferentiation studies, hMSCs were cultured in
adipogenic medium for 12 days, allowing full differentiation into
adipocytes. Oleuropein (80 nM) was added to the culture medium for
48 hours. De-differentiation of adipocytes was detected by the
disappearance of the accumulated lipid by Oil Red O staining.
Oleuropein-treated cells were collected by trypsinization and
cultured in osteogenic medium containing 10 nM dexamethasone, 10
.mu.M .beta.-glycerophosphate, 50 .mu.g/ml L-ascorbate 2-phosphate,
and 10 nM 1.alpha., 25-dihydroxyvitamin D3 for 6 days.
Results
[0306] As seen in FIG. 2, fully differentiated adipocytes from hMSC
lost their Oil Red 0 staining after the addition of oleuropein.
Furthermore, treatment with osteogenic medium resulted in
transdifferentiation into osteoblasts. In contrast, hMSC-derived
adipocytes treated with osteogenic medium, but not with oleuropein,
showed little change at day 6. 48 hours after the addition of
oleuropein, the expression of adipogenic marker genes PPAR.gamma.2,
LPL, and .alpha.P2 were down-regulated, while PPAR.delta. was
up-regulated. Upon the switch to osteogenic medium, expression of
osteocalcin (OC) and alkaline phosphatase (ALP) are observed.
Summary of Results
[0307] It was determined that oleuropein can modulate adipogenic
differentiation of cultured hMSC, and can de-differentiate
hMSC-derived adipocytes, allowing transdifferentiation. These
results are important because they show that oleuropein can
modulate the differentiation and commitment steps in adipogenesis,
and offer a possible cellular mechanism for anti-obesity
effects.
Example 2
Modulation of Endothelial Dysfunction and Atherosclerosis by
Oleuropein
[0308] Oleuropein Reduces Diet-Induced Atherosclerosis in the
Western Diet-Fed apoE ko Mouse
Methods
[0309] It was previously demonstrated that oleuropein and OLE,
following administration to mice, could be detected in the blood
and urine by LC-MS. To test whether oleuropein reduces diet-induced
atherosclerosis, oleuropein was administered to apoE ko mice at
various doses from 0.25 mg/ml, 2.5 mg/ml, and 25 mg/ml in the
drinking water followed by feeding them with a Western diet
containing 42% of calories from fat (Harlan-Teklad). At the 4 month
time-point for the 0.25 mg/ml dose (daily dose 1.25 mg), serum
levels of oleuropein achieved with this dose are comparable to
those in animals fed a diet supplemented with olive oil.
[0310] The mice were sacrificed, blood and tissues collected.
Aortas were dissected from the aortic valve to the iliac
bifurcation, opened longitudinally, and pinned to a black wax
surface. Atherosclerotic lesions were stained with Oil Red O. The
total area of the aorta and the atherosclerotic lesion areas were
determined by planimetry using Image Pro software. FIG. 3 shows
representative aortas stained with Oil Red 0 from untreated and
oleuropein treated apoE ko mice.
Results
[0311] As seen in FIG. 4, there was a significant reduction in
lesion area at 4 months in both male and female apoE ko mice that
were treated with oleuropein. Female mice had more lesions than
male mice in both control and oleuropein-treated groups, consistent
with the known gender effects in the apoE ko model. Error bars
indicate SEM; differences between control and oleuropein-treated
mice were significant for both genders (*) at p<0.05.
Summary of these Results
[0312] It was demonstrated that oleuropein modulates diet-induced
atherogenesis in the Western diet-fed apoE ko mouse model, at doses
comparable to those achievable by diet. These results suggest that
the beneficial effects of a Mediterranean diet may not be due
solely to effects of olive oil on the lipid profile, but may also
be mediated in part by non-lipid components such as oleuropein.
Example 3
Computational Approaches to New Treatments for Obesity
Methods
[0313] Detailed theoretical and computational analysis for binding
of oleuropein to PPAR.delta., PPAR.gamma. and PPAR.alpha. was
performed, using an approach that combines AutoDock, MD and MM-PBSA
calculations. We used the crystal structure of PPAR.delta. and
PPAR.gamma. ligand binding domains. The large ligand-binding site
is formed by several .alpha.-helices, including the C-terminal AF-2
helix. We first used molecular docking to generate several distinct
binding orientations, and performed molecular dynamics simulation
to further relax the complex. Then, we applied MM-PBSA to estimate
the affinity for each binding mode. The binding modes with the
lowest free energy are expected to be the most favorable. We then
analyzed the detailed interactions based on these binding
modes.
Oleuropein Mimics the Binding Mode of High Affinity Ligands for
PPAR.delta.
[0314] FIG. 5a shows docking of oleuropein to PPAR.delta. in the
energetically most favorable binding mode (II), with free energy of
-52.73 kcal/mol. In the predicted structure, oleuropein occupies
the Y-shaped ligand-binding pocket, identical to two known ligands
of PPAR.delta.: the synthetic agonist GW2433, which is a
high-affinity ligand, and the natural fatty acid eicosapentanoic
acid (EPA), which binds efficiently to all three PPARs.
[0315] FIG. 5 b shows an overlay of oleuropein (red and blue),
GW2433 (green) and EPA (purple) in their bound configurations.
Oleuropein's sugar group is located in a similar position and
orientation as the carboxyl groups in GW2433 and EPA. The two
hydroxyls on the sugar ring are oriented toward the AF-2 helix and
held in place through a network of hydrogen bonds with Y473, and
hydrophobic interactions with L469 and T84, as seen in FIG. 5d.
Both Y473 and L469 are part of the AF-2 helix. The same network of
hydrogen bonds occurs in the binding of ligands to PPAR.delta. and
y, and is believed to be important for ligand-mediated activation
of these receptors.
Oleuropein Shows Different Binding Modes than High Affinity Ligands
for PPAR.gamma.
[0316] FIG. 6 shows docking of oleuropein with PPAR.gamma. in the
most favorable binding mode (I), with free energy of -27.09
kcal/mol. In the predicted structure, oleuropein binds into the
large PPAR.gamma. pocket in an extended (up-down) conformation
rather than the U-shaped conformation of other known ligands such
as G1262570 and rosiglitazone. FIG. 6b shows an overlay of
oleuropein (red and blue), rosiglitazone (yellow) and the
PPAR.gamma. agonist G1262570 (green). Unlike known ligands,
oleuropein does not interact directly with AF-2 helix. At one end
of the molecule, the dihydroxyl phenol group is directed into the
solvent-accessible channel between H3 and the .beta. strands. At
the other end, the oleuropein sugar headgroup is inserted 4.0 .ANG.
deeper than the phenyloxazole tail of GI262570 into the lipophilic
pocket adjacent to the .beta. sheet, forming two strong hydrogen
bonds with the carboxyl group of E259 (shown in FIG. 6d).
Oleuropein Shows Different Binding Modes than High Affinity Ligands
for PPAR.alpha.
[0317] FIG. 7a shows the binding structure of
PPAR.alpha.-oleuropein complex with free energy of -24.71
kcal/mole. The PPAR.alpha. backbone is represented by the yellow
ribbon, and oleuropein is represented with vdw and is color coded
as follows: carbon, cyan and oxygen, red. In the predicted
structure as shown, oleuropein adopts a Y-shaped configuration when
it binds to PPAR.alpha., similar to how it binds to PPAR.delta..
This is consistent with the similarities between the binding sites
of PPAR.alpha. and PPAR.delta.. This conformation differs from the
U-shaped conformation of other known ligands of PPAR.alpha., such
as GW409544. FIG. 7b represents superposition of the structures of
GW409544 (green) and oleuropein (red and blue) bound to
PPAR.alpha.. FIG. 7c shows the chemical structures of oleuropein
and GW409544. FIG. 7d shows hydrogen bonds formed by oleuropein and
the surroundings (indicated as green dotted lines).
Summary of these Results
[0318] The known ligand binding domains of PPAR .delta. is large
and Y-shaped. Our molecular modeling results show binding modes for
oleuropein that are as favorable as the currently known ligands.
Oleuropein mimics precisely the binding mode of high affinity
ligands for PPAR.delta., indicates that oleuropein is capable of
modulating PPAR.delta. activity similar to that of know PPAR.delta.
agonist. Oleruopein has the potential to increase fat oxidation,
increase energy uncoupling and thermogenesis and thus decrease fat
accumulation, reduce body weight and prevent obesity. For
PPAR.gamma., on the other hand, in the predicted structure,
oleuropein binds to PPAR.gamma. in an extended (up-down)
conformation rather than the U-shaped conformation of other known
ligands such as G1262570 and rosiglitazone. Unlike these ligands,
oleuropein does not interact directly with AF-2 helix. These
results predicate that oleuropein has the potential to interact
with PPAR.gamma. distinct from PPAR.gamma. known agonists including
TDZ. Oleruopein does not act on the AF2 helix of PPAR.gamma., the
region that involves in adipogenesis, thus suggesting an important
mechanism for regulation of PPAR.gamma. activity in uncoupling
adipogenesis from insulin sensitivity and other activities. For
PPAR.alpha., in the predicted structure, oleuropein adopts a
Y-shaped configuration when it binds to PPAR.alpha.. This is
similar to how it binds to PPAR.delta.. This is consistent with the
similarities between the binding sites of PPAR.alpha. and
PPAR.delta. as well as their functions. Both PPAR.alpha. and
PPAR.delta. are involved in fat oxidation and energy utilization.
In this context, the energetically favorable Y configuration of
oleuropein may offer insight into its biological effects. Studies
are underway to confirm the biological significance of these
modeling results, and correlating them with in vitro and in vivo
data. We are studying the experimental binding and
co-crystallization of oleuropein with the ligand binding domains of
PPAR .alpha., .delta. and .gamma.. We are analyzing the expression
and activity of PPAR .alpha., .delta. and .gamma. in tissues from
oleuropein-treated apoE ko mice that show reduction in diet-induced
atherosclerosis, to identify the importance of these isoforms to
the observed biological activity.
Example 4
Preparation of Oleuropein from Olive Leaf Extract
LC-MS Standardization of Olive Leaf Extract (OLE) Containing
Oleuropein as the Active Moiety
A. Preparation of Olive Leaf Extract
[0319] Selection cleaning and processessing of the olive leaves.
Healthy whole olive leaves were selected and cleaned to remove
dust, residual insecticides and contaminating material. The leaves
were cut into small pieces or powdered into fine powders and placed
in a sterile flask just before extraction, with sterile distilled
water and pre-heated to 80.degree. C.
[0320] Extraction media and ratio. A comparison was made between
the extraction with water, PBS (Phosphate Buffered Saline, 10 mM
sodium phosphate buffer, pH 6.8, containing 0.15% NaCl) and organic
solvents (50% methanol, or 40% ethanol). No significant differences
were found in biological activity using the different extraction
methods. Since the therapeutic ingredients in olive leaf are
apparently water-soluble, a decision was made to use water
extraction, because it gives stable extracts that can be
concentrated with ease. For efficient extraction, it was determined
that the optimal ratio is one gram of dry leaves to 40 ml of
water.
[0321] Extraction conditions: The extraction mixture was covered
and incubated with agitation for 10-12 hours at 80.degree. C. in a
water bath. It is important that the extraction temperature be kept
under 85.degree. C. Heating above 86.degree. C. inactivates
oleuropein, one of the principle ingredients of OLE. Alternative
methods for extraction including but not limited to microwave for
two repeats of 5-10 min, or ultrasound for 25 min.
[0322] Concentration of the extract by lyophilization. At the end
of 10-12 hours at 80.degree. C., proper extraction results in a
medium brown colored OLE with about 70-80% of the original volume.
The liquid was poured off and collected. The leaves were extracted
again for a second time under the same conditions to ensure
complete extraction of the active ingredients. Small pieces of
leaves and insoluble material in the extract were removed by
centrifugation at 20,000.times.g for 30 min. The clear supernatant
was collected and labeled as Step 1 OLE. Step 1 OLE was
concentrated by freeze-drying using a Lyophilizer. The dried OLE
was collected, and labeled as Step 2 OLE.
[0323] Sterilization by Millipore filtration. Step 2 OLE was
dissolved in sterile water at 10-20 mg/ml, sterilized by Millipore
filtration with a 0.45 micron filter, distributed at small lots in
sterile cryo-tubes under aseptic conditions and stored at
-80.degree. C. This is Step 3 OLE and its oleuropein content was
quantitated by LC-MS using known oleuropein standard. Standardized
OLE was used in all of our experiments.
[0324] Preparation and LC-MS Standardization of Oleuropein
[0325] Oleuropein was prepared and quantitated by LC-MS as noted
above. This process was performed using an HP1100 equipped with
diode array detector and ESI-mass spectrometer. LC was done using a
C18 column, using a gradient of 5-95% CH.sub.3CN--H.sub.2O
containing 1% acetic acid. The diode array recordings were made at
280 nm and 230 nm, and the ESI mass spectrum was made in negative
detection mode. Purified oleuropein, was used as a standard.
Experiments were optimized by infusion of the standards in negative
scan mode to investigate the [M-H] ion of oleuropein glycoside (m/z
539). Oleuropein fractionates as a single peak by HPLC at 1.846
min, and contains predominantly oleuropein glycoside (m/z 539). The
oleuropein content in the lyophilized OLE ranges from 20-25% as
standardized by LC-MS.
[0326] Preparation and LC-MS Standardization of Hydroxytyrosol
[0327] Hydroxytyrosol was prepared from homogeneous oleuropein
isolated from OLE. The procedure involves treatment of oleuropein
with .beta.-glycosidase (including but not limited to Sigma G4511)
in 80 mM sodium acetate pH 5.0 using 1 Unit of enzyme/.mu.mole of
substrate at 37.degree. C. for 1 hr to remove the glucose moiety
and to yield oleuropein aglycone. The oleuropein aglycone was then
be treated with esterase (including but not limited to Sigma E0887)
in sodium phosphate buffer at pH 7.5 using 1 Unit of
enzyme/.mu.mole of substrate at 25.degree. C. for 1 hr to yield
hydroxytyrosol and elonelic acid. The final products were resolved
and quantitated by LC-MS as described above. Detection and
quantification were performed at 280 and 320 nm for hydroxytyrosol
at 0.698 min (m/z 153) and oleuropein aglycone at 2.087 min (m/z
377), while 240 nm was used for the detection of elenolic acid at
1.846 min (m/z 241).
[0328] LC-MS Standards
Synthesis of Hydroxytyrosol
[0329] Hydroxytyrosol, the major metabolite of oleuropein is
biological active. We have designed a unique method for chemical
synthesis of hydroxytyrosol. Chemically synthesized hydroxytyrosol
is as active as its natural counterpart from OLE. Major steps in
chemical synthesis of hydroxytyrosol are briefly outlined
below:
[0330] We started our hydroxytyrosol synthesis from
3,4-dihydroxylphenylacetic ester. It was prepared by the following
procedure. The reaction mixture of 3,4-dihydroxylphenylacetic acid
168 mg in CH.sub.3OH was treated with Acetyl chloride and
CH.sub.3OH mixture. The reaction mixture was stirred at room
temperature overnight. The reaction mixture was purified by column
chromatography. A total of 140 mg of oily compound was obtained
##STR00001##
[0331] Hydroxytyrosol was prepared from 3,4-dihydroxylphenylacetic
ester by LiAlH.sub.4 reduction. 140 mg of
3,4-dihydroxylphenylacetic ester was dissolved in tetrahydrofuran
(THF). 1M of LiAlH.sub.4 (4 ml) solution was added to the reaction
mixture. The mixture was stirred at 0.degree. C. for 2 hours. The
reaction mixture was purified by column chromatography to give
hydroxyltyrosol and characterized by LC-MS.
##STR00002##
B. HPLC Analysis of OLE Components
[0332] Step 3 OLE was subjected to HPLC using a Waters two solvents
delivery system with photodiode array detector. A Symmetry C18
column (5 .mu.m, 3.9.times.250 mm) column with a Sentry Guard
3.9.times.20 mm insert was used. Data acquisition and quantitation
were performed with Millennium 32 software (version 3.0). The
mobile phase was 79% distilled water and 21% acetonitrile (HPLC
grade), both acidified to pH 3 with 0.1 M orthophosphoric acid.
This solvent system is designed for resolution and quantitation of
polypheolic compounds. The flow rate was 1 ml/min, and the
injection volume was 20 .mu.l. Polyphenolic compounds were
monitored by absorbance at 280 in. The run time was 35 minutes. The
structure of oleuropein is shown in FIG. 8. The elution profile is
shown in FIG. 9. A total of seven major peaks were resolved from
the included material. The solvent front and excluded material
appear before peak 1, with retention time (Rt) less than 6
minutes.
C. Identification of OLE Components
[0333] The major OLE components were identified by TLC and HPLC
with known standards. TLC was carried out on silica gel 60 F254
(Merck) with chloroform/methanol/acetic acid (70:30:10).
Secoiridoids and flavonoids were detected by visualization under UV
light at 254 nm with 10% ferric chloride and 10% aminoethyl
diphenylborate spray, using known standards. HPLC verification was
conducted by repeated HPLC of peak material with known amounts of
standards. The identities of the polyphenolic compounds in each
peak are shown in Table 5, along with their relative levels in the
OLE and their cytotoxicity.
TABLE-US-00005 TABLE 5 Identity and characterizations of phenolic
compounds in OLE Peak Rt (min) Compound % (w/w) Cytotoxicity Excl.
<6 Solvent front -- -- 1 6.6 Rutin 0.34 -- 2 7.8 Verbascoside
0.38 -- 3 8.9 Luteolin7-glucoside 0.68 -- 4 16.0
Apigenin7-glucoside 0.18 -- 5 17.0 Flavonid x 0.56 -- 6 22.8
Oleuropein 12.8 -- 7 34.0 Oleuroside 0.51 --
D. LC-MS Analysis
[0334] Samples: The standard and peak 6 oleuropein were prepared in
sterile water with a final concentration of 1 mg/ml. Step 3 OLE was
prepared as described above at a final concentration of 10 mg/ml.
All samples were Millipore filtered and stored at -20.degree. C.
until use.
[0335] Experimental protocol: LC-MS was performed using an HP1100
equipped with diodearray detector and ESI-mass spectrometer. LC was
done using a C18 column (20.times.4.0 mm), with 4 minute elution
using a gradient of 5-95% CH.sub.3CN (containing 1% acetic
acid)-H2O (containing 1% acetic acid). The diodearray records were
made at 280 nm and 230 nm, and ESI mass spectrum was made in
negative detection mode. The standard and peak 6 oleuropein were
diluted to a final concentration of 0.5 mg/ml, and OLE was diluted
to 5 mg/ml. The injection volumes were all 5 .mu.l. Experiments
were optimized by infusion of the standards in negative scan mode
to investigate the [M-H] ion of oleuropein (m/z 539).
Results
[0336] Peak 6 Oleuropein Showed Similar Purity to the Oleuropein
Standard by LC
[0337] FIG. 11 shows the LC-MS results of oleuropein standard
(11A), peak 6 oleuropein (11B) and OLE (11C). As seen in FIGS.
11A-LC and 11B-LC, peak 6 oleuropein is as pure as the standard. A
single major peak was observed at 1.827 min and a minor peak at
1.696 min (less than 1%) in both samples.
[0338] Peak 6 Oleuropein Showed a Single Mass as the Oleuropein
Standard by MS
[0339] MS of the standard and the peak 6 oleuropein are seen in
FIGS. 11A-MS and 11B-MS. A single mass peak at [M-H].sup.-=539
corresponds to oleuropein was detected in both samples. A minor
peaks was observed at mass [M-H].sup.-=1079 it represents
[2M-H].
[0340] OLE Contains Several Components by LC
[0341] FIG. 11C-LC represents the LC profile of OLE. Several peaks
were detected. Peaks A at 1.82 min is the major peak, peaks B, and
C are moderate and minor respectively.
[0342] MS of OLE Identified Oleuropein, Oleuropein Aglycone and
Hydroxytyrosol.
[0343] FIGS. 11C-MS of OLE A, OLE B, and OLE C represent MS of LC
peaks A, B, C respectively. MS of peaks A, B, and C identified mass
peaks at [M-H].sup.-=539, 377 and 153 correspond to oleuropein,
oleuropein aglycone and hydroxytyrosol respectively.
[0344] Heavy metals, pesticides, fungicides & herbicides were
analyzed and not detected in the samples.
Summary and Discussion of Results:
[0345] LC-MS results show that peak 6 oleuropein consists of a
single major peak at [M-H].sup.-=539 corresponds to oleuropein. The
purity of this sample is equal to or even better than the standard.
The relative size of the peak [M-H].sup.-=1079 is less in peak 6
oleuropein than in the standard.
[0346] LC-MS of OLE indicates that in addition to oleuropein,
oleuropein aglycone and hydroxytyrosol are also present. Oleuropein
is a heterosidic ester of elenolic acid and hydroxytyrosol
(3,4-dihydroxy-phenylethanol), containing a molecule of glucose,
upon .beta.-glycosidase action it yields oleuropein agylcone.
Hydrolysis of oleuropein aglycon yields hydroxytyrosol and elenolic
acid. The structural relationships among these compounds are shown
in FIG. 10. These molecules are major metabolites of oleuropein
thus their presence in the OLE is expected. Elenolic acid has no UV
absorbance thus it cannot be detected in LC-MS analysis.
[0347] Oleuropein can also be prepared from olive leaves by
extracting with 50% aqueous methanol. After evaporation of
methanol, the aqueous phase can be extracted with chloroform and
then saturated with NaCl and filtered. Oleuropein and other
phenolic compounds can be extracted with ethyl acetate. The ethyl
acetate phase was totally evaporated to dryness. Oleuropein can be
further purified and characterized by LC-MS as described above.
Example 5
Oleuropein is Bioavailable: it is Absorbed and Well Tolerated in
Animals
A. Bioavailability Studies
[0348] It is important to establish that chronic administration of
oleuropein results in the absorption of the biologically active
material in vivo. In addition, it is necessary to determine the
physiologically achievable and relevant concentrations in tissue,
blood, and urine, and to compare these with concentrations that
show activity in vitro or in cell culture.
[0349] To verify that we can administer oleuropein chronically to
mice, we added purified oleuropein to drinking water at
concentrations of 5 .mu.g/ml, 50 .mu.g/ml, 500 .mu.g/ml, and 5
mg/ml. The water intake of mice averages 5 ml per mouse per day,
these dosages of oleuropein result in daily doses of 0.025 mg, 0.25
mg, 2.5 mg, and 25 mg per mouse per day (equivalent to 1, 10, 100,
and 1000 mg/kg body weight). 5 male and 5 female C57BL/6 wild-type
mice were used for each dose of oleuropein, and housed them in
metabolic cages that allow precise determination of water intake
and quantitative urine collection.
B. LC-MS Quantitation
[0350] After seven days to 4 months of chronic steady-state
administration, mice blood and urine were collected. 100 .mu.l of
serum or urine were extracted with 1 ml ethyl acetate, evaporated
to dryness, and reconstituted with 25 .mu.l water and 5 .mu.l was
injected for LC-MS analysis.
[0351] LC-MS quantitation was performed using an HP1100 equipped
with diode array detector and ESI-mass spectrometer. LC was done
using a C18 column (4.times.20 mm), with a 4 minute elution using a
gradient of 5-95% CH.sub.3CN--H.sub.2O containing 1% acetic acid.
The diode array recordings were made at 280 nm and 230 nm, and the
ESI mass spectrum was made in negative detection mode. Purified
oleuropein, and hydroxytyrosol (synthesized by us) were used as
standards. The injection volumes were 5 .mu.l. Experiments were
optimized by infusion of the standards in negative scan mode to
investigate the [M-H] ion of oleuropein glycoside (m/z 539),
oleuropein aglycone (m/z 377), and hydroxytyrosol (m/z 153).
C. Summary of Results
[0352] FIG. 12 shows results from LC-MS analysis of mice blood and
urine after 4 months of chronic steady-state administration.
Oleuropein standard shows a single peak by LC (12A) with mass of
539 (m/z 539) (12B). In comparison, the LC profile of serum (12C-D)
and urine (12E-F) contain mainly hydroxytyrosol (m/z 153) as well
as other derivatives and metabolites. Our results show that 1) we
can administer oleuropein in a dose-dependent manner chronically to
mice, 2) oleuropein is absorbed by the mice and metabolized to
hydroxytyrosol, and 3) we can quantitate the amount of oleuropein
and hydroxytyrosol in the serum and urine of mice by LC-MS.
[0353] In summary, our results show that oleuropein is
bioavailable. It is absorbed and well tolerated in animals. It is
metabolized to hydroxytyrosol, which is secreted in urine.
Example 6
Inhibition of HIV-1 Fusion by Oleuropein and Hydroxytyrosol
Materials and Methods
Oleuropein (Ole) and Hydroxytyrosol (HT)
[0354] Oleuropein (Ole) was purified from olive leaf extract,
characterized, and standardized by liquid chromatography-coupled
mass spectrometry (LC-MS) [3]. Hydroxytyrosol (HT) was prepared by
stepwise hydrolysis of Ole with .beta.-glucosidase (Sigma G4511) in
80 .mu.M sodium acetate, pH 5.0 using 1 Unit/.mu.mole substrate at
37.degree. C. for 1 hr. This treatment removes the glucose moiety
from Ole and yields oleuropein aglycone (Ole-AG). Ole-AG was
subsequently hydrolyzed with esterase (Sigma E0887) in 50 mM sodium
phosphate buffer at pH 7.5 at 1 Unit/.mu.mole substrate at
25.degree. C. for 1 hr to yield hydroxytyrosol and elenolic acid.
The mixture was separated by HPLC and standardized by LC-MS. HT was
also prepared by chemical synthesis from 3,4-dihydroxylphenylacetic
ester (DHPA).
Cell Lines and HIV-1
[0355] Uninfected MT2 and H9 cell lines, and HIV-1.sub.IIIB
chronically infected H9 (H9/HIV-1.sub.IIIB) and HIV-1/IIIB virus,
were obtained through the AIDS Research and Reference Reagent
Program, NIAID, NIH. MT-2 cells [4,5] were obtained from D.
Richman, and H9 and HIV-1.sub.IIIB virus stocks [6,7] from R.
Gallo. The cell lines were cultured in RPMI medium 1640 containing
100 U/ml penicillin, 100 .mu.g/ml streptomycin, 2 mM L-glutamine,
and 10% heat-inactivated fetal calf serum. Viral stocks were
prepared and standardized as described [8].
Anti-HIV and Cytotoxicity Assays
[0356] The effects of Ole and HT on acute HIV infection and viral
replication were measured by assays on syncytial formation in
cell-cell HIV-1 transmission and on HIV-1 core protein p24
expression as described [8]. Cytotoxicity was evaluated by the MTT
assay [8].
Molecular Modeling
[0357] Molecular modeling was performed by molecular docking,
molecular dynamics (MD) simulation and free energy calculations
[9,10]. Docking was performed with Autodock version 3.0.5 [11]. The
relaxation of docking structure obtained was then implemented under
Discovery from Insight II (Accelrys Inc., San Diego, Calif.,
U.S.A.) using 500 steps of Steepest Descent followed by Conjugate
Gradient until the root mean square of the energy gradient reaches
a value of 0.01 kcal/mol.ANG..
HIV-1 gp41 Fusion Peptides C34 and N36
[0358] HIV-1 gp41 fusion peptides, N36 and C34 were synthesized by
solid phase FMOC method (GeneMed, CA) and purified by HPLC. The
sequences of these peptides are
(Ac-SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH.sub.2) and
(Ac-WMEWDRFINNYTSLIHSLIEESQNQQEKNEQELL-NH.sub.2) respectively.
Corresponding viral peptides N36 and C34 prepared from HIV-1 were
obtained through the NIH AIDS Research and Reference Reagent
Program, NIAID, NIH [12] and used as standards for purification and
bioassays.
Fusion Complex Formation
[0359] Fusion complex formation was carried out by incubating
equimolar amounts of HIV-1 gp41 fusion peptides N36 and C34 in PBS
(Phosphate Buffer Saline, containing 50 mM sodium phosphate, pH 7.2
and 150 mM NaCl) at 10 or 20 .mu.M each at 37.degree. C. Peptide
N36 was first incubated either alone or with various concentrations
of Ole or HT for 30 min. Next, an equimolar amount of C34 was added
and the samples incubated for 30 min.
Native Polyacrylamide Gel Electrophoresis (N-PAGE)
[0360] N-PAGE was carried out as previously reported [13] with
modifications that involve fusion peptide concentration, order of
reactions, and time of incubation in fusion complex formation.
Tris-glycine gels (18%, Invitrogen, Carlsbad, Calif.) were
electrophoresed at 120 V for 2 h, stained with Coomassie Blue and
analyzed by densitometry.
Circular Dichroism (CD) Spectroscopy
[0361] CD spectra were recorded on an AVIV 62-DS CD spectrometer,
using 1 mm sample cells and a fixed temperature of 4.degree. C.
[8]. Each spectrum is a smoothed average of 10 scans. The bandwidth
for each measurement was 1 nm. CD intensities are expressed as mean
residue ellipticities [0] (degrees cm.sup.2/dmol). Prior to
calculation of the final ellipticity, all spectra were corrected by
subtracting the reference spectra of PBS without peptides.
Results
Preparation and LC-MS Analysis of Oleuropein (Ole) and
Hydroxytyrosol (HT)
[0362] FIGS. 13A-1B and 13C-1D represent LC-MS analysis of Ole and
HT. Ole fractionates as a single peak by HPLC at 1.848 min with m/z
of 539 and HT as a single peak by HPLC at 1.108 min with m/z of
153. FIG. 13E shows the major steps in Ole metabolism. These are
also the basic reactions in HT preparation from Ole. First, the
glucose moiety is removed by .beta.-glycosidase to yield oleuropein
aglycone (Ole-AG). Next, Ole-AG is hydrolyzed by esterase to yield
HT and elenolic acid (EA). FIG. 13F shows chemical synthesis of HT
using 3,4-dihydroxylphenylacetic acid (DHPAA) as the starting
material. Two major steps are involved: 1) acetylation with acetyl
chloride (AcCl), and 2) reduction by LiAlH.sub.4 to yield HT.
Chemically synthesized HT was purified and characterized by LC-MS,
and demonstrates identical biological, chemical and physical
properties as natural HT prepared from Ole from olive leaf
extract.
OLE and HT Inhibit HIV-1 Infection and Replication, but are not
Toxic to Target Cells
[0363] Ole and HT exhibit dose dependent inhibition of HIV-1
infection and replication as measured by syncytial formation and
p24 production (Table 6). The average EC.sub.50 for Ole is 55 .mu.M
for syncytial formation and 73 nM for p24 production. The
corresponding EC.sub.50s are 61 nM and 68 nM for HT. No
cytotoxicity was detected, either by MTT assay or trypan blue dye
exclusion, over a 10,000-fold concentration range from 1 nM to 10
.mu.M. The EC.sub.50s for inhibition on fusion complex, 6HB
formation are also presented in Table 6.
Ole and HT Bind to the Conserved Hydrophobic Pocket on the Surface
of the Central Trimeric Coiled-Coil of HIV-1 gp41
[0364] Inhibition of syncytial formation by Ole and HT reflects
effects on early events during viral infection/entry, including CD4
receptor and coreceptor binding as well as viral fusion. To probe
anti-HIV mechanisms of Ole and HT, we carried out molecular docking
and MD calculations of these small molecules with viral targets. We
found that Ole and HT bind to the conserved hydrophobic pocket on
the surface of the central trimeric coiled-coil of the HIV-1 gp41
fusion domain.
[0365] HIV-1 envelope glycoprotein (Env) mediates viral entry by
fusing virus to target cells. Env is trimeric on the virion
surface. Each monomer contains a surface subunit, gp120, for virus
binding to CD4 receptor and coreceptors [14-17] and a noncovalently
associated transmembrane subunit, gp41, that mediates fusion of the
virus with the target cell [18,19]. FIG. 14A shows the structure of
HIV-1 gp41. Like other type I transmembrane proteins, HIV-1 gp41
consists of extracellular (ectodomain), transmembrane, and
cytoplasmic domains. The ectodomain contains four functional
regions: the fusion peptide, N-terminal heptad repeat (NHR),
C-terminal heptad repeat (CHR), and a tryptophan-rich region.
Binding of gp120 to the cellular receptor CD4 and co-receptor
triggers conformational changes in gp41 that induce fusion [20-23].
This increases exposure of two heptad repeat motifs, NHR and CHR,
and insertion of the fusion peptide into the target membrane
[20-23]. Subsequently NHR and CHR fold in an antiparallel manner to
create the six-helix bundle 6HB composed of a trimeric NHR
coiled-coil core surrounded by three CHR helices that pack in the
grooves of the coiled-coil as seen in FIG. 14B [24-26]. Formation
of the 6HB promotes fusion between viral and cellular membranes and
is essential for viral entry and infection [25,26]. Ole and HT
interact with the NHR coiled-coil trimer N36 helices and interfere
with the formation of 6HB with the CHR, C34, as shown in FIGS. 14C
and 14D.
[0366] We used the crystal structure of the HIV-1 gp41 fusion
complex, PDB code 1 AIK [20] as a reference for our modeling work.
To provide a ligand binding site, one of the C34 helices was
removed from the 6HB (FIG. 15A). FIG. 15B shows the chemical
structure of Ole with the 9 free rotatable bonds selected in our
modeling interaction.
[0367] Molecular simulations suggest that the conserved hydrophobic
cavity of the gp41 N36 trimer coiled-coil is the most likely
binding site for Ole and HT. This cavity is mainly occupied by
W628, W631 and neighboring 1635 and D632. The predicted binding
structures of Ole and HT are shown in FIGS. 15C and 15D
respectively. Ole and HT form stable hydrogen bonds with Q577 on
the N36 peptide. FIGS. 15E and 15F are ribbon representations of
the predicted binding site of Ole and HT. 5HB, consisting of three
N36-peptides (pink, residues 546-581) and two C34 peptides (green,
residues 628-661), is used for docking calculations. Only one
groove is exposed for the binding of small molecules. Both Ole and
HT occupy the binding site similarly, with the diphenol ring
forming stable hydrogen bonds with Q577. This blocks the close
contacts between the hydrophobic groove in the gp41 NHR and the
indole rings of W631 and W628, thus interfering with the formation
of 6HB. In addition to hydrogen binding, hydrophobic interactions
with I573, G 572, and L 568 also play important roles in the
interaction.
Native PAGE Shows that Ole and HT Inhibit HIV-1 Fusion Core 6HB
Formation
[0368] To test the predictions from molecular modeling, we examined
the effect of Ole and HT on the formation of fusion complex 6HB by
electrophoretic mobility shift using native-PAGE (FIG. 16A). The
electrodes were connected from cathode (negative terminal on top)
to anode (positive terminal on bottom) and the peptides move in the
electric field according to their charge and size. Peptides
carrying net negative charges, such as C34, moves toward the
positive terminal (bottom) whereas peptides carrying net positive
charges, such as N36, moves toward the negative terminal or remain
at the top of the well. Incubation of N36 and C34 resulted in the
formation of the 6HB fusion complex which is larger than C34, moves
slower than free C34, and thus migrates to the middle of the gel.
Pre-incubation of N36 with Ole or HT results in inhibition of 6HB
formation. A dose-dependent disappearance of 6HB band was detected
with concomitant appearance of the free C34 band. Total inhibition
is achieved at 100 nM Ole or HT with EC.sub.50s around 66 and 58
nM. These results confirm the predictions from molecular
modeling.
CD Analysis Indicates that Ole and HT Inhibit HIV-1 Fusion Core 6HB
Formation
[0369] We also used CD analysis to confirm molecular modeling
predictions (FIG. 16B). Because N36 and C34 are single stranded
random coils, they do not assume ordered structure in solution, so
they display characteristic random coil CD spectra. However,
formation of fusion complex 6HB results in a distinctive CD
spectrum, including a saddle-shaped negative peak between 210-220
nm in the far UV region and a significant increase in molar
ellipticity (.theta.) at 222 nm. Preincubation of N36 with Ole or
HT interrupts 6HB formation and results in a dose dependent shift
of the CD spectra from helical to random coil with EC.sub.50s of 62
nM for Ole and 60 nM for HT.
TABLE-US-00006 TABLE 6 Anti-HIV Activity and Inhibition on HIV-1
Fusion Core Formation Inhibitory Anti-HIV Activity.sup.a
Activity.sup.b EC.sub.50 (nM) EC.sub.50 (nM) IC.sub.50 (nM) Fusion
Core Formation Syn- Cyto- 6HB (N36 + C34) Compound cytium P24
toxicity N-PAGE CDSA Ole 55 .+-. 5 73 .+-. 8 >10,000 66 .+-. 5
62 .+-. 6 HT 61 .+-. 6 68 .+-. 8 >10,000 58 .+-. 8 60 .+-. 4
.sup.aEC.sub.50, effective concentration at 50% inhibition;
IC.sub.50, cytotoxicity concentration at 50% inhibition.
.sup.bN-PAGE, native polyacrylamide gel electrophoresis; CDSA,
circular dichroism spectroscopy analysis. Values are means .+-. SD
of triplicates in three independent determinations.
Discussion
[0370] Ole and HT are small molecules with molecular weights of 539
and 153 respectively. Their inhibition of the fusion-promoting
refolding of gp41 is an excellent example of how small molecules
can block formation of protein-protein complexes. We narrowed down
the target of binding to a hydrophobic pocket on the gp41 inner
core. This pocket is highly conserved among the different HIV
clades. Consistent with this, we found that Ole and HT are active
against a panel of HIV-1 primary isolates that includes both M and
T tropic strains from different clades. Our results suggest that
Ole and HT may be useful against other viruses with type I
transmembrane envelope glycoprotein, including severe acute
respiratory syndrome associated coronavirus [23,27], respiratory
syncytial virus, Ebola virus [28], measles virus [29], and avian
flu [30,31].
[0371] Fuzeon (T-20 or Enfuvirtide) is the only FDA approved HIV
fusion inhibitor [32,33]. It is a peptide derived from the CHR
region of gp41 that partially overlaps with the C34 sequence.
Fuzeon is commercially produced by chemical synthesis. Because of
its large size--it consists of 36 amino acids with a molecular
weight of 4492--its manufacturing process is very complex,
involving 106 chemical steps [34,35]. In contrast, our typical
process for chemical synthesis of HT involves only two steps:
acetylation and reduction (FIG. 13F). In addition, Ole and HT can
also be easily prepared from natural olive leaf extract in only two
steps: deglycosylation and oxidation (FIG. 13E). The fact that Ole
and HT act both outside and inside of the cellular environments in
viral entry and integration offers unique benefits to these small
molecules against viral resistance.
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Example 7
Effect of Oleuropein and Hydroxytyrosol on HIV-1 Integrase
Material and Methods
Oleuropein (Ole) and Hydroxytyrosol (HT)
[0407] Ole and HT used in this study were prepared and purified
from olive leaf extract as described in the preceding article.
[0408] Target Cells, HIV-1, Anti-HIV and Cytotoxicity Assays
[0409] Target cells MT2, H9, HIV-1.sub.IIIB chronically infected H9
(H9/HIV-1.sub.IIIB) and HIV-1/IIIB virus, were obtained through the
AIDS Research and Reference Reagent Program, NIAID, NIH. MT-2 cells
were from D. Richman [7,8]. H9 cells and HIV-1IIIB virus stocks
were from R. Gallo [9,10]. The cell lines were cultured in RPMI
medium 1640 containing 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 2 mM L-glutamine, and 10% heat-inactivated fetal calf
serum. Viral stocks were prepared and standardized as described
previously [11]. Anti-HIV activity was measured by the microtiter
syncytial formation and HIV-1 p24 production assays [11].
Cytotoxicity was determined by the MTT assay [11].
Molecular Modeling
[0410] A combination of molecular docking, molecular dynamics (MD)
simulation and free energy calculations [12,13] were performed to
probe the interactions of Ole or HT with viral targets. Docking was
performed with AutoDock version 3.0.5 [14]. Relaxation of docking
structure obtained was carried out by the program Discovery from
Insight II (Accelrys Inc., San Diego, Calif., U.S.A.), using 500
steps of Steepest Descent followed by Conjugate Gradient until the
root mean square (RMS) of the energy gradient reaches 0.01
kcal/mol.ANG..
HIV-1 Integrase
[0411] HIV-1 integrase was expressed in E. coli from pIN
(F185H/C280S) and purified according to previously reported method
[15]. This recombinant clone makes the integrase protein more
soluble and stable without affecting in vitro activity. HIV-1
integrase protein (F185H/C280S) was used as a standard for
purification and assay. The integrase clone pIN (F185H/C280S) and
the standard integrase protein were obtained through the NIH AIDS
Research and Reference Reagent Program, NIAID, NIH from Dr. Robert
Craigie [16].
Integrase Substrates
[0412] Oligonucleotide substrates were synthesized and purified as
described previously [17]. Three types of substrates were
synthesized with sequences that correspond to the U3 and U5 ends of
HIV-LTR: (i) the 21-nucleotide minus strand of U3 end HIV-LTR
5'-GAGTGAATTAGCCCTTCCAGT-3' (SEQ ID NO: 41), and the U5 HIV-LTR,
5'-GTGTGGAAAATCTCTAGCAGT-3' (SEQ ID NO: 42), as well as their
complementary strands, for assaying the 3'-processing reaction;
(ii) the 19-mer U3-GT, and U5-GT (i.e., U3 and U5 minus the 3'-end
dinucleotide GT) for assaying heterologous integration (strand
transfer); and (iii) a 38-mer dumbbell substrate with the sequence
5'-TGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-3' (SEQ ID NO: 43), for
assaying the dis-integration.
Radiolabeling and Preparation of the Substrates
[0413] The integrase substrates were 5'-end radiolabeled as
reported previously [17]. Briefly, 1 g of the oligonucleotide was
5'-end labeled with 100 .mu.Ci of [.alpha.-.sup.32P]ATP (3000
Ci/mmol; 1 Ci=37 GBq; A-mersham) by 20 units of polynucleotide T4
kinase in a final volume of 40 .mu.l of kinase buffer (Boehringer
Mannheim) at 37.degree. C. for 60 min. The reaction was stopped by
EDTA (25 mM) and heat inactivation. Unincorporated label was
removed by two passages through a Sephadex G-25 spin column
(Boehringer Mannheim). The purified labeled oligonucleotide was
then annealed with an equimolar amount of unlabeled complementary
strand in 10 mM Tris HCl, pH 7.5/1 mM EDTA/100 mM NaCl at
95.degree. C. for 5 min followed by slow cooling. The dumbbell
substrate was self-annealed under the same conditions.
Integrase Assays
[0414] Integrase assays were carried out in 20 mM Hepes, pH 7.5/10
mM MgCl.sub.2 or MnCl.sub.2/10 mM dithiothreitol/0.05% Nonidet P-40
(integrase buffer) with 40 .mu.mol of HIV-1 integrase and 20 ng of
5'-end radiolabeled substrates specific for 3'-processing (21-mer
U3), strand-transfer (19-mer U3-GT)) or disintegration (38-mer
dumbbell) in the presence or absence of Ole or HT in a final volume
of 10 .mu.l at 37.degree. C. for 60 min. For 3'-processing and
disintegration, the reactions were stopped by the addition of 10
.mu.l of 90% formamide/0.025% bromophenol blue/0.025% xylene
cyanol/89 nM Tris/89 mM boric acid/2 mM EDTA, pH 8.0. Samples were
heated at 75.degree. C. for 3 min, load at 10 .mu.l/well onto 18%
polyacrylamide denaturing (7.5 M urea) gels in TBE buffer and
electrophoresed at 200 V constant voltage at room temperature for 2
h. The results were visualized by autoradiography of wet gels. For
strand-transfer (integration), pUC18 plasmid DNA (50 ng) was used
as the target for the integration of viral DNA into heterologous
plasmid DNA. The reaction was stopped by 0.1% SDS and integration
products monitored on 1% agarose gels at 10 ul/well in TBE buffer
at 100 V constant voltage at room temperature for 45 min. The
results were visualized by autoradiography of dried gels.
Results
Ole and HT Bind to the Catalytic Site of HIV-1 Integrase
[0415] To examine the molecular interactions of Ole and HT with HIV
integrase, we performed a series of docking simulations. In these
studies, we focused on the catalytic core domain (CCD) of the viral
enzyme, because the linkages between the CCD and both the N- and
C-terminal domains (NTD and CTD) are flexible and have been not
precisely determined. We used the crystal structure 1QS4 (pdb ID)
as the starting structure [18], because it is the only structure
has an inhibitor (5CITEP) bound in the active site. To keep the
binding pocket available, we removed the ligand (5CITEP) from the
CCD. Before docking, the missing residues in the loop region and
the point mutation at position 185 were restored with AMBER
software. The K185 mutation was converted to the native F185.
[0416] FIGS. 17A and 17B represent the predicted bound
conformations of Ole and HT within the active site of HIV-1
integrase. Two unique binding regions have been identified within
the integrase active site [2, 4], referred to as regions I and II
(FIG. 17B). Region I, near the active site center, encompasses the
conserved DDE motif, D64-D116-E152 in HIV-1 integrase. These
residues are highly conserved in all integrases, retrotransposases
and other DNA-processing enzymes (polynucleotide transferases).
Mutation of any of these acidic amino acids abolishes integrase
activities and viral replication. D64 and D116 are involved in the
formation of coordination complex with divalent metal (Mg2+ or
Mn2+). A second metal (Mg2+ or Mn2+) can be coordinated between
D116 and E152 once HIV-1 integrase binds its DNA substrates (20,
21). Metal ion coordination with viral integrase and the
phosphodiester backbone of the DNA substrates are likely to occur
during 3'-processing and strand-transfer reactions. Region II is
close to the active catalytic loop (amino acid residues 139-147),
and involves the flexible loop formed by amino acid residues
140-149. This loop region has been identified as the DNA binding
site which is important for integrase action [19].
[0417] As seen in FIG. 17A, Ole binds to both regions I and II. The
.beta.-glycopyranose moiety of Ole interacts with residues in
region II whereas the dihydroxyphenol ring occupies region I. FIGS.
17C and 17D are ribbon representation of the HIV-integrase CCD
showing major strong H-bonding sites with Ole and HT. The flexible
loop widens the active site region and allows the sugar ring of Ole
to dock with strong H-bond with P142 and Q148 as well as form weak
interactions with S147. The dihydroxyphenol moiety of Ole binds to
region I with a strong H-bond interaction with D64 and a weak H
bond-network interactions with K156 and K159. This suggests that
Ole would be a strong integrase binding inhibitor because it
interacts with residues in both regions I and II. On the other
hand, the dihydroxyphenol ring of HT binds to region II with strong
H bond interaction with F139 and nearby T115, and weak interactions
with E138 and Q 148. Since the dihydroxyphenol ring is capable of
binding both regions I and II, HT maintains the ability to bind the
integrase active site even if mutations occur. Thus the likelihood
of resistance development should be less than inhibitors that bind
to a single site. Thus, interaction modeling suggests that Ole and
HT bind to both regions I and II, so they would be expected to be
effective against metal coordination as well as substrate binding.
Modeling results therefore predict that Ole and HT would inhibit
all of the three HIV-1 integrase activities.
Ole and HT Inhibit 3'-Processing Activity of HIV-1 Integrase
[0418] Modeling predictions that Ole and HT may affect
HIV-integrase activity were tested in all of the three activities,
namely 3'-processing, strand transfer (integration) and
disintegration. Results of HIV-1 integrase inhibitory activities of
Ole and HT are summarized in Table 7 with their anti-HIV
activities.
[0419] FIG. 18A shows the 3'-processing reaction and results. 5'
[.sup.32P] labeled 21-mer double-stranded oligonucleotide that
mimics the U3HIV-1 LTR was used as a substrate. 3'-processing by
HIV-1 integrase, removes the dinucleotide GT from the 3' end of the
labeled minus strand of the 21-mer substrate and yields a 3'
recessed product (U3-GT) with 19 nucleotides in length. Ole or HT
inhibits the 3'-processing activity of HIV-1 integrase. FIG. 18A
demonstrates dose-dependent inhibition of 3'-processing by Ole and
HT as detected by 7.5 M urea denaturating polyacrylamide gel
electrophoresis. Inhibition of the formation of the 19-mer product
from the 21-mer substrate increases with the increase of Ole or HT
concentration from 25 to 100 nM. The degree of inhibition depends
on the concentrations of the HIV-1 integrase, the substrate and the
inhibitor. Under our assay conditions, EC.sub.50s of 46 and 54 nM
were obtained for Ole and HT respectively, and total inhibition was
observed at 100 nM. Substrate U5 HIV-LTR showed similar
results.
Ole and HT Inhibit Strand-Transfer Activity of HIV-1 Integrase
[0420] The effect of Ole and HT on the strand-transfer activity of
HIV-1 integrase was tested by a quantitative heterologous
integration assay. FIG. 18B shows the design and results of this
assay: U3-GT, a 5' [.sup.32P] labeled and 3'-recessed 19-mer was
used as the viral substrate. To focus on strand transfer, a
supercoiled pUC18 plasmid DNA of 2.69 kb was used as the
heterologous target. Incubation of the 5' [.sup.32P] labeled viral
substrate with unlabeled target in the presence of HIV-1 integrase
results in the integration of the labeled 19-mer viral substrate
into the 2.69 kb target plasmid. Integration was monitored by the
conversion of unlabeled plasmid to labeled DNA in agarose gel
electrophoresis as seen in the figure. Under these conditions, any
inhibition detected must be specific for strand transfer and not
for 3' processing. Ole and HT demonstrated dose-dependent
inhibition of the strand-transfer activity of HIV-1 integrase with
EC.sub.50s of 56 and 43 nM respectively.
Ole and HT are Effective Against Disintegration Activity of HIV-1
Integrase
[0421] FIG. 18C shows a schematic representation of disintegration
reaction and assay results. Disintegration is the reverse of
integration and involves concerted strand-cleavage and ligation
reactions [20]. Strand cleavage takes place precisely at the
junction between the viral and the target sequences and is coupled
with the rejoining of the cleaved target sequences. The
disintegration substrate is a dumbbell shaped 38-mer
oligonucleotide that mimics the recombination intermediate of HIV-1
integration. It contains 5' [.sup.32P] labeled virus-specific
U5-LTR sequence of 14-mer in the stem of the hairpin loop and
arbitrary target DNA sequences of 24-mer in the base of the
dumbbell [21,22]. The folded structure of the annealed substrate
shown is based on reports obtained from hairpin formation by
similar sequences [23,24]. Dis-integration of the 5' [.sup.32P]
labeled dumbbell by HIV-1 integrase is expected to give two
products, a 5' [.sup.32P] labeled 14-mer hairpin loop viral
sequence and an unlabeled 24-mer closed circular target DNA. In the
presence of HIV-integrase, the production of labeled 14-mer hairpin
loop was detected by 7.5 M urea denaturating polyacrylamide gel
electrophoresis and autoradiography of the gel, whereas the 24-mer
product, the target DNA, was not seen in the autoradiography
because it is unlabeled. This product can be detected by UV
shadowing of the gel or by the use of 3'-labeled substrate. In the
presence of Ole or HT, dose-dependent inhibition on the formation
of the labeled 14-mer disintegration product was observed with
EC.sub.50s of 28 or 18 .mu.M respectively.
TABLE-US-00007 TABLE 7 Anti-HIV Activity and Inhibition on HIV-1
Integrase Activity Anti-HIV Activity.sup.a Inhibitory EC.sub.50
(nM) IC.sub.50 (nM) Activity.sup.b EC.sub.50 (nM) Syn- Cyto- HIV-1
Integrase Activities Compound cytium P24 toxicity 3'-Proc ST Dis-In
Ole 55 .+-. 5 73 .+-. 8 >10,000 46 .+-. 6 56 .+-. 5 28 .+-. 3 HT
61 .+-. 6 68 .+-. 8 >10,000 54 .+-. 5 43 .+-. 5 18 .+-. 2
.sup.aEC.sub.50, effective concentration at 50% inhibition;
IC.sub.50, cytotoxicity concentration at 50% inhibition.
.sup.b3'-Proc, 3'-processing; ST, strand transfer; Dis-In,
disintegration. Values are means .+-. SD of triplicates in three
independent determinations
Discussion
[0422] Several classes of HIV integrase inhibitors have been
reported [4,23], but none is clinically available yet. Lack of
structural information for the intact protein, issues regarding
different active site conformations dependent on crystal structure,
and uncertain oligo-meric character of the enzyme protein have
impeded the discovery of a clinically useful HIV-integrase
inhibitor [4,23]. Thus, molecular modeling becomes a key component
in both the design of new integrase inhibitors and the
identification of important protein-ligand interactions. There are
14 crystal structures of HIV-integrase available from the Protein
Data Bank (PDB); however, only one has an inhibitor bound in the
active site: 1QS4 [18]. We believe that this crystal structure
contains the inhibitor would be the most relevant active site
conformation on which to conduct the docking simulations with our
anti-HIV small molecules, Ole and HT, despite previously reported
crystal-packing effects associated with this structure [24].
[0423] The docking results reported here show good correlation with
experimental data and provide a valuable tool for both evaluating
compounds and designing more potent inhibitors. Ole and HT exhibit
dose dependent inhibition in all of the three activities of HIV-1
integrase: 3'-processing, strand transfer and disintegration with
EC.sub.50s all in the nM range. These compounds also showed good
antiviral efficacy both in cell-to-cell transmission of HIV-1 as
assayed by syncytial formation and in HIV-1 replication as assayed
by p24 production. However, they are not toxic in the effective
dose ranges and even at the concentration of 1,000 times EC.sub.50
(Table 7).
[0424] To our knowledge, Ole and HT are the first group of small
molecules capable of multiple actions against the AIDS virus,
inhibiting both viral entry and integration. To act both outside
and inside of the cellular environments represents a great
advantage of this novel class of drugs. The structure-function
information described here should facilitate the design of
innovative multi-functional HIV-1 inhibitors.
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Sequence CWU 1
1
43120DNAArtificial Sequenceprimer 1ggatgtcgtg tctgtggaga
20220DNAArtificial Sequenceprimer 2tgaggagagt tacttggtcg
20321DNAArtificial Sequenceprimer 3gagatttctc tgtatggcac c
21421DNAArtificial Sequenceprimer 4ctgcaaatga gacactttct c
21520DNAArtificial Sequenceprimer 5gtacctggaa acttgtctcc
20620DNAArtificial Sequenceprimer 6gttcaatgcg aacttcagtc
20724DNAArtificial Sequenceprimer 7ggtgaatggc ctgcctccct acaa
24824DNAArtificial Sequenceprimer 8cacagaatga tggccgcaat gaat
24924DNAArtificial Sequenceprimer 9tggagcttca gaagctcaac acca
241024DNAArtificial Sequenceprimer 10atctcgttgt ctgagtacca gtcc
241116DNAArtificial Sequenceprimer 11catgagagcc ctcaca
161216DNAArtificial Sequenceprimer 12agagcgacac ctagac
161323DNAArtificial Sequenceprimer 13gtgcagatct tggtggtagt agc
231424DNAArtificial Sequenceprimer 14agagccaatc cttatcccga agtt
2415505PRTHomo sapiens 15Met Gly Glu Thr Leu Gly Asp Ser Pro Ile
Asp Pro Glu Ser Asp Ser1 5 10 15Phe Thr Asp Thr Leu Ser Ala Asn Ile
Ser Gln Glu Met Thr Met Val20 25 30Asp Thr Glu Met Pro Phe Trp Pro
Thr Asn Phe Gly Ile Ser Ser Val35 40 45Asp Leu Ser Val Met Glu Asp
His Ser His Ser Phe Asp Ile Lys Pro50 55 60Phe Thr Thr Val Asp Phe
Ser Ser Ile Ser Thr Pro His Tyr Glu Asp65 70 75 80Ile Pro Phe Thr
Arg Thr Asp Pro Val Val Ala Asp Tyr Lys Tyr Asp85 90 95Leu Lys Leu
Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro Ala Ser100 105 110Pro
Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Lys Pro His Glu115 120
125Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg Val Cys Gly
Asp130 135 140Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu
Gly Cys Lys145 150 155 160Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys
Leu Ile Tyr Asp Arg Cys165 170 175Asp Leu Asn Cys Arg Ile His Lys
Lys Ser Arg Asn Lys Cys Gln Tyr180 185 190Cys Arg Phe Gln Lys Cys
Leu Ala Val Gly Met Ser His Asn Ala Ile195 200 205Arg Phe Gly Arg
Met Pro Gln Ala Glu Lys Glu Lys Leu Leu Ala Glu210 215 220Ile Ser
Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg225 230 235
240Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro
Leu245 250 255Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr
Thr Asp Lys260 265 270Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu
Met Met Gly Glu Asp275 280 285Lys Ile Lys Phe Lys His Ile Thr Pro
Leu Gln Glu Gln Ser Lys Glu290 295 300Val Ala Ile Arg Ile Phe Gln
Gly Cys Gln Phe Arg Ser Val Glu Ala305 310 315 320Val Gln Glu Ile
Thr Glu Tyr Ala Lys Ser Ile Pro Gly Phe Val Asn325 330 335Leu Asp
Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu340 345
350Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val
Leu355 360 365Ile Ser Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu
Lys Ser Leu370 375 380Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys
Phe Glu Phe Ala Val385 390 395 400Lys Phe Asn Ala Leu Glu Leu Asp
Asp Ser Asp Leu Ala Ile Phe Ile405 410 415Ala Val Ile Ile Leu Ser
Gly Asp Arg Pro Gly Leu Leu Asn Val Lys420 425 430Pro Ile Glu Asp
Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln435 440 445Leu Lys
Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys Leu Leu450 455
460Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val Gln
Leu465 470 475 480Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser
Leu His Pro Leu485 490 495Leu Gln Glu Ile Tyr Lys Asp Leu Tyr500
50516225DNAHomo sapiens 16atgcaggcgc aggacaggca gctggcaggg
cagctgctgc ggctgcgggc ccagctgcac 60cgactgaaga tggaccaagc ctgtcacctg
caccaggagc tgctggatga ggccgagctg 120gagctggagc tggagcccgg
ggccggccta gccctggccc cgctgctgcg gcacctgggc 180ctcacgcgca
tgaacatcag cgcccggcgc ttcaccctct gctga 22517303DNAHomo sapiens
17atggttgagc ccttcttggg aacctggaag ctggtctcca gtgaaaactt tgaggattac
60atgaaagaac tgggagtgaa tttcgcagcc cggaacatgg cagggttagt gaaaccgaca
120gtaactatta gtgttgatgg gaaaatgatg accataagaa cagaaagttc
tttccaggac 180actaagatct ccttcaagct gggggaagaa tttgatgaaa
ctacagcaga caaccggaaa 240gtaaaggtca gaactaattc ttcctggtgt
tcaacaatca ggaaaaagtt agaaggtggt 300tag 303181071DNAHomo sapiens
18gcagttctgg tcctcctagg agcggccgcc tgcgcggcgc ggccccgtgg tcggatgctg
60ggcggcagag aggccgaggc gcacgcgcgg ccctacatgg cgtcggtgca gctgaacggc
120gcgcacctgt gcgcaggcgt cctggtggcg gagcggtggg tgctgagcgc
ggcgcactgc 180ctggaggacg cggccgacgg gaaggtgcag gttctcctgg
gcgcgcactc cctgtcgcag 240ccggagccct ccaagcgcct gtacgacgtg
ctccgcgcag tgccccaccc ggacagccag 300cccgacacca tcgaccacga
cctcctgctg ctacagctgt cggagaaggc cacactgggc 360cctgctgtgc
gccccctgcc ctggcagcgc gtggaccgcg acgtggcacc gggaactctc
420tgcgacgtgg ccggctgggg catagtcaac cacgcgggcc gccgcccgga
cagcctgcag 480cacgtgctct tgccagtgct ggaccgcgcc acctgcaacc
ggcgcacgca ccacgacggc 540gccatcaccg agcgcttgat gtgcgcggag
agcaatcgcc gggacagctg caagggtgac 600tccgggggcc cgctggtgtg
cgggggcgtg ctcgagggcg tggtcacctc gggctcgcgc 660gtttgcggca
accgcaagaa gcccgggatc tacacccgcg tggcgagcta tgcggcctgg
720atcgacagcg tcctggccta gggtgccggg gcctgaaggt cagggtcacc
caagcaacaa 780agtcccgagc aatgaagtca tccactcctg catctggttg
gtctttattg agcacctact 840atatgcagaa ggggaggccg aggtgggagg
atcattggat ctcaggagtt ggagatcagc 900atgggccacg tagcgcgact
ccatctctac aaataaataa aaattagctg ggcaattggc 960gggcatggag
gtgggtgctt gtagttccag ctactcagga ggctgaggtg ggaggatgac
1020ttgaacgcag gaggctgagg ctgcagtgag ttgtgattgc accactgccc t
107119118PRTHomo sapiens 19Asp Ala His Lys Val Leu Ser Ala Leu Gln
Ala Val Gln Gly Leu Leu1 5 10 15Val Ala Gln Gly Arg Ala Asp Ser Gln
Ala Gln Leu Leu Leu Ser Thr20 25 30Val Val Gly Val Phe Thr Ala Pro
Gly Leu His Leu Lys Gln Pro Phe35 40 45Val Gln Gly Leu Ala Leu Tyr
Thr Pro Val Val Leu Pro Arg Ser Leu50 55 60Asp Phe Thr Glu Arg Asp
Val Ala Ala Glu Lys Ile Asp Arg Phe Met65 70 75 80Gln Ala Val Thr
Gly Trp Lys Thr Gly Cys Ser Leu Met Gly Ala Ser85 90 95Val Asp Ser
Thr Leu Ala Phe Asn Thr Tyr Val His Phe Gln Gly Lys100 105 110Ala
Asn Leu Ser Ala Gly115201926DNAHomo sapiens 20gttgttctgg gtactacagc
agaagggtat gcggaagcga gcaccccagt ctgagatggc 60tcctgccggt gtgagcctga
gggccaccat cctctgcctc ctggcctggg ctggcctggc 120tgcaggtgac
cgggtgtaca tacacccctt ccacctcgtc atccacaatg agagtacctg
180tgagcagctg gcaaaggcca atgccgggaa gcccaaagac cccaccttca
tacctgctcc 240aattcaggcc aagacatccc ctgtggatga aaaggcccta
caggaccagc tggtgctagt 300cgctgcaaaa cttgacaccg aagacaagtt
gagggccgca atggtcggga tgctggccaa 360cttcttgggc ttccgtatat
atggcatgca cagtgagcta tggggcgtgg tccatggggc 420caccgtcctc
tccccaacgg ctgtctttgg caccctggcc tctctctatc tgggagcctt
480ggaccacaca gctgacaggc tacaggcaat cctgggtgtt ccttggaagg
acaagaactg 540cacctcccgg ctggatgcgc acaaggtcct gtctgccctg
caggctgtac agggcctgct 600agtggcccag ggcagggctg atagccaggc
ccagctgctg ctgtccacgg tggtgggcgt 660gttcacagcc ccaggcctgc
acctgaagca gccgtttgtg cagggcctgg ctctctatac 720ccctgtggtc
ctcccacgct ctctggactt cacagaactg gatgttgctg ctgagaagat
780tgacaggttc atgcaggctg tgacaggatg gaagactggc tgctccctga
cgggagccag 840tgtggacagc accctggctt tcaacaccta cgtccacttc
caagggaaga tgaagggctt 900ctccctgctg gccgagcccc aggagttctg
ggtggacaac agcacctcag tgtctgttcc 960catgctctct ggcatgggca
ccttccagca ctggagtgac atccaggaca acttctcggt 1020gactcaagtg
tccttcactg agagcgcctg cctgctgctg atccagcctc actatgcctc
1080tgacctggac aaggtggagg gtctcacttt ccagcaaaac tccctcaact
ggatgaagaa 1140actgtctccc cggaccatcc acctgaccat gccccaactg
gtgctgcaag gatcttatga 1200cctgcaggac ctgctcgccc aggctgagct
gcccgccatt ctgcacaccg agctgaacct 1260gcaaaaattg agcaatgacc
gcatcagggt gggggaggtg ctgaacagca ttttttttga 1320gcttgaagcg
gatgagagag agcccacaga gtctacccaa cagcttaaca agcctgaggt
1380cttggaggtg accctgaacc gcccattcct gtttgctgtg tatgatcaaa
gcgccactgc 1440cctgcacttc ctgggccgcg tggccaaccc gctgagcaca
gcatgaggcc agggccccag 1500aacacagtgc ctggcaaggc ctctgcccct
ggcctttgag gcaaaggcca gcagcagata 1560acaaccccgg acaaatcagc
gatgtgtcac ccccagtctc ccaccttttc ttctaatgag 1620tcgactttga
gctggaaagc agccgtttct ccttggtcta agtgtgctgc atggagtgag
1680cagtagaagc ctgcagcggc acaaatgcac ctcccagttt gctgggttta
ttttagagaa 1740tgggggtggg gaggcaagaa ccagtgttta gcgcgggact
actgttccaa aaagaattcc 1800aaccgaccag cttgtttgtg aaacaaaaaa
gtgttccctt ttcaagttga gaacaaaaat 1860tgggttttaa aattaaagta
tacatttttg cattgcaaaa aaaaaaaaaa aaaaaaaaaa 1920aaaaaa
192621330PRTHomo sapiens 21Met Leu Leu Leu Ile Asn Val Ile Leu Thr
Leu Trp Val Ser Cys Ala1 5 10 15Asn Gly Gln Val Lys Pro Cys Asp Phe
Pro Asp Ile Lys His Gly Gly20 25 30Leu Phe His Glu Asn Met Arg Arg
Pro Tyr Phe Pro Val Ala Val Gly35 40 45Lys Tyr Tyr Ser Tyr Tyr Cys
Asp Glu His Phe Glu Thr Pro Ser Gly50 55 60Ser Tyr Trp Asp Tyr Ile
His Cys Thr Gln Asn Gly Trp Ser Pro Ala65 70 75 80Val Pro Cys Leu
Arg Lys Cys Tyr Phe Pro Tyr Leu Glu Asn Gly Tyr85 90 95Asn Gln Asn
Tyr Gly Arg Lys Phe Val Gln Gly Asn Ser Thr Glu Val100 105 110Ala
Cys His Pro Gly Tyr Gly Leu Pro Lys Ala Gln Thr Thr Val Thr115 120
125Cys Thr Glu Lys Gly Trp Ser Pro Thr Pro Arg Cys Ile Arg Val
Arg130 135 140Thr Cys Ser Lys Ser Asp Ile Glu Ile Glu Asn Gly Phe
Ile Ser Glu145 150 155 160Ser Ser Ser Ile Tyr Ile Leu Asn Lys Glu
Ile Gln Tyr Lys Cys Lys165 170 175Pro Gly Tyr Ala Thr Ala Asp Gly
Asn Ser Ser Gly Ser Ile Thr Cys180 185 190Leu Gln Asn Gly Trp Ser
Ala Gln Pro Ile Cys Ile Asn Ser Ser Glu195 200 205Lys Cys Gly Pro
Pro Pro Pro Ile Ser Asn Gly Asp Thr Thr Ser Phe210 215 220Leu Leu
Lys Val Tyr Val Pro Gln Ser Arg Val Glu Tyr Gln Cys Gln225 230 235
240Pro Tyr Tyr Glu Leu Gln Gly Ser Asn Tyr Val Thr Cys Ser Asn
Gly245 250 255Glu Trp Ser Glu Pro Pro Arg Cys Ile His Pro Cys Ile
Ile Thr Glu260 265 270Glu Asn Met Asn Lys Asn Asn Ile Lys Leu Lys
Gly Arg Ser Asp Arg275 280 285Lys Tyr Tyr Ala Lys Thr Gly Asp Thr
Ile Glu Phe Met Cys Lys Leu290 295 300Gly Tyr Asn Ala Asn Thr Ser
Ile Leu Ser Phe Gln Ala Val Cys Arg305 310 315 320Glu Gly Ile Val
Glu Tyr Pro Arg Cys Glu325 33022878DNAHomo sapiens 22agtcggtgtc
tcagccacag cggcttcacc atgcacagct gggagcgcct ggcagttctg 60gtcctcctag
gagcggccgc ctgcgcggcg ccgccccgtg gtcggatcct gggcggcaga
120gaggccgagg cgcacgcgcg gccctacatg gcgtcggtgc agctgaacgg
cgcgcacctg 180tgcggcggcg tcctggtggc ggagcagtgg gtgctgagcg
cggcgcactg cctggaggac 240gcggccgacg ggaaggtgca ggttctcctg
ggcgcgcact ccctgtcgca gccggagccc 300tccaagcgcc tgtacgacgt
gctccgcgca gtgccccacc cggacagcca gcccgacacc 360atcgaccacg
acctcctgct gctacagctg tcggagaagg ccacactggg ccctgctgtg
420cgccccctgc cctggcagcg cgtggaccgc gacgtggcac cgggaactct
ctgcgacgtg 480gccggctggg gcatagtcaa ccacgcgggc cgccgcccgg
acagcctgca gcacgtgctc 540ttgccagtgc tggaccgcgc cacctgcaac
cggcgcacgc accacgacgg cgccatcacc 600gagcgcttga tgtgcgcgga
gagcaatcgc cgggacagct gcaagggtga ctccgggggc 660ccgctggtgt
gcgggggcgt gctcgagggc gtggtcacct cgggctcgcg cgtttgcggc
720aaccgcaaga agcccgggat ctacacccgc gtggcgagct atgcggcctg
gatcgacagc 780gtcctggcct agggtgccgg ggcctgaagg tcagggtcac
ccaagcaaca aagtcccgag 840cattgaagtc atccactcct gcaaaaaaaa aaaaaaaa
878233549DNAHomo sapiens 23cccctcttcc tcctcctcaa gggaaagctg
cccacttcta gctgccctgc catccccttt 60aaagggcgac ttgctcagcg ccaaaccgcg
gctccagccc tctccagcct ccggctcagc 120cggctcatca gtcggtccgc
gccttgcagc tcctccagag ggacgcgccc cgagatggag 180agcaaagccc
tgctcgtgct gactctggcc gtgtggctcc agagtctgac cgcctcccgc
240ggaggggtgg ccgccgccga ccaaagaaga gattttatcg acatcgaaag
taaatttgcc 300ctaaggaccc ctgaagacac agctgaggac acttgccacc
tcattcccgg agtagcagag 360tccgtggcta cctgtcattt caatcacagc
agcaaaacct tcatggtgat ccatggctgg 420acggtaacag gaatgtatga
gagttgggtg ccaaaacttg tggccgccct gtacaagaga 480gaaccagact
ccaatgtcat tgtggtggac tggctgtcac gggctcagga gcattaccca
540gtgtccgcgg gctacaccaa actggtggga caggatgtgg cccggtttat
caactggatg 600gaggaggagt ttaactaccc tctggacaat gtccatctct
tgggatacag ccttggagcc 660catgctgctg gcattgcagg aagtctgacc
aataagaaag tcaacagaat tactggcctc 720gatccagctg gacctaactt
tgagtatgca gaagccccga gtcgtctttc tcctgatgat 780gcagattttg
tagacgtctt acacacattc accagagggt cccctggtcg aagcattgga
840atccagaaac cagttgggca tgttgacatt tacccgaatg gaggtacttt
tcagccagga 900tgtaacattg gagaagctat ccgcgtgatt gcagagagag
gacttggaga tgtggaccag 960ctagtgaagt gctcccacga gcgctccatt
catctcttca tcgactctct gttgaatgaa 1020gaaaatccaa gtaaggccta
caggtgcagt tccaaggaag cctttgagaa agggctctgc 1080ttgagttgta
gaaagaaccg ctgcaacaat ctgggctatg agatcaataa agtcagagcc
1140aaaagaagca gcaaaatgta cctgaagact cgttctcaga tgccctacaa
agtcttccat 1200taccaagtaa agattcattt ttctgggact gagagtgaaa
cccataccaa tcaggccttt 1260gagatttctc tgtatggcac cgtggccgag
agtgagaaca tcccattcac tctgcctgaa 1320gtttccacaa ataagaccta
ctccttccta atttacacag aggtagatat tggagaacta 1380ctcatgttga
agctcaaatg gaagagtgat tcatacttta gctggtcaga ctggtggagc
1440agtcccggct tcgccattca gaagatcaga gtaaaagcag gagagactca
gaaaaaggtg 1500atcttctgtt ctagggagaa agtgtctcat ttgcagaaag
gaaaggcacc tgcggtattt 1560gtgaaatgcc atgacaagtc tctgaataag
aagtcaggct gaaactgggc gaatctacag 1620aacaaagaac ggcatgtgaa
ttctgtgaag aatgaagtgg aggaagtaac ttttacaaaa 1680catacccagt
gtttggggtg tttcaaaagt ggattttcct gaatattaat cccagcccta
1740cccttgttag ttattttagg agacagtctc aagcactaaa aagtggctaa
ttcaatttat 1800ggggtatagt ggccaaatag cacatcctcc aacgttaaaa
gacagtggat catgaaaagt 1860gctgttttgt cctttgagaa agaaataatt
gtttgagcgc agagtaaaat aaggctcctt 1920catgtggcgt attgggccat
agcctataat tggttagaac ctcctatttt aattggaatt 1980ctggatcttt
cggactgagg ccttctcaaa ctttactcta agtctccaag aatacagaaa
2040atgcttttcc gcggcacgaa tcagactcat ctacacagca gtatgaatga
tgttttagaa 2100tgattccctc ttgctattgg aatgtggtcc agacgtcaac
caggaacatg taacttggag 2160agggacgaag aaagggtctg ataaacacag
aggttttaaa cagtccctac cattggcctg 2220catcatgaca aagttacaaa
ttcaaggaga tataaaatct agatcaatta attcttaata 2280ggctttatcg
tttattgctt aatccctctc tcccccttct tttttgtctc aagattatat
2340tataataatg ttctctgggt aggtgttgaa aatgagcctg taatcctcag
ctgacacata 2400atttgaatgg tgcagaaaaa aaaaagatac cgtaatttta
ttattagatt ctccaaatga 2460ttttcatcaa tttaaaatca ttcaatatct
gacagttact cttcagtttt aggcttacct 2520tggtcatgct tcagttgtac
ttccagtgcg tctcttttgt tcctggcttt gacatgaaaa 2580gataggtttg
agttcaaatt ttgcattgtg tgagcttcta cagattttag acaaggaccg
2640tttttactaa gtaaaagggt ggagaggttc ctggggtgga ttcctaagca
gtgcttgtaa 2700accatcgcgt gcaatgagcc agatggagta ccatgagggt
tgttatttgt tgtttttaac 2760aactaatcaa gagtgagtga acaactattt
ataaactaga tctcctattt ttcagaatgc 2820tcttctacgt ataaatatga
aatgataaag atgtcaaata tctcagaggc tatagctggg 2880aacccgactg
tgaaagtatg tgatatctga acacatacta gaaagctctg catgtgtgtt
2940gtccttcagc ataattcgga agggaaaaca gtcgatcaag ggatgtattg
gaacatgtcg 3000gagtagaaat tgttcctgat gtgccagaac ttcgaccctt
tctctgagag agatgatcgt 3060gcctataaat agtaggacca atgttgtgat
taacatcatc aggcttggaa tgaattctct 3120ctaaaaataa aatgatgtat
gatttgttgt tggcatcccc tttattaatt cattaaattt 3180ctggatttgg
gttgtgaccc agggtgcatt aacttaaaag attcactaaa gcagcacata
3240gcactgggaa ctctggctcc gaaaaacttt gttatatata tcaaggatgt
tctggcttta 3300cattttattt attagctgta aatacatgtg tggatgtgta
aatggagctt gtacatattg 3360gaaaggtcat tgtggctatc tgcatttata
aatgtgtggt gctaactgta tgtgtcttta 3420tcagtgatgg tctcacagag
ccaactcact cttatgaaat gggctttaac aaaacaagaa 3480agaaacgtac
ttaactgtgt gaagaaatgg aatcagcttt taataaaatt gacaacattt
3540tattaccac 3549242315DNAHomo sapiens 24gctcagccgg ctcatcagtc
ggtccgcgcc ttgcagctcc tccagaggga
cgcgccccga 60gatggagagc aaagccctgc tcgtgctgac tctggccgtg tggctccaga
gtctgaccgc 120ctcccgcgga ggggtggccg ccgccgacca aagaagagat
tttatcgaca tcgaaagtaa 180atttgcccta aggacccctg aagacacagc
tgaggacact tgccacctca ttcccggagt 240agcagagtcc gtggctacct
gtcatttcaa tcacagcagc aaaaccttca tggtgatcca 300tggctggacg
gtaacaggaa tgtatgagag ttgggtgcca aaacttgtgg ccgccctgta
360caagagagaa ccagactcca atgtcattgt ggtggactgg ctgtcacggg
ctcaggagca 420ttacccagtg tccgcgggct acaccaaact ggtgggacag
gatgtggccc ggtttatcaa 480ctggatggag gaggagttta actaccctct
ggacaatgtc catctcttgg gatacagcct 540tggagcccat gctgctggca
ttgcaggaag tctgaccaat aagaaagtca acagaattac 600tggcctcgat
ccagctggac ctaactttga gtatgcagaa gccccgagtc gtctttctcc
660tgatgatgca gattttgtag acgtcttaca cacattcacc agagggtccc
ctggtcgaag 720cattggaatc cagaaaccag ttgggcatgt tgacatttac
ccgaatggag gtacttttca 780gccaggatgt aacattggag aagctatccg
cgtgattgca gagagaggac ttggagatgt 840ggaccagcta gtgaagtgct
cccacgagcg ctccattcat ctcttcatcg actctctgtt 900gaatgaagaa
aatccaagta aggcctacag gtgcagttcc aaggaagcct ttgagaaagg
960gctctgcttg agttgtagaa agaaccgctg caacaatctg ggctatgaga
tcagtaaagt 1020cagagccaaa agaagcagca aaatgtacct gaagactcgt
tctcagatgc cctacaaagt 1080cttccattac caagtaaaga ttcatttttc
tgggactgag agtgaaaccc ataccaatca 1140ggcctttgag atttctctgt
atggcaccgt ggccgagagt gagaacatcc cattcactct 1200gcctgaagtt
tccacaaata agacctactc cttcctaatt tacacagagg tagatattgg
1260agaactactc atgttgaagc tcaaatggaa gagtgattca tactttagct
ggtcagactg 1320gtggagcagt cccggcttcg ccattcagaa gatcagagta
aaagcaggag agactcagaa 1380aaaggtgatc ttctgttcta gggagaaagt
gtctcatttg cagaaaggaa aggcacctgc 1440ggtatttgtg aaatgccatg
acaagtctct gaataagaag tcaggctgaa actgggcgaa 1500tctacagaac
aaagaacggc atgtgaattc tgtgaagaat gaagtggagg aagtaacttt
1560tacaaaacat acccagtgtt tggggtgttt caaaagtgga ttttcctgaa
tattaatccc 1620agccctaccc ttgttagtta ttttaggaga cagtctcaag
cactaaaaag tggctaattc 1680aatttatggg gtatagtggc caaatagcac
atcctccaac gttaaaagac agtggatcat 1740gaaaagtgct gttttgtcct
ttgagaaaga aataattgtt tgagcgcaga gtaaaataag 1800gctccttcat
gtggcgtatt gggccatagc ctataattgg ttagaacctc ctattttaat
1860tggaattctg gatctttcgg actgaggcct tctcaaactt tactctaagt
ctccaagaat 1920acagaaaatg cttttccgcg gcacgaatca gactcatcta
cacagcagta tgaatgatgt 1980tttagaatga ttccctcttg ctattggaat
gtggtccaga cgtcaaccag gaacatgtaa 2040cttggagagg gacgaagaaa
gggtctgata aacacagagg ttttaaacag tccctaccat 2100tggcctgcat
catgacaaag ttacaaattc aaggagatat aaaatctaga tcaattaatt
2160cttaataggc tttatcgttt attgcttaat ccctctctcc cccttctttt
ttgtctcaag 2220attatattat aataatgttc tctgggtagg tgttgaaaat
gagcctgtaa tcctcagctg 2280acacataatt tgaatggtgc aaaaaaaaaa aaaaa
2315252922DNAHomo sapiens 25agcctgggct ctgtgagact gaggtggcgg
tcagccggag tgagtgttgg ggtcctgggg 60cacctgcctt acatggcttg tttatgaaca
ttaaagggaa gaagttgaag cttgaggagc 120gaggatggca gtcaacaaag
gcctcacctt gctggatgga gacctccctg agcaggagaa 180tgtgctgcag
cgggtcctgc agctgccggt ggtgagtggc acctgcgaat gcttccagaa
240gacctacacc agcactaagg aagcccaccc cctggtggcc tctgtgtgca
atgcctatga 300gaagggcgtg cagagcgcca gtagcttggc tgcctggagc
atggagccgg tggtccgcag 360gctgtccacc cagttcacag ctgccaatga
gctggcctgc cgaggcttgg accacctgga 420ggaaaagatc cccgccctcc
agtacccccc tgaaaagatt gcttctgagc tgaaggacac 480catctccacc
cgcctccgca gtgccagaaa cagcatcagc gttcccatcg cgagcacttc
540agacaaggtc ctgggggccg ctttggccgg gtgcgagctt gcctgggggg
tggccagaga 600cactgcggaa tttgctgcca acactcgagc tggccgactg
gcttctggag gggccgactt 660ggccttgggc agcattgaga aggtggtgga
gtacctcctc cctgcagaca aggaagagtc 720agcccctgct cctggacacc
agcaagccca gaagtctccc aaggccaagc caagcctctt 780gagcagggtt
ggggctctga ccaacaccct ctctcgatac accgtgcaga ccatggcccg
840ggccctggag cagggccaca ccgtggccat gtggatccca ggcgtggtgc
ccctgagcag 900cctggcccag tggggtgcct cagtggccat gcaggcggtg
tcccggcgga ggagcgaagt 960gcgggtaccc tggctgcaca gcctcgcagc
cgcccaggag gaggatcatg aggaccagac 1020agacacggag ggagaggaca
cggaggagga ggaagaattg gagactgagg agaacaagtt 1080cagtgaggta
gcagccctgc caggccctcg aggcctcctg ggtggtgtgg cacataccct
1140gcagaagacc ctccagacca ccatctcggc tgtgacatgg gcacctgcag
ctgtgctggg 1200catggcaggg agggtgctgc acctcacacc agcccctgct
gtctcctcaa ccaaggggag 1260ggccatgtcc ctatcagatg ccctgaaggg
cgttactgac aacgtggtgg acacagtggt 1320gcattacgtg ccgctcccca
ggctgtcgct gatggagccc gagagcgaat tccgggacat 1380cgacaaccca
ccagccgagg tcgagcgccg ggaggcggag cgcagagcgt ctggggcgcc
1440gtccgccggc ccggagcccg ccccgcgtct cgcacagccc cgccgcagcc
tgcgcagcgc 1500gcagagcccc ggcgcgcccc ccggcccggg cctggaggac
gaagtcgcca cgcccgcagc 1560gccgcgcccg ggcttcccgg ccgtgccccg
cgagaagcca aagcgcaggg tcagcgacag 1620cttcttccgg cccagcgtca
tggagcccat cctgggccgc acgcattaca gccagctgcg 1680caagaagagc
tgagtcgccg caccagccgc cgcgccccgg gccggcgggt ttctctaaca
1740aataaacaga acccgcactg cccaggcgag cgttgccact ttcaaagtgg
tcccctgggg 1800agctcagcct catcctgatg atgctgccaa ggcgcacttt
ttatttttat tttattttta 1860tttttttttt agcatccttt tggggcttca
ctctcagagc cagtttttaa gggacaccag 1920agccgcagcc tgctctgatt
ctatggcttg gttgttacta taagagtaat tgcctaactt 1980gatttttcat
ctctttaacc aaacttgtgg ccaaaagata tttgaccgtt tccaaaattc
2040agattctgcc tctgcggata aatatttgcc acgaatgagt aactcctgtc
accactctga 2100aggtccagac agaaggtttt gacacattct tagcactgaa
ctcctctgtg atctaggatg 2160atctgttccc cctctgatga acatcctctg
atgatctagg ctcccagcag gctactttga 2220agggaacaat cagatgcaaa
agctcttggg tgtttattta aaatactagt gtcactttct 2280gagtacccgc
cgcttcacag gctgagtcca ggcctgtgtg ctttgtagag ccagctgctt
2340gctcacagcc acatttccat ttgcatcatt actgccttca cctgcatagt
cactcttttg 2400atgctgggga accaaaatgg tgatgatata tagactttat
gtatagccac agttcatccc 2460caaccctagt cttcgaaatg ttaatatttg
ataaatctag aaaatgcatt catacaatta 2520cagaattcaa atattgcaaa
aggatgtgtg tctttctccc cgagctcccc tgttcccctt 2580cattgaaaac
caccacggtg ccatctcttg tgtatgcagg gctatgcacc tgcaggcacg
2640tgtgtatgca ctccccgctt gtgtttacac aagctgtggg gtgttacgca
tgcctgcttt 2700tttcacttaa taatacagct tggagagatt tttgtatcac
attataaatc ccactcgctc 2760tttttgatgg ccacataata actactgcat
aatatggata cgccttattt gatttaacta 2820gttccctaat gatggacttt
taagttgttt cctttttttt tcttttttgc tactgcaaac 2880gatgctataa
taaatgtcct tatcaaaaaa aaaaaaaaaa aa 292226509PRTHomo sapiens 26Met
Pro Ser Gly Phe Gln Gln Ile Gly Ser Glu Asp Gly Glu Pro Pro1 5 10
15Gln Gln Arg Val Thr Gly Thr Leu Val Leu Ala Val Phe Ser Ala Val20
25 30Leu Gly Ser Leu Gln Phe Gly Tyr Asn Ile Gly Val Ile Asn Ala
Pro35 40 45Gln Lys Val Ile Glu Gln Ser Tyr Asn Glu Thr Trp Leu Gly
Arg Gln50 55 60Gly Pro Glu Gly Pro Ser Ser Ile Pro Pro Gly Thr Leu
Thr Thr Leu65 70 75 80Trp Ala Leu Ser Val Ala Ile Phe Ser Val Gly
Gly Met Ile Ser Ser85 90 95Phe Leu Ile Gly Ile Ile Ser Gln Trp Leu
Gly Arg Lys Arg Ala Met100 105 110Leu Val Asn Asn Val Leu Ala Val
Leu Gly Gly Ser Leu Met Gly Leu115 120 125Ala Asn Ala Ala Ala Ser
Tyr Glu Met Leu Ile Leu Gly Arg Phe Leu130 135 140Ile Gly Ala Tyr
Ser Gly Leu Thr Ser Gly Leu Val Pro Met Tyr Val145 150 155 160Gly
Glu Ile Ala Pro Thr His Leu Arg Gly Ala Leu Gly Thr Leu Asn165 170
175Gln Leu Ala Ile Val Ile Gly Ile Leu Ile Ala Gln Val Leu Gly
Leu180 185 190Glu Ser Leu Leu Gly Thr Ala Ser Leu Trp Pro Leu Leu
Leu Gly Leu195 200 205Thr Val Leu Pro Ala Leu Leu Gln Leu Val Leu
Leu Pro Phe Cys Pro210 215 220Glu Ser Pro Arg Tyr Leu Tyr Ile Ile
Gln Asn Leu Glu Gly Pro Ala225 230 235 240Arg Lys Ser Leu Lys Arg
Leu Thr Gly Trp Ala Asp Val Ser Gly Val245 250 255Leu Ala Glu Leu
Lys Asp Glu Lys Arg Lys Leu Glu Arg Glu Arg Pro260 265 270Leu Ser
Leu Leu Gln Leu Leu Gly Ser Arg Thr His Arg Gln Pro Leu275 280
285Ile Ile Ala Val Val Leu Gln Leu Ser Gln Gln Leu Ser Gly Ile
Asn290 295 300Ala Val Phe Tyr Tyr Ser Thr Ser Ile Phe Glu Thr Ala
Gly Val Gly305 310 315 320Gln Pro Ala Tyr Ala Thr Ile Gly Ala Gly
Val Val Asn Thr Val Phe325 330 335Thr Leu Val Ser Val Leu Leu Val
Glu Arg Ala Gly Arg Arg Thr Leu340 345 350His Leu Leu Gly Leu Ala
Gly Met Cys Gly Cys Ala Ile Leu Met Thr355 360 365Val Ala Leu Leu
Leu Leu Glu Arg Val Pro Ala Met Ser Tyr Val Ser370 375 380Ile Val
Ala Ile Phe Gly Phe Val Ala Phe Phe Glu Ile Gly Pro Gly385 390 395
400Pro Ile Pro Trp Phe Ile Val Ala Glu Leu Phe Ser Gln Gly Pro
Arg405 410 415Pro Ala Ala Met Ala Val Ala Gly Phe Ser Asn Trp Thr
Ser Asn Phe420 425 430Ile Ile Gly Met Gly Phe Gln Tyr Val Ala Glu
Ala Met Gly Pro Tyr435 440 445Val Phe Leu Leu Phe Ala Val Leu Leu
Leu Gly Phe Phe Ile Phe Thr450 455 460Phe Leu Arg Val Pro Glu Thr
Arg Gly Arg Thr Phe Asp Gln Ile Ser465 470 475 480Ala Ala Phe His
Arg Thr Pro Ser Leu Leu Glu Gln Glu Val Lys Pro485 490 495Ser Thr
Glu Leu Glu Tyr Leu Gly Pro Asp Glu Asn Asp500 505274194DNAHomo
sapiens 27agcagagctg cggccggggg aacccagttt ccgaggaact tttcgccggc
gccgggccgc 60ctctgaggcc agggcaggac acgaacgcgc ggagcggcgg cggcgactga
gagccggggc 120cgcggcggcg ctccctagga agggccgtac gaggcggcgg
gcccggcggg cctcccggag 180gaggcggctg cgccatggac gagccaccct
tcagcgaggc ggctttggag caggcgctgg 240gcgagccgtg cgatctggac
gcggcgctgc tgaccgacat cgaagacatg cttcagctta 300tcaacaacca
agacagtgac ttccctggcc tatttgaccc accctatgct gggagtgggg
360cagggggcac agaccctgcc agccccgata ccagctcccc aggcagcttg
tctccacctc 420ctgccacatt gagctcctct cttgaagcct tcctgagcgg
gccgcaggca gcgccctcac 480ccctgtcccc tccccagcct gcacccactc
cattgaagat gtacccgtcc atgcccgctt 540tctcccctgg gcctggtatc
aaggaagagt cagtgccact gagcatcctg cagaccccca 600ccccacagcc
cctgccaggg gccctcctgc cacagagctt cccagcccca gccccaccgc
660agttcagctc cacccctgtg ttaggctacc ccagccctcc gggaggcttc
tctacaggaa 720gccctcccgg gaacacccag cagccgctgc ctggcctgcc
actggcttcc ccgccagggg 780tcccgcccgt ctccttgcac acccaggtcc
agagtgtggt cccccagcag ctactgacag 840tcacagctgc ccccacggca
gcccctgtaa cgaccactgt gacctcgcag atccagcagg 900tcccggtcct
gctgcagccc cacttcatca aggcagactc gctgcttctg acagccatga
960agacagacgg agccactgtg aaggcggcag gtctcagtcc cctggtctct
ggcaccactg 1020tgcagacagg gcctttgccg accctggtga gtggcggaac
catcttggca acagtcccac 1080tggtcgtaga tgcggagaag ctgcctatca
accggctcgc agctggcagc aaggccccgg 1140cctctgccca gagccgtgga
gagaagcgca cagcccacaa cgccattgag aagcgctacc 1200gctcctccat
caatgacaaa atcattgagc tcaaggatct ggtggtgggc actgaggcaa
1260agctgaataa atctgctgtc ttgcgcaagg ccatcgacta cattcgcttt
ctgcaacaca 1320gcaaccagaa actcaagcag gagaacctaa gtctgcgcac
tgctgtccac aaaagcaaat 1380ctctgaagga tctggtgtcg gcctgtggca
gtggagggaa cacagacgtg ctcatggagg 1440gcgtgaagac tgaggtggag
gacacactga ccccaccccc ctcggatgct ggctcacctt 1500tccagagcag
ccccttgtcc cttggcagca ggggcagtgg cagcggtggc agtggcagtg
1560actcggagcc tgacagccca gtctttgagg acagcaaggc aaagccagag
cagcggccgt 1620ctctgcacag ccggggcatg ctggaccgct cccgcctggc
cctgtgcacg ctcgtcttcc 1680tctgcctgtc ctgcaacccc ttggcctcct
tgctgggggc ccgggggctt cccagcccct 1740cagataccac cagcgtctac
catagccctg ggcgcaacgt gctgggcacc gagagcagag 1800atggccctgg
ctgggcccag tggctgctgc ccccagtggt ctggctgctc aatgggctgt
1860tggtgctcgt ctccttggtg cttctctttg tctacggtga gccagtcaca
cggccccact 1920caggccccgc cgtgtacttc tggaggcatc gcaagcaggc
tgacctggac ctggcccggg 1980gagactttgc ccaggctgcc cagcagctgt
ggctggccct gcgggcactg ggccggcccc 2040tgcccacctc ccacctggac
ctggcttgta gcctcctctg gaacctcatc cgtcacctgc 2100tgcagcgtct
ctgggtgggc cgctggctgg caggccgggc agggggcctg cagcaggact
2160gtgctctgcg agtggatgct agcgccagcg cccgagacgc agccctggtc
taccataagc 2220tgcaccagct gcacaccatg gggaagcaca caggcgggca
cctcactgcc accaacctgg 2280cgctgagtgc cctgaacctg gcagagtgtg
caggggatgc cgtgtctgtg gcgacgctgg 2340ccgagatcta tgtggcggct
gcattgagag tgaagaccag tctcccacgg gccttgcatt 2400ttctgacacg
cttcttcctg agcagtgccc gccaggcctg cctggcacag agtggctcag
2460tgcctcctgc catgcagtgg ctctgccacc ccgtgggcca ccgtttcttc
gtggatgggg 2520actggtccgt gctcagtacc ccatgggaga gcctgtacag
cttggccggg aacccagtgg 2580accccctggc ccaggtgact cagctattcc
gggaacatct cttagagcga gcactgaact 2640gtgtgaccca gcccaacccc
agccctgggt cagctgatgg ggacaaggaa ttctcggatg 2700ccctcgggta
cctgcagctg ctgaacagct gttctgatgc tgcgggggct cctgcctaca
2760gcttctccat cagttccagc atggccacca ccaccggcgt agacccggtg
gccaagtggt 2820gggcctctct gacagctgtg gtgatccact ggctgcggcg
ggatgaggag gcggctgagc 2880ggctgtgccc gctggtggag cacctgcccc
gggtgctgca ggagtctgag agacccctgc 2940ccagggcagc tctgcactcc
ttcaaggctg cccgggccct gctgggctgt gccaaggcag 3000agtctggtcc
agccagcctg accatctgtg agaaggccag tgggtacctg caggacagcc
3060tggctaccac accagccagc agctccattg acaaggccgt gcagctgttc
ctgtgtgacc 3120tgcttcttgt ggtgcgcacc agcctgtggc ggcagcagca
gcccccggcc ccggccccag 3180cagcccaggg caccagcagc aggccccagg
cttccgccct tgagctgcgt ggcttccaac 3240gggacctgag cagcctgagg
cggctggcac agagcttccg gcccgccatg cggagggtgt 3300tcctacatga
ggccacggcc cggctgatgg cgggggccag ccccacacgg acacaccagc
3360tcctcgaccg cagtctgagg cggcgggcag gccccggtgg caaaggaggc
gcggtggcgg 3420agctggagcc gcggcccacg cggcgggagc acgcggaggc
cttgctgctg gcctcctgct 3480acctgccccc cggcttcctg tcggcgcccg
ggcagcgcgt gggcatgctg gctgaggcgg 3540cgcgcacact cgagaagctt
ggcgatcgcc ggctgctgca cgactgtcag cagatgctca 3600tgcgcctggg
cggtgggacc actgtcactt ccagctagac cccgtgtccc cggcctcagc
3660acccctgtct ctagccactt tggtcccgtg cagcttctgt cctgcgtcga
agctttgaag 3720gccgaaggca gtgcaagaga ctctggcctc cacagttcga
cctgcggctg ctgtgtgcct 3780tcgcggtgga aggcccgagg ggcgcgatct
tgaccctaag accggcggcc atgatggtgc 3840tgacctctgg tggccgatcg
gggcactgca ggggccgagc cattttgggg ggcccccctc 3900cttgctctgc
aggcacctta gtggcttttt tcctcctgtg tacagggaag agaggggtac
3960atttccctgt gctgacggaa gccaacttgg ctttcccgga ctgcaagcag
ggctctgccc 4020cagaggcctc tctctccgtc gtgggagaga gacgtgtaca
tagtgtaggt cagcgtgctt 4080agcctcctga cctgaggctc ctgtgctact
ttgccttttg caaactttat tttcatagat 4140tgagaagttt tgtacagaga
attaaaaatg aaattattta taatctggaa aaaa 4194281837DNAHomo sapiens
28gagccgcgca cgggactggg aaggggaccc acccgagggt ccagccacca gccccctcac
60taatagcggc caccccggca gcggcggcag cagcagcagc gacgcagcgg cgacagctca
120gagcagggag gccgcgccac ctgcgggccg gccggagcgg gcagccccag
gccccctccc 180cgggcacccg cgttcatgca acgcctggtg gcctgggacc
cagcatgtct ccccctgccg 240ccgccgccgc ctgcctttaa atccatggaa
gtggccaact tctactacga ggcggactgc 300ttggctgctg cgtacggcgg
caaggcggcc cccgcggcgc cccccgcggc cagacccggg 360ccgcgccccc
ccgccggcga gctgggcagc atcggcgacc acgagcgcgc catcgacttc
420agcccgtacc tggagccgct gggcgcgccg caggccccgg cgcccgccac
ggccacggac 480accttcgagg cggctccgcc cgcgcccgcc cccgcgcccg
cctcctccgg gcagcaccac 540gacttcctct ccgacctctt ctccgacgac
tacgggggca agaactgcaa gaagccggcc 600gagtacggct acgtgagcct
ggggcgcctg ggggccgcca agggcgcgct gcaccccggc 660tgcttcgcgc
ccctgcaccc accgcccccg ccgccgccgc cgcccgccga gctcaaggcg
720gagccgggct tcgagcccgc ggactgcaag cggaaggagg aggccggggc
gccgggcggc 780ggcgcaggca tggcggcggg cttcccgtac gcgctgcgcg
cttacctcgg ctaccaggcg 840gtgccgagcg gcagcagcgg gagcctctcc
acgtcctcct cgtccagccc gcccggcacg 900ccgagccccg ctgacgccaa
ggcgcccccg accgcctgct acgcgggggc cgcgccggcg 960ccctcgcagg
tcaagagcaa ggccaagaag accgtggaca agcacagcga cgagtacaag
1020atccggcgcg agcgcaacaa catcgccgtg cgcaagagcc gcgacaaggc
caagatgcgc 1080aacctggaga cgcagcacaa ggtcctggag ctcacggccg
agaacgagcg gctgcagaag 1140aaggtggagc agctgtcgcg cgagctcagc
accctgcgga acttgttcaa gcagctgccc 1200gagcccctgc tcgcctcctc
cggccactgc tagcgcggcc cccgcgcgcg tccccctgcc 1260ggccggggct
gagactccgg ggagcgcccg cgcccgcgcc ctcgcccccg cccccggcgg
1320cgccggcaaa actttggcac tggggcactt ggcagcgcgg ggagcccgtc
ggtaatttta 1380atattttatt atatatatat atctatattt ttgtccaaac
caaccgcaca tgcagatggg 1440gctcccgccc gtggtgttat ttaaagaaga
aacgtctatg tgtacagatg aatgataaac 1500tctctgcttc tccctctgcc
cctctccagg cgccggcggg cgggccggtt tcgaagttga 1560tgcaatcggt
ttaaacatgg ctgaacgcgt gtgtacacgg gactgacgca acccacgtgt
1620aactgtcagc cgggccctga gtaatcgctt aaagatgttc ctacgggctt
gttgctgttg 1680atgttttgtt ttgttttgtt ttttggtctt tttttgtatt
ataaaaaata atctatttct 1740atgagaaaag aggcgtctgt atattttggg
aatcttttcc gtttcaagca ttaagaacac 1800ttttaataaa cttttttttg
agaatggtta caaagcc 1837293549DNAHomo sapiens 29cccctcttcc
tcctcctcaa gggaaagctg cccacttcta gctgccctgc catccccttt 60aaagggcgac
ttgctcagcg ccaaaccgcg gctccagccc tctccagcct ccggctcagc
120cggctcatca gtcggtccgc gccttgcagc tcctccagag ggacgcgccc
cgagatggag 180agcaaagccc tgctcgtgct gactctggcc gtgtggctcc
agagtctgac cgcctcccgc 240ggaggggtgg ccgccgccga ccaaagaaga
gattttatcg acatcgaaag taaatttgcc 300ctaaggaccc ctgaagacac
agctgaggac acttgccacc tcattcccgg agtagcagag 360tccgtggcta
cctgtcattt caatcacagc agcaaaacct tcatggtgat ccatggctgg
420acggtaacag gaatgtatga gagttgggtg ccaaaacttg tggccgccct
gtacaagaga 480gaaccagact ccaatgtcat tgtggtggac tggctgtcac
gggctcagga gcattaccca 540gtgtccgcgg gctacaccaa actggtggga
caggatgtgg cccggtttat caactggatg 600gaggaggagt ttaactaccc
tctggacaat gtccatctct tgggatacag ccttggagcc 660catgctgctg
gcattgcagg aagtctgacc aataagaaag tcaacagaat tactggcctc
720gatccagctg gacctaactt tgagtatgca gaagccccga gtcgtctttc
tcctgatgat 780gcagattttg tagacgtctt
acacacattc accagagggt cccctggtcg aagcattgga 840atccagaaac
cagttgggca tgttgacatt tacccgaatg gaggtacttt tcagccagga
900tgtaacattg gagaagctat ccgcgtgatt gcagagagag gacttggaga
tgtggaccag 960ctagtgaagt gctcccacga gcgctccatt catctcttca
tcgactctct gttgaatgaa 1020gaaaatccaa gtaaggccta caggtgcagt
tccaaggaag cctttgagaa agggctctgc 1080ttgagttgta gaaagaaccg
ctgcaacaat ctgggctatg agatcaataa agtcagagcc 1140aaaagaagca
gcaaaatgta cctgaagact cgttctcaga tgccctacaa agtcttccat
1200taccaagtaa agattcattt ttctgggact gagagtgaaa cccataccaa
tcaggccttt 1260gagatttctc tgtatggcac cgtggccgag agtgagaaca
tcccattcac tctgcctgaa 1320gtttccacaa ataagaccta ctccttccta
atttacacag aggtagatat tggagaacta 1380ctcatgttga agctcaaatg
gaagagtgat tcatacttta gctggtcaga ctggtggagc 1440agtcccggct
tcgccattca gaagatcaga gtaaaagcag gagagactca gaaaaaggtg
1500atcttctgtt ctagggagaa agtgtctcat ttgcagaaag gaaaggcacc
tgcggtattt 1560gtgaaatgcc atgacaagtc tctgaataag aagtcaggct
gaaactgggc gaatctacag 1620aacaaagaac ggcatgtgaa ttctgtgaag
aatgaagtgg aggaagtaac ttttacaaaa 1680catacccagt gtttggggtg
tttcaaaagt ggattttcct gaatattaat cccagcccta 1740cccttgttag
ttattttagg agacagtctc aagcactaaa aagtggctaa ttcaatttat
1800ggggtatagt ggccaaatag cacatcctcc aacgttaaaa gacagtggat
catgaaaagt 1860gctgttttgt cctttgagaa agaaataatt gtttgagcgc
agagtaaaat aaggctcctt 1920catgtggcgt attgggccat agcctataat
tggttagaac ctcctatttt aattggaatt 1980ctggatcttt cggactgagg
ccttctcaaa ctttactcta agtctccaag aatacagaaa 2040atgcttttcc
gcggcacgaa tcagactcat ctacacagca gtatgaatga tgttttagaa
2100tgattccctc ttgctattgg aatgtggtcc agacgtcaac caggaacatg
taacttggag 2160agggacgaag aaagggtctg ataaacacag aggttttaaa
cagtccctac cattggcctg 2220catcatgaca aagttacaaa ttcaaggaga
tataaaatct agatcaatta attcttaata 2280ggctttatcg tttattgctt
aatccctctc tcccccttct tttttgtctc aagattatat 2340tataataatg
ttctctgggt aggtgttgaa aatgagcctg taatcctcag ctgacacata
2400atttgaatgg tgcagaaaaa aaaaagatac cgtaatttta ttattagatt
ctccaaatga 2460ttttcatcaa tttaaaatca ttcaatatct gacagttact
cttcagtttt aggcttacct 2520tggtcatgct tcagttgtac ttccagtgcg
tctcttttgt tcctggcttt gacatgaaaa 2580gataggtttg agttcaaatt
ttgcattgtg tgagcttcta cagattttag acaaggaccg 2640tttttactaa
gtaaaagggt ggagaggttc ctggggtgga ttcctaagca gtgcttgtaa
2700accatcgcgt gcaatgagcc agatggagta ccatgagggt tgttatttgt
tgtttttaac 2760aactaatcaa gagtgagtga acaactattt ataaactaga
tctcctattt ttcagaatgc 2820tcttctacgt ataaatatga aatgataaag
atgtcaaata tctcagaggc tatagctggg 2880aacccgactg tgaaagtatg
tgatatctga acacatacta gaaagctctg catgtgtgtt 2940gtccttcagc
ataattcgga agggaaaaca gtcgatcaag ggatgtattg gaacatgtcg
3000gagtagaaat tgttcctgat gtgccagaac ttcgaccctt tctctgagag
agatgatcgt 3060gcctataaat agtaggacca atgttgtgat taacatcatc
aggcttggaa tgaattctct 3120ctaaaaataa aatgatgtat gatttgttgt
tggcatcccc tttattaatt cattaaattt 3180ctggatttgg gttgtgaccc
agggtgcatt aacttaaaag attcactaaa gcagcacata 3240gcactgggaa
ctctggctcc gaaaaacttt gttatatata tcaaggatgt tctggcttta
3300cattttattt attagctgta aatacatgtg tggatgtgta aatggagctt
gtacatattg 3360gaaaggtcat tgtggctatc tgcatttata aatgtgtggt
gctaactgta tgtgtcttta 3420tcagtgatgg tctcacagag ccaactcact
cttatgaaat gggctttaac aaaacaagaa 3480agaaacgtac ttaactgtgt
gaagaaatgg aatcagcttt taataaaatt gacaacattt 3540tattaccac
354930402PRTHomo sapiens 30Met Gln Met Ser Pro Ala Leu Thr Cys Leu
Val Leu Gly Leu Ala Leu1 5 10 15Val Phe Gly Glu Gly Ser Ala Val His
His Pro Pro Ser Tyr Val Ala20 25 30His Leu Ala Ser Asp Phe Gly Val
Arg Val Phe Gln Gln Val Ala Gln35 40 45Ala Ser Lys Asp Arg Asn Val
Val Phe Ser Pro Tyr Gly Val Ala Ser50 55 60Val Leu Ala Met Leu Gln
Leu Thr Thr Gly Gly Glu Thr Gln Gln Gln65 70 75 80Ile Gln Ala Ala
Met Gly Phe Lys Ile Asp Asp Lys Gly Met Ala Pro85 90 95Ala Leu Arg
His Leu Tyr Lys Glu Leu Met Gly Pro Trp Asn Lys Asp100 105 110Glu
Ile Ser Thr Thr Asp Ala Ile Phe Val Gln Arg Asp Leu Lys Leu115 120
125Val Gln Gly Phe Met Pro His Phe Phe Arg Leu Phe Arg Ser Thr
Val130 135 140Lys Gln Val Asp Phe Ser Glu Val Glu Arg Ala Arg Phe
Ile Ile Asn145 150 155 160Asp Trp Val Lys Thr His Thr Lys Gly Met
Ile Ser Asn Leu Leu Gly165 170 175Lys Gly Ala Val Asp Gln Leu Thr
Arg Leu Val Leu Val Asn Ala Leu180 185 190Tyr Phe Asn Gly Gln Trp
Lys Thr Pro Phe Pro Asp Ser Ser Thr His195 200 205Arg Arg Leu Phe
His Lys Ser Asp Gly Ser Thr Val Ser Val Pro Met210 215 220Met Ala
Gln Thr Asn Lys Phe Asn Tyr Thr Glu Phe Thr Thr Pro Asp225 230 235
240Gly His Tyr Tyr Asp Ile Leu Glu Leu Pro Tyr His Gly Asp Thr
Leu245 250 255Ser Met Phe Ile Ala Ala Pro Tyr Glu Lys Glu Val Pro
Leu Ser Ala260 265 270Leu Thr Asn Ile Leu Ser Ala Gln Leu Ile Ser
His Trp Lys Gly Asn275 280 285Met Thr Arg Leu Pro Arg Leu Leu Val
Leu Pro Lys Phe Ser Leu Glu290 295 300Thr Glu Val Asp Leu Arg Lys
Pro Leu Glu Asn Leu Gly Met Thr Asp305 310 315 320Met Phe Arg Gln
Phe Gln Ala Asp Phe Thr Ser Leu Ser Asp Gln Glu325 330 335Pro Leu
His Val Ala Gln Ala Leu Gln Lys Val Lys Ile Glu Val Asn340 345
350Glu Ser Gly Thr Val Ala Ser Ser Ser Thr Ala Val Ile Val Ser
Ala355 360 365Arg Met Ala Pro Glu Glu Ile Ile Met Asp Arg Pro Phe
Leu Phe Val370 375 380Val Arg His Asn Pro Thr Gly Thr Val Leu Phe
Met Gly Gln Val Met385 390 395 400Glu Pro314592DNAHomo sapiens
31aggctgttga ggctgggcca tctcctcctc acttccattc tgactgcagt ctgtggttct
60gattccatac cagaggggct caggatgctg ttgctgggag ctgttctact gctattagct
120ctgcccggtc atgaccagga aaccacgact caagggcccg gagtcctgct
tcccctgccc 180aagggggcct gcacaggttg gatggcgggc atcccagggc
atccgggcca taatggggcc 240ccaggccgtg atggcagaga tggcacccct
ggtgagaagg gtgagaaagg agatccaggt 300cttattggtc ctaagggaga
catcggtgaa accggagtac ccggggctga aggtccccga 360ggctttccgg
gaatccaagg caggaaagga gaacctggag aaggtgccta tgtataccgc
420tcagcattca gtgtgggatt ggagacttac gttactatcc ccaacatgcc
cattcgcttt 480accaagatct tctacaatca gcaaaaccac tatgatggct
ccactggtaa attccactgc 540aacattcctg ggctgtacta ctttgcctac
cacatcacag tctatatgaa ggatgtgaag 600gtcagcctct tcaagaagga
caaggctatg ctcttcacct atgatcagta ccaggaaaat 660aatgtggacc
aggcctccgg ctctgtgctc ctgcatctgg aggtgggcga ccaagtctgg
720ctccaggtgt atggggaagg agagcgtaat ggactctatg ctgataatga
caatgactcc 780accttcacag gctttcttct ctaccatgac accaactgat
caccactaac tcagagcctc 840ctccaggcca aacagcccca aagtcaatta
aaggctttca gtacggttag gaagttgatt 900attatttagt tggaggcctt
tagatattat tcattcattt actcattcat ttattcattc 960attcatcaag
taactttaaa aaaatcatat gctatgttcc cagtcctggg gagcttcaca
1020aacatgacca gataactgac tagaaagaag tagttgacag tgctattttg
tgcccactgt 1080ctctcctgat gctcatatca atcctataag gcacagggaa
caagcattct cctgttttta 1140cagattgtat cctgaggctg agagagttaa
gtgaatgtct aaggtcacac agtattaagt 1200gacagtgcta gaaatcaaac
ccagagctgt ggactttgtt cactagactg tgccctttta 1260tagaggtaca
tgttctcttt ggagtgttgg taggtgtctg tttcccacct cacctgagag
1320ccattgaatt tgccttcctc atgaattaaa acctccccca agcagagctt
cctcagagaa 1380agtggttcta tgatgaagtc ctgtcttgga aggactacta
ctcaatggcc cctgcactac 1440tctacttcct cttacctatg tcccttctca
tgcctttccc tccaacgggg aaagccaact 1500ccatctctaa gtgctgaact
catccctgtt cctcaaggcc acctggccag gagcttctct 1560gatgtgatat
ccactttttt tttttttgag atggagtctc actctgtcac ccaggctgga
1620gtacagtgac acgacctcgg ctcactgcag cctccttctc ctgggtccaa
gcaattattg 1680tgcctcagcc tcccgagtag ctgagacttc aggtgcattc
caccacacat ggctaatttt 1740tgtattttta gtagaaatgg ggtttcgtca
tgttggccag gctggtctcg aactcctggc 1800ctaggtgatc cacccgcctc
gacctcccaa agtgctggga ttacaggcat gagccaccat 1860gcccagtcga
tatctcactt tttattttgc catggatgag agtcctgggt gtgaggaaca
1920cctcccacca ggctagaggc aactgcccag gaaggactgt gcttccgtca
cctctaaatc 1980ccttgcagat ccttgataaa tgcctcatga agaccaatct
cttgaatccc atatctaccc 2040agaattaact ccattccagt ctctgcatgt
aatcagtttt atccacagaa acattttcat 2100tttaggaaat ccctggtttt
aagtatcaat ccttgttcag ctggacaata tgaatctttt 2160ccactgaagt
tagggatgac tgtgattttc agaacacgtc cagaattttt catcaagaag
2220gtagcttgag cctgaaatgc aaaacccatg gaggaattct gaagccattg
tctccttgag 2280taccaacagg gtcagggaag actgggcctc ctgaatttat
tattgttctt taagaattac 2340aggttgaggt agttgatggt ggtaaacatt
ctctcaggag acaataactc cagtgatgtt 2400cttcaaagat tttagcaaaa
acagagtaaa tagcattctc tatcaatata taaatttaaa 2460aaactatctt
tttgcttaca gttttaaatt ctgaacaatt ctctcttata tgtgtattgc
2520taatcattaa ggtattattt tttccacata taaagctttg tctttttgtt
gttgttgttg 2580tttttaagat ggagtttccc tctgttgcca ggctagagtg
cagtggcatg atctcggctt 2640actgcaacct ttgcctccca ggttcaagcg
attcttctgc ctcagcctcc cgagtagctg 2700ggaccacagg tgcctaccac
catgccaggc taatttttgt atttttagta aagacagggt 2760ttcaccatat
tggccaggct ggtctcgaac tcctgacctt gtgatctgcc cgcctccatt
2820tttgttgtta ttttttgaga aagatagata tgaggtttag agagggatga
agaggtgaga 2880gtaagccttg tgttagtcag aactctgtgt tgtgaatgtc
attcacaaca gaaaacccaa 2940aatattatgc aaactactgt aagcaagaaa
aataaaggaa aaatggaaac atttattcct 3000ttgcataata gaaattacca
gagttgttct gtctttagat aaggtttgaa ccaaagctca 3060aaacaatcaa
gacccttttc tgtatgtcct tctgttctgc cttccgcagt gtaggcttta
3120ccctcaggtg ctacacagta tagttctagg gtttccctcc cgatatcaaa
aagactgtgg 3180cctgcccagc tctcgtatcc ccaagccaca ccatctggct
aaatggacat catgttttct 3240ggtgatgccc aaagaggaga gaggaagctc
tctttcccag atgccccagc aagtgtaacc 3300ttgcatctca ttgctctggc
tgagttgtgt gcctgtttct gaccaatcac tgagtcagga 3360ggatgaaata
ttcatattga cttaattgca gcttaagtta ggggtatgta gaggtatttt
3420ccctaaagca aaattgggac actgttatca gaaataggag agtggatgat
agatgcaaaa 3480taatacctgt ccacaacaaa ctcttaatgc tgtgtttgag
ctttcatgag tttcccagag 3540agacatagct ggaaaattcc tattgatttt
ctctaaaatt tcaacaagta gctaaagtct 3600ggctatgctc acagtctcac
atctggttgg ggtgggctcc ttacagaaca cgctttcaca 3660gttaccctaa
actctctggg gcagggttat tcctttgtgg aaccagaggc acagagagag
3720tcaactgagg ccaaaagagg cctgagagaa actgaggtca agatttcagg
attaatggtc 3780ctgtgatgct ttgaagtaca attgtggatt tgtccaattc
tctttagttc tgtcagcttt 3840tgcttcatat attttagcgc tctattatta
gatatataca tgtttagtat tatgtcttat 3900tggtgcattt actctcttat
cattatgtaa tgtccttctt tatctgtgat aattttctgt 3960gttctgaagt
ctactttgtc taaaaataac atacgcactc aacttccttt tctttcttcc
4020ttcctttctt tcttccttcc tttctttctc tctctctctc tttccttcct
tccttcctcc 4080ttttctttct ctctctctct ctctctcttt ttttgacaga
ctctcgttct gtggccctgg 4140ctggagttca gtggtgtgat cttggctcac
tgctacctct accatgagca attctcctgc 4200ctcagcctcc caagtagctg
gaactacagg ctcatgccac tgcgcccagc taatttttgt 4260atttttcgta
gagacggggt ttcaccacat tcgtcaggtt ggtttcaaac tcctgacttt
4320gtgatccacc cgcctcggcc tcccaaagtg ctgggattac aggcatgagc
catcacacct 4380ggtcaacttt cttttgatta gtgtttttgt ggtatatctt
tttccatcat gttactttaa 4440atatatctat attattgtat ttaaaatgtg
tttcttacag actgcatgta gttgggtata 4500atttttatcc agtctaaaaa
tatctgtctt ttaattggtg tttagacaat ttatatttaa 4560taaaattgtt
gaatttaaaa aaaaaaaaaa aa 4592321125DNAHomo sapiens 32ttctgccctc
gagcccaccg ggaacgaaag agaagctcta tctcgcctcc aggagcccag 60ctatgaactc
cttctccaca agcgccttcg gtccagttgc cttctccctg gggctgctcc
120tggtgttgcc tgctgccttc cctgccccag tacccccagg agaagattcc
aaagatgtag 180ccgccccaca cagacagcca ctcacctctt cagaacgaat
tgacaaacaa attcggtaca 240tcctcgacgg catctcagcc ctgagaaagg
agacatgtaa caagagtaac atgtgtgaaa 300gcagcaaaga ggcactggca
gaaaacaacc tgaaccttcc aaagatggct gaaaaagatg 360gatgcttcca
atctggattc aatgaggaga cttgcctggt gaaaatcatc actggtcttt
420tggagtttga ggtataccta gagtacctcc agaacagatt tgagagtagt
gaggaacaag 480ccagagctgt gcagatgagt acaaaagtcc tgatccagtt
cctgcagaaa aaggcaaaga 540atctagatgc aataaccacc cctgacccaa
ccacaaatgc cagcctgctg acgaagctgc 600aggcacagaa ccagtggctg
caggacatga caactcatct cattctgcgc agctttaagg 660agttcctgca
gtccagcctg agggctcttc ggcaaatgta gcatgggcac ctcagattgt
720tgttgttaat gggcattcct tcttctggtc agaaacctgt ccactgggca
cagaacttat 780gttgttctct atggagaact aaaagtatga gcgttaggac
actattttaa ttatttttaa 840tttattaata tttaaatatg tgaagctgag
ttaatttatg taagtcatat ttatattttt 900aagaagtacc acttgaaaca
ttttatgtat tagttttgaa ataataatgg aaagtggcta 960tgcagtttga
atatcctttg tttcagagcc agatcatttc ttggaaagtg taggcttacc
1020tcaaataaat ggctaactta tacatatttt taaagaaata tttatattgt
atttatataa 1080tgtataaatg gtttttatac caataaatgg cattttaaaa aattc
112533212PRTHomo sapiens 33Met Asn Ser Phe Ser Thr Ser Ala Phe Gly
Pro Val Ala Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val Leu Pro Ala Ala
Phe Pro Ala Pro Val Pro Pro20 25 30Gly Glu Asp Ser Lys Asp Val Ala
Ala Pro His Arg Gln Pro Leu Thr35 40 45Ser Ser Glu Arg Ile Asp Lys
Gln Ile Arg Tyr Ile Leu Asp Gly Ile50 55 60Ser Ala Leu Arg Lys Glu
Thr Cys Asn Lys Ser Asn Met Cys Glu Ser65 70 75 80Ser Lys Glu Ala
Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala85 90 95Glu Lys Asp
Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu100 105 110Val
Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr115 120
125Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val
Gln130 135 140Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys
Ala Lys Asn145 150 155 160Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr
Thr Asn Ala Ser Leu Leu165 170 175Thr Lys Leu Gln Ala Gln Asn Gln
Trp Leu Gln Asp Met Thr Thr His180 185 190Leu Ile Leu Arg Ser Phe
Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala195 200 205Leu Arg Gln
Met21034233PRTHomo sapiens 34Met Ser Thr Glu Ser Met Ile Arg Asp
Val Glu Leu Ala Glu Glu Ala1 5 10 15Leu Pro Lys Lys Thr Gly Gly Pro
Gln Gly Ser Arg Arg Cys Leu Phe20 25 30Leu Ser Leu Phe Ser Phe Leu
Ile Val Ala Gly Ala Thr Thr Leu Phe35 40 45Cys Leu Leu His Phe Gly
Val Ile Gly Pro Gln Arg Glu Glu Phe Pro50 55 60Arg Asp Leu Ser Leu
Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser65 70 75 80Ser Arg Thr
Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro85 90 95Gln Ala
Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu100 105
110Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro
Ser115 120 125Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys
Gly Gln Gly130 135 140Cys Pro Ser Thr His Val Leu Leu Thr His Thr
Ile Ser Arg Ile Ala145 150 155 160Val Ser Tyr Gln Thr Lys Val Asn
Leu Leu Ser Ala Ile Lys Ser Pro165 170 175Cys Gln Arg Glu Thr Pro
Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu180 185 190Pro Ile Tyr Leu
Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu195 200 205Ser Ala
Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly210 215
220Gln Val Tyr Phe Gly Ile Ile Ala Leu225 2303510049DNAHomo sapiens
35gcgccgcctc cttcggcgtt cgccccacgg accggcaggc ggcggaccgc ggcccaggct
60gaagctcagg gccctgtctg ctctgtggac tcaacagttt gtggcaagac aagctcagaa
120ctgagaagct gtcaccacag ttctggaggc tgggaagttc aagatcaaag
tgccagcaga 180ttcagtgtca tgtgaggacg tgcttcctgc ttcatagata
agagtagctt ggagctcggc 240ggcacaacca gcaccatctg gtcgcgatgg
tggacacgga aagcccactc tgccccctct 300ccccactcga ggccggcgat
ctagagagcc cgttatctga agagttcctg caagaaatgg 360gaaacatcca
agagatttcg caatccatcg gcgaggatag ttctggaagc tttggcttta
420cggaatacca gtatttagga agctgtcctg gctcagatgg ctcggtcatc
acggacacgc 480tttcaccagc ttcgagcccc tcctcggtga cttatcctgt
ggtccccggc agcgtggacg 540agtctcccag tggagcattg aacatcgaat
gtagaatctg cggggacaag gcctcaggct 600atcattacgg agtccacgcg
tgtgaaggct gcaagggctt ctttcggcga acgattcgac 660tcaagctggt
gtatgacaag tgcgaccgca gctgcaagat ccagaaaaag aacagaaaca
720aatgccagta ttgtcgattt cacaagtgcc tttctgtcgg gatgtcacac
aacgcgattc 780gttttggacg aatgccaaga tctgagaaag caaaactgaa
agcagaaatt cttacctgtg 840aacatgacat agaagattct gaaactgcag
atctcaaatc tctggccaag agaatctacg 900aggcctactt gaagaacttc
aacatgaaca aggtcaaagc ccgggtcatc ctctcaggaa 960aggccagtaa
caatccacct tttgtcatac atgatatgga gacactgtgt atggctgaga
1020agacgctggt ggccaagctg gtggccaatg gcatccagaa caaggaggcg
gaggtccgca 1080tctttcactg ctgccagtgc acgtcagtgg agaccgtcac
ggagctcacg gaattcgcca 1140aggccatccc aggcttcgca aacttggacc
tgaacgatca agtgacattg ctaaaatacg 1200gagtttatga ggccatattc
gccatgctgt cttctgtgat gaacaaagac gggatgctgg 1260tagcgtatgg
aaatgggttt ataactcgtg aattcctaaa aagcctaagg aaaccgttct
1320gtgatatcat ggaacccaag tttgattttg ccatgaagtt caatgcactg
gaactggatg 1380acagtgatat ctcccttttt gtggctgcta tcatttgctg
tggagatcgt cctggccttc 1440taaacgtagg acacattgaa aaaatgcagg
agggtattgt acatgtgctc agactccacc 1500tgcagagcaa ccacccggac
gatatctttc tcttcccaaa acttcttcaa aaaatggcag 1560acctccggca
gctggtgacg gagcatgcgc
agctggtgca gatcatcaag aagacggagt 1620cggatgctgc gctgcacccg
ctactgcagg agatctacag ggacatgtac tgagttcctt 1680cagatcagcc
acaccttttc caggagttct gaagctgaca gcactacaaa ggagacgggg
1740gagcagcacg attttgcaca aatatccacc actttaacct tagagcttgg
acagtctgag 1800ctgtaggtaa ccggcatatt attccatatc tttgttttaa
ccagtacttc taagagcata 1860gaactcaaat gctgggggta ggtggctaat
ctcaggactg ggaagattac ggcgaattat 1920gctcaatggt ctgattttaa
ctcacccgat gttaatcaat gcacattgct ttagatcaca 1980ttcgtgattt
accatttaat taactggtaa cctcaaaatt cgtggcctgt cttcccattc
2040accccgcttt tgactattgt gctcctttat aattctgaaa actaatcagc
actttttaac 2100aatgtttata atcctataag tctagatgta tccaaaggtg
aagtatgtaa aaagcagcaa 2160aatatttatt tcaaagactt cacttctgtt
tcctgaatct aaagaaagac aacatgctgc 2220tttttaatca taggatggag
aattttaaag aactgtttgg gccaggcaca gtcgctcata 2280cttgtaatcc
cagcactttg ggaggccgag gcgggtggat cacaaggtca gcagatcgag
2340accatcctgg ccaacatggt gaaaccctgt ctctactaaa aatacaaaaa
ttagccgggt 2400gtggtggcac atgcctgtaa tcccagctac tcgggaagct
gaggcaggag aattgcttga 2460accagggagt tggaggttgc agtgagctaa
gactgcacca ctgcactcca gcctggtgac 2520agaacgagac tctgtcttaa
aaacaaacaa acaaaaaaaa aatctgttag ataagctatc 2580aaaatgcagc
tgttgttttg tttttggctc actgttttcg tggttgtaac taatatgtgg
2640aaaggcccat ttccaggttt gcgtagaaga gcccagaaaa cagagtctca
agacccccgc 2700tctggactgt cataagctag cacccgtggt aagcgggacg
agacaagctc ccgaagcccg 2760ccagcttcct gctccactca gctccgtcca
gtcaacctga acccacccag tccagctgtc 2820tgtgggaatg gtggtgttct
tagggacaga ctgacacctt acttgtcagt gttcctccgg 2880gccccatttg
gcagctcccg tatcttttgt tatgttgctt ttaaagatat gatgttttat
2940tgttttaact cttggtgaca gtagatgctc tctggagcgc agacgaggca
catgtgtctt 3000catagcctgg gctgggtggg agccagtcac cctgcggatc
gagagagggg gtagagtctt 3060cttcaaatgg cagttttact tcaaatggca
gatttcacaa gagttggtta ttttttacaa 3120tggtttaggt tgttaagtct
cctttgtatg taaggtagtt ttttcaacat ctaaaatttt 3180tgttttagcc
ttcaaaacca acttaccaac ctcagtccag ctgggaaggc agcgttgatt
3240atggtagttt gtcaagaata tatggacctg gaaacacttt ctctctctgt
ccacctggta 3300gataaattgt cctgttgaga atttttagat ctggactgga
actgccagga ccaccgcctc 3360cagggagtcg ctgggcacct ggaggtatcg
tcgatgcctc tcccccatct ttagaaaatt 3420tggctcttct gaggtcatta
ttattttaag aatgattagg attgataagg gtcccatgac 3480cagcattatg
aaaatgcgag agtgggaagg acacagtgtg agacttccac tagaaaaaag
3540tgaaagttag ggttaggaca tcctttttta aaaattacaa atttagtccg
ttttggtttt 3600tgtaatcagg ctaggcacag tggctcacac atggaatccc
agcactttgg gaggccgagg 3660tgggaggatc acttgagccc aggagttcga
gaccagccta ggcaacatag caagaccctg 3720tctgtacaca aaatttaaaa
attagttcat cggggtggca cacatcagta gtcccagcta 3780ctctgcaggc
tgaggtggga ggattgcttg aacccaggag gtcgaggctg cagtgagctg
3840tgatctcacc actgcattcc agcctgggtg acagagttag attccaccct
ctcccacccc 3900ggcaaaaaaa aaaaaaaaag atgcaatcaa aggggctgtt
ggccagcaat ggcagcagca 3960gcggcgggca gtctgcccaa gtgtcttagg
aaccaaaagc aaataaaagt gtttccatat 4020atgccaccag ccaagtggcc
atcctaattc agaaagaagc tagcctttga gtgtctgtca 4080tggtgcatcc
gtttcagtat tatttcctaa aatgagaagc ccctgtgtca acaagatcca
4140ggggctggag cccaatgcca agcctgtgtt gtccccagcg accctgcagc
tgctcgctct 4200gatgtaccct gtgccattca aggagatgtg gtccaggaaa
gtgagcctca tggttttcag 4260agaagtcatt gttctgttta cattttcata
aaacctgttt aaaatagctc cccgtctcag 4320gctttcagca gtaacagtga
gctgactggc aagttcgatg ttagctcccg ggacactcag 4380cagcgatggt
gagcattttg gtttccttaa ggcccagcaa gacttccagg gacatctctg
4440gtgaagccag aatggagaca cccgtgacct caggctgaaa gtcactcgac
attggtctct 4500tgtgttgata gggaaggaaa tcaggcattc ctatttcttt
aaataacaaa accactaatt 4560gccactcaat gctggaatat tttgggtcac
ctaatcatag atttctcagg gcatcaatac 4620tcaaatatag gctgattatg
ccccagttca aatgggaact attaacagag tgcatttctt 4680gcttgctggg
tttcaacaga catcagccaa aagaacaaaa gagatgtcag gacagattcc
4740aggagtgtcg gagcacatgt gtggcacccg ctccctctgg cagcgaatgt
aggaagtcgc 4800caaatttacc cactcttcaa caagtcattg tttaaacacg
gtttttcatt ttctcaactt 4860ttaatagcaa aaagtgccaa agtcctcaga
gacctaacag ccttggtcta ccgtgctgac 4920cagggtgaag gcacggcgag
ggactcctcc cagacgtgcc tcttgtgtgc cagctggctg 4980tggctcggga
gcagacgcag gcctctccat tgtccagggg agcctggcgg cgcatccctc
5040ctctcccacc tcctggcact tccagctggg tgtcccacat gttggattcc
gtccccacca 5100cacttccaga gaccggagaa ctgtgcaggg cctaaggccg
tttggatgaa ttgtcaaaac 5160aagatgcttc cagttacagc ggcaggagcg
ggactgggag cacgggctga cggctgctgg 5220tgcctttctt cccacctcgc
ttgcctgttt ccgcttgacc cttcctccag ctccgatgag 5280aagagtataa
agcatcttcc taacgggtgt gtttgctata cgaacataat ggacgtgaag
5340tggggcagaa acccagaact cagcattcaa ggatgcccag gagagctgtc
cctgttttaa 5400agagctgtgt tttgttttgt ttcgcattta gagagcagac
aaggcaccct tctgctgcgc 5460tgatacgttt cttacactgg gccattttag
acccccaggg aaacagcctt cctggagcgt 5520tgtctggagg ttccagggac
agggcagcct cccagagccg agcaagagct caaggtacaa 5580atgagagatt
tgctataccg tgagaagtca acaacttagc caccacttcc ccgcaatgga
5640ccatgtaaca aatacctcag caggccctgc aaaaggccat gctagagctg
aggcgcacag 5700cctgtggcct ctgtagttag ggcaggtggg atggagactc
cttgagtgca cacacctgag 5760cctgcccaca cacaggggag cagcatctcg
tatgacgtct ggaaggaact tcggttgtgt 5820aaagggagcc ttgaagatac
gtgcaaaagg tgctacccca atttggtgaa actgacattg 5880ggcacgtctt
gggcttagga gaagcggccg atggtcccgg cctgcagtga caaacccccc
5940tccccgcacc gcccccagca ccccctctcc tcttcacctc ttcctgctgg
ccacgaggaa 6000gccacttcct cagagagacc ctaccagatg cggatggaaa
cagatgcacc aaagcaagcc 6060ctgatgaaac cgcgacttcc taaggtctgt
ctcctctgaa cttgcacctg ggcctctctg 6120tgtttggttc caagcacttc
ccacctcaaa ctcccatttt caaaccactg tatctctgcg 6180cacatctgct
acttaccagc cgcatacatg atggagggtt ttttggtcct gatccagtgg
6240ccacacctgt ctttgaaatg tctcactgaa ctccagtttt aaaatagatt
cattgcttca 6300acacagcaag cccaatgcac ccagctaaga ctggcttgac
cgacagcctg gcctttggtg 6360gggggcttcc tggggcctgg ggaaagctgg
ccaccttcaa cagctggtac ctcttcaaca 6420gtgtggcctt tcaaaatgca
gatgccacca ggagaacatg cccacagctc accacctatg 6480gatgccatgg
ctctgggcag ctttcaaagc aggttcctgt ggtctcctca gctgtttgag
6540ggggtaacag caaatcagcc tccattttaa aatgaaaaca ccagcctcca
gatgtagggc 6600ctgctgggtg ttgctagccg ctggtcccca ggcacggtgc
actttctcca cctcctgcag 6660cctccctgtt gtttctagac tcttgcacct
ggtgagtgca aggataggtg acccaggggc 6720ctgcagcctt gtcctcagct
cccatctcct ggactgccag cctcaccctc tgcagttagc 6780atggttggcc
tgatgcaggg atcccgaggg attacttttt agaccttctt tcacattcag
6840aaaagtagta tagattcagg agaggcaaga aaattatgct gtccatagaa
gtcacccatg 6900aagactgatg ccaccacctg aaggctcatg attgttaaaa
atgtccacgg gaacctctcg 6960tccacaggag gtttgtctca acacttccca
tttttacggc attggcattg ccaagcatgg 7020ggaagtatct gctcttctca
tgttaaaagt ggcccagctt ttcttaactc agtccaagct 7080gacttgttta
gctgcactgg aatttcttac caaccaaata tttgcatcga gcaaaggggg
7140ctgtgtgcac ctccctaatg gcagcgatga tggctgctgt cattcaagcc
catcttcaga 7200cgtcacagtc tggaagtgaa atgtccacaa acatctgtgg
cagaaaaggc tatacggacc 7260acccagttgt gctgcagctt tacagagcaa
ggaagggttg tggcaaataa atgattaacc 7320tgcctcgact gtgctgaggg
caacaaaggc catctcacca aaggattatt cgatgccatt 7380aaatcatccc
gtgaccttcc tgcttccgag tccatggcct ttgcccaggg catgtactcc
7440cctgagaggc cttctgccta gaaagatcta tgactgggtt ccaaagttga
ggcctaggtt 7500tttgctggga tttagatatt ttcaggcacc attttgacag
cattcaggaa aacggttatt 7560gaccccatag actagggtaa gaataaaggc
aataaatttg gtctgactca gaatatagga 7620gatccatata tttctctgga
aaccacagtg tacactaaaa tgtgaaattg aaggttttgt 7680taaaaagaaa
aagataatga gcttcatgct ttgtttaatt acataatgat ttccattacg
7740ctatttctgt gaaatgcagc aggttcttaa acgttatttc agtggcatgg
gctggaagct 7800tatcacaaaa agccatgtgt gtggccttat cagaacagaa
agagacaggc tggtgcccaa 7860ggctgctgcc tgctccacct tttgccagct
ctggacatct gaggacgtcc cggcagatct 7920ggaatggggc cctcaactga
ccatttgctt ctcagaattt cagtttgaga catgagaggt 7980ataatcagtt
acttttctcc ccccagagaa acccttttgt gaggggagag gagctatggt
8040atgtggttca gctgaaacac atacaactgc atccttttgg agtcctttgc
caacaaaaac 8100agaccaacag accagatggt gtccatgttc aatatcatgt
cttgatggac gcagctgatg 8160acctcaaata cttgagtggt ctcatggctg
ttagatggat tatttgaaaa aaaaaaaaaa 8220aaaagagaga aaaaataatt
gatttttaca tcagagatag caaactaaga cctggggagg 8280ggggtcagct
tttattttat tttatttttt ttaagtttgc tagttgggtc aaatgtgagg
8340aggagggagt ctacctgcca cctcttctct tgcccctctt ctgcccacac
atccagcatc 8400caaaatccat tcatttaatg aattgataaa gtgccgtgca
aactggtgca caaacaggcc 8460cccagtccac gcagcctggc tcctaggaaa
agtggtgacc gggcgtgggg gggcatgccg 8520cagccctggg acacagtcgg
gcaccttccc cggaccccca ggccttggct gtgcctcaag 8580tcagagaggg
tcagccttca ggccccggag acgagtgact ggccgatcat ttcacaataa
8640aatcactcac ttttggcaac ttcacttttt ttaaggcaca gtcagttcct
tttctcatgt 8700acctcacaaa agatgaagac catgtagtac tctttttggt
aaagttacag tgttcatgtt 8760aaatatcact tttttctaca ttgtgtggta
aaaagaacta cgttaatagc tatatcttaa 8820atactgtgat ttgacttttt
gaaaaatatc ctaatacaaa tattttacta acttacaatc 8880actcatttaa
taagaaacat ttggattctt ttgaaatcag tgttaattga ctcatattct
8940taaaagcctg gctcttgacc ctattggaaa cacaaaggaa gctgaaatca
aacatctaaa 9000atacactgcg tacacgtgtg cgtgcacaca cacacacaca
cacacacaca cacagctctt 9060catttctcct gagccatgca gaatttactt
tcaatgtgga aatctgttcc ctttaccaca 9120ctgtatatgc acagagcaca
agagaggcta tctctagtca cttccaccag cgaggcctta 9180gactccgtat
tagaggccac cgatttcata caacagtgtt tcgctaaaga cccttcacta
9240ttcttgttta gtaaatagct gtctgctctt cagggaactg ttacctatgg
gttattacca 9300aagaacgctg gcaattggaa atgtcctgat ggaaattctt
tgcacgtgcc ggttctctgg 9360catcctccag gtggcccaac ccaaagcaga
aagcagaaac cacagacccc gtgagtctcc 9420ccataccttg tttccaataa
cttggcaaaa cttcttggtg catattggtt acaccctctg 9480ggattcataa
tgccattagg ctaaaaccct aagagagagg gttgacagaa acacacgcga
9540gaatgaggca gatcccagag caaggactgg gcccagactc tccacatgtg
ctctactagt 9600gagtgcctta tactctcagt attttggggc ttacagcttc
ttatttgtgc taaaaaggtg 9660cagttccaaa gtaggaactg ccacacaggc
cccagcatcc tctctccaac ttcatacctc 9720tctcctggtg gggggagcgg
gcatccagga cctccggaat caaggatgtg cagagaagag 9780cgaaagtaat
ttttctagtc acatgaactg attggttcca ggcaattaga aaatggctat
9840aaaataacct taattttaaa aaaaaatctt gggtcttcgt tttcctatta
ggagactgaa 9900ctgaccacat gtattgattt atatcctgaa tatatgggaa
cttctgtgtt tgggatgtcc 9960tactgtaaga ctgatgaatg tacagagtta
atttcagggt acagttttgc cttaatggtt 10020ttaaaaaata aactattttt
taaaatttt 1004936468PRTHomo sapiens 36Met Val Asp Thr Glu Ser Pro
Leu Cys Pro Leu Ser Pro Leu Glu Ala1 5 10 15Gly Asp Leu Glu Ser Pro
Leu Ser Glu Glu Phe Leu Gln Glu Met Gly20 25 30Asn Ile Gln Glu Ile
Ser Gln Ser Ile Gly Glu Asp Ser Ser Gly Ser35 40 45Phe Gly Phe Thr
Glu Tyr Gln Tyr Leu Gly Ser Cys Pro Gly Ser Asp50 55 60Gly Ser Val
Ile Thr Asp Thr Leu Ser Pro Ala Ser Ser Pro Ser Ser65 70 75 80Val
Thr Tyr Pro Val Val Pro Gly Ser Val Asp Glu Ser Pro Ser Gly85 90
95Ala Leu Asn Ile Glu Cys Arg Ile Cys Gly Asp Lys Ala Ser Gly
Tyr100 105 110His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly Phe
Phe Arg Arg115 120 125Thr Ile Arg Leu Lys Leu Val Tyr Asp Lys Cys
Asp Arg Ser Cys Lys130 135 140Ile Gln Lys Lys Asn Arg Asn Lys Cys
Gln Tyr Cys Arg Phe His Lys145 150 155 160Cys Leu Ser Val Gly Met
Ser His Asn Ala Ile Arg Phe Gly Arg Met165 170 175Pro Arg Ser Glu
Lys Ala Lys Leu Lys Ala Glu Ile Leu Thr Cys Glu180 185 190His Asp
Ile Glu Asp Ser Glu Thr Ala Asp Leu Lys Ser Leu Ala Lys195 200
205Arg Ile Tyr Glu Ala Tyr Leu Lys Asn Phe Asn Met Asn Lys Val
Lys210 215 220Ala Arg Val Ile Leu Ser Gly Lys Ala Ser Asn Asn Pro
Pro Phe Val225 230 235 240Ile His Asp Met Glu Thr Leu Cys Met Ala
Glu Lys Thr Leu Val Ala245 250 255Lys Leu Val Ala Asn Gly Ile Gln
Asn Lys Glu Ala Glu Val Arg Ile260 265 270Phe His Cys Cys Gln Cys
Thr Ser Val Glu Thr Val Thr Glu Leu Thr275 280 285Glu Phe Ala Lys
Ala Ile Pro Gly Phe Ala Asn Leu Asp Leu Asn Asp290 295 300Gln Val
Thr Leu Leu Lys Tyr Gly Val Tyr Glu Ala Ile Phe Ala Met305 310 315
320Leu Ser Ser Val Met Asn Lys Asp Gly Met Leu Val Ala Tyr Gly
Asn325 330 335Gly Phe Ile Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys
Pro Phe Cys340 345 350Asp Ile Met Glu Pro Lys Phe Asp Phe Ala Met
Lys Phe Asn Ala Leu355 360 365Glu Leu Asp Asp Ser Asp Ile Ser Leu
Phe Val Ala Ala Ile Ile Cys370 375 380Cys Gly Asp Arg Pro Gly Leu
Leu Asn Val Gly His Ile Glu Lys Met385 390 395 400Gln Glu Gly Ile
Val His Val Leu Arg Leu His Leu Gln Ser Asn His405 410 415Pro Asp
Asp Ile Phe Leu Phe Pro Lys Leu Leu Gln Lys Met Ala Asp420 425
430Leu Arg Gln Leu Val Thr Glu His Ala Gln Leu Val Gln Ile Ile
Lys435 440 445Lys Thr Glu Ser Asp Ala Ala Leu His Pro Leu Leu Gln
Glu Ile Tyr450 455 460Arg Asp Met Tyr465371863DNAHomo sapiens
37actgatgtct tgactcatgg gtgtattcac aaattctgtt acttcaagtc tttttctttt
60aacggattga tcttttgcta gatagagaca aaatatcagt gtgaattaca gcaaacccct
120attccatgct gttatgggtg aaactctggg agattctcct attgacccag
aaagcgattc 180cttcactgat acactgtctg caaacatatc acaagaaatg
accatggttg acacagagat 240gccattctgg cccaccaact ttgggatcag
ctccgtggat ctctccgtaa tggaagacca 300ctcccactcc tttgatatca
agcccttcac tactgttgac ttctccagca tttctactcc 360acattacgaa
gacattccat tcacaagaac agatccagtg gttgcagatt acaagtatga
420cctgaaactt caagagtacc aaagtgcaat caaagtggag cctgcatctc
caccttatta 480ttctgagaag actcagctct acaataagcc tcatgaagag
ccttccaact ccctcatggc 540aattgaatgt cgtgtctgtg gagataaagc
ttctggattt cactatggag ttcatgcttg 600tgaaggatgc aagggtttct
tccggagaac aatcagattg aagcttatct atgacagatg 660tgatcttaac
tgtcggatcc acaaaaaaag tagaaataaa tgtcagtact gtcggtttca
720gaaatgcctt gcagtgggga tgtctcataa tgccatcagg tttgggcgga
tgccacaggc 780cgagaaggag aagctgttgg cggagatctc cagtgatatc
gaccagctga atccagagtc 840cgctgacctc cgggccctgg caaaacattt
gtatgactca tacataaagt ccttcccgct 900gaccaaagca aaggcgaggg
cgatcttgac aggaaagaca acagacaaat caccattcgt 960tatctatgac
atgaattcct taatgatggg agaagataaa atcaagttca aacacatcac
1020ccccctgcag gagcagagca aagaggtggc catccgcatc tttcagggct
gccagtttcg 1080ctccgtggag gctgtgcagg agatcacaga gtatgccaaa
agcattcctg gttttgtaaa 1140tcttgacttg aacgaccaag taactctcct
caaatatgga gtccacgaga tcatttacac 1200aatgctggcc tccttgatga
ataaagatgg ggttctcata tccgagggcc aaggcttcat 1260gacaagggag
tttctaaaga gcctgcgaaa gccttttggt gactttatgg agcccaagtt
1320tgagtttgct gtgaagttca atgcactgga attagatgac agcgacttgg
caatatttat 1380tgctgtcatt attctcagtg gagaccgccc aggtttgctg
aatgtgaagc ccattgaaga 1440cattcaagac aacctgctac aagccctgga
gctccagctg aagctgaacc accctgagtc 1500ctcacagctg tttgccaagc
tgctccagaa aatgacagac ctcagacaga ttgtcacgga 1560acacgtgcag
ctactgcagg tgatcaagaa gacggagaca gacatgagtc ttcacccgct
1620cctgcaggag atctacaagg acttgtacta gcagagagtc ctgagccact
gccaacattt 1680cccttcttcc agttgcacta ttctgaggga aaatctgaca
cctaagaaat ttactgtgaa 1740aaagcatttt aaaaagaaaa ggttttagaa
tatgatctat tttatgcata ttgtttataa 1800agacacattt acaatttact
tttaatatta aaaattacca tattatgaaa aaaaaaaaaa 1860aaa
186338505PRTHomo sapiens 38Met Gly Glu Thr Leu Gly Asp Ser Pro Ile
Asp Pro Glu Ser Asp Ser1 5 10 15Phe Thr Asp Thr Leu Ser Ala Asn Ile
Ser Gln Glu Met Thr Met Val20 25 30Asp Thr Glu Met Pro Phe Trp Pro
Thr Asn Phe Gly Ile Ser Ser Val35 40 45Asp Leu Ser Val Met Glu Asp
His Ser His Ser Phe Asp Ile Lys Pro50 55 60Phe Thr Thr Val Asp Phe
Ser Ser Ile Ser Thr Pro His Tyr Glu Asp65 70 75 80Ile Pro Phe Thr
Arg Thr Asp Pro Val Val Ala Asp Tyr Lys Tyr Asp85 90 95Leu Lys Leu
Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro Ala Ser100 105 110Pro
Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Lys Pro His Glu115 120
125Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg Val Cys Gly
Asp130 135 140Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu
Gly Cys Lys145 150 155 160Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys
Leu Ile Tyr Asp Arg Cys165 170 175Asp Leu Asn Cys Arg Ile His Lys
Lys Ser Arg Asn Lys Cys Gln Tyr180 185 190Cys Arg Phe Gln Lys Cys
Leu Ala Val Gly Met Ser His Asn Ala Ile195 200 205Arg Phe Gly Arg
Met Pro Gln Ala Glu Lys Glu Lys Leu Leu Ala Glu210 215 220Ile Ser
Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg225 230 235
240Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro
Leu245 250 255Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr
Thr Asp Lys260 265 270Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu
Met Met Gly Glu Asp275 280 285Lys Ile Lys Phe Lys His Ile Thr Pro
Leu Gln Glu Gln Ser Lys Glu290 295 300Val Ala Ile Arg Ile Phe Gln
Gly Cys Gln Phe Arg Ser Val Glu Ala305 310 315 320Val Gln Glu Ile
Thr Glu Tyr Ala Lys Ser Ile Pro Gly Phe Val Asn325 330 335Leu Asp
Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val
His Glu340 345 350Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys
Asp Gly Val Leu355 360 365Ile Ser Glu Gly Gln Gly Phe Met Thr Arg
Glu Phe Leu Lys Ser Leu370 375 380Arg Lys Pro Phe Gly Asp Phe Met
Glu Pro Lys Phe Glu Phe Ala Val385 390 395 400Lys Phe Asn Ala Leu
Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile405 410 415Ala Val Ile
Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys420 425 430Pro
Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln435 440
445Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys Leu
Leu450 455 460Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His
Val Gln Leu465 470 475 480Leu Gln Val Ile Lys Lys Thr Glu Thr Asp
Met Ser Leu His Pro Leu485 490 495Leu Gln Glu Ile Tyr Lys Asp Leu
Tyr500 505393734DNAHomo sapiens 39gcggagcgtg tgacgctgcg gccgccgcgg
acctggggat taatgggaaa agttttggca 60ggagcgggag aattctgcgg agcctgcggg
acggcggcgg tggcgccgta ggcagccggg 120acagtgttgt acagtgtttt
gggcatgcac gtgatactca cacagtggct tctgctcacc 180aacagatgaa
gacagatgca ccaacgaggc tgatgggaac caccctgtag aggtccatct
240gcgttcagac ccagacgatg ccagagctat gactgggcct gcaggtgtgg
cgccgagggg 300agatcagcca tggagcagcc acaggaggaa gcccctgagg
tccgggaaga ggaggagaaa 360gaggaagtgg cagaggcaga aggagcccca
gagctcaatg ggggaccaca gcatgcactt 420ccttccagca gctacacaga
cctctcccgg agctcctcgc caccctcact gctggaccaa 480ctgcagatgg
gctgtgacgg ggcctcatgc ggcagcctca acatggagtg ccgggtgtgc
540ggggacaagg catcgggctt ccactacggt gttcatgcat gtgaggggtg
caagggcttc 600ttccgtcgta cgatccgcat gaagctggag tacgagaagt
gtgagcgcag ctgcaagatt 660cagaagaaga accgcaacaa gtgccagtac
tgccgcttcc agaagtgcct ggcactgggc 720atgtcacaca acgctatccg
ttttggtcgg atgccggagg ctgagaagag gaagctggtg 780gcagggctga
ctgcaaacga ggggagccag tacaacccac aggtggccga cctgaaggcc
840ttctccaagc acatctacaa tgcctacctg aaaaacttca acatgaccaa
aaagaaggcc 900cgcagcatcc tcaccggcaa agccagccac acggcgccct
ttgtgatcca cgacatcgag 960acattgtggc aggcagagaa ggggctggtg
tggaagcagt tggtgaatgg cctgcctccc 1020tacaaggaga tcagcgtgca
cgtcttctac cgctgccagt gcaccacagt ggagaccgtg 1080cgggagctca
ctgagttcgc caagagcatc cccagcttca gcagcctctt cctcaacgac
1140caggttaccc ttctcaagta tggcgtgcac gaggccatct tcgccatgct
ggcctctatc 1200gtcaacaagg acgggctgct ggtagccaac ggcagtggct
ttgtcacccg tgagttcctg 1260cgcagcctcc gcaaaccctt cagtgatatc
attgagccta agtttgaatt tgctgtcaag 1320ttcaacgccc tggaacttga
tgacagtgac ctggccctat tcattgcggc catcattctg 1380tgtggagacc
ggccaggcct catgaacgtt ccacgggtgg aggctatcca ggacaccatc
1440ctgcgtgccc tcgaattcca cctgcaggcc aaccaccctg atgcccagta
cctcttcccc 1500aagctgctgc agaagatggc tgacctgcgg caactggtca
ccgagcacgc ccagatgatg 1560cagcggatca agaagaccga aaccgagacc
tcgctgcacc ctctgctcca ggagatctac 1620aaggacatgt actaacggcg
gcacccaggc ctccctgcag actccaatgg ggccagcact 1680ggaggggccc
acccacatga cttttccatt gaccagccct tgagcacccg gcctggagca
1740gcagagtccc acgatcgccc tcagacacat gacacccacg gcctctggct
ccctgtgccc 1800tctctcccgc ttcctccagc cagctctctt cctgtctttg
ttgtctccct ctttctcagt 1860tcctctttct tttctaattc ctgttgctct
gtttcttcct ttctgtaggt ttctctcttc 1920ccttctccct tgccctccct
ttctctctcc accccccacg tctgtcctcc tttcttattc 1980tgtgagatgt
tttgtattat ttcaccagca gcatagaaca ggacctctgc ttttgcacac
2040cttttcccca ggagcagaag agagtggggc ctgccctctg ccccatcatt
gcacctgcag 2100gcttaggtcc tcacttctgt ctcctgtctt cagagcaaaa
gacttgagcc atccaaagaa 2160acactaagct ctctgggcct gggttccagg
gaaggctaag catggcctgg actgactgca 2220gccccctata gtcatggggt
ccctgctgca aaggacagtg ggcaggaggc cccaggctga 2280gagccagatg
cctccccaag actgtcattg cccctccgat gctgaggcca cccactgacc
2340caactgatcc tgctccagca gcacacctca gccccactga cacccagtgt
ccttccatct 2400tcacactggt ttgccaggcc aatgttgctg atggccccct
gcactggccg ctggacggca 2460ctctcccagc ttggaagtag gcagggttcc
ctccaggtgg gcccccacct cactgaagag 2520gagcaagtct caagagaagg
aggggggatt ggtggttgga ggaagcagca cacccaattc 2580tgcccctagg
actcggggtc tgagtcctgg ggtcaggcca gggagagctc ggggcaggcc
2640ttccgccagc actcccactg cccccctgcc cagtagcagc cgcccacatt
gtgtcagcat 2700ccagggccag ggcctggcct cacatccccc tgctcctttc
tctagctggc tccacgggag 2760ttcaggcccc actccccctg aagctgcccc
tccagcacac acacataagc actgaaatca 2820ctttacctgc aggctccatg
cacctccctt ccctccctga ggcaggtgag aacccagaga 2880gaggggcctg
caggtgagca ggcagggctg ggccaggtct ccggggaggc aggggtcctg
2940caggtcctgg tgggtcagcc cagcacctgc tcccagtggg agcttcccgg
gataaactga 3000gcctgttcat tctgatgtcc atttgtccca atagctctac
tgccctcccc ttccccttta 3060ctcagcccag ctggccacct agaagtctcc
ctgcacagcc tctagtgtcc ggggaccttg 3120tgggaccagt cccacaccgc
tggtccctgc cctcccctgc tcccaggttg aggtgcgctc 3180acctcagagc
agggccaaag cacagctggg catgccatgt ctgagcggcg cagagccctc
3240caggcctgca ggggcaaggg gctggctgga gtctcagagc acagaggtag
gagaactggg 3300gttcaagccc aggcttcctg ggtcctgcct ggtcctccct
cccaaggagc cattctgtgt 3360gtgactctgg gtggaagtgc ccagcccctg
cccctacggg cgctgcagcc tcccttccat 3420gccccaggat cactctctgc
tggcaggatt cttcccgctc cccacctacc cagctgatgg 3480gggttggggt
gcttcctttc aggccaaggc tatgaaggga cagctgctgg gacccacctc
3540cccctccccg gccacatgcc gcgtccctgc cccgacccgg gtctggtgct
gaggatacag 3600ctcttctcag tgtctgaaca atctccaaaa ttgaaatgta
tatttttgct aggagcccca 3660gcttcctgtg tttttaatat aaatagtgta
cacagactga cgaaacttta aataaatggg 3720aattaaatat ttaa
373440441PRTHomo sapiens 40Met Glu Gln Pro Gln Glu Glu Ala Pro Glu
Val Arg Glu Glu Glu Glu1 5 10 15Lys Glu Glu Val Ala Glu Ala Glu Gly
Ala Pro Glu Leu Asn Gly Gly20 25 30Pro Gln His Ala Leu Pro Ser Ser
Ser Tyr Thr Asp Leu Ser Arg Ser35 40 45Ser Ser Pro Pro Ser Leu Leu
Asp Gln Leu Gln Met Gly Cys Asp Gly50 55 60Ala Ser Cys Gly Ser Leu
Asn Met Glu Cys Arg Val Cys Gly Asp Lys65 70 75 80Ala Ser Gly Phe
His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly85 90 95Phe Phe Arg
Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys Glu100 105 110Arg
Ser Cys Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys115 120
125Arg Phe Gln Lys Cys Leu Ala Leu Gly Met Ser His Asn Ala Ile
Arg130 135 140Phe Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val
Ala Gly Leu145 150 155 160Thr Ala Asn Glu Gly Ser Gln Tyr Asn Pro
Gln Val Ala Asp Leu Lys165 170 175Ala Phe Ser Lys His Ile Tyr Asn
Ala Tyr Leu Lys Asn Phe Asn Met180 185 190Thr Lys Lys Lys Ala Arg
Ser Ile Leu Thr Gly Lys Ala Ser His Thr195 200 205Ala Pro Phe Val
Ile His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys210 215 220Gly Leu
Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Lys Glu225 230 235
240Ile Ser Val His Val Phe Tyr Arg Cys Gln Cys Thr Thr Val Glu
Thr245 250 255Val Arg Glu Leu Thr Glu Phe Ala Lys Ser Ile Pro Ser
Phe Ser Ser260 265 270Leu Phe Leu Asn Asp Gln Val Thr Leu Leu Lys
Tyr Gly Val His Glu275 280 285Ala Ile Phe Ala Met Leu Ala Ser Ile
Val Asn Lys Asp Gly Leu Leu290 295 300Val Ala Asn Gly Ser Gly Phe
Val Thr Arg Glu Phe Leu Arg Ser Leu305 310 315 320Arg Lys Pro Phe
Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val325 330 335Lys Phe
Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile340 345
350Ala Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val
Pro355 360 365Arg Val Glu Ala Ile Gln Asp Thr Ile Leu Arg Ala Leu
Glu Phe His370 375 380Leu Gln Ala Asn His Pro Asp Ala Gln Tyr Leu
Phe Pro Lys Leu Leu385 390 395 400Gln Lys Met Ala Asp Leu Arg Gln
Leu Val Thr Glu His Ala Gln Met405 410 415Met Gln Arg Ile Lys Lys
Thr Glu Thr Glu Thr Ser Leu His Pro Leu420 425 430Leu Gln Glu Ile
Tyr Lys Asp Met Tyr435 4404121DNAArtificial Sequencesynthetic
oligonucleotide 41gagtgaatta gcccttccag t 214221DNAArtificial
Sequencesynthetic oligonucleotide 42gtgtggaaaa tctctagcag t
214338DNAArtificial Sequencesynthetic oligonucleotide 43tgctagttct
agcaggccct tgggccggcg cttgcgcc 38
* * * * *
References