U.S. patent application number 13/826270 was filed with the patent office on 2013-11-07 for use of chloroquine to treat metabolic syndrome.
The applicant listed for this patent is St. Jude Children's Research Hospital, The Washington University. Invention is credited to Michael B. Kastan, Jochen Schneider, Clay F. Semenkovich.
Application Number | 20130296237 13/826270 |
Document ID | / |
Family ID | 38049340 |
Filed Date | 2013-11-07 |
United States Patent
Application |
20130296237 |
Kind Code |
A1 |
Kastan; Michael B. ; et
al. |
November 7, 2013 |
Use Of Chloroquine To Treat Metabolic Syndrome
Abstract
The present invention provides methods and compositions for
modulating certain metabolic processes and for treating a variety
of disorders associated with metabolic syndrome, including insulin
related disorders, ischemia, oxidative stress, atherosclerosis,
hypertension, obesity, abnormal lipid metabolism, and stroke by
administering an effective dose of a chloroquine compound. The
invention also provides methods and compositions relating to
administering an effective dose of a chloroquine compound in
combination with at least a second pharmaceutically active
ingredient or compound including an antihyperglycemic diabetes
treatment, an antihypertensive agent, an antithrombotic agent,
and/or an inhibitor of cholesterol synthesis or absorption.
Inventors: |
Kastan; Michael B.; (Chapel
Hill, NC) ; Semenkovich; Clay F.; (Ladue, MO)
; Schneider; Jochen; (Ensdorf/Saar, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Washington University
St. Jude Children's Research Hospital |
St. Louis
Memphis |
MO
TN |
US
US |
|
|
Family ID: |
38049340 |
Appl. No.: |
13/826270 |
Filed: |
March 14, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12093198 |
May 30, 2008 |
8440695 |
|
|
PCT/US06/60391 |
Oct 31, 2006 |
|
|
|
13826270 |
|
|
|
|
60736192 |
Nov 9, 2005 |
|
|
|
Current U.S.
Class: |
514/6.5 ;
514/14.8; 514/143; 514/161; 514/171; 514/211.07; 514/236.2;
514/292; 514/305; 514/313; 514/56 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61K 31/4706 20130101; Y02A 50/411 20180101; A61P 3/00 20180101;
A61K 45/06 20130101 |
Class at
Publication: |
514/6.5 ;
514/313; 514/236.2; 514/211.07; 514/171; 514/143; 514/56; 514/161;
514/14.8; 514/292; 514/305 |
International
Class: |
A61K 31/4706 20060101
A61K031/4706; A61K 45/06 20060101 A61K045/06 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERAL SPONSORED
RESEARCH OR DEVELOPMENT
[0002] This invention was made in part in the course of research
sponsored by the National Institutes of Health (NIH Grant Nos. P50
HL083762, CA71387, CA21765, HL58427, AG20091, ES05777, and HL57278)
as well as the Clinical Nutrition Research Unit (DK56341), the
Diabetes Research and Training Center (DK20579). The U.S.
government may have certain rights in this invention.
Claims
1-31. (canceled)
32. A pharmaceutical composition which comprises an amount of a
chloroquine compound effective to increase Ataxia-Telangiectasia
mutated (ATM) protein kinase activity in a mammal with metabolic
syndrome and a pharmaceutically acceptable carrier.
33. The pharmaceutical composition of claim 32 which further
comprises an effective amount of a second pharmaceutically active
ingredient.
34. The pharmaceutical composition of claim 33, wherein the second
pharmaceutically active ingredient is a member selected from the
group consisting of an antihyperglycemic diabetes treatment, an
antihypertensive agent, an antithrombotic agent, and an inhibitor
of cholesterol synthesis or absorption.
35. The pharmaceutical composition of claim 34, wherein the
antihypertensive agent is selected from the group consisting of an
angiotensin converting enzyme inhibitor (ace inhibitor), an
angiotensin receptor blocker (ARB), a beta-blocker, and a calcium
channel blocker.
36. The pharmaceutical composition of claim 35, wherein the beta
blocker is selected from the group consisting of propranolol,
atenolol, esmolol, metoprolol, labetalol, talinolol, timolol,
carvedilol and acebutolol.
37. The pharmaceutical composition of claim 35, wherein the calcium
channel blocker is selected from the group consisting of
nifedipine, verapamil, diltiazem, and nimodipine.
38. The pharmaceutical composition of claim 34, wherein the
inhibitor of cholesterol synthesis is selected from the group
consisting of an inhibitor of HMG CoA reductase, squalene
epoxidase, squalene synthetase, cholesterol sulfate, and phosphate
synthetase.
39. The pharmaceutical composition of claim 38, wherein the
inhibitor of HMG CoA reductase is selected from the group
consisting of atorvastatin, cerivastatin, simvastatin, lovastatin,
pravastatin, compactin, fluvastatin, mevastatin, fluindostatin, and
dalvastatin, cholesterol sulfate, cholesterol phosphate, 25-OH or
26-OH cholesterol, and oxygenated sterols.
40. The pharmaceutical composition of claim 34 wherein the
antihyperglycemic diabetes treatment is selected from the group
consisting of insulin, an insulin analog, actos (pioglitazone),
avandia (rosiglitazone), a sulfonylurea, tolbutamide, a
biguanide-type medication agent, phentolamine, and tolazemide.
41. The pharmaceutical composition of claim 34, wherein the
antithrombotic agent is selected from the group consisting of
unfractionated heparin, low molecular weight (LMW) heparin,
heparinoid, aspirin, ticlopidine, clopidogrel, dipyridamole,
hirudin, and glycoprotein IIb/IIIa antagonists.
42. The pharmaceutical composition of claim 32 comprising about 1
mg to about 140 mg of the chloroquine compound.
43. The pharmaceutical composition of claim 32 comprising about 80
mg of the chloroquine compound.
44. The pharmaceutical composition of claim 32, wherein the
chloroquine compound is selected from the group consisting of
chloroquine, chloroquine phosphate, hydroxychloroquine, chloroquine
diphosphate, chloroquine sulphate, hydroxychloroquine sulphate,
enantiomers thereof, pharmaceutically acceptable salts thereof, and
mixtures thereof.
45. The pharmaceutical composition of claim 44, wherein the
chloroquine compound is selected from the group consisting of
chloroquine, chloroquine phosphate, hydroxychloroquine, chloroquine
diphosphate, and mixtures thereof.
46. The pharmaceutical composition of claim 45, wherein the
chloroquine compound is chloroquine.
47. The pharmaceutical composition of claim 45, wherein the
chloroquine compound is hydroxychloroquine.
48. The pharmaceutical composition of claim 32, wherein the
chloroquine compound is an essentially pure (+) isomer.
49. The pharmaceutical composition of claim 32, wherein the
chloroquine compound is an essentially pure (-) isomer.
50. The pharmaceutical composition of claim 32, wherein the
chloroquine compound is a mixture of isomers.
51. The pharmaceutical composition of claim 32, wherein the
chloroquine compound is selected from the group consisting of
mefloquine, pyronaridine, piperaquine, amodiaquine, and quinidine,
enantiomers thereof, pharmaceutically acceptable salts thereof, and
mixtures thereof, or has a structure selected from the group
consisting of: ##STR00002## wherein R.sub.1 to R.sub.6 are
independently selected from hydrogen or alkyl, provided that no
more than two substituents from R.sub.1 to R.sub.5 may be
simultaneously alkyl; R.sub.7 and R.sub.8 is hydrogen, alkyl,
alkenyl or aralkyl, or together with the N atom signify pyrrolidine
or piperidine, which optionally can be substituted by alkyl, or
octahydroindole or 3-azabicyclo[3,2,2]nonane; R.sub.9 is hydrogen
or halogen; R.sub.10 is halogen or trifluoromethyl; and n=0 or 1;
or wherein the symbols R.sub.1 and R.sub.3 is tri- or
tetramethylene; R.sub.2 and R.sub.4 to R.sub.6 is hydrogen; n=0;
and R.sub.7 and R.sub.8 are defined as above; or wherein the
symbols R.sub.1 and R.sub.7 is methylene or dimethylene and n=1, or
R.sub.1 and R.sub.7 are di- or trimethylene and n=0, or R.sub.3 and
R.sub.7 are di- or trimethylene and n=1, or R.sub.3 and R.sub.7 are
tri- or tetramethylene and n=0, or R.sub.5 and R.sub.7 are tri- or
tetramethylene and n=1, or R.sub.1 and R.sub.5 are di- or
tri-methylene and n=1, and the remaining substituents are hydrogen,
except R.sub.8 which is alkyl, alkenyl or alkynyl; wherein the
symbols R.sub.3 and R.sub.5 is tri- or tetramethylene and n=1; all
remaining substituents to R.sub.6 are hydrogen; and R.sub.7 and
R.sub.8 are alkyl, alkenyl or aralkyl or together with the n atom
are pyrrolidine or piperidine, which optionally can be substituted
by alkyl; R.sub.9 is hydrogen or halogen; and R.sub.10 is halogen
or trifluoromethyl, ##STR00003## wherein R' is an alkylene group
which is ##STR00004## wherein n is 3 or 4, wherein R.sub.1' and
R.sub.2' are methyl or ethyl; wherein R.sub.3' is hydrogen or
isopropyl, wherein R.sub.4' and R.sub.5' are hydrogen, chloro,
bromo, fluoro, trifluoromethyl or methoxy groups, and ##STR00005##
Wherein R.sub.1'' and R.sub.2'' are each hydrogen, or join to form
a cyclic structure of formula ##STR00006## and R.sub.3'' and
R.sub.4'' are independently selected from hydrogen, C1-C.sub.8
lower alkyl or hydroxy substituted C.sub.1-C.sub.8 lower alkyl, and
enantiomers thereof, pharmaceutically acceptable salts thereof, and
mixtures thereof.
52. The pharmaceutical composition of claim 32, wherein the
chloroquine compound is formulated in a sustained release
formulation.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit under 35 U.S.C.
119(e), of U.S. Provisional Application 60/736,192, filed Nov. 9,
2005, which is hereby incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to methods and
compositions for modulating certain metabolic processes. The
invention further relates to treating a variety of symptoms
associated with metabolic syndrome, including insulin related
disorders, ischemia, oxidative stress, atherosclerosis,
hypertension, obesity, abnormal lipid metabolism, and stroke by
administering an effective dose of a chloroquine compound. In
particular, the invention relates to increasing ATM activity in a
mammal suffering from metabolic syndrome by administering an
effective dose of a chloroquine compound.
BACKGROUND OF THE INVENTION
[0004] It is estimated that between 20-25% of American adults
(about 47 million) have metabolic syndrome, a complex condition
associated with an increased risk of vascular disease. Metabolic
syndrome is also known as Syndrome X, metabolic syndrome X, insulin
resistance syndrome, or Reaven's syndrome, after Dr. Gerald M.
Reaven, who first described the disorder. Metabolic syndrome is
generally believed to be a combination of disorders that affect a
large number of people in a clustered fashion. The symptoms and
features of the syndrome include at least three of the following
conditions: diabetes mellitus type II; impaired glucose tolerance
or insulin resistance; high blood pressure; central obesity and
difficulty losing weight; high cholesterol; combined
hyperlipidemia; including elevated LDL; decreased HDL; elevated
triglycerides; and fatty liver (especially in concurrent obesity).
Insulin resistance is typical of metabolic syndrome and leads to
several of its features, including glucose intolerance,
dyslipidemia, and hypertension. Obesity is commonly associated with
the syndrome as is increased abdominal girth, highlighting the fact
that abnormal lipid metabolism likely contributes to the underlying
pathophysiology of metabolic syndrome.
[0005] Metabolic syndrome was codified in the United States with
the publication of the National Cholesterol Education Program Adult
Treatment Panel III (ATP III) guidelines in 2001. On a physiologic
basis, insulin resistance appears to be responsible for the
syndrome. However, insulin resistance can be defined in a myriad of
different ways, including impaired glucose metabolism (reduced
clearance of glucose and/or the failure to suppress glucose
production), the inability to suppress lipolysis in tissues,
defective protein synthesis, altered cell differentiation, aberrant
nitric oxide synthesis affecting regional blood flow, as well as
abnormal cell cycle control and proliferation, all of which have
been implicated in the cardiovascular disease associated with
metabolic syndrome. At least at present, there is no obvious
molecular mechanism causing the syndrome, probably because the
condition represents a failure of one or more of the many
compensatory mechanisms that are activated in response to energy
excess and the accumulation of fat.
[0006] According to ATP III, the diagnosis of metabolic syndrome
requires the presence of three or more of the following: elevated
fasting triglycerides (greater than or equal to 150 mg/dI), low
HDL, cholesterol (less than 50 mg/dI in women, less than 40 mg/dI
in men), hypertension (blood pressure greater than or equal to
130/85 mm Hg), increased waist circumference (due to excess
visceral adiposity, greater than 35 inches in women, greater than
40 inches in men) and elevated fasting glucose (greater than or
equal to 100 mg/dI). The presence of three components is not a
perfect predictor of insulin resistance, and the World Health
Organization has established somewhat different criteria that
include microalbuminuria (i.e., slightly elevated albumin excretion
in the urine), and some groups modify the ATP III criteria to
include a body mass index (BMI) of greater than or equal to 30
kg/M.sup.2 and abnormal nonfasting glucose and lipid values.
Regardless of the definition, the syndrome identifies a group of
individuals at increased risk for vascular disease. In an analysis
of the Third National Health and Nutrition Examination Survey
(NHANES III) participants over the age of 50 with metabolic
syndrome showed a coronary heart disease prevalence exceeding that
of diabetes. NHANES II data indicate total mortality as well as
death from coronary heart disease and cardiovascular disease are
increased in adults with metabolic syndrome.
[0007] Individuals at risk for metabolic syndrome include those who
exhibit central obesity with increased abdominal girth (due to
excess visceral adiposity) of about more than 35 inches in women
and more than 40 inches in men. Individuals at risk for metabolic
syndrome also include those that have a BMI greater than or equal
to 30 kg/M.sup.2 and may also have abnormal levels of nonfasting
glucose, lipids, and blood pressure.
Oxidative Stress, Metabolic Syndrome, and Vascular Disease
[0008] Reductionist systems, animal models and studies in humans
are consistent with a role for reactive oxygen species in the
development of vascular dysfunction. Chronic inflammation is one
source of reactive oxygen species and an emerging body of evidence
implicates inflammation and oxidative stress in vascular disease
associated with the metabolic syndrome. CRP (C reactive protein), a
circulating marker of inflammation, is higher in those with more
components of the metabolic syndrome, and elevated CRP levels may
be predictive of vascular disease events in women with the
syndrome. Cytokines associated with insulin resistance, such as
IL-6 and TNF.alpha., also increase with obesity. Obesity promotes
the accumulation of macrophages and an induction of an inflammatory
pattern of gene expression in adipose tissue. Inhibition of
oxidative stress pathways decreases reactive oxygen species in
adipose tissue and improves metabolic syndrome in obese mouse
models. Obesity increases expression of monocyte-chemoattractant
protein-1, a proatherogenic molecule that also promotes insulin
resistance.
JNK and the Metabolic Syndrome
[0009] While oxidative stress can affect multiple potential
molecular mediators of the metabolic syndrome, c-Jun N-terminal
kinase (JNK) may be particularly important. JNK, a member of the
mitogen activated protein kinase superfamily of signaling
molecules, is activated by stressors that include reactive oxygen
species, fatty acids, and inflammatory cytokines. Activated JNK
phosphorylates cJun, which in combination with cFos constitutes the
AP-1 transcription factor complex. Several lines of evidence link
JNK activity to features of the metabolic syndrome. JNK activity is
increased by endoplasmic reticulum stress caused by obesity. There
are three known JNK genes and multiple isoforms, JNK1 and 2 are
widely distributed, while JNK3 expression is more limited. Mice
deficient in JNK1 are protected from obesity and insulin
resistance. Expression of wild type JNK in liver decreases insulin
sensitivity and overexpression of a dominant negative JNK in liver
increases insulin sensitivity in obese mice. JNK decreases the
expression of adiponectin, which has insulin sensitizing effects,
in adipocyte cell lines. Treatment of animals with a cell-permeable
inhibitor of JNK enhances insulin sensitivity. At least part of the
mechanism underlying the JNK effect is understood since JNK has
been shown to interfere with insulin signaling by phosphorylating
serine residue 307 on insulin receptor substrate 1 (IRS1).
Pharmacologic inhibition of JNK activity and genetic JNK2
deficiency in apoE null mice decreases atherosclerosis, in part due
to decreased activity of scavenger receptor A in the absence of
JNK-dependent phosphorylation.
ATM and Stress Responses
[0010] Ataxia telangiectasia (AT) is an autosomal recessive
disorder presenting in early childhood that is characterized by
progressive cerebellar ataxia, skin and eye telangiectasias, a
predisposition to malignancies (especially lymphomas), and immune
deficiency. AT patients also manifest impaired growth, accelerated
aging, and other signs of insulin resistance including glucose
intolerance. The likely mechanism of AT was provided when the
single gene responsible for this disease. ATM (Ataxia
Telangiectasia Mutated) was identified and found to be a member of
the phosphoinositol-3 kinase family. ATM was subsequently shown to
be important for insulin signaling leading to translation
initiation by Yang and Kastan in Nature Cell Bio., 2000, 2:
893-898. The frequency of ATM heterozygotes may be as high as 2% in
the general population, and a study of these ATM carriers (405
grandparents of AT children) reported an increased risk of death
from ischemic heart disease.
[0011] The primary function of ATM is to respond to DNA damage,
much of which is caused by reactive oxygen species. After genotoxic
stress, ATM initiates pathways that interfere with cell cycle
progression, permitting DNA to be repaired before errors are
propagated through replication. ATM is recognized to be important
for restoring homeostasis in response to oxidative stress. Levels
of manganese superoxide dismutase, catalase, and thioredoxin
reflective of increased oxidative stress are present in cerebellae
from ATM null mice. ATM resides at sites in the cytoplasm as well
as the nucleus and co-localizes with catalase in peroxisomes;
increased lipid peroxidation and decreased catalase has been
detected in AT-deficient cells. Markers of oxidative stress are
increased in both AT heterozygotes and homozygotes. Activation JNK
and the AP-1 pathway is present in the brains of ATM-deficient
mice. Self-renewal of hematopoietic stem cells requires the
inhibition of reactive oxygen species generation by ATM.
p53 and Atherosclerosis
[0012] How ATM modulates responses to oxidative stress is unknown
but it is reasonable to assume that p53 is involved. The tumor
suppressor p53 responds to DNA damage by inducing an increasingly
complex series of events, including apoptosis and cell cycle arrest
(which appear to be transcription-dependent), as well as control of
DNA repair and recombination (which may be
transcription-independent). In response to stress, activated ATM
phosphorylates p53 and MDM2, which leads to an increase in p53
protein levels and activity. While a role for ATM in
atherosclerosis has not been firmly established, several studies
suggest that p53 may be involved in vascular disease. p53 (and
MDM2) are present in human atherosclerotic lesions. In rabbits,
diet-induces atherosclerosis is associated with oxidation-induced
DNA damage and the induction of p53 in the vasculature. In the apoE
null model. p53.sup.-/- mice have increased atherosclerosis
associated with accelerated cell proliferation without an effect on
apoptosis. The p53 effect appears to be mediated in part by
macrophages, since the transplantation of p53 null bone marrow in
both apoE*3-Leiden mice (an animal model for human-like
atherosclerosis described van Vlijmen et al., Circ Res., 2001,
88(8):780-6) as well as LDL receptor null mice results in more
atherosclerosis. Recent evidence suggests that the
anti-atherosclerotic effect of p53 in dietary models may be
complex, promoting apoptosis in macrophages and preventing
apoptosis in smooth muscle cells. The protective effect may be
limited to diet-induced atherosclerosis models. Using a
plaque-rupture model involving phenylephrine administration, one
group reported that adenoviral-mediated overexpression of p53 in
smooth muscle cells increased apoptosis and destabilized lesions.
Another growth suppressor, p27 (a cyclin-dependent kinase
inhibitor), has been shown to decrease diet-induced atherosclerosis
by decreasing macrophage proliferation, but p21 (a different member
of the same cyclin-dependent kinase inhibitor family) increases
atherosclerosis. In short, recent studies have shown that ATM is an
important activator of p53, and p53 is likely to have
anti-atherogenic effects in the vasculature.
ATM and the Antimalarial Drug Chloroquine
[0013] Inactive ATM exists as a dimer in cells. In response to
stress, ATM phosphorylates itself, a modification that does not
affect the intrinsic kinase activity of the molecule but instead
dissociates the dimer and allows substrates access to the kinase
domain of the molecule. This phosphorylation, representing ATM
activation, occurs at serine 1981 of ATM and is sensitive to
cellular stress. Low dose irradiation producing as few as four
strand breaks in the entire genome and experiments using
manipulated mammalian cells following induction of only two
well-defined DNA strand breaks have been shown to cause ATM
phosphorylation. Thus, it is believed that ATM activation does not
require physical contact with DNA. Instead, since strand breaks
alter chromatin structure, ATM is probably capable of sensing
subtle changes in chromatin structure. Chromatin structure can be
altered without inducing DNA strand breaks by several manipulations
including exposure to mildly hypotonic media, inhibitors of histone
deacetylase, and exposure to the antimalarial drug chloroquine.
Chloroquine
[0014] Chloroquine is a DNA intercalating agent that functions as a
mild topoisomerase II inhibitor. Chloroquine is used to prevent and
treat malaria, a red blood cell infection with plasmodium species
of protozoa transmitted by the bite of a mosquito, and to treat
parasitic conditions such as liver disease caused by other protozoa
(tiny one-celled animals).
[0015] Chloroquine and related aminoquinolines have also found use
in the treatment of other chronic inflammatory diseases.
Antimalarials were first described as treatments for rheumatologic
disease in 1894, and the class gained acceptance for use in
inflammatory conditions following a 1951 report to the Lancet.
Chloroquine and related aminoquinolines have been shown to be
effective for treating systemic lupus erythematosus (SLE) and
rheumatoid arthritis (RA). These anti-inflammatory effects have led
to their use as prophylaxis for deep venous thrombosis and
treatment of sacroidosis.
Chloroquine and Metabolism
[0016] Effects of chloroquine on insulin sensitivity were reported
in 1984, when high dose chloroquine (1000 mg/day) was used in a
patient to successfully reverse severe insulin resistance thought
to be due to accelerated degradation of insulin. Subsequent studies
confirmed a modest effect of glucose lowering that was proportional
to the degree of insulin resistance, i.e., there was essentially no
effect in non-diabetic subjects and the greatest effect was seen in
those who were most insulin resistant, when administering high dose
chloroquine. Chloroquine has been reported to increase the affinity
of the insulin receptor and increase insulin secretion by isolated
islets, both of which may reflect a global increase in insulin
signaling. There is also a report of a hyperinsulinemic-euglycemic
clamp study in patients with type 2 diabetes that demonstrated that
administering high dose chloroquine (1000 mg/day) for three days
modestly decreased insulin resistance in peripheral tissues without
affecting endogenous glucose production (Powrie et al., Am. J.
Physiol., 1991, 260:897-904). Rahman et al. (J. Rheumatol., 1999,
26(2):325-30), reported that antimalarials lower total cholesterol
in patients also receiving steroids and may minimize steroid
induced hypercholesterolemia in patients with systemic lupus
erythematosus. Munro et al. (Ann. Rheum. Dis., 1997, 56:374-377)
reported that for a test group of 100 rheumatoid arthritis
patients, the group treated with oral hydroxychloroquine had a
significant overall improvement in their lipid profile. Wallace
(Lupus, 1996, 5 Suppl 1:S59-64) concluded that chloroquines are
safe and effective as a therapy for selected patients having any
one of the following disorders: porphyria cutanea tarda, cutaneous
sarcoidosis, cutaneous manifestations of dermatomyositis,
hyperlipidemia, and thromboembolic prophylaxis for patients with
antiphospholipid antibodies.
[0017] Insulin resistance is widely held to explain the association
between the metabolic syndrome and vascular disease. However,
insulin resistance may not directly cause atherosclerosis
(Semenkovich, 2006, J. Clin. Invest. 116:1813-1822). Insulin
resistance is not related to vascular lesions after correcting for
glucose tolerance. Pioglitazone, an insulin sensitizer, does not
decrease cardiovascular events in patients with insulin resistance.
Studies directly addressing the role of macrophage insulin
resistance in atherosclerosis are conflicting As opposed to
representing a unique entity, the metabolic syndrome may simply
reflect the cumulative contribution of its components to
atherosclerotic risk.
[0018] Historically, chloroquine has been used with caution because
it can cause retinal toxicity. There are numerous reports
addressing chloroquine toxicity that can be summarized as follows:
reports that retinopathy is rare and the drug can be taken for
years without toxicity; hydroxychloroquine, a less effective
anti-inflammatory agent, can also cause retinopathy, although the
risk is lower than with chloroquine; retinopathy risk is
dose-dependent with the lowest cumulative dose associated with
retinopathy being 125 grams (several groups have shown no effects
despite cumulative doses of 300 grams); and doses of about 250 mg
per day or 3.5 mg/kg per day are considered safe for chronic
treatment.
[0019] Presently there is no one treatment for the combination of
symptoms that make up metabolic syndrome. Thus, there is a need for
an effective, safe treatment for the combination of disorders
associated with metabolic syndrome. The present invention provides
novel methods and composition comprising low doses of a chloroquine
compound to modulate ATM activity and to alleviate or prevent the
numerous symptoms of metabolic syndrome. Compounds and methods of
the present invention may also be utilized in combination with one
or more other treatments such as antihyperglycemic diabetes
treatment, an antihypertensive agent, an antithrombic agent, and/or
an inhibitor of cholesterol synthesis or absorption to augment
these treatments.
SUMMARY OF THE INVENTION
[0020] The present invention provides methods of treating metabolic
syndrome as well as methods of prophylaxis for metabolic syndrome
and related disorders.
[0021] The invention also encompasses methods for treating
metabolic syndrome by administering an effective amount of a
chloroquine compound to a mammal in need of such treatment. In
certain embodiments, the mammal is a human. The invention also
includes a method for increasing Ataxia-Telangiectasia Mutated
(ATM) protein kinase activity in a mammal with metabolic syndrome
by administering an effective amount of a chloroquine compound to a
mammal in need of such treatment. The invention includes the method
where the mammal with metabolic syndrome exhibits improvement in
symptoms associated with metabolic syndrome when compared to
symptoms prior to administering the effective amount of a
chloroquine compound.
[0022] In certain methods the metabolic syndrome comprises at least
three symptoms selected from the group consisting of elevated
fasting triglycerides (greater than or equal to 150 mg/dI), low HDL
cholesterol (less than 50 mg/dI in women, less than 40 mg/dI in
men), hypertension (blood pressure greater than or equal to 130/85
mm Hg), increased waist circumference (greater than 35 inches in
women, greater than 40 inches in men) and elevated fasting glucose
(greater than or equal to 100 mg/dI).
[0023] The methods also comprise administering to a subject at risk
of metabolic syndrome, an effective amount of chloroquine compound.
Individuals at risk for metabolic syndrome include those who
exhibit central obesity with increased abdominal girth of about
more than 35 inches in women and about more than 40 inches in men.
Individuals at risk for metabolic syndrome also include those that
have a BMI greater than or equal to 30 kg/M.sup.2 and may also have
abnormal levels of fasting glucose, lipids and blood pressure.
[0024] In some methods, there is an additional step of determining
presence of a genetic variation in an ATM gene of the subject at
risk of the metabolic syndrome.
[0025] In some methods, the subject/mammal is free of diseases of
the immune system, infectious diseases, and neurological diseases.
In some methods, the subject is free of psoriasis, malaria,
protozoal infections, Epstein Barr virus infection, Alzheimer's
disease, Parkinson's disease, lupus erythematosus, rheumatism,
hypercalcemia, multiple sclerosis, and migraine.
[0026] In some methods, the chloroquine is administered
intravenously. In some methods, the chloroquine is administered
orally.
[0027] In some methods, the chloroquine compound is selected from
the group consisting of chloroquine, chloroquine phosphate,
hydroxychloroquine, chloroquine diphosphate, chloroquine sulphate,
hydroxychloroquine sulphate, or enantiomers, derivatives, analogs,
metabolites, pharmaceutically acceptable salts, and mixtures
thereof. Optionally, the chloroquine compound is chloroquine,
chloroquine phosphate, hydroxychloroquine, or chloroquine
diphosphate, or mixtures any of these compounds. In certain methods
the compound is chloroquine or hydroxychloroquine.
[0028] In some methods, the chloroquine compound is a mixture of
isomers. In some methods the chloroquine compound is an essentially
pure (+) isomer. In some methods the chloroquine compound is an
essentially pure (-) isomer.
[0029] In some methods, the chloroquine compound has a systemic
effect, In some methods, the amount of chloroquine compound
administered is at least about 0.1 mg/kg per day. In some methods,
the amount of chloroquine ranges from about 0.6 mg/kg/day to about
3.0 mg/kg/day. In some methods, the amount of chloroquine ranges
from about 0.8 mg/kg/day to about 1.2 mg/kg/day. In some methods,
the amount of the compound administered ranges from about 3.5 mg/kg
to about 7 mg/kg per week. In some methods, the amount of the
compound administered ranges from about 0.1 mg/kg/day to about 0.2
mg/kg/day. In some methods, the amount of the compound administered
is about 80 mg/day. In some methods, the amount of the compound
administered ranges from about 0.1 mg/kg to about 9 mg/kg once a
week.
[0030] In some methods, the cumulative amount of the compound
administered is less than about 30 grams (cumulative does). In some
methods, the cumulative amount of the compound administered is less
than about 100 grams.
[0031] In some methods, the chloroquine compound is administered
more than once a week. In some methods the chloroquine compound is
administered daily, In some methods, the chloroquine compound is
administered once a month. In come methods, the chloroquine
compound is formulated in a sustained release formulation. In some
methods, the patient is human.
[0032] The invention also provides methods and compositions
relating to administering an effective dose of a chloroquine
compound in combination with at least a second pharmaceutically
active ingredient or compound. This second pharmaceutically active
compound may include any of the treatments including an
antihyperglycemic diabetes treatment, an antihypertensive agent, an
antithrombotic agent, and an inhibitor of cholesterol synthesis or
absorption.
[0033] An embodiment of the invention includes a composition for
treating metabolic syndrome comprising an effective amount of a
chloroquine compound and an effective amount of at least a second
pharmaceutically active ingredient.
[0034] Another embodiment of the invention includes a
pharmaceutical composition which comprises an amount chloroquine
effective to increase Ataxia-Telangiectasia Mutated (ATM) protein
kinase activity in a mammal with metabolic syndrome and a
pharmaceutically acceptable carrier.
[0035] A further embodiment of the invention includes a
pharmaceutical composition which comprises an effective amount of
chloroquine for increasing Ataxia-Telangiectasia Mutated (ATM)
protein kinase activity in a mammal with metabolic syndrome, an
effective amount of a second pharmaceutically active ingredient,
and a pharmaceutically acceptable carrier. The pharmaceutical
composition may include a second pharmaceutically active ingredient
that is an antihyperglycemic diabetes treatment, an
antihypertensive agent, an antithrombotic agent, and an inhibitor
of cholesterol synthesis or absorption.
[0036] In certain embodiments the antihypertensive agent includes
any of the following agents: an angiotensin converting enzyme
inhibitor (ACE inhibitor), an angiotensin receptor blocker (ARB), a
beta-blocker, and a calcium channel blocker. In certain embodiments
the beta blocker includes any of the following agents: propranolol,
atenolol, esmolol, metoprolol, labetalol, talinolol, timolol,
carvedilol and acebutolol. In certain embodiments the calcium
channel blocker includes any of the following agents: nifedipine,
verapamil, diltiazem, and nimodipine. In certain embodiments the
inhibitor of cholesterol synthesis includes any of the following
agents: an inhibitor of HMG CoA reductase, squalene epoxidase,
squalene synthetase, cholesterol sulfate, and phosphate
synthetase.
[0037] In certain embodiments the inhibitor of HMG CoA reductase
includes any of the following agents: atorvastatin, cerivastatin,
simvastatin, lovastatin, pravastatin, compactin, fluvastatin,
mevastatin, fluindostatin, and dalvastatin, cholesterol sulfate,
cholesterol phosphate, 25-OH or 26-OH cholesterol, and oxygenated
sterols.
[0038] In certain embodiments the antihyperglycemic diabetes
treatment includes any of the following agents: insulin, an insulin
analog, actos (pioglitazone), avandia (rosiglitazone), a
sulfonylurea tolbutamide, abiguanide-type medication/agent,
phentolamine, and tolazemide.
[0039] In certain embodiments the antithrombotic agent includes any
of the following agents: unfractionated heparin, low molecular
weight (LMW) heparin, heparinoid, aspirin, ticlopidine,
clopidogrel, dipyridamole, hirudin, and glycoprotein IIb/IIIa
antagonists.
[0040] Embodiments of the present invention include a
pharmaceutical composition comprising about 1 mg to about 140 mg of
a chloroquine compound and a pharmaceutically acceptable
carrier.
[0041] A further embodiment of the present invention includes a
pharmaceutical composition comprising about 80 mg of a chloroquine
compound and a pharmaceutically acceptable carrier. In certain
embodiments the chloroquine compound is at least one compound
selected from chloroquine, chloroquine phosphate,
hydroxychloroquine, chloroquine diphosphate, chloroquine sulphate,
hydroxychloroquine sulphate, or enantiomers, derivatives, analogs,
metabolites, pharmaceutically acceptable salts, or mixtures
thereof. In additional embodiments, the compound may be any
compound selected from chloroquine, chloroquine phosphate,
hydroxychloroquine, chloroquine diphosphate, or mixtures thereof.
In a preferred embodiment the compound is chloroquine. In yet
further embodiments the compound is hydroxychloroquine.
[0042] In additional embodiments the chloroquine compound may be an
essentially pure (+) isomer, an essentially pure (-) isomer, or a
mixture of isomers.
[0043] In yet additional embodiments, the invention is directed to
use of an effective amount of any of the aforementioned chloroquine
compounds or compositions in the manufacture of a medicament to
increase Ataxia-Telangiectasia Mutated (ATM) protein kinase
activity in a mammal with metabolic syndrome, including for
conditions associated with metabolic syndrome such as elevated
fasting triglycerides, low HDL cholesterol, hypertension, increased
weight circumference, elevated fasting glucose, or any combinations
thereof.
[0044] In yet additional embodiments, the invention is directed to
use of any of the aforementioned chloroquine compounds or
compositions in the manufacture of a medicament for the therapeutic
and/or prophylactic treatment of metabolic syndrome, including for
treating conditions associated with metabolic syndrome such as
elevated fasting triglycerides, low HDL cholesterol, hypertension,
increased weight circumference, elevated fasting glucose, or any
combinations thereof.
[0045] In yet additional embodiments, the invention is directed to
use of any of the aforementioned chloroquine compounds or
compositions in the manufacture of a medicament for the prophylaxis
of metabolic syndrome, including for prevention symptoms or
conditions associated with metabolic syndrome such as elevated
fasting triglycerides, low HDL cholesterol, hypertension, increased
weight circumference, elevated fasting glucose, or any combinations
thereof.
[0046] In yet additional embodiments, the invention is directed to
use of any of the aforementioned chloroquine compounds or
compositions in combination with an effective amount of at least a
second pharmaceutically active ingredient in the manufacture of a
medicament for the therapeutic and/or prophylactic treatment of
metabolic syndrome, including for treating conditions associated
with metabolic syndrome such as elevated fasting triglycerides, low
HDL cholesterol, hypertension, increased weight circumference,
elevated fasting glucose, or any combinations thereof.
DETAILED DESCRIPTION
[0047] The present invention provides methods, compositions, and
kits for the prevention, prophylaxis and/or treatment of metabolic
syndrome and its related disorders. The disorders associated with
metabolic syndrome include insulin resistance, glucose intolerance,
hypertension, obesity, abnormal lipid metabolism (e.g.
dyslipidemia), central adiposity, oxidative stress and its many
manifestations including, stroke, ischemia, and atherosclerosis.
Although there is some overlap between these disorders (for
example, atherosclerosis is a common cause of ischemia and ischemia
often gives rise to stroke), the different symptoms are not
coextensive. For example, atherosclerosis can cause problems by
aneurysm as well as ischemia. Effective low dose amounts of
chloroquine compounds will be useful for prevention, prophylaxis
and/or treatment of a combination of these symptoms associated with
metabolic syndrome.
[0048] The invention is based, in part, on experiments with mouse
ATM/Apo-E models and from results in humans with metabolic
syndrome. The ApoE-null model mice lacking a single allele of ATM
(models for metabolic syndrome) had markedly worse glucose
tolerance, increased hypertension, and worse atherosclerosis than
normal ApoE-null mice. ApoE-null mice lacking both ATM alleles had
even worse symptoms, so much so that many mice failed to survive
development or the neonatal period. Treating ApoE-null model mice
lacking a single allele of ATM (being fed a high fat diet) with 7
mg/kg of chloroquine per week significantly reduced
atherosclerosis, hypertension, glucose tolerance, and central
adiposity.
[0049] Similarly, preliminary trials in humans with metabolic
syndrome showed surprisingly effective low dose amounts of
chloroquine compounds (80 mg/day) activated ATM kinase and induced
p53 in peripheral blood monocytes of subjects. This low dose
chloroquine treatment also improved a number of symptoms in humans
with metabolic syndrome including glucose intolerance,
atherosclerosis, elevated LDL cholesterol levels, triglyceride
levels, and total cholesterol. These experiments also showed that
low doses of chloroquine activate ATM and p53 in cultured cells and
in isolated patient monocytes.
[0050] Aspects of the present invention relate to a number of terms
used in accordance with the following definitions.
[0051] The term "increasing ATM activity" refers to increasing the
level of ATM activity in a mammal with metabolic syndrome, above
the level of ATM activity prior to treatment with chloroquine.
Typically, the increase in ATM activity will correspond to the
prevention of or alleviation of one or more of the symptoms
associated with metabolic syndrome.
[0052] The term "metabolic syndrome" refers to the following
symptoms and features including at least three of the following
symptoms: diabetes mellitus type II; impaired glucose tolerance or
insulin resistance; high blood pressure; central obesity and
difficulty losing weight; high cholesterol; combined
hyperlipidemia; including elevated LDL; decreased HDL; elevated
triglycerides; and fatty liver (especially in concurrent
obesity).
Chloroquine Compounds
[0053] The term "chloroquine compound" as used herein means related
aminoquinolines, including chloroquine-like compounds, chloroquine
and enantioners, analogs, derivatives, metabolites,
pharmaceutically acceptable salts, and mixtures thereof. Examples
of chloroquine compounds include, but are not limited to,
chloroquine phosphate, hydroxychloroquine, chloroquine diphosphate,
chloroquine sulphate, hydroxychloroquine sulphate, and enantiomers,
analogs, derivatives, metabolites, pharmaceutically acceptable
salts, and mixtures thereof. The term "chloroquine-like compounds"
as used herein means compounds that mimic chloroquine's biological
and/or chemical properties.
[0054] In a specific embodiment, the invention is practiced with
chloroquine. The chemical structure of chloroquine,
N.sub.4-(7-Chloro-4-quinolinly)-N.sub.1,N.sub.1-diethyl-1,4-pentanediamin-
e or 7-chloro-4-(4-diethylamino-1-methylbutylamino) quinoline, is
as follows:
##STR00001##
[0055] Chloroquine (The Merck index, p. 2220, 1996) is a
synthetically manufactured drug containing a quinoline nucleus.
Suitable synthesis techniques for chloroquine are well known in the
art. For example see U.S. Pat. No. 2,233,970.
[0056] As mentioned above, the chloroquine compounds useful herein
include chloroquine analogs and derivatives. A number of
chloroquine analogs and and derivatives are well known. For
example, suitable compounds and methods for synthesizing the same
are described in U.S. Pat. Nos. 6,417,177; 6,127,111; 5,639,737;
5,624,938; 5,736,557; 5,596,002; 5,948,791; 5,510,356; 2,653,940;
2,233,970; 5,668,149; 5,639,761, 4,431,807; and 4,421,920.
[0057] Examples of suitable chloroquine compounds include
chloroquine phosphate;
7-chloro-4-(4-diethylamino-1-butylamino)quinoline
(desmethylchloroquine);
7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;
7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;
7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;
7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;
7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;
7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline
(hydroxychloroquine;
7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methyl-1-butylamino)quinoli-
ne; hydroxychloroquine phosphate;
7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline
(desmethylhydroxychloroquine);
7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;
7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl-amino-1-butylamino)quinolin-
e;
7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quin-
oline;
7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyla-
mino)quinoline;
7-hydroxy-4-(1-carboxy-4-ethyl-(-2-hydroxyethyl)-amino-1-methylbutylamino-
)quinoline; 8-[(4-aminopentyl)amino]-6-methoxydihydrochloride
quinoline; 1-acetyl-1,2,3,4-tetrahydroquinoline;
8-[4-aminopentyl)amino]-6 methoxyquinoline dihydrochloride;
1-butyryl-1,2,3,4- tetrahydroquinoline;
7-chloro-2-(o-chlorostyryl)-4-[4-diethylamino-1-methylbutyl]aminoquinolin-
e phosphate;
3-chloro-4-(4-hydroxy-.alpha.,.alpha.'-bis(2-methyl-1-pyrrolidinyl)-2,5-x-
ylidinoquinoline,
4-[(4-diethylamino)-1-methylbutyl)amino]-6-methoxyquinoline;
3,4-dihydro-1 (2H)-quinolinecarboxyaldehyde;
1,1'-pentamethylenediquinoleinium diiodide; and 8-quinolinol
sulfate, enantiomers thereof, as well as suitable pharmaceutical
salts thereof.
[0058] Additional suitable chloroquine derivatives include
aminoquinoline derivatives and their pharmaceutically acceptable
salts such as those described in U.S. Pat. Nos. 5,948,791 and
5,596,002. Suitable examples include
(S)-N.sub.2-(7-Chloro-quinolin-4-yl)-N.sub.1,N.sub.1-dimethyl-pro-
pane-1,2-diamine;
(R)-N.sub.2-(7-chloro-quinolin-4-yl)-N.sub.2,N.sub.2-dimethyl-propane-1,2-
-diamine;
N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.2,N.sub.2-trimethyl-propa-
ne-1,2-diamine;
N.sub.3-(7-chloro-quinolin-4-yl)-N.sub.1,N.sub.1-diethyl-propane-1,3-diam-
ine; (RS)-(7-chloro-quinolin-4-yl)-(1-methyl-piperdine-3-yl)-amine;
(RS)-(7-chloro-quinolin-4-yl)-(1-methyl-pyrrolidin-3-yl)-amine;
(RS)-N.sub.2-(7-Chloro-quinolin-4-yl)-N.sub.1,N.sub.1-dimethyl-propane-1,-
2-diamine;
(RS)-N.sub.2-(7-chloro-quinolin-4-yl)-N.sub.1,N.sub.1-dimethyl--
propane-1,2-diamine;
(S)-N.sub.2(7-chloro-quinolin-4-yl)-N.sub.1N.sub.1-diethyl-propane-1,2-di-
amine;
(R)-N.sub.2-(7-chloro-quinolin-4-yl)-N.sub.1,N.sub.1-diethyl-propan-
e-1,2-diamine;
(RS)-7-chloro-quinolin-4-yl)-(1-methyl-2-pyrrolidin-1-yl)-ethyl-amine;
N.sub.2-(7-chloro-quinolin-4-yl)-N.sub.1,N.sub.1dimethyl-ethane-1,2-diami-
ne;
N.sub.2-(7-chloro-quinolin-4-yl)-N.sub.1,N.sub.1-diethyl-ethane-1,2-di-
amine;
N.sub.3-(7-chloro-quinolin-4-yl)-N.sub.1,N.sub.1-dimethyl-propane-1-
,3-diamine;
(R)-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.2,N.sub.2-dimethyl-propane-1,2-
-diamine;
(S)-N.sub.1-(7-chloro-quinoline-4-yl)-N.sub.2,N.sub.2-dimethyl-p-
ropane-1,2-diamine;
(RS)-(7-chloro-quinolin-4-yl)-(1-methyl-pyrrolidin-2-yl)-methyl)-amine;
N.sub.1-(7-Chloro-quinolin-4-yl)-N.sub.2-(3-chloro-benzyl)-2-methyl-propa-
ne-1,2-diamine;
N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.2-(benzyl)-2-methyl-2-methyl-propa-
ne-1,2-diamine;
N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.2-(2-hydroxy-3-methoxy-benzyl)-2-m-
ethyl-propane-1,2-diamine;
N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.2-(2-hydroxy-5-methoxy-benzyl)-2-m-
ethyl-propane-1,2-diamine; and
N.sub.1-(7-chloro-quinoline-4-yl)-N.sub.2-(4-hydroxy-3-methoxy-benzyl-2-m-
ethyl-propane-1,2-diamine;
(1S,2S)-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.2-(benzyl)-cyclohexane-1,2-
-diamine;
(1S,2S)-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.2-(4-chlorobenzyl-
)-cyclohexane-1,2-diamine;
(1S,2S)-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.2-(4-dimethylamino-benzyl)-
-cyclohexane-1,2-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(4-dimethylamino-benzyl)-cyc-
lohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(benzyl)-cyclohexane-1,4-dia-
mine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(3-chloro-benzyl)-cyclo-
hexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(2-hydroxy-4-methoxy-benzyl--
cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(3,5-dimethoxy-benzyl)-cyclo-
hexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(4-methylsulphanyl-benzyl)-c-
yclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(4-diethylamino-benzyl)-cycl-
ohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(biphenyl-4-yl)methyl-cycloh-
exane-1,4-diamine;
trans-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-[2-(3,5-dimethoxy-phenyl)--
ethyl]-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(4-methoxy-benzyl)-cyclohexa-
ne-1,4-diamine;
trans-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(4-dimethylamino-benzyl)-c-
yclohexane-1,4-diamine; and
trans-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(2,6-diflouro-benzyl)-cycl-
ohexane-1,4-diamine.
[0059] Chloroquine compounds, such as chloroquine, may exhibit the
phenomena of tautomerism, conformational isomerism, geometric
isomerism, and/or optical isomerism. The invention covers any
tautomeric, conformational isometric, optical isomeric and/or
geometric isomeric forms of the chloroquine compounds, as well as
mixtures of these various different forms.
[0060] Chloroquine and hydroxychloroquine are generally racemic
mixtures of (-)- and (+)-enantiomers. The (-)-enantiomers are also
knows as (R)-enantiomers (physical rotation) and 1-enantiomers
(optical rotation). The (+)-enantiomers are also known as
(S)-enantiomers (physical rotation) and r-enantiomers of
chloroquine are described in Augustijins and Verbeke, Clin.
Pharmacokin., 1993, 24(3):259-69; Augustijins, et al., Eur. J. Drug
Metabol. Pharmacokin., 1999, 24(1):105-8; DuCharme and Farinotti,
Clin. Pharmacokin., 1995, 31(4):257-74; Ducharme, et al., Br. J.
Clin. Pharmacol., 1995, 40(2):127-33. Preferably, the
(-)-enantiomer of chloroquine is used. The enantiomers of
chloroquine and hydroxychloroquine can be prepared by procedures
known to the art.
[0061] The chloroquine compounds may metabolize to produce active
metabolites. The use of active metabolites is also within the scope
of the present invention.
[0062] Although an understanding of mechanism is not required for
practice of the invention, and the present invention is not limited
by any particular mechanism, it is believed that one mechanism of
action of chloroquine compounds is to enhance the activity of
Ataxia-Telangiectasia Mutated (ATM) kinase. The agonistic
properties of chloroquine on ATM kinase have been demonstrated (see
see U.S. Pub. No. 20030077661 entitled "ATM Kinase Compositions and
Methods," filed Nov. 27, 2003, which is incorporated by reference
herein in its entirety). Hence, chloroquine-like compounds include
compounds that are agonists of ATM kinase. Agonists of ATM kinase
include compounds that promote the dissociation of ATM into active
monomers and/or compounds that promote phosphorylation of a serine
corresponding to the residue 1981 of ATM kinase of SEQ ID NO:1.
Chloroquine compounds may also be effective via one or other
mechanisms that do not involve interaction with ATM.
Use of Chloroquine Compounds
[0063] The invention provides methods of prophylaxis or therapeutic
treatment of an animal or mammalian subject including a human.
Although an understanding of the mechanism is not required for the
practice of the invention, it is believed that chloroquine
compounds act in part by protecting normal cells from radiation or
free radicals and by inhibiting the cellular damage caused by the
radiation or free radicals to normal cells enhancing the repair
process of the normal cells. The methods generally involve the
administration of effective amounts of chloroquine compounds for
the treatment of one or more the diseases or disorders described in
more detail below.
[0064] Genetic risk of metabolic syndrome can be associated with
either homozygous or heterozygous variations in genes. These
variations are present in the germline of the patient. Generally,
the most commonly occurring allele in a population is referred to
as the wildtype allele, and other less common alleles are referred
to as variant alleles. Variant forms associated with metabolic
syndrome can be recognized by comparing alleles in populations with
and without metabolic syndrome (usually the individuals in the
population with metabolic syndrome have the same type for purposes
of analysis). Alleles occurring significantly more frequently in
the population having metabolic syndrome are associated with
metabolic syndrome. A causative relationship can be conferred by
transforming the allele into cells or transgenic animals or
knocking out an endogenous allele and determining whether the
allele causes metabolic syndrome in the transformed cell or
animal.
[0065] ATM and/or p53 are examples of genes having variations
associated with cancer (see, e.g., Garber et al., Cancer Research,
1991, 51: 6094-6097; Olsen el al., J. Nat. Cancer Inst., 2001, 93:
121-127). As noted, these genes are also thought at least in part
to affect the prophylactic and therapeutic benefits of chloroquine
compounds. Optionally, subjects can be screened for variations in
these genes before commencing treatment. Subjects having wildtype
forms of ATM or p53, or heterozygous mutations or homozygous
mutations leaving residual activity of p53 or ATM are preferred for
treatment with chloroquine compounds. Subjects having mutations
that effectively eliminate ATM or p53 function are less preferred
for treatments with chloroquine compounds as described herein.
Individuals missing one allele of ATM or p53 are also preferred for
treatment with chloroquine compounds. Any subject with symptoms of
metabolic syndrome is envisioned to benefit from treatments with
chloroquine compounds as described herein.
[0066] Methods for determining presence of genetic variations in
individuals are described in "improvements to Analysis Methods for
Individual Genotyping", filed Feb. 24, 2004, U.S. Ser. No.
10/768,785, "Apparatus and Methods for Analyzing and Characterizing
Nucleic Acid Sequences", filed Jan. 30, 2003, and U.S. Ser. No.
10/042,819, "Genetic Analysis Systems and Methods", filed Jan. 7,
2002, and EPO 730 663, each of which is incorporated by
reference.
Therapeutic and Prophylactic Benefits
[0067] Low doses of chloroquine compounds can be used as
prophylactic agents. For prophylactic benefit, the chloroquine
compound can be administered to a subject at risk of developing
metabolic syndrome, but not presently showing symptoms. Individuals
at risk for metabolic syndrome include those who exhibit central
obesity with increased abdominal girth of about greater than 35
inches in women and about greater than 40 inches in men.
Individuals at risk for metabolic syndrome also include those that
have a BMI greater than or equal to 30 kg/M.sup.2 and may also have
abnormal levels of nonfasting glucose, lipids and blood pressure.
Genetic risk of metabolic syndrome can be associated with either
homozygous or heterozygous variations in genes as described above.
A prophylactic benefit is achieved when a disorder is delayed,
reduced in severity or prevented from afflicting a subject. A
prophylactic benefit can include a result in which the subject is
inflicted with a milder form of the disorder than in the absence of
treatment or the appearance of fewer or no symptoms of the
disorder, or the absence of the disorder in the subject being
treated.
[0068] Low doses of chloroquine compounds can be used for their
therapeutic benefits in treating the numerous or cluster of
symptoms associated with metabolic syndrome. A therapeutic benefit
includes eradication or amelioration of the underlying disorder
being treated. A therapeutic benefit also includes the eradication
or amelioration of one or more of the physiological symptoms
associated with the underlying disorder such that an improvement is
observed in the subject, notwithstanding that the subject may still
be afflicted with the underlying disorder. A therapeutic benefit
also includes elimination or reduction of consequences of the
underlying disorder, such as a reduction in the generation of free
radicals and the resulting damage to macromolecules and tissue in
atherosclerosis, stroke, ischemia and oxidative stress. A
therapeutic benefit can also result when administration of a
chloroquine compound inhibits or prevents further deterioration in
the patient's condition of an existing disorder.
Dosage and Dosage Regimen
[0069] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the chloroquine compound and
other optional active ingredients are present in an effective
amount for treating or preventing metabolic syndrome.
[0070] The effective amounts of compounds of the present invention
include doses that partially or completely achieve the desired
therapeutic, prophylactic, and/or biological effect. The actual
amount effective for a particular application depends on the
condition being treated and the route of administration. The
effective amount for use in humans can be determined from animal
models. For example, a dose for humans can be formulated to achieve
circulating and/or gastrointestinal concentrations that have been
found to be effective in mammals.
[0071] In some methods, the elective amount includes the dose
ranges, modes of administration, formulations, and so forth, that
have been recommended or approved by any of the various regulatory
or advisory organizations in the medical or pharmaceutical arts
(e.g., FDA, AMA) or by the manufacturer or supplier. Effective
amounts of chloroquine can be found, for example, in the Physicians
Desk Reference. Chloroquine can be administered orally, in the form
of chloroquine phosphate, or by injection or i.v. in the form
chloroquine hydrochloride. See generally, Physician's Desk
Reference, Medical Economics Company, Inc., Montvale N.J. (54th Ed.
2000), at pp. 2733-2735.
[0072] In some methods, the daily low dosage range of chloroquine,
can vary between about 3.5 mg/kg to about 7.0 mg/kg body weight. In
offset methods, the dose range may be even lower, from about 0.1
mg/kg/day through about 3.0 mg/kg/day. Some daily doses of
chloroquine diphosphate range from about 3.5 mg/kg through about
7.0 mg/kg.
[0073] The dosage can vary depending on the subject being treated.
For example, a preferred dosage in mice is about 3.5 mg/kg once or
twice a day. The equivalent dosages in monkeys and humans are shown
in the Table 1.
TABLE-US-00001 TABLE 1 Man (60 kg) Mouse (20 g) Monkey (3 kg) Man
(60 kg) CHQ Equivalent 3.5 mg/kg 0.875 mg/kg 0.292 mg/kg 17.5 mg
CHQ 7.0 mg/kg 1.75 mg/kg 0.583 mg/kg 35.0 mg CHQ
[0074] Preferred dosages ranges in humans include from about 0.1 to
about 3 mg/kg/day, more preferably from about 0.6 mg/kg/day to
about 3 mg/kg/day. Another preferred dosage range in humans
includes from about 0.8-1.2 mg/kg per day. Another preferred dosage
range in humans includes from about 0.1 mg/kg to about 9 mg/kg once
a week. A particularly preferred dosage in humans includes about 80
mg per day. Another dosage in humans includes about 40 mg per day.
The dosage can be administered daily, weekly, monthly or bimonthly
(every two months). For patients subject to a chronic risk (e.g.,
through genetic variation), the dosage is preferably administered
weekly, monthly or bimonthly for an indefinite period. The dosage
range can be lower e.g., about 0.1 to about 0.2 mg/kg per day or
per week of chloroquine if a purified (-) enantiomer is used. If
hydroxychloroquine is used the dosage range is usually higher than
if chloroquine is used.
[0075] Pharmaceutically active compounds suitable for use in
combination with chloroquine compounds of the present invention may
include an effective amount of compounds such as antihypertensive
agents including: .beta.-receptor blocker (beta-blockers),
angiotensin converting enzyme inhibitors (ACE inhibitors),
angiotensin receptor blockers (ARBs), and calcium channel blockers,
inhibitors of cholesterol synthesis and absorption, antithrombotic
agents, and antihyperglycemic diabetes treatments including insulin
or an insulin analog, actos (pioglitazone), avandia
(rosiglitazone), sulfonylureas such as acetohexamide, amaryl
(glimepiride), chlorpropamide, glipizide, glyburide, avandia
(rosiglitazone), or tolbutamide, a biguanide-type medication such
as glucophage, glucotrol, glucagon, or glucovance (a combination of
glyburide and metformin), phentolamine, or tolazemide. Mixtures of
any of the above agents including any pharmaceutically acceptable
salt form of these agents, may be used in combination with the
chloroquine compounds of the present invention. Each of these
agents may be produced by methods known in the art. These agents
may also be administered at the pharmaceutically or therapeutically
effective dosages or amounts known in the art for these compounds,
such as those described in the Physician's Desk Reference 2001, 55
Edition, Copyright 2001, published by Medical Economics Company,
Inc., the relevant portions describing each of these products being
incorporated herein by reference. U.S. Pat. Nos. 6,610,272 and
6,734,197 describe a number of diabetic treatments with typical
dosing and effective amounts, and each of these patients is hereby
incorporated by reference in its entirety.
[0076] In some methods, the effective amount of chloroquine or
other pharmaceutically active compound is administered at regular
intervals, such as every other week, once a week, more than once a
week, or once a day. The dose of chloroquine or other
pharmaceutically active compound can be administered once or more
than once a day. In some methods, the effective amount of a
chloroquine compound is an amount that produces the intended
beneficial effects but does not produce the side-effects associated
with chloroquine compounds, like retinoblastoma.
Kits
[0077] The invention provides a kit comprising a chloroquine
compounds packaged in association with instructions teaching a
method of using the compound according to one or more of the
above-described methods. The kit can contain the chloroquine
compound packaged in unit dosage form. The kit may also contain
additional pharmaceutically active agents or compounds including
unit dosage forms of compounds such as antihypertensive agents
including .beta.-receptor blockers (beta-blockers), angiotensin
converting enzyme inhibitors (ACE inhibitors), angiotensin receptor
blockers (ARBs), and calcium channel blockers, inhibitors of
cholesterol synthesis and absorption, antithrombotic agents, and
diabetes treatments. Typical unit dose forms of certain cholesterol
lowering drugs and ACE inhibitors are well known and described in
U.S. Pat. No. 5,190,970, which is hereby incorporated by reference
in its entirety. Typical unit dose forms of antithrombotic agents
and calcium channel blockers are well known and are described by
Coull et al., Stroke, 2002, 33:1934-1942. Typical unit dose forms
of beta-blockers are well known in the art, and are described in
U.S. Pat. Nos. 6,756,408 and 6,933,279. Typical diabetes treatments
and dosages are well known in the art and are described in U.S.
Pat. No. 6,610,272. Embodiments of various methods and combination
involving chloroquine treatment and chloroquine combination
treatments are described in more detail in the following
sections.
Routes of Administration and Formulation
[0078] The compounds useful in the present invention, or
pharmaceutically acceptable salts thereof, can be delivered to the
subject using a wide variety of routes or modes of administration.
Suitable routes of administration include, but are not limited to,
inhalation, transdermal, oral, rectal, transmucosal, intestinal,
and parenteral administration, including intramuscular,
subcutaneous and intravenous injections. A preferred mode of
administration includes in oral dosage form. An embodiment of the
present invention includes a dosage formulation containing about 1
mg to about 140 mg of chloroquine compound along with a
pharmaceutically acceptable salt. A preferred embodiment includes a
dosage formulation containing about 80 mg of a chloroquine
compounds along with a pharmaceutically acceptable salt. Any dosage
formulation containing less than about 140 mg, including about 40
mg of a chloroquine compound will useful for long term treatment of
individuals with metabolic syndrome. Long term treatment refers to
an extended period of time, typically longer than two weeks, and
includes any length of time whereby the individual/subject (mammal)
exhibits improvement in metabolic syndrome symptoms. This dosage
formulations will be beneficial for prophylactic treatment of
individuals who will be taking low dosages of chloroquine for
extended periods of time to prevent metabolic syndrome.
[0079] The chloroquine compounds can be administered topically or
systemically. Systemic administration is preferred. In some
methods, topical administration also has a systemic effect.
[0080] The term "pharmaceutically acceptable salt" means those
salts which retain the biological effectiveness and properties of
the compounds used in the present invention, and which are not
biologically or otherwise undesirable. Such salts include salts
with inorganic or organic acids, such as hydrochloric acid,
hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid,
methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric
acid, succinic acid, lactic acid, mandelic acid, malic acid, citric
acid, tartaric acid or maleic acid. In addition, if the compounds
used in the present invention contain a carboxy group or other
acidic group, it can be converted into a pharmaceutically
acceptable addition salt with inorganic or organic bases. Examples
of suitable bases include sodium hydroxide, potassium hydroxide,
ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine,
diethanolamine and triethanolamine.
[0081] Agents used in accordance with the methods of the invention
can be conveniently administered in a pharmaceutical composition
containing the active compound in combination with a suitable
carrier. Such pharmaceutical compositions can be prepared by
methods and contain carriers which are well-known in the art. A
generally recognized compendium of such methods and ingredients is
Remington: The Science and Practice of Pharmacy, Alfonso R.
Gennaro, editor, 20th ed. Lippingcott Williams and Wilkins;
Philadelphia, Pa., 2000. A pharmaceutically-acceptable carrier,
composition or vehicle, such as a liquid or solid filler, diluent,
excipient, or solvent encapsulating material, is involved in
carrying or transporting the subject compound from one organ, or
portion of the body, to another organ, or portion of the body. Each
carrier must be acceptable in the sense of being compatible with
the other ingredients of the formulation and not injurious to the
subject.
[0082] Examples of materials which can serve as
pharmaceutically-acceptable carriers include sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; lycols, such as propylene glycol, polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; pH buffered
solutions; polyesters, polycarbonates and/or polyanhydrides; and
other non-toxic compatible substances employed to pharmaceutical
formulations. Wetting agents, emulsifiers and lubricants, such as
sodium lauryl sulfate and magnesium stearate, as well as coloring
agents, release agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the compositions.
[0083] Agents of use in the invention can be administered
parenterally (for example, by intravenous, intraperitoneal,
subcutaneous or intramuscular injection), topically (including
buccal and sublingual), orally, intranasally, intravaginally, or
rectally, with oral administration being particularly
preferred.
[0084] For oral therapeutic administration, the composition can be
combined with one or more carriers and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, chewing gums, foods and the like.
Also, for oral consumption the active ingredient can be dissolved
or suspended in water or other edible oral solutions. Such
compositions and preparations should contain at least 0.1% of
active compound. The percentage of the compositions and
preparations can, of course, be varied and can conveniently be
between about 0.1 to about 100% of the weight of a given unit
dosage form. The amount of active agent in such therapeutically
useful compositions is such that an effective dosage level is
obtained.
[0085] The tablets, troches, pills, capsules, and the like can also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or as aspartame
or a flavoring agent such as peppermint, oil of wintergreen, or
cherry flavoring. The above lining is merely representative and one
skilled in the art could envision other binders, excipients,
sweetening agents and the like. When the unit dosage form is a
capsule, it can contain, in addition to materials of the above
type, a liquid carrier, such as a vegetable oil or a polyethylene
glycol. Various other materials can be present as coatings or to
otherwise modify the physical form of the solid unit dosage form.
For instance, tablets, pills, or capsules can be coated with
gelatin, wax, shellac or sugar and the like.
[0086] For administration orally, the compounds can be formulated
as a sustained release preparation. Numerous techniques for
formulating sustained release preparations are described in the
following references--U.S. Pat. Nos. 4,891,223; 6,004,582;
5,397,574; 5,419,917; 5,458,005; 5,458,887; 5,458,888; 5,472,708;
6,106,862; 6,103,263; 6,099,862; 6,099,859; 6,096,340; 6,077,541;
5,916,595; 5,837,379; 5,834,023; 5,885,616; 5,456,921; 5,603,956;
5,512,297; 5,399,362; 5,399,359; 5,399,358; 5,725,883; 5,773,025;
6,110,498; 5,952,004; 5,912,013; 5,897,876; 5,824,638; 5,464,633;
5,422,123; and 4,839,177; and WO 98/47491. These references are
hereby incorporated herein by reference in their entireties. In a
preferred embodiment, the sustained release formulation utilized
has an enteric coating.
[0087] For administration by inhalation the active compound(s) can
be conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebulizer, with the use of
a suitable propellant, e.g. dichlorodiflouromethane,
trichloroflouromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit can be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g. gelatin for use in
an inhaler or insufflator can be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch.
[0088] A syrup or elixir can contain the active agent, sucrose or
fructose as a sweetening agent, methyl and propylparabens as
preservatives, a dye and flavoring such a cherry or orange flavor.
Of course, any material used in preparing any unit dosage form
should be pharmaceutically acceptable and substantially non-toxic
in the amounts employed. In addition, the active components can be
incorporated into sustained-release preparations and devices
including, but not limited to, those relying on osmotic pressures
to obtain a desired release profile. Once daily formulations for
each of the active components are specifically included.
[0089] The compounds can also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0090] In addition to the formulations described previously, the
compounds can also be formulated as a depot preparation. Such long
acting formulations can be administered by implantation, or
transcutaneous delivery (for example subcutaneously or
intramuscularly), intramuscular injection or a transdermal patch.
Thus, for example, the compounds can be formulated with suitable
polymeric or hydrophobic materials (for example as an emulsion in
an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
[0091] The selected dosage level depends on a variety of factors
including the activity of the particular compound of the present
invention employed, the route of administration, the time of
administration, the rate of excretion or metabolism of the
particular compound being employed, the duration of the treatment,
other drugs, compounds and/or materials used in combination with
the particular compound employed, the age, sex, weight, condition,
general health and prior medical history of the subject being
treated, and like factors well-known in the medical arts.
[0092] If necessary the compounds as used herein can be
administered in combination with other therapeutic agents or
regimes as discussed. The choice of therapeutic agents that can be
administered with the compounds of the invention depends, in part,
on the condition being treated. For example the chloroquine
compounds of the present invention can be administered as the sole
pharmaceutical agent or in combination with one or more other
pharmaceutical agents or compounds where the combination provides
desired effects. Certain methods of the present invention also
include administering an effective amount of one or more additional
pharmaceutically active compound such as antihypertensive agents
including beta-blockers, angiotensin converting enzyme inhibitors
(ACE inhibitors), and calcium channel blockers, inhibitors of
cholesterol synthesis, antithrombotic agents, and diabetes drugs
such as insulin or actos (pioglitazone), sulfonylureas such a
amaryl (glimepiride), glipizide; avandia (rosiglitazone), a
biguanide-type medication such as glucophage; glucotrol; and
glucovance (a combination of glyburide and metformin).
[0093] "A combination amount sufficient," "an effective combination
amount" "therapeutically effective combination amount" or "an
effective amount of the combination of" all refer to combined
amounts of a chloroquine compound and any additional
pharmaceutically active ingredients (e.g., antihypertensive,
cholesterol lowering, and/or antidiabetic agents) that is effective
to ameliorate symptoms associated with metabolic syndrome. As used
herein, the term "combination" of a chloroquine compound and at
least a second pharmaceutically active ingredient means at least
two, but any desired combination of compounds can be delivered in a
simultaneous manner (meaning administered or delivered within a 24
hour period), in combination therapy wherein the chloroquine
compound is administered first, followed by the second or more
pharmaceutically active ingredient(s), as well as wherein the
second pharmaceutically active ingredient is delivered first,
followed by a chloroquine compound. The desired result can be
either a subjective relief of a symptom(s) or an objectively
identifiable improvement in the recipient of the dosage.
[0094] The terms "synergistic effective amount" may refer to a
combined amount of a chloroquine compound and at least one of a
antihypertensive, antithrombic, cholesterol lowering, and/or
antidiabetic agent that is effective to cause a synergistic effect
in the mammal in need of treatment. Synergy is a biological
phenomenon in which the effectiveness of two active components in a
mixture is more than additive, i.e., the effectiveness is greater
than the equivalent concentration of either component alone. For
example, the effectiveness of the combination therapy of a
chloroquine compound and an antidiabetic agent is expected to be
synergistic. Thus, synergism is a result, or function, that is more
than the sum of the results, or functions of individual
elements.
Administration of Chloroquine in Combination with Additional
Treatments
[0095] Chloroquine compounds may be used in conjunction with
virtually any inhibitors of cholesterol synthesis or absorption,
including any of the family of substances known as Hmg-CoA
reductase inhibitors. Hmg-CoA reductase inhibitors are taught for
example in U.S. Pat. Nos. 4,346,227 4,857,522, 5,190,970, and
5,461,039, each of which is hereby incorporated by reference in its
entirety. Hmg-CoA reductase inhibitors are exemplified by, but not
limited to atorvastatin and pravastatin. Particular Hmg-CoA
reductase inhibitors preferred for use in conjunction with the
present chloroquine formulation include: simvastatin, lovastatin,
pravastatin, compactin, fluvastatin, dalvastatin, HR-780, GR-95030,
CI-981, BMY 22089 and BMY 22566. U.S. Pat. No. 5,316,765 describes
a number of these Hmg-CoA reductase inhibitors and is hereby
incorporated by reference in its entirety. The preparation of
XU-62-320 (fluvastatin) is described in WIPO Patent WO84/02131. BMY
22089 is described in GB Patent No. 2,202,846. Pravastatin sodium
is described in U.S. Pat. No. 4,346,227 while simvastatin is
described in U.S. Pat. No. 4,444,784, each of which is hereby
incorporated by reference in its entirety.
[0096] Also included for use in conjunction with chloroquine
compounds of the present invention are the bio-active metabolites
of Hmg-CoA reductase inhibitors, such as pravastatin sodium (the
bioactive metabolite of mevastatin).
[0097] As used in methods or compositions of the present invention,
any one or several of the Hmg-CoA reductase inhibitor compounds may
be mixed with L-arginine or a substrate precursor to endogenous
nitric oxide, as described in U.S. Pat. Nos. 6,425,881 and
6,239,172, and 5,968,983 each of which is hereby incorporated by
reference to its entirety, to provide a therapeutically effective
mixture for use in conjunction with chloroquine compounds of the
present invention.
[0098] An embodiment of the present invention includes
administering a chloroquine compound along with second
pharmaceutically active ingredient that includes various diabetes
treatments such as insulin or insulin analogs, or other diabetic
drugs such as: actos (pioglitazone), sulfonylureas such a amaryl
(glimepiride), glipizide; avandia (rosiglitazone); a biguanide-type
medication such as glucophage; glucotrol; and glucovance (a
combination of glyburide and metformin). The term diabetic aid
includes natural, synthetic, semi-synthetic and recombinant
medicaments such as activin, glucagon, insulin, somatostatin,
proinsulin, amylin, and the like. Many of these diabetic treatments
are described in U.S. Pat. No. 6,610,272, which is herein
incorporated by reference in its entirety.
[0099] The term "insulin" encompasses natural extracted human
insulin, recombinantly produced human insulin, insulin extracted
from bovine and/or porcine sources, recombinantly produced porcine
and bovine insulin and mixtures of any of these insulin products.
The term is intended to encompass the polypeptide normally used in
the treatment of diabetics in substantially purified form but
encompasses the use of the term in its commercially available
pharmaceutical form, which includes additional excipients. The
insulin is preferably recombinantly produced and may be dehydrated
(completely dried) or in solution.
[0100] In accordance with the present invention, administering low
dose chloroquine in combination with insulin will lower the dose of
insulin required to manage the diabetic patient, while also
alleviating the symptoms of metabolic syndrome.
[0101] Additional pharmaceutically active ingredients or compounds
utilized in combination with the chloroquine compositions of the
present invention may include any of the following types of
compounds including anti-inflammatory agents, antioxidants,
antiarrhythmics, cytokines, antihypertensives, analgesics,
vasodilators and peptides.
Combination Treatments in View of Oxidative Stress
[0102] An embodiment of the present invention includes
administering an effective amount of a chloroquine compound along
with one or more measures to combat oxidative stress. Such measures
including changes in diet, for example, increased intake of fruit
and vegetables, and supplementation of the diet with
phyotochemicals or antioxidants, such as vitamin B12. Such measures
also include increased exercise, and decreased occupational stress.
Such measures also include administration of drugs with antioxidant
activity such as methylprednisolone, 21-aminosteroids,
2-methylaminochromans, pyrrolopyrimidines and thiazolidinones.
Although an understanding of mechanism is not required for practice
of the invention, it is believed that administration of a
chloroquine compound serves to stimulate the cellular response to
DNA damage and promote the repair of the cells exposed to radicals
generated by oxidative stress.
[0103] In some methods, a chloroquine compound is administered to a
subject exhibiting or at risk of ischemia in combination with a
second agent or a second pharmaceutically active ingredient
effective in prophylaxis or treatment of damage resulting from
ischemia and/or reperfusion. Such agents include antibodies to
adhesion molecules such as L-selectin, or CD18, tissue plasminogen
activator (see EP-B 0 093 619), activase, alteplase, duteplase,
silteplase, streptokinase, anistreplase, urokinase, heparin,
warfarin and coumarin. Additional thrombolytic agents include
saruplase and vampire bat plasminogen activator.
[0104] Subjects at risk of ischemia include those having previously
had heart disease, those having elevated biochemical markers of the
disease (e.g., C-reactive protein), those identified as having
blockage of blood vessels by angioplasty or MRI imaging, those with
deficiencies in or resistance to activated protein C, and those
undergoing a surgical procedure requiring temporary obstruction of
blood vessels. The presence or absence and the amount of myocardial
damage resulting from prolonged ischemia can be assessed by a
number of different means, including pathologic examination,
measurement of myocardial proteins in the blood, ECG recordings
(ST-T segment wave changes, Q waves), imaging modalities such as
myocardial perfusion imaging, echocardiography, contrast
ventriculography or positron emission tomography (see, e.g.,
Hanninen et al. Int. J. Bioelectromagnetism, 2000, No. 1 Vol. 2;
and Alpert, J. Am. College. Cardiol., 2000, 36-959-69). Myocardial
necrosis results in and can be recognized by the appearance in the
blood of different proteins released into the circulation due to
the damaged myocytes: myoglobin, cardiac troponins T and I,
creatine kinase, and lactate dehydrogenase. The response of the
subject to treatment with a chloroquine compound in combination
with a second agent or a second pharmaceutically active ingredient
effective in prophylaxis or treatment of damage resulting from
ischemia and/or reperfusion can be monitored by any of these tests.
Preferably, the amount of pathological damage or level of a marker
associated with the same shows a reduced increase, does not
increase, or even is reduced following administration of a
chloroquine compound relative to a placebo.
Combination Treatments Related to Stroke
[0105] Administration of chloroquine can be accompanied by
administration of additional agents or pharmaceutically active
ingredients to treat stroke. These include the same agents
discussed for treating ischemia and oxidative stress as described
above. Multiple symptoms may be present in a stroke patient, and it
is believed that a combination treatment with chloroquine and
another active agent effective in prophylaxis or treatment of
damage resulting from ischemia and/or reperfusion would provide
added benefits to the patient. Examples of these agents include
antithrombotic agents such as unfractionated heparin, low molecular
weight (LMW) heparin, heparinoids, aspirin, ticlopidine,
clopidogrel, dipyridamole, hirudin, and glycoprotein IIb/IIIa
antagonists, as described by Coull et al. Stroke, 2002,
33:1934-1942, which is hereby incorporated by reference in its
entirety.
[0106] Subjects at risk of stroke can be determined by presence of
one, and usually at least two of the following risk factors: high
blood pressure, heart disease, high cholesterol levels, sleep
apnea, previous occurrence of stroke, smoking, excessive alcohol
consumption and excessive weight. Alternatively, transcranial
doppler (TCD) testing uses sound waves to measure the speed with
which blood flows through the large blood vessels within the head.
The test can detect constriction (narrowing) of blood vessels as
well as blood flow abnormalities related cerebrovascular disease.
Damage to tissue from stroke can be monitored by MRI and/or by
cognitive testing. Monitoring of tissue damage, if any, can be
performed following administration of the combination chloroquine
treatments described herein.
Combination Treatments Related to Atherosclerosis
[0107] Administration of a chloroquine compound for treating
metabolic syndrome can also be combined with any desired agents
conventionally used in prophylaxis o treatment of atherosclerosis.
These include antithrombotic agents seen as unfractionated heparin,
low molecular weight (LMW) heparin, heparinoids, aspirin,
ticlopidine, clopidogrel, dipyridamole, hirudin, and glycoprotein
IIb/IIIa antagonists. Also included are lipid lowering agents such
as statins, ezetimibe, bile acid sequestrants, fibrates, HMG-CoA
reductase inhibitors, nicotinic acid derivatives, and blood
pressure lowering agents.
[0108] Additional pharmacologically active agents may be delivered
along with the primary active agents, e.g., the chloroquine
compounds of the invention. In one embodiment, such agents include,
but are not limited to agents that reduce the risk of
atherosclerotic events and/or complications thereof. Such agents
include, but are not limited to beta blockers, beta blockers
combined with thiazide diuretic combinations, statins, aspirin, ACE
inhibitors, ACE receptor blockers/inhibitors (ARBs), and the like,
as described in U.S. Pat. No. 6,933,279.
[0109] Suitable beta blockers include, but are not limited to
cardioselective (selective beta 1 blockers), e.g., acebutolol
(Sectral.TM.), atenolol (Tenormin.TM.), betaxolol (Kerlone.TM.),
bisoprolol (Zebeta.TM.), metoprolol (Lopressor.TM.), and the like.
Suitable non-selective blockers (block beta 1 and beta 2 equally)
include, but are not limited to carteolol (Cartrol.TM.), nadolol
(Corgard.TM.), penbutolol (Levatol.TM.), pindolol (Visken.TM.),
propranolol (Inderal.TM.), timolol (Blockadren.TM.), labetalol
(Normodyne.TM., Trandate.TM.), and the like. Suitable beta blocker
thiazide diuretic combinations include, but are not limited to
Lopressor HCT, ZIAC, Tenoretic, Corzide, Timolide, Inderal LA
40/25, Inderide, Normozide, and the like. The use of beta blockers
is also described in U.S. Pat. No. 6,756,408, which is hereby
incorporated by reference in its entirety.
[0110] Suitable ACE inhibitors include, but are not limited to
captopril (e.g. Capoten.TM. by Bristol-Myers Squibb), benazepril
(e.g., Lotensin #2122; by Novartis), enalapril (e.g., Vasotec.TM.
by Merck), fosinopril (e.g., Monopril.TM. by Bristol-Myers Squibb),
lisinopril (e.g. Prinivil.TM. by Merck or Zestril.TM. by
Astra-Zeneca), quinapril (e.g. Accupril.TM. by Parke-Davis)
ramipril (e.g., Altace.TM. by Hoechst Marion Roussel, King
Pharmaceutically), imidapril, perindopril erbumine (e.g., Aceon.TM.
by Rhone-Polenc Rorer), trandolapril (e.g., Mavik.TM. by Knoll
Pharmaceutical), and the like. Suitable ARBS (Ace Receptor
Blockers) include but are not limited to losartan (e.g. Cozaar.TM.
by Merck), irbesartan (e.g., Avapro.TM. by Sanofi), candesartan
(e.g., Atacand.TM. by Astra Merck), valsartan (e.g., Diovan.TM. by
Novartis), and the like.
[0111] Optionally, the methods are practiced on subjects that are
free of diseases of the immune system, infectious diseases,
neurological diseases, and multidrug resistance (i.e. resistance to
multiple drugs for treatment of the same conditions, such as two
anti-cancer drugs, or two antibiotics) before commencing
administration of the chloroquine compound. Optionally, the methods
are practiced on subjects free of psoriasis, malaria, protozoal
infections, Alzheimer's disease, Parkinson's disease, lupus
erythematosos, rheumatism, hypercalcemia, multiple sclerosis and
migraine before administering the chloroquine compound.
EXAMPLES
[0112] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
ATM Mouse Models
Methods
[0113] The ATM mutation was moved into the apoE null mouse model to
create an ATM.sup.-/- apoE.sup.-/- mouse model for evaluation the
role of ATM in vascular disease. The apoE null mouse model was
described by Plump and Breslow in Annu. Rev. Nurt., 1995,
15:495-518, which is hereby incorporated by reference in its
entirety. ATM.sup.-/- mice and conditions for breeding are
described by Spring, K. et al. in Nat. Genet., 2002, 32(1):185-90,
which is hereby incorporated by reference in its entirety. The
ATM-deficient mice carry a neo gene to interrupt exon 57 and
replace exon 58 of ATM (GenBank Accession No. U55702, SEQ ID NO:2),
deleting a region of the p13K domain that is also deleted in a
number of individuals with ataxia telangiectasia. Originally in a
mixed genetic background (C57BL/6 and 129Sv), these mice have been
backcrossed with BL/6 mice for more than 6 generations. These
backcrossed ATM.sup.-/- mice were intercrossed to obtain ATM
deficient littermates or bred with apoE-null or LDLR-null mice in
the C57BL/6 background Multiple different breeding pairs were
utilized to generate ATM.sup.+/+apoE.sup.-/-, ATM.sup.+/-
apoE.sup.-/-, and ATM.sup.-/-apoE.sup.-/- littermates that were
used for experiments. Animals were genotyped at the ATM locus using
a forward wild-type primer (5'-AAGATGCTGTCATGCAGCAGGTCTTCCAGA-3')
(SEQ ID NO:3), a reverse neo-specific primer
(5'-AATTCTCTAGAGCTCGCTGATCAGCCTCGA-3') (SEQ ID NO:4). and a reverse
wildtype primer (5'-TTGTCCAACCTGAGAGTGGCAATCCATAGC-3') (SEQ ID
NO:5). Mice housed in a specific pathogen-free barrier facility
were weaned at the age of three weeks to standard mouse chow
providing 6% calories as fat.
ATM Deficiency Phenocopies the Metabolic Syndrome
[0114] All experiments were performed using littermates. ATM
haploinsufficiency had no effect on breeding or viability in apoE
null mice, but ATM.sup.-/-apoE.sup.-/- animals were born at
decreased frequency (about 9% versus the expected 25% in
ATM.sup.+/- apoE.sup.-/-.times.ATM.sup.+/- apoE.sup.-/- matings).
This effect appeared to be due to fetal loss in utero; when
ATM.sup.-/-apoE.sup.-/- mice were born, they were viable and
resembled ATM heterozygotes.
[0115] For most experiments, sex-matched animals at the age of 6-8
weeks were started on a high fat Western-type diet consisting of a
semipurified diet (TD88137, from Harlan, Madison, Wis.) containing
0.15% cholesterol and providing 42% calories as fat. This diet is
typically used in murine models of atherosclerosis. After 8 weeks
on this diet, there was no difference in total body weight between
ATM, ATM.sup.+/+, ATM.sup.+/-, and ATM.sup.-/- mice in the apoE
model (determined for 12 ATM.sup.+/+, 17 ATM.sup.+/-, and 4
ATM.sup.-/- mice). However, ATM deficient mice had increased
adiposity (body fat of about 33% or 31%, compared about 23% for
ATM.sup.+/+ mice) due in part to increased intra-abdominal fat.
Adiposity was determined by DEXA with P<0.05 by ANOVA; and was
also observed through en bloc dissections of visceral fat (e.g.
whole mounts of visceral fat pads removed en bloc from age-matched
ATM.sup.+/+, ATM.sup.+/-, and ATM.sup.-/- mice). There was no
effect of genotype on metabolic rate measured by indirect
calorimetry or food intake. Consistent with the adiposity data,
adiponectin levels were decreased in ATM deficient mice. Serum
adiponectin levels in apoE.sup.-/- mice with different ATM
genotypes (.gtoreq.5 in each group) were determined before and
after Western diet feeding for 3 and 8 weeks. Individually tested,
ATM.sup.+/- levels were at borderline significant different from
ATM.sup.+/+ at baseline and 3 weeks, and significantly different
from ATM.sup.+/+ at 8 weeks.
[0116] Blood pressure was elevated in ATM-deficient apoE.sup.-/-
mice on the Western diet. Systolic blood pressure (SBP) and
diastolic blood pressure (DBP) were measured after 8 weeks of
Western diet feeding in ATM.sup.+/+, ATM.sup.+/-, and ATM.sup.-/-
mice. With Western diet feeding in the ATM deficient apoE null
mice, both systolic and diastolic blood pressures (determined by
tail cuff) were elevated in ATM-deficient mice. A plethysmography
device (such as those supplied by Kent Scientific, Litchfield,
Conn.) was used for tail cuff blood pressure determinations in
acclimated, warmed mice performed by a technician blinded to
treatment status, according to the manufacturer's instructions.
Results were reported as the mean of 5-8 measurements on each day
of 3 consecutive days. If appropriate, blood pressure
determinations are confirmed invasively under anesthesia or with
implantable telemetry sensors.
[0117] For the chloroquine experiments, mice were treated with
either chloroquine diphosphate (Sigma, St. Louis, Mo.) in 0.9%
saline or saline alone administered at 7 mg/kg body weight per week
after a loading dose of 7 mg/kg. Two mouth old male db/db
(Lepr.sup.db LEPR-deficient mice having excess adiposity) mice and
ob/ob (Lep.sup.ob, genetically obese) mice were purchased from
Harlan (Indianapolis, Ind.) and the Jackson Laboratory,
respectively.
[0118] Western diet-fed ATM.sup.+/-apoE.sup.-/- mice were glucose
intolerant and insulin resistant compared to
ATM.sup.+/+apoE.sup.-/- littermates. The intraperitoneal glucose
tolerance testing was conducted on 9 ATM.sup.+/+ and 10 ATM.sup.+/-
littermate mice after 8 weeks on Western diet. Effects on
adiposity, blood pressure and glucose metabolism were seen in both
genders and replicated in several littermate cohorts. Thus, the
ATM.sup.+/- genotype, which may be present in 0.5 to 2% of human
populations, models at least three components of the metabolic
syndrome in apoE null mice: increased adiposity, elevated blood
pressure, and glucose intolerance.
[0119] Fasting cholesterol, triglycerides, and free fatty acids
were not significantly affected by genotype at baseline (on chow
diet) or after 3 or 8 weeks of Western diet. The lipid chemistries
and atherosclerosis in apoE.sup.-/- mice with different ATM
genotypes were determined after Western diet feeding for 8 weeks.
Fasting glucose and insulin levels increased with Western diet
feeding and glucose was highest in ATM.sup.-/- animals. Serum
adiponectin levels were determined in apoE.sup.-/- mice with
different ATM genotypes (.gtoreq.5 in each group) before and after
Western diet feeding for 3 and 8 weeks. Individually tested,
ATM.sup.+/- levels were at borderline significance different from
ATM.sup.+/+ at baseline and 3 weeks, and significantly different
from ATM.sup.+/+ at 8 weeks. Lipoproteins were not affected by
genotype. Atherosclerosis after 8 weeks on the Western diet was
increased, in ATM-deficient mice inversely based on gene dosage.
Atherosclerosis data was assayed by the en face technique for the
mice. The P value indicates the overall results of the
Kruskal-Wallis test. In post tests, ATM.sup.+/+ mice showed
significant differences (P<0.05) as compared to each of the
other two genotypes. The same effects were seen in both males and
females. Atherosclerosis was measured in apoE.sup.-/- mice with
different ATM genotypes according to the gender after Western diet
feeding for 8 weeks. The P values indicate the overall results of
the Kruskal-Wallis test. These values are different from a similar
test group because this data set presents the analysis of 6 groups
as compared to 3 in the prior group. In post tests, male
ATM.sup.+/+ mice showed less lesion area vs. male ATM.sup.-/- mice
(P<0.05) at the arch, and vs. ATM.sup.+/- and ATM.sup.-/- mice
(P<0.05) in the thoracic and abdominal aorta. Female ATM.sup.+/+
mice showed less atherosclerosis as compared to ATM.sup.-/- mice
(P<0.05) in the abdominal aorta. In independent assessments,
ATM.sup.+/+ mice of both genders showed significant differences
(P<0.05) as compared to each of the other two genotypes
Insulin Resistance in ATM Deficiency
[0120] In hyperinsulinemic-euglycemic clam experiments conducted in
littermates eating the high fat Western diet, endogenous glucose
production was suppressed by insulin in ATM.sup.+/+apoE.sup.-/- but
not ATM.sup.+/-apoE.sup.-/- mice. Hyperinsulinemic euglycemic clamp
data was determined in ATM.sup.+/+ and ATM.sup.+/- mice for 4
animals of each genotype.
[0121] Consistent with the presence of hepatic insulin resistance,
IRS-2-associated PI 3-kinase activity was decreased in the livers
of insulin-stimulated high fat-fed ATM+/-mice. The IRS-2 (liver)
and IRS-1 (skeletal muscle)-associated PI 3-kinase activity was
determined in post-clamp tissues of ATM.sup.+/+ and ATM.sup.+/-
mice by ELISA after immunoprecipitation of the IRS-containing
complex. Phosphorylated Akt, a target of PI 3-kinase, was also
decreased in the livers of the same insulin-stimulated mice
(determined by Western blot analysis of phospo-Akt (Ser473) in
post-clamp liver of ATM.sup.+/+ and ATM.sup.+/- mice), consistent
with the presence of hepatic insulin resistance in high fat-fed
ATM+/-mice. Insulin-induced phosphorylation of Akt at both Ser 473
and Thr 308 was decreased in ATM-deficient livers of apoE null mice
as well as low density lipoprotein receptor null mice, another
model shown to have increased insulin resistance with ATM
deficiency. The baseline and insulin-stimulated Akt-phosphorylation
in livers of apoE null mice and LDLR null mice of each ATM genotype
were determined by Western blot analysis. The Western blot analysis
of phospho-Akt (Ser473) and phospo-Akt (Thr308) of one saline- and
one or two insulin-treated animals for each genotype showed
decreased liver Akt-phosphorylation with ATM deficiency in these
two different mouse backgrounds.
[0122] Impaired insulin signaling was present to other
ATM-deficient cell types. Decreased IRS expression causes insulin
resistance. mRNA as well as protein levels for IRS-2 were decreased
in macrophages elicited from ATM-deficient mice. Quantitative
RT-PCR determination of IRS-2 expression was performed in
thioglycollate-elicited macrophages from ATM.sup.+/+, ATM.sup.+/-,
and ATM.sup.-/- mice. Data were determined relative to GAPDH
expression (mean.+-.s.e.m.). *P<0.001. IRS-2 mRNA expression was
also analyzed in liver but did not detect consistent genotype
effects.
[0123] Activated Akt and the related kinases p38 and ERK were
decreased in the aortas of young chow-fed ATM-deficient mice.
Western blots showed aortic content of phospho-p38, phospho-Akt,
and phospho-ERK compared with total p38 protein. The same results
were detected in several samples. Young, chow-fed mice without
atherosclerotic lesions were used for the aortic analyses. Insulin
signaling can be disrupted by a serine phosphorylation at residue
307 of IRS-1. Aortic tissue from ATM.sup.-/- mice had the highest
level of IRS-1 Ser 307 phosphorylation, ATM.sup.+/- aortas were
intermediate, and ATM.sup.+/+ vessels lowest. IRS-1 phosphorylation
was detected at position Ser307, normalized to total IRS-1 protein,
as acquired by densitometric image analysis or the Western blots.
The same results were observed in 2 or more independent experiments
for each assay.
[0124] Jun N-terminal kinase (JNK) phosphorylates IRS-1 at serine
307 to cause insulin resistance. JNK was activated in aortas in
proportion to the degree of ATM deficiency. Phospho-JNK, normalized
to total JNK, was detected in whole aorta as determined by
quantitative Western blot analysis.
[0125] When cultured aortic smooth muscle cells were treated with a
morpholino antisense oligonucleotide to ATM, JNK activity was
increased, providing direct evidence that interfering with ATM
expression induces JNK activity, a mediator of insulin resistance.
Analysis of phospho-JNK in vascular smooth muscle cells cultured
from ATM.sup.+/+apoE.sup.-/- mice. primary active smooth muscle
cells were transfected with an anti-ATM oligonucleotide, or a
control oligonucleotide or the vehicle ethoxylated polyethylenimine
solution only. Cells were assayed by ELISA for phospho-JNK and
total JNK as measured in U/ml/well of a 96-well plate. The results
showed phospho-JNK normalized to total INK of wells with the same
treatment condition. These insults are consistent with the presence
of systemic insulin resistance, JNK activity (as well as
phosphorylation of c-Jun, the major JNK substrate) was also
increased in adipose tissue, skeletal muscle, and liver of
ATM-deficient mice. Increased expression of phospho-JNK was
detected in tissues involved in systemic insulin action in all
three ATM genotypes in apoE.sup.-/-, as determined by analysis of
Western blots for fat, skeletal muscle and liver. Comparisons were
made between ATM-deficient and ATM wild-type animals.
Analytical Procedures
[0126] Cholesterol, triglycerides and nonesterified fatty acids
(NEFA) were assayed as described following a 4 h fast. For glucose
tolerance and insulin tolerance tests, mice were fasted and
injected with 10% D-glucose (1 g/kg) or human regular insulin in
(0.75 units/kg body weight, Eli Lilly and Co., Indianapolis, Ind.)
in procedures separated by at least one week. Tail vein blood (5-10
.mu.l) was assayed for glucose at 0, 30, 60, and 120 minutes. Blood
glucose was assayed using a glucose meter (Hemocue, Mission Viejo,
Calif. or Becton Dickinson, San Jose, Calif.). The area under the
glucose and insulin curves (AUC) dosing the OGTT and ITT were
calculated using the formula: 0.25 (fasting value)+0.5 (1/2 h
value)+0.75 (1 h value)+0.5 (2 hr value). Body composition was
performed on anesthetized, living mice by dual energy X-ray
absorptiometry (PIXImus, GE Corporation).
Atherosclerosis Quantification
[0127] Atherosclerosis was measured using the en face or aortic
origin technique. For the former, pinned aortas were imaged with a
digital camera and analyzed by using an image processing program.
The percentage of involvement of the intimal surface area is
reported for the arch (encompassing the surface from the aortic
valve to the left subclavian artery), the thoracic aorta (extending
to the final intercostal artery), and the abdominal aorta (to the
ileal bifurcation). For the aortic origin technique, mean lesion
size for each animal was quantified by the degree of lipid staining
of cross-sections of aortic tissue as described. Anesthetized mice
were exsanguinated, the heart and aortic arch were perfused, then
tissues were removed en bloc and frozen immediately in tissue
freeze medium. For each sample, 64 sections were made on a cryostat
beginning just caudal to the aortic sinus and extending into the
proximal aorta at 10 .mu.m intervals. Slides were fixed with 60%
isopropanol and stained with Oil Red O. For each heart, lesions in
eight sections at 80 .mu.m intervals were quantified using image
analysis software calibrated using a hemocytometer grid.
[0128] ATM heterozygotes (+/-) in the ApoE null modal also had
glucose intolerance and were insulin resistant as measured in
insulin tolerance tests. The glucose tolerance and insulin
tolerance tests were performed on mice that were fasted for 12
hours and then injected with 10% D-glucose (1 g/kg) or human
regular insulin (0.75 units/kg body weight, Eli Lilly and Co.,
Indianapolis, Ind.) in procedures separated by at least one week.
Tail vein blood (5-10 .mu.l) was assayed for glucose at 0, 30, 60,
and 120 minutes. Blood glucose was assayed using a glucose meter
(Hemocue, Mission Viejo, Calif.). The area under the glucose and
insulin curves (AUC) during the OGTT and ITT were calculated using
the formula: 0.25 (fasting value)+0.5 (1/2 h value)+0.75 (1 h
value)+0.5 (2 h value), as described by Haffner et al., N. Engl. J.
Med., 1986, 315:220-224.
[0129] The phenotype was unaffected by gender and was observed in
multiple cohorts of littermates. Thus, the ATM.sup.+/- genotype,
which may be present in up to 2% of the general population, models
at least three components of metabolic syndrome in ApoE null mice:
increased visceral adiposity, elevated blood pressure, and glucose
intolerance.
[0130] Fasting cholesterol, triglycerides, and free fatty acids
were not affected by genotype at baseline (on chow diet) or after 3
or 8 weeks of Western diet. Fasting glucose and insulin levels
increased with Western diet feeding and glucose was highest in
ATM.sup.-/- animals, as shown in Table 2. Lipoproteins as assayed
by size exclusion chromatography were also unaffected by
genotype.
TABLE-US-00002 TABLE 2 Fasting Glucose and Insulin Values. Glucose
(mg/dl) Insulin (pg/ml) Parameter Baseline 8 weeks Baseline 8 weeks
ATM.sup.+/+ 157.4 .+-. 12 211.2 .+-. 13* 940 .+-. 200 .sup. 1750
.+-. 700.sup.# ATM.sup.+/- 178.0 .+-. 12 222.0 .+-. 12.sup.# 1870
.+-. 400 2840 .+-. 500 ATM.sup.-/- 180.1 .+-. 12 236.8 .+-. 29 1590
.+-. 200 2110 .+-. 900 Data are presented as mean .+-. s.e.m. for
.gtoreq.5 apoE.sup.-/- animals per ATM genotype. Differences
between glucose levels in this table and those in glucose tolerance
tests are explained in part by different assays (chemical
determinations using serum samples in this table vs. glucose
monitor determination using whole blood in glucose tolerance tests)
and different periods of fasting (4 hr. in this table vs. 12 hr.
for glucose tolerance tests). P values indicate comparisons of
values at baseline vs. 8 weeks of high fat-feeding. *P < 0.001,
.sup.#P < 0.05.
[0131] However, atherosclerosis assayed by the en face technique
(according to Semenkovich et al., J. Lipid Res., 1998, 39:
1141-1151) after 8 weeks on the Western diet was increased in
ATM-deficient mice in a manner that was inversely related to gene
dosage. At the arch, lesions were about 2-fold greater in
ATM.sup.+/- and about 3-folder greater in ATM.sup.-/- as compared
ATM.sup.+/+ mice. Relative differences were similar in the
abdominal aorta and more pronounced in the thoracic aorta. These
results illustrate that atherosclerosis is increased in
ATM-deficient mice.
Insulin Signaling Pathways in Mutant Mice
[0132] To investigate the accelerated atherosclerosis observed in
the ATM deficient mice with systemic insulin resistance and other
features of metabolic syndrome, the insulin signaling pathways in
the aortas of apoE.sup.-/- mice were studied. The protein kinase
Akt is known to mediate the metabolic effects of insulin in many
tissues, and defective Akt activity is common in insulin
resistance. Phospho-Akt, the activated form of this kinase, was
decreased in a gene dosage-dependent manner in the aortas of
ATM-deficient mice (in the apoE null model). Phospho-Akt activity
was lowest in ATM.sup.-/- vessels, intermediate in ATM.sup.+/-, and
highest in wild type mice. Similar decreases with ATM deficiency
were seen for Phospho-ERK, a MAP kinase usually stimulated by
stress, while total p38 mass was unaffected by genotype. These
results were obtained using aortas isolated from 8 week old mice
eating chow and subjected to Western blotting.
[0133] Downstream events in insulin signaling such as Akt
activation can be blocked by covalent modification of insulin
receptor substrates such as phosphorylation at serine 307 of IRS-1.
Aortic tissue from ATM.sup.-/- mice had the highest level of IRS-1
Ser 307 phosphorylation, ATM.sup.+/- aortas were intermediate, and
exhibited the lowest amount IRS-1 Ser 307 phosphorylation in the
apoE null mouse model. These data are consistent with presence of
vascular insulin resistance in atherosclerosis-prone ATM-deficient
mice due in part to disruption of normal insulin signaling by a
specific serine phosphorylation of IRS-1.
[0134] The stress-related kinase JNK is known to mediate insulin
resistance in part by phosphorylating IRS-1 at serine 307. JNK was
activated in the aortas of ATM-deficient mice in proportion to the
degree of ATM deficiency. Aortic phospho-JNK in chow fed mice of
various ATM genotypes in the ApoE null model was measured by
western blot.
[0135] Western blotting was performed by standard techniques using
antibodies purchased from Cell Signaling (Beverly, Mass.): total
Akt, phosphoAkt (Ser472 and Thr308), total p38 and phospho-p38
(Thr180/Tyr182), phospho-ERK (Thr202/Thr204), total JNK,
phospho-JNK (Thr183/Tyr185), total c-Jun, phospho-c-Jun (Ser63),
total p53 and phospho-p53 (Ser18). Anti-HSC70 which was purchased
from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-IRS-2 was
purchased from Upstate Biotechnology (Lake Placid, N.Y.). Smooth
muscle cells were assayed using a cell-based ELISA kit for
phospho-JNK from Activemotif (Carlsbad, Calif.).
Thioglycolate-elicited macrophages were assayed using a phospho-JNK
ELISA from Sigma (St. Louis, Mo.). Insulin and adiponectin were
assayed by ELISA using commercial reagents (Crystal Chem, Downers
Grove, Ill. and Alpco Diagnostics, Salem, N.H.).
Insulin Signaling
[0136] IRS-2 associated PI 3-kinase activity was determined by
immunoprecipitating IRS-2 from post-clamp liver protein extracts
using an anti-IRS-2 antibody (Upstate Biotechnology) and assessing
PI 3-kinase activity of the IRS-2-containing complex using an ELISA
based assay (Echelon Biosciences Inc.). Total and
Ser473-phosphorylated Akt levels in whole-cell protein extracts
from mice after hyperinsulinemic clamp studies were measured by
immunoblotting using antibodies from Cell Signaling. For some
studies, mice were fasted overnight, injected with human insulin
(10 mU/g), sacrificed 5 min later, then tissues dissected and snap
frozen.
Hyperinsulinemic-Euglycemic Clamps
[0137] Clamp experiments were performed as described
(Bernal-Mizrachi et al., 2003; Nat. Med. 9:1069-1075; Tordjman et
al., 2001; J. Clin. Invest. 107:1025-1034). Double lumen catheters
were placed and 3-[.sup.3H] glucose was infused to steady state.
Regular lumen insulin was infused at 10 mU/kg/min with 25%
D-glucose to maintain blood glucose at 120 mg/dI for at least 90
minutes. the 3-[.sup.3H] glucose infusion was continued during the
clamp with labeled glucose included in the 25% D-glucose infusion
to match blood specific activity at steady state. The rate of
appearance of glucose (R.sub.a), equal to glucose utilization
(R.sub.d) at steady state, was determined by dividing the infusion
rate of labeled glucose by specific activity. Engogenous glucose
production was calculated by subtracting the cold glucose infusion
rate from the clamp R.sub.d.
[0138] Most of the mass of the aorta is composed of smooth muscle
cells. Smooth muscle cells were cultured from the aortas of
apoE.sup.-/- mice by the explant technique as described by Weng et
al., Proc. Natl. Acad. Sci. USA, 2003, 100:6730-6735. Smooth muscle
cells growing from the explants were passaged, expanded, and their
identity verified by staining with a fluorescent smooth muscle
.alpha.-actin antibody (Vector Laboratories, Burlingame, Ga.).
Morpholino antisense oligonucleotides directed against mouse ATM
and a control were made by Gene Tools (Philomath, Oreg.). The
lissamine-tagged antisense oligonucleotide against ATM had the
following sequence: 5'-GTGCTAGACTCATGGTTTAAGATTT-3' (SEQ ID NO:6).
Subconfluent mouse primary aortic smooth muscle cells were seeded
at a concentration of 1.times.10.sup.6 per well in a 6-well tissue
culture plate. After culture in serum-reduced DMEM (0.4% FBS)
overnight, cells were incubated in a 5-mL solution containing 1.4
.mu.mol/L antisense ATM oligo or control oligo with 0.56 .mu.mol/L
ethoxylated polyethlenimine for 3 hours and subsequently switched
to normal growth medium for at least 24 hours. Successful
transfection was confirmed by fluorescence microscopy and
phospho-JNK and total JNK were assayed 72 hours later using a
cell-based ELISA kit.
[0139] When cultured aortic smooth muscle cells from
ATM.sup.+/+apoE.sup.-/- mice were treated with a Morpholino
antisense oligonucleotide to ATM (anti-ATM), JNK activity was
increased, as shown by ELISA. Control oligos had no effect on JNK
activity. These data show that interference with ATM expression
induces JNK activity, a mediator of insulin resistance and
atherosclerosis, and JNK activity would serve as a good target to
modulate in order to prevent or treat the symptoms associated with
metabolic syndrome (including insulin resistance and/or
atherosclerosis).
[0140] Macrophages are often involved in initiating early
atherosclerotic lesions, and the presence of JNK in macrophages is
proatherogenic. Total JNK activity was increased in
thioglycolate-elicited macrophages from ATM-deficient mice.
Expression of IRS-2, the predominant insulin receptor substrate in
macrophages, was decreased in ATM-deficient cells, indicating
impaired insulin signaling.
[0141] For LPL assays, cells were fed on day 6. On day 7, cells
were washed and incubated in DMEM plus 0.5% BSA for 4 hr, followed
by incubation with 10 U/ml heparin, collection of media, and
subsequent determination of LPL activity as described (Coleman et
al., 1995; J. Bio. Chem 270:12518-12525). For JNK inhibition
studies, cells were incubated in cell culture medium for 15 hr in
the presence of a JNK-specific inhibitor (SP600125, Sigma, St.
Louis, Mo.) or vehicle (DMSO). Cells were further incubated for 1
hr in DMEM plus 0.5% BSA plus SP600125 or vehicle in 10 U/ml
heparin, then media were assayed.
[0142] IRS-2 message levels were determined using total RNA (1
.mu.g) that was treated with DNase and reverse transcribed using
Superscript II (Invitrogen, Carlsbad, Calif.) and oligo-dT as
primer. PCR was performed with the GeneAmp.RTM. 7000 Sequence
Detection System using the TaqMan.RTM. Universal Master reagent kit
(Applied Biosystems, Foster City, Calif.). A negative control using
RNA not subjected to reverse transcription was included in each
assay.
[0143] Mouse IRS-2 was detected with the follow primer/probe set:
forward primer 5'-CAGTCCCACATCAGGCTTGAG-3' (SEQ ID NO:7), reverse
5'-GGACTGCACGGATGACCTTAG-3' (SEQ ID NO:8), labeled probe with
5'-carboxyfluorescein (FAM) and carboxytetramethylrhodamine (TAMRA)
5'FAM-CCTCAAGTCAGCCAGCCCCCTG-TAMRA-3' (SEQ ID NO:9); mouse LPL
forward 5'-TTC ACT TTT CTG GGA CTG AGA ATG-3' (SEQ ID NO:10),
reverse 5'-GCC ACT GTG CCG TAC AGA CA-3' (SEQ ID NO:11), probe
5'-FAM-TCC AGC CAG GAT GCA ACA-TAMRA-3' (SEQ ID NO:12), mouse
EGR-1: forward 5'-GCC TCG TGA GCA TGA CCA AT-3' (SEQ ID NO:13),
reverse 5' GCA GAG GAA GAC GAT GAA GCA-3' (SEQ ID NO:14), probe
5'-FAM-CTC CGA CCT CTT CAT CCT CGG CG-BH-3' (SEQ ID NO:15).
Sequence specific amplification was detected with an increasing
fluorescence signal of FAM (reported dye) during the amplification
cycle. Co-amplification of the mRNA for the rodent GAPDH control
reagent (Applied Biosystems, Foster City, Calif.) or mouse
ribosomal protein L32 (forward 5'-AAGCGAAACTGGCGGAAAC-3' (SEQ ID
NO:16), reverse 5'-GATCTGGCCCTTGAACC-3' (SEQ ID NO:17), probe
5'-HEX-CAGAGGCATTGACAACAGGGTGCG-BH-3' (SEQ ID NO:18)) was performed
in the same tube for all samples. All assays were run in
triplicate. The results are expressed as relative expression of
mRNA normalized to GAPDH or L32 levels that were not affected by
genotype. A VIC TaqMan.RTM. murine GAPDH control reagent (Applied
Biosystems, Foster City, Calif.) was used to normalize results.
GAPDH was not affected by genotype.
[0144] The comparative Cr method was employed for quantification.
JNK increases AP-1 activity, and AP-1 is known to increase the
expression of lipoprotein lipase, which is known to be
proatherogenic in macrophages. LPL enzyme activity was increased in
ATM-deficient macrophages, providing one likely mechanism for the
increased diet-induced atherosclerosis in ATM deficiency.
Blood Pressure Determinations
[0145] Systolic and diastolic blood pressures were measured over
several days in conscious mice using a tail-cuff system (Kent
Scientific, Litchfield, Conn.) as described (Bernal-Mizrachi et
al., 2003; Nat. Med. 9:1069-1075). Animals were habituated to the
blood pressure apparatus for several days before data were
collected. Five to eight measurements were recorded for each mouse
at each of three daily sessions. Results are presented as the mean
of those measurements on consecutive days.
[0146] These results demonstrate that ATM deficiency is a potential
cause of, or is at least associated with the metabolic syndrome,
resulting in insulin resistance and increased atherosclerosis. ATM
deficiency increases JNK activity, which impairs glucose tolerance
by interfering with insulin signaling through serine 307
phosphorylation of IRS-1 and promotes atherosclerosis by increasing
macrophage LPL activity through AP-1. Macrophage JNK activity is
gauged by assaying activated JNK1 and 2 (pThr.sup.183/pThr.sup.185)
by ELISA (Sigma, St. Louis) according to the manufacture's
instructions. Briefly, lipoprotein lipase (LPL) enzyme activity is
determined by measuring the release of labeled fatty acids from a
triolein emulsion in macrophages. Cells were fed on day 6. On day
7, cells were washed and incubated in DMEM plus 0.5% BSA for 4
hours, followed by incubation with 10 U/ml heparin, collection of
media, and subsequent determination of LPL activity as described by
Coleman et al., J. Biol. Chem., 1995, 270:12518-12525. Macrophages
from the same sources were used in the assays.
Reduction of Vascular Disease with Low-Dose Chloroquine Treatment
in ATM Deficient Mice
[0147] Since deficiency of ATM can cause vascular disease,
modulating or increasing ATM activity represents a novel approach
for treating vascular dysfunction. The inventors have been able to
show that providing chloroquine at low concentrations activates ATM
in cultured cells. Chloroquine dose-response experiments were
performed in mice using a variety of endpoints including
phosphorylation of p53 as a surrogate marker of ATM activation.
Using activated p53 as a marker is preferred, since it is a more
sensitive measure of ATM activity. However, low level activation of
ATM as detected with an antibody that recognizes serine
phosphorylation at residue 1981, may also be used in the assay.
Effects on p53 activation and the vascular phenotype were observed
after using 3.5 mg/kg of chloroquine (a low dose) administered by
intraperitoneal injection twice a week (a total weekly dose of 7.0
mg/kg).
[0148] ATM.sup.+/+ apoE.sup.-/- mice (of both sexes on a chow diet)
at the age of 8 weeks were treated with either chloroquine
diphosphate (Sigma, St. Louis, Mo.) dissolved in 0.9% saline or
saline alone administered at 7 mg/kg body weight per week after a
loading dose of 7 mg/kg. The mice were then fed the Western-type
diet as described. Body composition was performed on anaesthetized,
living mice by dual energy X-ray absorptiometry (PIXImus, GE
Corporation). Chloroquine had no effect on body weight or metabolic
rate (measured by indirect calorimetry). However, after 8 weeks,
glucose tolerance was improved compared to saline-treated mice.
These results demonstrate that low dose chloroquine improves
glucose tolerance in high fat-fed mice.
[0149] p53 is antiatherogenic and activated in diseased vessels,
such as those in patients with metabolic syndrome. Detection of
activated p53 phosphorylated at serine residue 18 is a robust
marker of ATM activation. Chloroquine treatment increased p53
activation in the aortas of mice on Western diet, consistent with
ATM activation by the drug. The results show increased activated
p53 in the aortic tissue of mice treated with chloroquine (CHQ, 7
mg/kg/week) as compared to saline injected mice.
[0150] Activation of the ATM-p53 axis by low dose chloroquine had
no effect on serum lipids, including fasting serum cholesterol,
triglycerides and non-esterified fatty acids (NEFA), determined at
baseline for mice on a chow diet without drug treatment and
following 4 and 8 weeks of Western diet feeding with chloroquine (7
mg/kg/week) or saline injections. All of these experiments were
performed using ATM.sup.+/+ apoE.sup.-/- littermate mice beginning
at the age of 8 weeks.
[0151] Serum is generally obtained after a four hour fast and
assayed for routine chemistries (glucose, triglycerides,
cholesterol, and non-esterified fatty acids) using an automated
plate reader connected to a computer, as described by Li et al.,
Nat. Med., 2000, 6:1115-1120. Insulin assays are performed on 5
.mu.l of serum by ELISA (Crystal Chem., Chicago). For measurement
of lipoproteins, serum from multiple animals will be pooled and
separated using superose columns. Additional aliquots of serum are
frozen for subsequent determination of leptin, adiponectin, etc.,
if appropriate.
[0152] Chloroquine treatment decreased atherosclerosis by 37% at
the arch, 25% at the thoracic aorta, and 25% at the abdominal
aorta. Atherosclerosis was assayed by using the en face technique
on the same mice, following 8 weeks of Western diet feeding with
chloroquine (CHQ) or saline treatment. This experiment was
performed with 25 animals in the chloroquine group and 21 animals
in the saline group. Equal numbers of males and females were
studied; chloroquine was effective in both genders.
[0153] Thus, the ATM-dependence of the chloroquine effect has been
demonstrated in several ways. First, a group of ATM.sup.-/-
apoE.sup.-/- mice and a group of ATM.sup.+/+ apoE.sup.-/- mice were
randomized to saline treatment or chloroquine injections (7
mg/kg/week, a described above) and fed the Western diet for 8
weeks. As expected, systolic and diastolic blood pressures were
increased in the ATM-deficient mice. Chronic (long-term, or
extended) low dose chloroquine treatment significantly lowered
systolic pressure by 9 mm Hg in ATM.sup.+/+ mice, as compared to
saline treated ATM.sup.+/+ mice. There was a similar trend for
diastolic blood pressure in these animals with functional ATM.
There was no effect of chloroquine in the ATM-deficient
animals.
[0154] Additional evidence of the ATM-dependence of the chloroquine
effect, was shown in isolated peritoneal macrophages from
ATM.sup.+/+apoE.sup.-/- and ATM.sup.-/-apoE.sup.-/- mice that were
treated chronically for six weeks with low dose chloroquine in the
setting of the Western diet. Macrophages were isolated as described
by Febbraio et al., J. Clin. Invest., 2000, 105:1049-1056. The mice
were injected intraperitoneally with a 4% solution of thioglycolate
media (Sigma, St. Louis, Mo.) on day 1. On day 5, peritoneal
macrophages were isolated, washed, counted, and plated in DMEM plus
10% FBS. Macrophages were subsequently harvested at various times
to isolate RNA or proton, respectively, according to standard
methods.
[0155] As compared to saline-treated mice, chloroquine
significantly decreased phospho-JNK activity in macrophages from
ATM.sup.+/+ mice, but had no effect in ATM null macrophages. The
results measured total phospho-JNK activity in
thioglycolate-elicited peritoneal macrophages from
ATM.sup.+/+apoE.sup.-/- and ATM.sup.-/- apoE.sup.-/- mice treated
with chloroquine (CHQ, 7 mg/kg/week) or saline after 6 weeks on
Western diet. The percentage difference in activated JNK for
macrophages from CHQ-treated was compared to saline-treated mice.
These results suggest that the vascular effects of chloroquine are
ATM-dependent and are at least partially mediated by suppression of
JNK activity, a know contributor to insulin resistance and lesion
development.
[0156] To further illustrate that the chloroquine effect is
dependent on the presence of ATM, Western diet-fed
ATM.sup.-/-apoE.sup.-/- mice were treated with saline or
chloroquine at a does of 7 mg/kg/week for 8 weeks and assaying for
atherosclerosis using the en face technique. The quantification of
aortic lesions involved cleaning the aortas and pinning them on a
wax matrix with the intimal surface facing upward (en face). Images
were captured with a digital camera, edited for artifacts, and data
were reported as the percent involvement of the arch (encompassing
the surface from the aortic valve to the left subclavian artery),
thoracic aorta (extending to the last intercostal artery), and
abdominal aorta (to the ideal bifurcation). At the time of
isolation, hearts were embedded in optical coherence tomography
(OCT) media and frozen to permit quantification of lesion formation
by staining of serial cryostat-cut sections of the aortic sinus, if
necessary. These samples were also used for
immunocytochemistry.
Bone Marrow Transplantation
[0157] Marrow was isolated from the femurs and tibias of 8-10 week
old ATM.sup.+/+apoE.sup.-/- and ATM.sup.-/-apoE.sup.-/- mice by
flushing the bones with cold PBS. Total bone marrow was washed,
triturated using a 24 gauge needle (Benson Dickson, Franklin Lakes,
N.J.), collected by centrifugation at 1250 rpm for 4 min and
diluted with PBS. After lysis of erythrocytes using 0.05% sodium
azide, cells were counted to obtain a defined concentration of
unfractionated bone marrow. Recipient mice were lethally irradiated
with 10 Gy from a cesium 137 gamma cell irradiator. Within 6 hours
after irradiation, recipients were reconstituted with
.about.5.times.106 donor marrow cells via a single injection. The
donor marrow was allowed to repopulate for 4 weeks after the
transplantation. The resultant null allele was detected by
genotyping of DNA from blood (Qiagen Blood Kit, Germantown, Md.).
For estimating engraftment, total bone marrow was isolated from the
femurs and tibias from C57BL/6-TgN(ACTbEGFP) (GFP) mice that were
crossed onto an apoE null background. The marrow was processed as
described and .about.5.times.10.sup.6 cells of unfractionated bone
marrow in PBS were injected into a cohort of apoE null mice in
parallel to those described above. Four weeks later, blood was
drawn from the recipient mice, from GFP apoE null mice and from
non-irradiated apoE null mice. The degree of engraftment as
assessed by FACS quantification of the percentage of GFP in
peripheral leucocytes as compared to non-transplanted controls and
the transgenic donor mice was 66-92%. Recipient mice were then
placed on a Western-type diet for 8 weeks followed by measurement
of serum chemistries and determination of atherosclerosis lesion
extent by the aortic origin technique.
ATM-Deficient Bone Marrow-Derived Cells Increase
Atherosclerosis
[0158] Since macrophages initiate early atherosclerotic lesions,
ATM.sup.+/+apoE.sup.-/- mice were transplanted with bone marrow
from ATM.sup.+/+apoE.sup.-/- or ATM.sup.-/-apoE.sup.-/- animals.
After four weeks of recovery (and verification of (and verification
of engraftment), animals were started on the Western diet, then
lesions were quantified by lipid staining of the aortic origin 8
weeks later. lesions were 80% larger in in animals transplanted
with ATM null marrow. There was no marrow genotype effect on
fasting cholesterol (1,110.+-.69 mg/dI for ATM.sup.+/+ vs.
1,183.+-.74 mg/dI for ATM.sup.-/-), glucose or triglycerides. ATM
deficiency was associated with increased JNK activity and increased
phosphorylation of e-Jun in ATM.sup.-/- macrophages. JNK, by
increasing activity of the transcription factor AP-1, increases
expression of early growth response gene-1 (Egr-1) and lipoprotein
lipase (LPL), mRNA levels for both Egr-1 and LPL (data were
expressed relative to L32 expression) were increased several-fold
in ATM null macrophages from transplanted mice. Macrophages
isolated from littermate mice not subjected to transplantation also
had increased JNK activity and LPL enzyme activity with ATM
deficiency.
[0159] Since JNK appears to be involved in the vascular disease of
ATM deficiency, it should be induced with Western diet feeding in
apoE null mice. Western diet-fed but not chow-fed apoE null mice
(wild type at the ATM locus) showed a robust increase in activated
aortic JNK. Phosphorylated ATM is difficult to detect in mouse
tissues, but detection is straightforward for p53, a major
substrate of activated ATM. Activated p53 (phospho-p53(Ser18) was
also detected in the aortas of Western diet-fed but not chow-fed
apoE null mice, consistent with ATM activation. This finding could
reflect a protective response to limit vascular disease or
implicate the ATM-p53 axis in atherogenesis.
Reduction of Metabolic Abnormalities with the ATM-Activating Drug
Chloroquine
[0160] To determine if promoting ATM activity decreases vascular
dysfunction, we treated mice with the anti-malarial drug
chloroquine, which activated ATM in cultured cells. Administration
of low does chloroquine (determined in dose response experiments)
to mice resulted in p53 activation in fat and muscle as well as
liver, aorta and cultured macrophages. ATM+/+apoE-/- mice were
randomized to treatment with saline injections or 3.5 mg/kg of
chloroquine administered by intraperitoneal injection twice a week
(a total weekly does of 7.0 mg/kg) and fed a Western diet. After
eight weeks, chloroquine had no effect on body weight or metabolic
rate determined by indirect calorimetry. Animals were treated with
intraperitoneal injections of chloroquine at 3.5 mg/kg per
injection or the same volume of saline as control. Western blots
showed phospho-p53 in fate and muscle.
[0161] Activation of the ATM-p53 axis by low dose chloroquine also
had no effect on serum lipids, but decreased atherosclerosis.
Animals were treated with twice-weekly intraperitoneal injections
of chloroquine at 3.5 mg/kg per injection for a total weekly does
of 7.0 mg/kg. Fasting serum lipids at baseline on chow diet and
following Western diet. feeding for 4 and 8 weeks in CHQ- and
saline-treated mice. Lesion area was 37% less at the arch, 25% less
at the thoracic aorta, and 25% less at the abdominal aorta in
chloroquine-treated mice. Equal numbers of males and females were
studied and chloroquine was effective in both genders.
[0162] Chloroquine also improved metabolic abnormalities in
established models of insulin resistance. At the same dose that
decreased atherosclerosis in apoE null mice, chloroquine lowered
both fasting and fed glucose levels as well as fasting insulin
levels (13,360.+-.996 pg/ml for chloroquine vs. 16,810.+-.1,120 for
saline, P<0.05) in ob/ob mice and fed glucose levels in db/db
mice. Fasting blood glucose with CHQ were lower compared to
baseline and compared to the vehicle (saline) controls 3 weeks
after initiation of the treatment. Fed blood glucose levels on two
different days were lower as compared to baseline and lower than
the saline-treated controls. Saline-treated controls did not differ
from baseline in the fasting or fed state. Blood glucose in male
db/db mice were treated with either CHQ or saline. Baseline
represents the average fed blood glucose levels of the 12 db/db
mice. CHQ treated db/db mice had lower blood glucose levels as
compared to the baseline and as compared to the saline-treated
controls on two different days of the treatment period. The saline
treated animals did not differ from baseline.
[0163] ATM deficiency also affected glucose metabolism in mouse
models without spoil deficiency. In aged (>1 year old)
littermate low density lipoprotein receptor (LDLR) mice,
heterozygous ATM deficiency resulted in impaired glucose tolerance
and resistance to glucose lowering by insulin. Western diet feeding
of ATM.sup.+/- and littermate ATM.sup.+/+ mice in a wild type
C57BL/6 background also produced greater glucose intolerance in the
ATM heterozygotes. The intraperitoneal glucose tolerance testing
was conducted on 12 aged ATM.sup.+/+ and 11 aged ATM.sup.+/- (>1
year of age littermate mice on the LDLR null background eating a
chow diet. The intraperitoneal insulin tolerance test (ITT) was
also conducted on these mice. The intraperitoneal glucose tolerance
testing was also conducted for 8 ATM.sup.+/+ and 8 ATM.sup.+/-
littermate mice on a C57BL/6 background after 4 weeks on Western
diet. These data suggest that increasing ATM activity in models
other apoE null mice decreases insulin resistance.
[0164] Several lines of evidence were pursued to investigate the
ATM-dependence of the chloroquine effect. In vivo chloroquine
treatment decreased LPL enzyme activity (for mice treated
systemically with chloroquine, compared to saline treated animals
with P<0.05) and phospho-JNK activity (for mice treated
systemically with chloroquine, compared to saline treated animals
with P<0.01) in peritoneal macrophages from ATM.sup.+/+ mice but
had no effect in ATM null macrophages. Chloroquine treatment
lowered systolic pressure by 9 mm Hg in ATM mice as compared to
saline-treated ATM.sup.+/+ mice.
[0165] There was no effect of chloroquine in the ATM-deficient
animals. Chloroquine improved glucose tolerance in Western diet-fed
ATM.sup.+/+apoE.sup.-/- mice but not ATM.sup.-/-apoE.sup.-/- mice.
The glycemic response in the ATM null mice was atypical, showing a
late drop in glucose levels that could reflect abnormally delayed
release of insulin. ATM.sup.-/- pancreatic islets manifested a
prominent induction of ATM expression with lipid loading,
suggesting a role for ATM in beta cell homeostasis. Taken together,
these data suggest that several of the effects of chloroquine in
this model are ATM-dependent.
[0166] Since JNK is the proposed link between ATM and its
downstream effects, we inhibited JNK activity in elicited
macrophages (using the compound SP600125) and assayed LPL enzyme
activity. Elevated LPL activity in ATM null cells was decreased to
control levels with JNK inhibition. Increased LPL activity was also
reduced to control levels with SP600125 treatment in ATM.sup.+/-
macrophages. LPL enzyme activity of thioglycollate-elicited
macrophages obtained from ATM.sup.+/+ and ATM.sup.-/- mice (n=3 for
each) that were treated in culture with a JNK inhibitor (SP6000125)
or vehicle (DMSO). These results are consistent with a role for JNK
in the mediation of some of the effects induced by ATM deficiency
in this model.
Statistical Analyses
[0167] Statistical significance of differences was calculated
using: the Student unpaired t test for parametric data involving
two groups, ANOVA for parametric data usually with Dunnett'or
Bonferroni post tests, the Mann-Whitney test for non-parametric
data involving two groups, and the Kruskal-Wallis test for
non-parametric data with Dunn's post test.
Example 2
Human Studies
[0168] Subjects meeting the ATP III criteria for the metabolic
syndrome have been enrolled and will continue to be enrolled in a
study consisting of four limbs to test whether the administration
of low dose and very low dose chloroquine to humans activates the
ATM-p53 axis and leads to an improvement in the symptoms of
metabolic syndrome.
[0169] Subjects between the age of 18 and 55 meet the ATP III
criteria for the metabolic syndrome and do not have retinal
abnormalities, G6PD deficiency, o certain other major active
medical conditions, as described in more detail below. Each has at
least three of the following five characteristics: elevated fasting
triglycerides (greater than or equal to about 150 mg/dI), low HDL
cholesterol (less than about 50 mg/dI in women, less than about 40
mg/dI in men), hypertension (blood pressure greater than or equal
to about 130/85 mm Hg), increased waist circumference (greater than
about 35 inches in women, greater than about 40 inches in men) and
elevated fasting glucose (greater than or equal to about 100
mg/dI). If an individual takes one of the antihypertensive agents
recommended by JNC 7 for hypertension, this is accepted as meeting
the blood pressure requirement even if the measured blood pressure
is below about 130/35. Enrollment is open to children (those
between the ages of 18 and 21), males and females of all racial and
ethnic groups. Participants are screened in two stages.
[0170] First, potential subjects are interviewed, the protocol is
be explained in detail, then risks and benefits of involvement are
discussed. Interested subjects have a complete history and physical
examination (including several measurements of blood pressure
according to a standard protocol and determination of waist size,
both performed by experienced personnel in the Washington
University GCRC, St. Louis, Mo.). Fasting blood sugar and fasting
serum lipids are measured. Individuals with the following criteria
are excluded: prior travel to countries requiring malarial
prophylaxis or prior know treatment with chloroquine or
hydroxychloroquine, morbid obesity (BMI greater than or equal to
about 45), coronary artery disease, peripheral artery disease
(including carotid disease), history of stroke, chronic renal
insufficiency, diabetes, seizure disorder, psoriasis (chloroquine
has been reported to cause exacerbations of this disease through
unclear mechanisms), hematologic disorders, current malignancies,
asthma or other active respiratory disease, liver disease, active
infections, or any other serious ongoing illness (including alcohol
or drug abuse) requiring medical care. Pregnant women or those
trying to become pregnant are also excluded.
[0171] Individuals taking any prescription medications are excluded
(including lipid lowering medications) with the exception of
antihypertensive agents in men and women and oral contraceptives in
women. In terms of antihypertensive agents, individuals taking
stable doses of any single medication currently recommended by JNC
7 as appropriate initial therapy may be enrolled. Those taking more
than one agent and those with uncontrolled hypertension (defined
for this study as having blood pressure greater than or equal to
about 160/100) will be excluded. Participants are asked not to take
over-the-counter medications during the study, with the exception
of acetaminophen for minor pain. They are specifically instructed
to avoid cimetidine (which is known to increase chloroquine levels)
and vitamin B (which may decrease chloroquine uptake by cells).
[0172] Second, those meeting the entrance criteria undergo the
following tests: (including liver function tests and creatinine),
CBC, TSH, a test for glucose-6-phosphate deydrogenase, EKG, hearing
test, and a complete ophthalmology evaluation including visual
field testing and slip lamp examination. Provided all of these
results are normal and informed consent is obtained, subjects begin
the protocol as described.
[0173] Using a factor of 1/12 for converting doses expressed in
mg/kg in mice to an equivalent surface area dose expressed in mg/kg
for humans (described by Friereich et al., Cancer Chemother. Rep.,
1966, 50:219-244) corresponds to a dosage of about 40 mg per week
in a 70 kg human, or about 80 mg per week in a 120 kg human. The
calculation, based on the 7 mg/kg/week dosage used in mice with
features of metabolic syndrome to reverse atherosclerosis and
diminish symptoms of metabolic syndrome is: 120 kg.times.7
mg/kg/week[mouse does] divided by 12 [correction factor for surface
area] divided by 0.9 [correction for oral absorption] equals about
78 mg. Since the results with 80 mg/day in humans have been
surprisingly good, even lower doses such as 80 mg/week or the
equivalent are being tested.
[0174] Enrolled subjects with the metabolic syndrome are treated
with a placebo for three weeks for limb 1, oral chloroquine at 80
mg/day (8% of the dose used in previous studies to define the
metabolic effects of chloroquine) for three weeks for limb 2, oral
chloroquine at 80 mg/week for three weeks for limb 3, and
chloroquine at 250 mg/day (25% of the dose used in previous
metabolic studies) for three weeks for limb 4. At the end of each
treatment period, participants have measurements of fasting serum
lipids, glucose tolerance testing, a variety of other serum
measurements including chemistries and markers of vascular disease,
undergo ambulatory blood pressure monitoring, and insulin
resistance is quantified by means of the
hyperinsulinemic-euglycemic clamp procedure. Glucose and insulin
tolerance tests are generally performed using D-glucose (1 g/kg)
and insulin (0.75 U/kg), which are administered in separate
procedures followed by serial measurements of blood glucose. If
necessary, an oral glucose tolerance test (OGTT) will be performed
the day after completing the clamp. After an overnight fast, an
intravenous catheter will be placed, baseline labs will be
obtained, 75 g of glucose will be consumed, and blood glucose,
insulin, C peptide, and glucagon will be determined every half hour
for 2 hours.
[0175] Two subjects with the metabolic syndrome have completed the
protocol. The mean glucose infusion rate to maintain euglycemia (90
mg/dI) at steady state during the first stage of the clamp (using
an insulin infusion rate of 15 mU/m.sup.2/min) for these two
subjects was determined. Chloroquine at a dose of 80 mg per day
resulted in a 63% increase in glucose infusion rate to maintain the
blood glucose, indicating a substantial increase in glucose
infusion rate to maintain the blood glucose, indicating a
substantial increase in insulin sensitivity. This 63% increase was
a surprising result considering the low does of chloroquine
administered. (Previous studies using high chloroquine doses of
1000 mg/kg for three days detected only a 13% increase in the
glucose infusion rate required to maintain euglycemia in patients
with type 2 diabetes.) Individual responses were nearly identical
for each subject. Glucose infusion rates were appropriately higher
for the euglycemia second stage of the clamp (insulin at 40
mU/M.sup.2/min in each limb), with 80 mg dose of chloroquine
increasing the glucose infusion rate by 23% compared to the
placebo, and the 250 mg dose having a blunted effect, the same
pattern seen for the first stage of the clamps.
[0176] Clamps are performed with infusion of stable isotope-labeled
glucose to allow the determination of both glucose disposal rates
and hepatic glucose output during hyperinsulinemic-euglycemic
clamps in subjects with the metabolic syndrome. The results are
presented as the mean for two subjects. Baseline glucose disposal
(at the absence of insulin infusion) under these conditions was
undetectable. Low dose chloroquine in these human subjects with the
metabolic syndrome had an insulin sensitizing effect on both
peripheral tissues and the liver. Chloroquine at the low dose of 86
mg/day increased glucose disposal in peripheral tissues by 62%, a
surprisingly high amount, and decreased hepatic glucose production
by 58% during the first stage of the clamp.
[0177] Fasting lipids and lipoproteins in subjects with the
metabolic syndrome were determined after 3 weeks of treatment with
placebo, chloroquine 80 mg/day, or chloroquine 250 mg/day. Data are
the average of two subjects in mg/dI.
[0178] Chloroquine also had beneficial effects on fasting lipids in
these two subjects with metabolic syndrome. Chloroquine at 80
mg/day for three weeks was associated with a 25% decrease in LDL
cholesterol, a 17% decrease in total cholesterol and a 10% decrease
in triglycerides (as shown in Table 3). Ambulatory blood pressures
(systolic, diastolic, mean arterial for the entire study period and
for the 6 am to midnight and midnight to 6 am periods) did not
appear to be affected in these tow subjects. Glucose tolerance
tests were also unaffected. body weight and diet did not change
during this protocol.
TABLE-US-00003 TABLE 3 Placebo CHQ 80 CHQ 250 Total Cholesterol 206
172 169 LDL cholesterol 132 100 99 HDL cholesterol 47 47 51
Triglycerides 139 125 129
[0179] Monocyte/macrophages were also successfully isolated from
these subjects during each limb of the protocol. Anticoagulated
blood was added to commercially available tubes containing
Histopaque, and mononuclear cells were collected after
centrifugation. These cells were subjected to a brief second spin
using a discontinuous sucrose gradient to remove platelets (which
can interfere with the adherence of monocytes to culture dishes),
then the mononuclear cells were placed in tissue culture dishes.
Two hours later, lymphocytes were washed away, and adherent
monocytes were collected and frozen in appropriate media so that
cells from different limbs of the protocol can subsequently be
studies in the same assays. Chloroquine treatment resulted in
activation of ATM and p53 in monocytes, the cell type responsible
for the initiation of atherosclerotic lesions. These western blots
showed activated ATM and activated p53 in monocytes from the same
subject following either three weeks of 80 mg/day chloroquine or
three weeks of 250 mg/day chloroquine. These western blots showed
no activation of ATM or p53 in monocytes isolated from a subject
receiving placebo. For activated ATM determinations, lysates were
immunoprecipitated using a total ATM antibody (D1611). The
immunoprecipitated samples were blotted for activated ATM using a
monoclonal antibody recognizing Ser1981P-ATM. The gels included a
positive control of 293 cells treated with camptothecin. Total
lysates were used to blot activated p53 using a polyclonal antibody
to Ser15P-p53 (cell signaling). To confirm the p53 data, total
lysates were tested using an ELISA-based p53 DNA binding assay
(TransAM p53 activity assay from Active Motif). These results
showed a near-linear increase in p53 binding to the consensus
binding site with increasing chloroquine dose. the same results
were also seen using monocytes from another subjects.
[0180] The pattern for ATM mirrored that for insulin sensitivity,
with greater activation at the low 80 mg/day dose and a blunted
effect at the higher 250 mg/day dose. For p53, activation was
directly related tot he chloroquine dose. Using the same lysates,
phopho-JNK activity (by ELISA) was decreased by chloroquine in a
dose-dependent fashion.
[0181] These data show that ATM deficiency promotes metabolic
syndrome and vascular disease in a mouse model, and low dose
chloroquine treatment in the mouse model decreases vascular disease
and has metabolic effects (including suppression of JNK activity)
that are ATM-dependent, and low dose chloroquine in humans improves
insulin sensitivity and dyslipidemia. The effects in humans appear
to be associated with activation of ATM and p53 in monocytes as
well as decreased JNK activity.
Long Term Studies
[0182] These data show that administration of low dose chloroquine
in mice decreases atherosclerosis, lowers blood pressure and
improves glucose intolerance and suggests that analogous
administration of low dose chloroquine in humans (80 mg/day)
improves insulin sensitivity and dyslipidemia. A randomized,
parallel group, placebo-controlled study of the effect of
chloroquine in human subjects with the metabolic syndrome is
ongoing to determine whether chronic or prolonged low dose
chloroquine treatment also decreases vascular disease in people
with the metabolic syndrome. In the active treatment group for this
study, the total chloroquine dose is 29 grams (assuming complete
compliance).
[0183] Retinal toxicity is known to be a problem associated with
chronic chloroquine therapy. It is believed that the lowest
cumulative dose associated with this side effect is about 125
grams. In the active treatment groups for the present study, the
total cumulative chloroquine dose is proposed to be 29 grams
(assuming complete compliance. Additionally, every participant
undergoes baseline eye exams and repeat exams at 6 and 12 months.
Plasma chloroquine levels are also monitored during the active
treatment portion of the study and at the 24 month time point.
[0184] A consideration for prolonged treatment is that chloroquine
remains in tissues for a long time. Beneficial effects of the drug
may persist after discontinuation of treatment. To address this
issue, subjects from both the placebo and chloroquine arms of the
human study will stop taking capsules at 12 months but return at 24
months for carotid IMT, OGTT and blood draws. If enhanced insulin
sensitivity and less carotid progression are seen in the
chloroquine group, this observation could provide time foundation
for intermittent, very low dose chloroquine as an appropriate
treatment for the metabolic syndrome.
Summary
[0185] Metabolic syndrome is a common disorder associated with
insulin resistance and atherosclerosis. The present data show that
deficiency of one or two alleles of ATM, the protein mutated in the
cancer-prone disorder ataxia telangiectasia, worsens features of
the metabolic syndrome and accelerates atherosclerosis in Western
diet-fed apoE-/- mice. Hyperinsulinemic-euglycemic clamps showed
these animals to have hepatic insulin resistance. Atherosclerosis
was greater when animals were transplanted with ATM-/- as compared
to ATM+/+ marrow, consistent with a role for marrow-derived cells
such as macrophages in ATM-deficient vascular disease. Jun
N-terminal kinase (JNK) activity was increased in ATM-deficient
cells including macrophages. Treatment of ATM+/+apoE-/- mice with
low dose chloroquine, an ATM activator, decreased atherosclerosis.
In an ATM-dependent manner, chloroquine decreased macrophage JNK
activity, decreased macrophage lipoprotein lipase activity (a
proatherogenic consequence of JNK activation), decreased blood
pressure, and improved glucose tolerance. Chloroquine also improved
metabolic abnormalities in ob/ob and db/db mice. These results
suggest that a ATM-dependent stress pathways mediate susceptibility
to the metabolic syndrome and that chloroquine or related agents
promoting ATM activity could represent a novel approach for
modulating insulin resistance and decreasing vascular disease.
Discussion
[0186] Metabolic syndrome is common and potentially lethal.
Treatment is limited to therapies for the disparate components of
the syndrome; hypertension, hyperglycemia, obesity, and
dyslipidemia. The current data demonstrate that apoE null mice with
ATM haploinsufficiency have accelerated features of the metabolic
syndrome. They are insulin resistant with increased JNK activity.
Treatment of ATM+/+ mice in the apoE null background with low doses
of the ATM-activating drug chloroquine lowers blood pressure,
improves glucose tolerances, suppresses JNK activity, and decreases
diet-induced atherosclerosis. Chloroquine also improves metabolic
abnormalities in ob/ob and db/db mice. Collectively, these findings
suggest that modulation of ATM-dependent signaling with chloroquine
represents a potential treatment for the metabolic syndrome.
[0187] Cholesterol levels were modestly higher th ATM+/-apoE-/- as
compared to ATM+/+apoE-/- mice in the current study on a chow diet
at baseline but this difference was not significant, and the
inventors did not detect significant genotype effects on serum
cholesterol at subsequent time points (3 and 8 weeks) in mice fed a
Western diet. These results suggest that in the setting of a
Western diet, events independent of differences in cholesterol
promote atherogenesis in ATM deficiency.
[0188] ATM has been linked to insulin signaling in certain cell
types and it has been suggested that individuals heterozygous for
ATM deficiency are prone to cardiovascular disease. No mechanistic
insights have existed to explain these associations. The current
data link ATM and signaling pathways known to affect atherogenesis
and responses to insulin and elucidates novel mechanisms for these
pathways. JNK is an attractive potential mediator of the metabolic
effects caused by ATM deficiency. Activated JNK phosphorylates
cJun, which combines with cFos to form the AP-1 transcription
factor complex. Activation of both JNK and the AP-1 pathway are
present in the brains of ATM-deficient mice. JNK activity is linked
to several features of the metabolic syndrome. JNK activity is
increased by endoplasmic reticulum stress caused by obesity. Mice
deficient in JNK1, one of three isoforms, are protected from
obesity and insulin resistance. Expressing wild type JNK decreases
insulin sensitivity in liver, while expressing dominant negative
JNK increases insulin sensitivity in liver. JNK decreases the
expression of adiponectin, which has insulin sensitizing effects,
in adipocyte cell lines. Treatment of animals with a JNK inhibitor
enhances insulin sensitivity. INK interferes with insulin signaling
by phosphorylating serine residue 307 on IRS-1. Inhibition of JNK
activity and genetic JNK2 deficiency in apoE null mice decreases
atherosclerosis.
[0189] The present data show that genetic deficiency of ATM
increases JNK activity in multiple tissues, and is associated with
systemic insulin resistance, serine 307 phosphorylation of IRS-1
and accelerated atherosclerosis. Disruption of ATM expression in
cultured cells induces JNK, and transplantation of mice with
ATM-deficient marrow increases atherosclerosis in the setting of
macrophages with increased JNK activity. JNK induces Egr-1 and LPL
and both were increased in ATM-deficient macrophages. Egr-1 has
been shown to promote atherosclerosis in apoE null mice and a
proatherogenic role for LPL in macrophages has been demonstrated,
thus providing a mechanism for increased atherosclerosis. Treatment
with chloroquine suppresses JNK activity and LPL activity in
macrophages from ATM+/+ but not ATM-/- mice, and increased LPL
activity in ATM-deficient macrophages is normalized by JNK
inhibition, suggesting that ATM-dependent suppression of JNK
mediates susceptibility to metabolic effects. Thus,
atherosclerosis, insulin resistance, and the component clinical
features of the metabolic syndrome may share a common unifying
mechanism related to ATM deficiency.
[0190] ATM signals to the p53 tumor suppressor gene product.
Interestingly, p53 has been reported to reduce atherogenesis in
mouse models, to activate JNK, and to mediate an antioxidant
function, potentially relevant to ATM and the metabolic syndrome.
Markers of oxidative stress are increased in mice and humans with
ATM deficiency and inhibition of oxidative stress pathways
decreases reactive oxygen species and improves metabolic syndrome
in obese mice.
[0191] The present inventors explored the relationship between ATM
and metabolic syndrome in part because the loss of ATM worsens
metabolic parameters in apoE null mice, and the inventors
previously found that chloroquine can activate the ATM kinase
(Bakkenist and Kastan, 2003). The linking and testing of these
observations allowed the present inventors to demonstrate the
ATM-dependence of many of the metabolic effects of chloroquine, a
dependence not previously suspected.
[0192] In summary, ATM deficiency is a novel mechanism contributing
to development of the metabolic syndrome. The present results
provide evidence that modulating ATM-dependent signaling with low
doses of chloroquine, comparable to .about.40 mg/week in a 70 kg
human, reduces vascular disease.
[0193] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of specific
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the scope of the invention as defined by the appended
claims.
[0194] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
1813056PRTHomo sapiens 1Met Ser Leu Val Leu Asn Asp Leu Leu Ile Cys
Cys Arg Gln Leu Glu 1 5 10 15 His Asp Arg Ala Thr Glu Arg Lys Lys
Glu Val Glu Lys Phe Lys Arg 20 25 30 Leu Ile Arg Asp Pro Glu Thr
Ile Lys His Leu Asp Arg His Ser Asp 35 40 45 Ser Lys Gln Gly Lys
Tyr Leu Asn Trp Asp Ala Val Phe Arg Phe Leu 50 55 60 Gln Lys Tyr
Ile Gln Lys Glu Thr Glu Cys Leu Arg Ile Ala Lys Pro 65 70 75 80 Asn
Val Ser Ala Ser Thr Gln Ala Ser Arg Gln Lys Lys Met Gln Glu 85 90
95 Ile Ser Ser Leu Val Lys Tyr Phe Ile Lys Cys Ala Asn Arg Arg Ala
100 105 110 Pro Arg Leu Lys Cys Gln Glu Leu Leu Asn Tyr Ile Met Asp
Thr Val 115 120 125 Lys Asp Ser Ser Asn Gly Ala Ile Tyr Gly Ala Asp
Cys Ser Asn Ile 130 135 140 Leu Leu Lys Asp Ile Leu Ser Val Arg Lys
Tyr Trp Cys Glu Ile Ser 145 150 155 160 Gln Gln Gln Trp Leu Glu Leu
Phe Ser Val Tyr Phe Arg Leu Tyr Leu 165 170 175 Lys Pro Ser Gln Asp
Val His Arg Val Leu Val Ala Arg Ile Ile His 180 185 190 Ala Val Thr
Lys Gly Cys Cys Ser Gln Thr Asp Gly Leu Asn Ser Lys 195 200 205 Phe
Leu Asp Phe Phe Ser Lys Ala Ile Gln Cys Ala Arg Gln Glu Lys 210 215
220 Ser Ser Ser Gly Leu Asn His Ile Leu Ala Ala Leu Thr Ile Phe Leu
225 230 235 240 Lys Thr Leu Ala Val Asn Phe Arg Ile Arg Val Cys Glu
Leu Gly Asp 245 250 255 Glu Ile Leu Pro Thr Leu Leu Tyr Ile Trp Thr
Gln His Arg Leu Asn 260 265 270 Asp Ser Leu Lys Glu Val Ile Ile Glu
Leu Phe Gln Leu Gln Ile Tyr 275 280 285 Ile His His Pro Lys Gly Ala
Lys Thr Gln Glu Lys Gly Ala Tyr Glu 290 295 300 Ser Thr Lys Trp Arg
Ser Ile Leu Tyr Asn Leu Tyr Asp Leu Leu Val 305 310 315 320 Asn Glu
Ile Ser His Ile Gly Ser Arg Gly Lys Tyr Ser Ser Gly Phe 325 330 335
Arg Asn Ile Ala Val Lys Glu Asn Leu Ile Glu Leu Met Ala Asp Ile 340
345 350 Cys His Gln Val Phe Asn Glu Asp Thr Arg Ser Leu Glu Ile Ser
Gln 355 360 365 Ser Tyr Thr Thr Thr Gln Arg Glu Ser Ser Asp Tyr Ser
Val Pro Cys 370 375 380 Lys Arg Lys Lys Ile Glu Leu Gly Trp Glu Val
Ile Lys Asp His Leu 385 390 395 400 Gln Lys Ser Gln Asn Asp Phe Asp
Leu Val Pro Trp Leu Gln Ile Ala 405 410 415 Thr Gln Leu Ile Ser Lys
Tyr Pro Ala Ser Leu Pro Asn Cys Glu Leu 420 425 430 Ser Pro Leu Leu
Met Ile Leu Ser Gln Leu Leu Pro Gln Gln Arg His 435 440 445 Gly Glu
Arg Thr Pro Tyr Val Leu Arg Cys Leu Thr Glu Val Ala Leu 450 455 460
Cys Gln Asp Lys Arg Ser Asn Leu Glu Ser Ser Gln Lys Ser Asp Leu 465
470 475 480 Leu Lys Leu Trp Asn Lys Ile Trp Cys Ile Thr Phe Arg Gly
Ile Ser 485 490 495 Ser Glu Gln Ile Gln Ala Glu Asn Phe Gly Leu Leu
Gly Ala Ile Ile 500 505 510 Gln Gly Ser Leu Val Glu Val Asp Arg Glu
Phe Trp Lys Leu Phe Thr 515 520 525 Gly Ser Ala Cys Arg Pro Ser Cys
Pro Ala Val Cys Cys Leu Thr Leu 530 535 540 Ala Leu Thr Thr Ser Ile
Val Pro Gly Ala Val Lys Met Gly Ile Glu 545 550 555 560 Gln Asn Met
Cys Glu Val Asn Arg Ser Phe Ser Leu Lys Glu Ser Ile 565 570 575 Met
Lys Trp Leu Leu Phe Tyr Gln Leu Glu Gly Asp Leu Glu Asn Ser 580 585
590 Thr Glu Val Pro Pro Ile Leu His Ser Asn Phe Pro His Leu Val Leu
595 600 605 Glu Lys Ile Leu Val Ser Leu Thr Met Lys Asn Cys Lys Ala
Ala Met 610 615 620 Asn Phe Phe Gln Ser Val Pro Glu Cys Glu His His
Gln Lys Asp Lys 625 630 635 640 Glu Glu Leu Ser Phe Ser Glu Val Glu
Glu Leu Phe Leu Gln Thr Thr 645 650 655 Phe Asp Lys Met Asp Phe Leu
Thr Ile Val Arg Glu Cys Gly Ile Glu 660 665 670 Lys His Gln Ser Ser
Ile Gly Phe Ser Val His Gln Asn Leu Lys Glu 675 680 685 Ser Leu Asp
Arg Cys Leu Leu Gly Leu Ser Glu Gln Leu Leu Asn Asn 690 695 700 Tyr
Ser Ser Glu Ile Thr Asn Ser Glu Thr Leu Val Arg Cys Ser Arg 705 710
715 720 Leu Leu Val Gly Val Leu Gly Cys Tyr Cys Tyr Met Gly Val Ile
Ala 725 730 735 Glu Glu Glu Ala Tyr Lys Ser Glu Leu Phe Gln Lys Ala
Asn Ser Leu 740 745 750 Met Gln Cys Ala Gly Glu Ser Ile Thr Leu Phe
Lys Asn Lys Thr Asn 755 760 765 Glu Glu Phe Arg Ile Gly Ser Leu Arg
Asn Met Met Gln Leu Cys Thr 770 775 780 Arg Cys Leu Ser Asn Cys Thr
Lys Lys Ser Pro Asn Lys Ile Ala Ser 785 790 795 800 Gly Phe Phe Leu
Arg Leu Leu Thr Ser Lys Leu Met Asn Asp Ile Ala 805 810 815 Asp Ile
Cys Lys Ser Leu Ala Ser Phe Ile Lys Lys Pro Phe Asp Arg 820 825 830
Gly Glu Val Glu Ser Met Glu Asp Asp Thr Asn Gly Asn Leu Met Glu 835
840 845 Val Glu Asp Gln Ser Ser Met Asn Leu Phe Asn Asp Tyr Pro Asp
Ser 850 855 860 Ser Val Ser Asp Ala Asn Glu Pro Gly Glu Ser Gln Ser
Thr Ile Gly 865 870 875 880 Ala Ile Asn Pro Leu Ala Glu Glu Tyr Leu
Ser Lys Gln Asp Leu Leu 885 890 895 Phe Leu Asp Met Leu Lys Phe Leu
Cys Leu Cys Val Thr Thr Ala Gln 900 905 910 Thr Asn Thr Val Ser Phe
Arg Ala Ala Asp Ile Arg Arg Lys Leu Leu 915 920 925 Met Leu Ile Asp
Ser Ser Thr Leu Glu Pro Thr Lys Ser Leu His Leu 930 935 940 His Met
Tyr Leu Met Leu Leu Lys Glu Leu Pro Gly Glu Glu Tyr Pro 945 950 955
960 Leu Pro Met Glu Asp Val Leu Glu Leu Leu Lys Pro Leu Ser Asn Val
965 970 975 Cys Ser Leu Tyr Arg Arg Asp Gln Asp Val Cys Lys Thr Ile
Leu Asn 980 985 990 His Val Leu His Val Val Lys Asn Leu Gly Gln Ser
Asn Met Asp Ser 995 1000 1005 Glu Asn Thr Arg Asp Ala Gln Gly Gln
Phe Leu Thr Val Ile Gly 1010 1015 1020 Ala Phe Trp His Leu Thr Lys
Glu Arg Lys Tyr Ile Phe Ser Val 1025 1030 1035 Arg Met Ala Leu Val
Asn Cys Leu Lys Thr Leu Leu Glu Ala Asp 1040 1045 1050 Pro Tyr Ser
Lys Trp Ala Ile Leu Asn Val Met Gly Lys Asp Phe 1055 1060 1065 Pro
Val Asn Glu Val Phe Thr Gln Phe Leu Ala Asp Asn His His 1070 1075
1080 Gln Val Arg Met Leu Ala Ala Glu Ser Ile Asn Arg Leu Phe Gln
1085 1090 1095 Asp Thr Lys Gly Asp Ser Ser Arg Leu Leu Lys Ala Leu
Pro Leu 1100 1105 1110 Lys Leu Gln Gln Thr Ala Phe Glu Asn Ala Tyr
Leu Lys Ala Gln 1115 1120 1125 Glu Gly Met Arg Glu Met Ser His Ser
Ala Glu Asn Pro Glu Thr 1130 1135 1140 Leu Asp Glu Ile Tyr Asn Arg
Lys Ser Val Leu Leu Thr Leu Ile 1145 1150 1155 Ala Val Val Leu Ser
Cys Ser Pro Ile Cys Glu Lys Gln Ala Leu 1160 1165 1170 Phe Ala Leu
Cys Lys Ser Val Lys Glu Asn Gly Leu Glu Pro His 1175 1180 1185 Leu
Val Lys Lys Val Leu Glu Lys Val Ser Glu Thr Phe Gly Tyr 1190 1195
1200 Arg Arg Leu Glu Asp Phe Met Ala Ser His Leu Asp Tyr Leu Val
1205 1210 1215 Leu Glu Trp Leu Asn Leu Gln Asp Thr Glu Tyr Asn Leu
Ser Ser 1220 1225 1230 Phe Pro Phe Ile Leu Leu Asn Tyr Thr Asn Ile
Glu Asp Phe Tyr 1235 1240 1245 Arg Ser Cys Tyr Lys Val Leu Ile Pro
His Leu Val Ile Arg Ser 1250 1255 1260 His Phe Asp Glu Val Lys Ser
Ile Ala Asn Gln Ile Gln Glu Asp 1265 1270 1275 Trp Lys Ser Leu Leu
Thr Asp Cys Phe Pro Lys Ile Leu Val Asn 1280 1285 1290 Ile Leu Pro
Tyr Phe Ala Tyr Glu Gly Thr Arg Asp Ser Gly Met 1295 1300 1305 Ala
Gln Gln Arg Glu Thr Ala Thr Lys Val Tyr Asp Met Leu Lys 1310 1315
1320 Ser Glu Asn Leu Leu Gly Lys Gln Ile Asp His Leu Phe Ile Ser
1325 1330 1335 Asn Leu Pro Glu Ile Val Val Glu Leu Leu Met Thr Leu
His Glu 1340 1345 1350 Pro Ala Asn Ser Ser Ala Ser Gln Ser Thr Asp
Leu Cys Asp Phe 1355 1360 1365 Ser Gly Asp Leu Asp Pro Ala Pro Asn
Pro Pro His Phe Pro Ser 1370 1375 1380 His Val Ile Lys Ala Thr Phe
Ala Tyr Ile Ser Asn Cys His Lys 1385 1390 1395 Thr Lys Leu Lys Ser
Ile Leu Glu Ile Leu Ser Lys Ser Pro Asp 1400 1405 1410 Ser Tyr Gln
Lys Ile Leu Leu Ala Ile Cys Glu Gln Ala Ala Glu 1415 1420 1425 Thr
Asn Asn Val Tyr Lys Lys His Arg Ile Leu Lys Ile Tyr His 1430 1435
1440 Leu Phe Val Ser Leu Leu Leu Lys Asp Ile Lys Ser Gly Leu Gly
1445 1450 1455 Gly Ala Trp Ala Phe Val Leu Arg Asp Val Ile Tyr Thr
Leu Ile 1460 1465 1470 His Tyr Ile Asn Gln Arg Pro Ser Cys Ile Met
Asp Val Ser Leu 1475 1480 1485 Arg Ser Phe Ser Leu Cys Cys Asp Leu
Leu Ser Gln Val Cys Gln 1490 1495 1500 Thr Ala Val Thr Tyr Cys Lys
Asp Ala Leu Glu Asn His Leu His 1505 1510 1515 Val Ile Val Gly Thr
Leu Ile Pro Leu Val Tyr Glu Gln Val Glu 1520 1525 1530 Val Gln Lys
Gln Val Leu Asp Leu Leu Lys Tyr Leu Val Ile Asp 1535 1540 1545 Asn
Lys Asp Asn Glu Asn Leu Tyr Ile Thr Ile Lys Leu Leu Asp 1550 1555
1560 Pro Phe Pro Asp His Val Val Phe Lys Asp Leu Arg Ile Thr Gln
1565 1570 1575 Gln Lys Ile Lys Tyr Ser Arg Gly Pro Phe Ser Leu Leu
Glu Glu 1580 1585 1590 Ile Asn His Phe Leu Ser Val Ser Val Tyr Asp
Ala Leu Pro Leu 1595 1600 1605 Thr Arg Leu Glu Gly Leu Lys Asp Leu
Arg Arg Gln Leu Glu Leu 1610 1615 1620 His Lys Asp Gln Met Val Asp
Ile Met Arg Ala Ser Gln Asp Asn 1625 1630 1635 Pro Gln Asp Gly Ile
Met Val Lys Leu Val Val Asn Leu Leu Gln 1640 1645 1650 Leu Ser Lys
Met Ala Ile Asn His Thr Gly Glu Lys Glu Val Leu 1655 1660 1665 Glu
Ala Val Gly Ser Cys Leu Gly Glu Val Gly Pro Ile Asp Phe 1670 1675
1680 Ser Thr Ile Ala Ile Gln His Ser Lys Asp Ala Ser Tyr Thr Lys
1685 1690 1695 Ala Leu Lys Leu Phe Glu Asp Lys Glu Leu Gln Trp Thr
Phe Ile 1700 1705 1710 Met Leu Thr Tyr Leu Asn Asn Thr Leu Val Glu
Asp Cys Val Lys 1715 1720 1725 Val Arg Ser Ala Ala Val Thr Cys Leu
Lys Asn Ile Leu Ala Thr 1730 1735 1740 Lys Thr Gly His Ser Phe Trp
Glu Ile Tyr Lys Met Thr Thr Asp 1745 1750 1755 Pro Met Leu Ala Tyr
Leu Gln Pro Phe Arg Thr Ser Arg Lys Lys 1760 1765 1770 Phe Leu Glu
Val Pro Arg Phe Asp Lys Glu Asn Pro Phe Glu Gly 1775 1780 1785 Leu
Asp Asp Ile Asn Leu Trp Ile Pro Leu Ser Glu Asn His Asp 1790 1795
1800 Ile Trp Ile Lys Thr Leu Thr Cys Ala Phe Leu Asp Ser Gly Gly
1805 1810 1815 Thr Lys Cys Glu Ile Leu Gln Leu Leu Lys Pro Met Cys
Glu Val 1820 1825 1830 Lys Thr Asp Phe Cys Gln Thr Val Leu Pro Tyr
Leu Ile His Asp 1835 1840 1845 Ile Leu Leu Gln Asp Thr Asn Glu Ser
Trp Arg Asn Leu Leu Ser 1850 1855 1860 Thr His Val Gln Gly Phe Phe
Thr Ser Cys Leu Arg His Phe Ser 1865 1870 1875 Gln Thr Ser Arg Ser
Thr Thr Pro Ala Asn Leu Asp Ser Glu Ser 1880 1885 1890 Glu His Phe
Phe Arg Cys Cys Leu Asp Lys Lys Ser Gln Arg Thr 1895 1900 1905 Met
Leu Ala Val Val Asp Tyr Met Arg Arg Gln Lys Arg Pro Ser 1910 1915
1920 Ser Gly Thr Ile Phe Asn Asp Ala Phe Trp Leu Asp Leu Asn Tyr
1925 1930 1935 Leu Glu Val Ala Lys Val Ala Gln Ser Cys Ala Ala His
Phe Thr 1940 1945 1950 Ala Leu Leu Tyr Ala Glu Ile Tyr Ala Asp Lys
Lys Ser Met Asp 1955 1960 1965 Asp Gln Glu Lys Arg Ser Leu Ala Phe
Glu Glu Gly Ser Gln Ser 1970 1975 1980 Thr Thr Ile Ser Ser Leu Ser
Glu Lys Ser Lys Glu Glu Thr Gly 1985 1990 1995 Ile Ser Leu Gln Asp
Leu Leu Leu Glu Ile Tyr Arg Ser Ile Gly 2000 2005 2010 Glu Pro Asp
Ser Leu Tyr Gly Cys Gly Gly Gly Lys Met Leu Gln 2015 2020 2025 Pro
Ile Thr Arg Leu Arg Thr Tyr Glu His Glu Ala Met Trp Gly 2030 2035
2040 Lys Ala Leu Val Thr Tyr Asp Leu Glu Thr Ala Ile Pro Ser Ser
2045 2050 2055 Thr Arg Gln Ala Gly Ile Ile Gln Ala Leu Gln Asn Leu
Gly Leu 2060 2065 2070 Cys His Ile Leu Ser Val Tyr Leu Lys Gly Leu
Asp Tyr Glu Asn 2075 2080 2085 Lys Asp Trp Cys Pro Glu Leu Glu Glu
Leu His Tyr Gln Ala Ala 2090 2095 2100 Trp Arg Asn Met Gln Trp Asp
His Cys Thr Ser Val Ser Lys Glu 2105 2110 2115 Val Glu Gly Thr Ser
Tyr His Glu Ser Leu Tyr Asn Ala Leu Gln 2120 2125 2130 Ser Leu Arg
Asp Arg Glu Phe Ser Thr Phe Tyr Glu Ser Leu Lys 2135 2140 2145 Tyr
Ala Arg Val Lys Glu Val Glu Glu Met Cys Lys Arg Ser Leu 2150 2155
2160 Glu Ser Val Tyr Ser Leu Tyr Pro Thr Leu Ser Arg Leu Gln Ala
2165 2170 2175 Ile Gly Glu Leu Glu Ser Ile Gly Glu Leu Phe Ser Arg
Ser Val 2180 2185 2190 Thr His Arg Gln Leu Ser Glu Val Tyr Ile Lys
Trp Gln Lys His 2195 2200 2205 Ser Gln Leu Leu Lys Asp Ser Asp Phe
Ser Phe Gln Glu Pro Ile 2210 2215 2220 Met Ala Leu Arg Thr Val Ile
Leu Glu Ile Leu Met Glu Lys Glu 2225 2230 2235 Met Asp Asn Ser Gln
Arg Glu
Cys Ile Lys Asp Ile Leu Thr Lys 2240 2245 2250 His Leu Val Glu Leu
Ser Ile Leu Ala Arg Thr Phe Lys Asn Thr 2255 2260 2265 Gln Leu Pro
Glu Arg Ala Ile Phe Gln Ile Lys Gln Tyr Asn Ser 2270 2275 2280 Val
Ser Cys Gly Val Ser Glu Trp Gln Leu Glu Glu Ala Gln Val 2285 2290
2295 Phe Trp Ala Lys Lys Glu Gln Ser Leu Ala Leu Ser Ile Leu Lys
2300 2305 2310 Gln Met Ile Lys Lys Leu Asp Ala Ser Cys Ala Ala Asn
Asn Pro 2315 2320 2325 Ser Leu Lys Leu Thr Tyr Thr Glu Cys Leu Arg
Val Cys Gly Asn 2330 2335 2340 Trp Leu Ala Glu Thr Cys Leu Glu Asn
Pro Ala Val Ile Met Gln 2345 2350 2355 Thr Tyr Leu Glu Lys Ala Val
Glu Val Ala Gly Asn Tyr Asp Gly 2360 2365 2370 Glu Ser Ser Asp Glu
Leu Arg Asn Gly Lys Met Lys Ala Phe Leu 2375 2380 2385 Ser Leu Ala
Arg Phe Ser Asp Thr Gln Tyr Gln Arg Ile Glu Asn 2390 2395 2400 Tyr
Met Lys Ser Ser Glu Phe Glu Asn Lys Gln Ala Leu Leu Lys 2405 2410
2415 Arg Ala Lys Glu Glu Val Gly Leu Leu Arg Glu His Lys Ile Gln
2420 2425 2430 Thr Asn Arg Tyr Thr Val Lys Val Gln Arg Glu Leu Glu
Leu Asp 2435 2440 2445 Glu Leu Ala Leu Arg Ala Leu Lys Glu Asp Arg
Lys Arg Phe Leu 2450 2455 2460 Cys Lys Ala Val Glu Asn Tyr Ile Asn
Cys Leu Leu Ser Gly Glu 2465 2470 2475 Glu His Asp Met Trp Val Phe
Arg Leu Cys Ser Leu Trp Leu Glu 2480 2485 2490 Asn Ser Gly Val Ser
Glu Val Asn Gly Met Met Lys Arg Asp Gly 2495 2500 2505 Met Lys Ile
Pro Thr Tyr Lys Phe Leu Pro Leu Met Tyr Gln Leu 2510 2515 2520 Ala
Ala Arg Met Gly Thr Lys Met Met Gly Gly Leu Gly Phe His 2525 2530
2535 Glu Val Leu Asn Asn Leu Ile Ser Arg Ile Ser Met Asp His Pro
2540 2545 2550 His His Thr Leu Phe Ile Ile Leu Ala Leu Ala Asn Ala
Asn Arg 2555 2560 2565 Asp Glu Phe Leu Thr Lys Pro Glu Val Ala Arg
Arg Ser Arg Ile 2570 2575 2580 Thr Lys Asn Val Pro Lys Gln Ser Ser
Gln Leu Asp Glu Asp Arg 2585 2590 2595 Thr Glu Ala Ala Asn Arg Ile
Ile Cys Thr Ile Arg Ser Arg Arg 2600 2605 2610 Pro Gln Met Val Arg
Ser Val Glu Ala Leu Cys Asp Ala Tyr Ile 2615 2620 2625 Ile Leu Ala
Asn Leu Asp Ala Thr Gln Trp Lys Thr Gln Arg Lys 2630 2635 2640 Gly
Ile Asn Ile Pro Ala Asp Gln Pro Ile Thr Lys Leu Lys Asn 2645 2650
2655 Leu Glu Asp Val Val Val Pro Thr Met Glu Ile Lys Val Asp His
2660 2665 2670 Thr Gly Glu Tyr Gly Asn Leu Val Thr Ile Gln Ser Phe
Lys Ala 2675 2680 2685 Glu Phe Arg Leu Ala Gly Gly Val Asn Leu Pro
Lys Ile Ile Asp 2690 2695 2700 Cys Val Gly Ser Asp Gly Lys Glu Arg
Arg Gln Leu Val Lys Gly 2705 2710 2715 Arg Asp Asp Leu Arg Gln Asp
Ala Val Met Gln Gln Val Phe Gln 2720 2725 2730 Met Cys Asn Thr Leu
Leu Gln Arg Asn Thr Glu Thr Arg Lys Arg 2735 2740 2745 Lys Leu Thr
Ile Cys Thr Tyr Lys Val Val Pro Leu Ser Gln Arg 2750 2755 2760 Ser
Gly Val Leu Glu Trp Cys Thr Gly Thr Val Pro Ile Gly Glu 2765 2770
2775 Phe Leu Val Asn Asn Glu Asp Gly Ala His Lys Arg Tyr Arg Pro
2780 2785 2790 Asn Asp Phe Ser Ala Phe Gln Cys Gln Lys Lys Met Met
Glu Val 2795 2800 2805 Gln Lys Lys Ser Phe Glu Glu Lys Tyr Glu Val
Phe Met Asp Val 2810 2815 2820 Cys Gln Asn Phe Gln Pro Val Phe Arg
Tyr Phe Cys Met Glu Lys 2825 2830 2835 Phe Leu Asp Pro Ala Ile Trp
Phe Glu Lys Arg Leu Ala Tyr Thr 2840 2845 2850 Arg Ser Val Ala Thr
Ser Ser Ile Val Gly Tyr Ile Leu Gly Leu 2855 2860 2865 Gly Asp Arg
His Val Gln Asn Ile Leu Ile Asn Glu Gln Ser Ala 2870 2875 2880 Glu
Leu Val His Ile Asp Leu Gly Val Ala Phe Glu Gln Gly Lys 2885 2890
2895 Ile Leu Pro Thr Pro Glu Thr Val Pro Phe Arg Leu Thr Arg Asp
2900 2905 2910 Ile Val Asp Gly Met Gly Ile Thr Gly Val Glu Gly Val
Phe Arg 2915 2920 2925 Arg Cys Cys Glu Lys Thr Met Glu Val Met Arg
Asn Ser Gln Glu 2930 2935 2940 Thr Leu Leu Thr Ile Val Glu Val Leu
Leu Tyr Asp Pro Leu Phe 2945 2950 2955 Asp Trp Thr Met Asn Pro Leu
Lys Ala Leu Tyr Leu Gln Gln Arg 2960 2965 2970 Pro Glu Asp Glu Thr
Glu Leu His Pro Thr Leu Asn Ala Asp Asp 2975 2980 2985 Gln Glu Cys
Lys Arg Asn Leu Ser Asp Ile Asp Gln Ser Phe Asp 2990 2995 3000 Lys
Val Ala Glu Arg Val Leu Met Arg Leu Gln Glu Lys Leu Lys 3005 3010
3015 Gly Val Glu Glu Gly Thr Val Leu Ser Val Gly Gly Gln Val Asn
3020 3025 3030 Leu Leu Ile Gln Gln Ala Ile Asp Pro Lys Asn Leu Ser
Arg Leu 3035 3040 3045 Phe Pro Gly Trp Lys Ala Trp Val 3050 3055
2620DNAHomo sapiens 2gcgagaggag tcgggatctg cgctgcagcc accgccgcgg
ttgatactac tttgaccttc 60cgagtgcagt ggtaggggcg cggaggcaac gcagcggctt
ctgcgctggg aaattcagtc 120gtgtgcgacc cagtctgtcc tctccccaga
ccgccaatct catgcacccc tccagagtgg 180cccttgactc ctccctctcc
tcactccatc tttcctggcc tctctccggg tgcttagcgg 240acttggccaa
taacctcctc cttttaaacg ccctgaattg aaccctgcct cctgcgcatc
300ctctttttgt gtcaccttag ggttcagatt taactacgcg acttgactag
tcatcttttg 360atctctctct cgtatttagt acttttagtc agcgagcatt
tattgatatt tcaacttcag 420cctcgcggtt aagagcttgg gctctggaat
catacggctg gaattggaat tctgtcagtc 480gtgtggccgc tctctactgt
cttgtgaaga taagtgagat aatcttgacc tgtggtgagc 540actcgtgagc
gttagctgct gtatttacca ggtacagata agacaactac agtggatgat
600aatgtatgtg gtgatagggg 620330DNAartificialprimer 3aagatgctgt
catgcagcag gtcttccaga 30430DNAartificialprimer 4aattctctag
agctcgctga tcagcctcga 30530DNAartificialprimer 5ttgtccaacc
tgagagtggc aatccatagc 30625DNAartificialprimer 6gtgctagact
catggtttaa gattt 25721DNAartificialprimer 7cagtcccaca tcaggcttga g
21821DNAartificialprimer 8ggactgcacg gatgacctta g
21923DNAartificialprimer 9ccttcaagtc agccagcccc ctg
231024DNAartificialprimer 10ttcacttttc tgggactgag aatg
241120DNAartificialprimer 11gccactgtgc cgtacagaga
201218DNAartificialprimer 12tccagccagg atgcaaca
181320DNAartificialprimer 13gcctcgtgag catgaccaat
201421DNAartificialprimer 14gcagaggaag acgatgaagc a
211523DNAartificialprimer 15ctccgacctc ttcatcctcg gcg
231619DNAartificialprimer 16aagcgaaact ggcggaaac
191717DNAartificialprimer 17gatctggccc ttgaacc
171824DNAartificialprimer 18cagaggcatt gacaacaggg tgcg 24
* * * * *