U.S. patent application number 10/856343 was filed with the patent office on 2005-02-10 for treatment of dna damage related disorders.
This patent application is currently assigned to St. Jude Children's Research Hospital. Invention is credited to Bakkenist, Christopher J., Kastan, Michael B..
Application Number | 20050032834 10/856343 |
Document ID | / |
Family ID | 32474121 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032834 |
Kind Code |
A1 |
Kastan, Michael B. ; et
al. |
February 10, 2005 |
Treatment of DNA damage related disorders
Abstract
The present invention provides methods and compositions for
prophylaxis and treatment of a variety of disorders including DNA
damage related disorders, cancer, ischemia, oxidative stress,
atherosclerosis, and stroke using a chloroquine compound.
Inventors: |
Kastan, Michael B.;
(Cordova, TN) ; Bakkenist, Christopher J.;
(Cordova, TN) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
St. Jude Children's Research
Hospital
|
Family ID: |
32474121 |
Appl. No.: |
10/856343 |
Filed: |
May 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10856343 |
May 27, 2004 |
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10496685 |
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10496685 |
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PCT/US03/37838 |
Nov 26, 2003 |
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10496685 |
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10351733 |
Jan 24, 2003 |
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10496685 |
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10307077 |
Nov 27, 2002 |
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Current U.S.
Class: |
514/313 |
Current CPC
Class: |
G01N 33/6893 20130101;
C12N 2501/06 20130101; A61K 31/675 20130101; G01N 33/6842 20130101;
C12N 5/0602 20130101; A61K 31/4706 20130101; Y02A 50/30 20180101;
G01N 2800/52 20130101; C07K 16/40 20130101; C12N 2501/999 20130101;
Y02A 50/411 20180101; G01N 33/6812 20130101; C12Q 1/485
20130101 |
Class at
Publication: |
514/313 |
International
Class: |
A61K 031/47 |
Claims
What is claimed is:
1. A method of prophylaxis of cancer comprising administering to a
subject at risk of cancer, an effective amount of a chloroquine
compound, provided the subject has been free of a localized skin
epithelialoma or carcinoma for at least a year before commencing
administration of the chloroquine compound.
2. The method of claim 1, wherein the subject has never suffered
from a localized skin epithelialoma or carcinoma basal cell before
commencing administration of the chloroquine compound.
3. The method of claim 1, wherein the subject has never suffered
from skin cancer.
4. The method of claim 1, wherein the subject has been free of
malaria for at least one year before commencing administration of
the chloroquine compound.
5. The method of claim 1, wherein the subject has never suffered
from malaria before commencing administration of the chloroquine
compound.
6. The method of claim 1, wherein the subject is free of detectable
antibodies against Epstein Barr virus.
7. The method of claim 1, further comprising monitoring the subject
for development of a cancer after administration of the chloroquine
compound.
8. The method of claim 7, wherein the monitoring comprising
monitoring the subject for a cancer other than a localized skin
epithelialoma or carcinoma basal cell.
9. The method of claim 7, wherein the monitoring comprising taking
a sample of a body fluid, or performing a scan of an internal
organ.
10. The method of claim 1, wherein the subject is at risk of cancer
by having a precancerous tissue.
11. The method of claim 1, wherein the subject has a first cancer,
and is at risk of a second cancer.
12. The method of claim 1, wherein the subject has a cancer and is
undergoing radiation therapy to treat the cancer, the radiation
therapy putting the subject at risk of a second cancer; and wherein
the administration effects prophylaxis of the second cancer.
13. The method of claim 1, wherein the chloroquine compound is
administered before or during the radiation therapy.
14. The method of claim 1, wherein the subject has a cancer and is
undergoing chemotherapy to treat the cancer, the chemotherapy
placing the subject at risk of a second cancer, and wherein the
administration effects prophylaxis of the second cancer.
15. The method of claim 14, wherein the chloroquine compound is
administered before or during the chemotherapy.
16. The method of claim 1, wherein the subject is at risk of cancer
due to a genetic variation associated with increased risk of
cancer.
17. The method of claim 1, wherein the subject is at risk of cancer
due to viral infection.
18. The method of claim 1, wherein the subject is at risk of caner
due to exposure to a carcinogen or irradiation.
19. The method of claim 1, wherein the subject is at risk of cancer
due to exposure to X-rays.
20. The method of claim 1, further comprising determining presence
of a genetic variation in an ATM gene of the subject associated
with cancer.
21. The method of claim 1, further comprising administering a
chemopreventive agent other than the chloroquine compound to the
subject.
22. The method of claim 1, further comprising determining the risk
of cancer in the subject before administering the chloroquine
compound.
23. The method of claim 1, wherein the chloroquine compound is
administered intravenously.
24. The method of claim 1, wherein the chloroquine compound is
administered orally.
25. The method of claim 1, wherein the subject is free of diseases
of the immune system, infectious diseases, multiple-drug
resistance, and neurological diseases.
26. The method of claim 1, wherein 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.
27. The method of claim 1, wherein the prophylaxis is effective to
prevent detectable development of cancer for at least six months
after administering the effective dosage.
28. The method of claim 1, wherein the administering is performed
before exposure of the subject to the risk of cancer.
29. The method of claim 1, wherein the administering is performed
at regular intervals for a period of at least six months.
30. The method of claims 1, wherein 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.
31. The method of claim 30, wherein the compound is chloroquine,
chloroquine phosphate or chloroquine diphosphate.
32. The method of claim 1, wherein chloroquine compound has a
systemic effect.
33. The method of claim 1, wherein the patient is human and the
dosage is 0.05 to 1 mg/kg per day.
34. The method of claim 1, wherein the patient is human and the
dosage is 0.2 to 0.6 mg/kg per day.
35. The method of claim 34, wherein the patient has been exposed to
a carcinogen or radiation, and the dosage is administered at least
on the day of exposure and the day following exposure.
36. The method of claim 34, wherein the patient has been exposed to
a carcinogen or radiation, and the dosage is administered at least
on the day before the exposure, on the day of the exposure, and at
least on the day following the exposure.
37. The method of claim 1, wherein the patient is human and has
genetic susceptible to cancer, and the dosage is 0.2 to 0.6 mg/kg
week.
38. The method of claims 1, wherein the amount of the compound
administered is up to about 10 mg/kg/day.
39. The method of claims 1, wherein the amount of the compound
administered is more than about 0.1 mg/kg/day.
40. The method of claim 1, wherein the amount of the compound
administered is more than about 1.0 mg/kg/day.
41. The method of claim 1, wherein the amount of the compound
administered is less than about 50 mg/kg/day.
42. The method of claim 1, wherein the amount of the compound
administered is less than about 10 mg/kg/day.
43. The method of claim 1, wherein the chloroquine compound is
administered more than once a week.
44. The method claim 1, wherein the chloroquine compound is
administered daily.
45. The method of claim 1, wherein the chloroquine compound is
formulated in a sustained release formulation.
46. The method of claim 1, wherein the subject is human.
47. A method of therapeutically treating cancer comprising
administering to a subject having a cancer, an effective amount of
a chloroquine compound whereby the cancer is therapeutically
treated.
48. The method of claim 47, wherein the cancer is other than a
localized skin epithelialoma or carcinoma basal cell.
49. The method of claim 47, wherein the cancer is other than skin
cancer.
50. The method of claim 47, wherein the treatment reduces or
eliminates further growth of the cancer.
51. The method of claim 47, wherein the treatment shrinks or
eliminates the tumor.
52. The method of claim 47, wherein the treatment inhibits invasion
of the cancer into tissues of the subject and/or inhibits
metastasis of the cancer.
53. The method of claim 47, further comprising monitoring changes
in the cancer responsive to the administering.
54. The method of claim 53, wherein the monitoring comprising
taking a sample of a body fluid, or performing a scan of an
internal organ.
55. The method of claim 47, further comprising identifying a
genetic variation in an ATM gene of the subject associated with
cancer.
56. The method of claim 47, further comprising administering a
chemotherapeutic agent other than the chloroquine compound to the
subject.
57. The method of claim 47, further comprising determining presence
of the cancer before the administering step.
58. The method of claim 47, wherein the subject is free of diseases
of the immune system, infectious diseases, multiple drug resistance
and neurological diseases.
59. The method of claim 47, wherein the patient is human and the
dosage is 0.05 to 1 mg/kg per week.
60. The method of claim 1, wherein the patient is human and the
dosage is 0.2 to 0.6 mg/kg per day.
61. The method of claim 47, wherein the subject is free of
psoriasis, malaria, protozoal infections, Alzheimer's disease,
Parkinson's disease, lupus erythematosus, rheumatism,
hypercalcemia, multiple sclerosis, and migraine.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of
attorney docket 29202-710.831 filed May 26th 2004, the US national
phase of PCT US03/37838 filed Nov. 26, 2003, which is a
continuation-in-part of 10/351,733 filed Jan. 24, 2003 and is also
a continuation-in-part of U.S. Ser. No. 10/307,077, filed Nov. 27,
2002, all of which are incorporated herein by reference in their
entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0002] This invention was made in the course of research sponsored
by the National Institutes of Health (NIH Grant Nos. CA71387). The
U.S. government may have certain rights in this invention.
BACKGROUND OF THE INVENTION
[0003] Cancer is now the second leading cause of death in the
United States. Over 1 million new cases of cancer are expected to
be diagnosed in 2003 and over 500,000 people are expected to die of
cancer.
[0004] Cancer is typically treated with one or a combination of
three types of therapies: surgery, radiation, and chemotherapy.
Overall costs for cancer, including treatments, were approximately
$170 billion dollars in 2002. The cancer treatments are not only
expensive; they are ineffective most of the time and also have many
side effects. Hence, there is a demand for more effective cancer
prevention and treatment agents, as well as for the prevention and
treatment of DNA damage related conditions.
BRIEF SUMMARY OF THE INVENTION
[0005] The invention provides methods of prophylaxis of cancer. The
methods comprise administering to a subject at risk of cancer, an
effective amount of a chloroquine compound, provided the subject
has been free of a localized skin epithelialoma or carcinoma for at
least a year before commencing administration of the chloroquine
compound. Optionally, the subject has never suffered from a
localized skin epithelialoma or carcinoma basal cell before
commencing administration of the chloroquine compound. Optionally,
the subject has never suffered from skin cancer.
[0006] In some methods, the subject is monitored for development of
a cancer after administration of the chloroquine compound.
Optionally, the monitoring comprising monitoring the subject for a
cancer other than a localized skin epithelialoma or carcinoma basal
cell. Optionally, the monitoring comprising taking a sample of a
body fluid, or performing a scan of an internal organ.
[0007] In some methods, the subject is at risk of cancer by having
a precancerous tissue. In some methods, the subject has a first
cancer, and is at risk of a second cancer. In some methods, the
subject has a cancer and is undergoing radiation therapy to treat
the cancer, the radiation therapy putting the subject at risk of a
second cancer; and wherein the administration effects prophylaxis
of the second cancer. In some methods, the chloroquine is
administered before or during the radiation therapy. In some
methods, the subject has a cancer and is undergoing chemotherapy to
treat the cancer, the chemotherapy placing the subject at risk of a
second cancer, and wherein the administration effects prophylaxis
of the second cancer. In some methods, the chloroquine compound is
administered before or during the chemotherapy.
[0008] In some methods, the subject is at risk of cancer due to a
genetic variation associated with increased risk of cancer. In some
methods, the subject is at risk of cancer due to viral infection.
In some methods, the subject is at risk of caner due to exposure to
a carcinogen or irradiation. In some methods, the subject is at
risk of cancer due to exposure to X-rays.
[0009] In some methods, there is an additional step of determining
presence of a genetic variation in an ATM gene of the subject
associated with cancer. In some methods, there is an additional
step of administering a chemopreventive agent other than the
chloroquine compound to the subject. In some methods there is a
further step of determining the risk of cancer in a subject before
administering the chloroquine compound.
[0010] In some methods, the chloroquine is administered
intravenously. In some methods, the chloroquine is administered
orally.
[0011] In some methods, the subject 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, Alzheimer's disease, Parkinson's disease, lupus
erythematosus, rheumatism, hypercalcemia, multiple sclerosis, and
migraine.
[0012] In some methods, the prophylaxis is effective to prevent
detectable development of cancer for at least six months after
administering the effective dosage. In some methods, the
administering is performed before exposure of the subject to the
risk of cancer. In some methods, the administering is performed at
regular intervals for a period of at least six months.
[0013] 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 or chloroquine diphosphate.
[0014] In some methods, the chloroquine compound has a systemic
effect. In some methods, the patient is human and the dosage is
0.05 to 1 mg/kg per day. In some methods, the patient is human and
the dosage is 0.2 to 0.6 mg/kg per day. In some methods, the
patient has been exposed to a carcinogen or radiation, and the
dosage is administered at least on the day of exposure and the day
following exposure. In some methods, the patient has been exposed
to a carcinogen or radiation, and the dosage is administered at
least on the day before the exposure, on the day of the exposure,
and at least on the day following the exposure. In some methods,
the patient is human and has genetic susceptible to cancer, and the
dosage is 0.2 to 0.6 mg/kg week. In some methods, the amount of the
compound administered is up to about 10 mg/kg/day. In some methods,
the amount of the compound administered is more than about 0.1
mg/kg/day. In some methods, the amount of the compound administered
is less than about 50 mg/kg/day. In some methods, the amount of the
compound administered is less than about 10 mg/kg/day. 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 formulated in a
sustained release formulation. In some methods, the patient is
human.
[0015] The invention further provides methods of therapeutically
treating cancer comprising administering to a subject having a
cancer an effective amount of a chloroquine compound whereby the
cancer is therapeutically treated. Optionally, the cancer is other
than a localized skin epithelialoma or carcinoma basal cell.
Optionally, the cancer is other than skin cancer. Optionally, the
treatment reduces or eliminates further growth of the cancer.
Optionally, the treatment shrinks or eliminates the tumor.
Optionally, the treatment inhibits invasion of the cancer into
tissues of the subject and/or inhibits metastasis of the
cancer.
[0016] Some methods involve a further step of monitoring changes in
the cancer responsive to the administering. Optionally, the
monitoring comprises taking a sample of a body fluid, or performing
a scan of an internal organ. Optionally, the monitoring comprises
identifying a genetic variation in an ATM gene of the subject
associated with cancer. Optionally, the methods involve an
additional step of administering a chemotherapeutic agent other
than the chloroquine compound to the subject. Optionally, the
methods involve an additional step of determining presence of the
cancer before the administering step.
[0017] In some methods, the subject is free of diseases of the
immune system, infectious diseases, and neurological diseases.
[0018] In some methods, the patient is human and the dosage is 0.05
to 1 mg/kg per week.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows a Kaplan-Meier survival curve of C57/BL6 mice
after exposure to 8 Gy total body irradiation (TBI). Half of the
cohort received a dose of chloroquine (dashed line) by either IP
injection (1.75 mg/kg or 3.5 mg/kg) or in their drinking water (3.5
mg/kg or 7 mg/kg) the day before the TBI. The one mouse which died
in the chloroquine-treated group received 1.75 mg/kg by i.p.
injection.
[0020] FIG. 2 shows that chloroquine treatment enhances survival
after TBI by enhancing recovery of hematopoietic progenitor cells.
Five mice received 3.5 mg/kg chloroquine (C) by i.p. injection 24
and 4 hours prior to TBI (bars with diagonal stripes). Five mice
received no chloroquine (stippled bars). Fourteen days after
irradiation, the cellularity (open bars) of hematopoietic tissues
(spleen, thymus, bone marrow) was assessed by a blinded observer on
a scale of 0-3 with 3 being normal cellularity. The bars represent
the average cellularity of the tissues from the 5 mice in each
group.
[0021] FIG. 3 shows a Kaplan-Meier survival curve of AT mice after
exposure to 8 Gy TBI. Half of the cohort received a dose of 3.5
mg/kg chloroquine (CHL; dashed line) by i.p. injection 24 and 4
hours prior to the TBI.
[0022] FIG. 4 demonstrates that chloroquine treatment prevents the
development of tumors in E.mu.-t-myc mice. After weaning, a cohort
of transgenic mice expressing the c-myc oncogene were started on
chloroquine (CHL) at 7.0 mg/kg in the drinking water ((+), solid
line). Within 100 days, all of the mice with no drug in the water
had died of leukemia, while none of the cohort of mice on drug had
succumbed. The latter group of mice was then divided into two
groups (timing of this event depicted by heavy arrow), one group of
which was taken off of chloroquine ((-), dashed line) and the other
group of which was started on i.p. injections of 3.5 mg/kg of
chloroquine once a week. Within a month, all of the mice taken off
of chloroquine had developed malignancies and all of the mice on
the weekly i.p. injections remained tumor-free for months.
[0023] FIG. 5 illustrates that chloroquine treatment reduces the
development of tumors in mice injected with the potent chemical
carcinogen, 3-methylcholanthrene (3-MC). Chloroquine (CHL, 3.5
mg/kg) was given by i.p. injection 24 and 4 hours prior to 3-MC
injection in 30 mice and 30 mice received the carcinogen with no
chloroquine pretreatment. The percentage of animals remaining
tumor-free is plotted. Statistical significance, log rank test
P<0.0001.
[0024] FIG. 6 demonstrates that chloroquine treatment reduces the
development of tumors in mice exposed to ionizing radiation in a
protocol that induces thymic lymphomas. Chloroquine (CHL, 3.5
mg/kg) was given by i.p. injection 24 and 4 hours prior to
irradiation with 1.75 Gy in four successive weeks and animals were
subsequently observed for the development of tumors. Statistical
significance, log rank test P=0.0012.
[0025] FIG. 7 shows tumor incidence in wildtype mice receiving
either placebo or CHQ before 3-MC injection. CHQ markedly protects
from tumor development.
[0026] FIG. 8 shows tumor incidence in ATM-null mice receiving
either placebo or CHQ before 3 MC injection. CHQ does not protect
from tumor development.
[0027] FIG. 9 shows tumor incidence in p53-null mice receiving
either placebo or CHQ before 3 MC injection. CHQ does not protect
from tumor development.
[0028] FIG. 10 shows protection against coat color loss in mice
treated with chloroquine or hydroxychloroquine.
DEFINITIONS
[0029] The term "animal subject" or "subject" includes humans as
well as other mammals. Nonhuman mammals can include veterinary
subjects as well as animal models in which a disorder has been
specifically induced to simulate a human disease. For example, a
transgenic mouse model of stroke is described in Stroke
27:1124-1129 (1996). Severe combined immunodeficient mice grafted
with human skin are useful as models of human skin cancers,
including melanomas.
[0030] A subject is at risk of a disorder, if the subject presently
lacks clinical symptoms of the disorder, but has a statistically
significantly higher probability of acquiring the disorder than
individuals in the general population. Such risk can be shown by
abnormal levels of a marker (e.g., a blood protein) or other sign
associated with the disorder. The risk can also arise through
exposure to a condition (e.g., radiation) associated with a
statistically significant increased risk of the disorder. The risk
can also arise through possession of a genetic variation associated
with the disorder, or first degree relatives having the disorder.
Levels of a marker are considered abnormal if outside the mean plus
at least one and preferably at least 2 standard deviations of the
level of marker in a population of individuals not known to be
suffering from a disorder. Likewise a condition is associated with
statistically significant increased risk of the disorder if the
number of individuals in a population of individual exposed to the
condition acquiring the disorder is greater than the number of
individuals in a population of individuals not exposed to the
condition acquiring the disorder to a statistically significant
extent.
[0031] Statistical significance is measure at the level of
p<0.05.
[0032] A symptom of a disorder means a phenomenon experienced by an
individual having the disorder indicating a departure from normal
function, sensation or appearance.
[0033] A sign of a disorder is any bodily manifestation that serves
to indicate presence or risk of a disorder.
[0034] The term "treating" includes achieving a therapeutic benefit
and/or a prophylactic benefit.
DETAILED DESCRIPTION OF THE INVENTION
[0035] I. GENERAL
[0036] The present invention provides methods, compositions, and
kits for the prevention, prophylaxis and/or treatment of several
disorders. These disorders include DNA damage related disorders,
cancers, oxidative stress and its many manifestations, 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 disorders are not coextensive. For example,
atherosclerosis can cause problems by aneurysm as well as ischemia.
Chloroquine compounds are useful for prevention, prophylaxis and/or
treatment of these disorders.
[0037] II. Chloroquine Compounds
[0038] The term "chloroquine compounds" as used herein means
chloroquine-like compounds, chloroquine and enantiomers, 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.
[0039] In a specific embodiment, the invention is practiced with
chloroquine. The chemical structure of chloroquine,
N.sup.4-(7-Chloro-4-quinolinyl)-N.sup.1,N.sup.1-diethyl-1,4-pentanediamin-
e or 7-chloro-4-(4-diethylamino-1-methylbutylamino) quinoline, is
as follows: 1
[0040] 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.
[0041] As mentioned above, the chloroquine compounds useful herein
include chloroquine analogs and derivatives. A number of
chloroquine analogs 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.
[0042] Examples of suitable chloroquine compounds include
chloroquine phosphate;
7-chloro-4-(4-diethylamino-1-butylamino)quinoline
(desmethylchloroquine);
7-hydroxy-4-(4-diethylamino-1-butylamino)quinolin- e;
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-methy-
lbutylamino)quinoline; hydroxychloroquine phosphate;
7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline
(desmethylhydroxychloroquine);
7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amin-
o-1-butylamino)quinoline;
7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-a-
mino-1-butylamino)quinoline;
7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethy-
l)-amino-1-butylamino)quinoline;
7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxye-
thyl)-amino-1-methylbutylamino)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-methoxyqu- inoline 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-pyrroli-
dinyl)-2,5-xylidinoquinoline,
4-[(4-diethylamino)-1-methylbutyl)amino]-6-m- ethoxyquinoline;
3,4-dihydro-1 (2H)-quinolinecarboxyaldehyde;
1,1'-pentamethylenediquinoleinium diiodide; and 8-quinolinol
sulfate, enantiomers thereof, as well as suitable pharmaceutical
salts thereof.
[0043] 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-pr-
opane-1,2-diamine;
(R)--N.sub.2-(7-chloro-quinolin-4-yl)-N.sub.1,N.sub.1-d-
imethyl-propane-1,2-diamine;
N.sub.1-(7-chloro-quinolin-4-yl)-2,N.sub.2,N.-
sub.2-trimethyl-propane-1,2-diamine;
N.sub.3-(7-chloro-quinolin-4-yl)-N.su-
b.1,N.sub.1-diethyl-propane-1,3-diamine;
(RS)-(7-chloro-quinolin-4-yl)-(1-- methyl-piperidin-3-yl)-amine;
(RS)-(7-choro-quinolin-4-yl)-(1-methyl-pyrro- lidin-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-diethyl-propane-1,2-diamine;
(S)--N.sub.2-(7-chloro-quinolin-4--
yl)-N.sub.1,N.sub.1-diethyl-propane-1,2-diamine;
(R)--N.sub.2-(7-chloro-qu-
inolin-4-yl)-N.sub.1,N.sub.1-diethyl-propane-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.1-dimethyl-ethane-1,2-diam-
ine;
N.sub.2-(7-chloro-quinolin-4-yl)-N.sub.1,N.sub.1-diethyl-ethane-1,2-d-
iamine;
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-dimethy-
l-propane-1,2-diamine;
(S)--N.sub.1-(7-chloro-quinoline-4-yl)-N.sub.2,N.su-
b.2-dimethyl-propane-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-propane-1,2-diamine;
N.sub.1-(7-chloro-quinolin-4-
-yl)-N.sub.2-(benzyl)-2-methyl-propane-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-hyd-
roxy-5-methoxy-benzyl)-2-methyl-propane-1,2-diamine; and
N.sub.1-(7-chloro-quinolin-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-y-
l)-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)-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quino-
lin-4-yl)-N.sub.4-(benzyl)-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(3-chloro-benzyl)-cyclohexan-
e-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(2-hydroxy-4-m-
ethoxy-benzyl)-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-y-
l)-N.sub.4-(3,5-dimethoxy-benzyl)-cyclohexane-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-di-
ethylamino-benzyl)-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-
-4-yl)-N.sub.4-(biphenyl-4-yl)methyl-cyclohexane-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.su-
b.4-(4-methoxy-benzyl)-cyclohexane-1,4-diamine;
trans-N.sub.1-(7-chloro-qu-
inolin-4-yl)-N.sub.4-(4-dimethylamino-benzyl)-cyclohexane-1,4-diamine;
and
trans-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(2,6-difluoro-benzyl)-cycl-
ohexane-1,4-diamine.
[0044] 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 isomeric, optical isomeric and/or
geometric isomeric forms of the chloroquine compounds, as well as
mixtures of these various different forms.
[0045] Chloroquine and hydroxychloroquine are generally racemic
mixtures of (-)- and (+)-enantiomers. The (-)-enantiomers are also
known as (R)-enantiomers (physical rotation) and 1-enantiomers
(optical rotation). The (+)-enantiomers are also known as
(S)-enantiomers (physical rotation) and r-enantiomers (optical
rotation). The metabolism of the (+)- and the (-)-enantiomers of
chloroquine are described in Augustijins and Verbeke (1993) Clin.
Pharmacokin. 24(3):259-69; Augustijins, et al. (1999) Eur. J. Drug
Metabol. Pharmacokin. 24(1):105-8; DuCharme and Farinotti (1996)
Clin. Pharmacokin. 31(4):257-74; Ducharme, et al. (1995) Br. J.
Clin. Pharmacol. 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.
[0046] The chloroquine compounds may metabolize to produce active
metabolites. The used of active metabolites is also within the
scope of the present invention.
[0047] Although an understanding of mechanism is not required for
practice of the invention, 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
US Pub. No. 20030077661 entitled "ATM Kinase Compositions and
Methods," filed Nov. 27, 2003, which is incorporated by reference
herein in its entirety). Hence, choroquine-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.
[0048] III. Use of Chloroquine Compounds
[0049] 1. General
[0050] The invention provides methods of prophylaxis or therapeutic
treatment of an animal subject, including a human. The methods
generally involve the administration of effective amounts of
chloroquine compounds including chloroquine like compounds for the
treatment of one or more the diseases or disorders described in
more detail below. The effects of the chloroquine compounds used in
the present methods are preferably systemic, but can be local, or
topical depending on the mode of administration. Although an
understanding of mechanism is not required for practice of the
invention, it is believed that chloroquine compounds act in part by
protecting normal cells from radiation or radicals and by
inhibiting the cellular damage caused by the radiation or radicals
to normal cells and enhancing the repair process of the normal
cells.
[0051] 2. DNA-Damage Related Disorders
[0052] The methods are generally applicable to "DNA damage related
disorders." This term includes cancer, aging, and other disorders
caused by damage to DNA due to exposure to carcinogens, toxins,
free radicals, like oxygen radicals, or DNA damaging radiations
like ionizing radiation and UV radiation. Cancer and other
disorders included in this definition are discussed individually
below. Although an understanding of mechanism is not required for
practice of the invention, it is believed that administration of
chloroquine compounds prevents DNA damage, inhibits the effects of
DNA damage, and/or stimulates cellular response to DNA damage, and
that the chloroquine compound acts at least in part through
agonizing ATM and p53.
[0053] The p53 gene spans 20 kbp in humans and has 11 exons, 10 of
which are protein coding (see Tominaga et al., 1992, Critical
Reviews in Oncogenesis 3:257 282, incorporated herein by
reference). The gene produces a 53 kilodalton phosphoprotein that
regulates DNA replication. The protein acts to halt replication at
the G1/S boundary in the cell cycle and is believed to act as a
"molecular policeman," shutting down replication when the DNA is
damaged or blocking the reproduction of DNA viruses (Kastan et al.
Cancer Res 51:6304-6311 (1991); Kuerbitz et al., Proc Natl Acad Sci
USA. 89:7491-7495 (1992); Kastan, Cell 71:587-597 (1992), Hartwell
et al., Science 266:1821-1828 (1994)).
[0054] In one embodiment, chloroquine compounds are used as
prophylactics to inhibit side effects of frequent exposure to
X-rays in individuals, such as athletes. This method is useful for
other subject populations that are frequently exposed to DNA
damaging radiations, such as X-ray technicians, police officers,
astronauts, and the like. It is known that exposure to X-rays
causes DNA damage. Administration of chloroquine compounds is
contemplated to inhibit the side-effects of frequent exposure to
DNA damaging radiations, including inhibit the damage to cells due
to damage to DNA.
[0055] The prophylactic benefits of chloroquine compounds can be
obtained by administering in advance of exposure to the DNA
damaging agent to provide the enhancing effect in one embodiment.
The amount of time prior to the exposure to the DNA damaging agent
that the chloroquine compound is administered can vary from days,
hours, to minutes. Also, the chloroquine compounds can be
administered during exposure to the DNA damaging agent or after
such exposure. Chloroquine compounds can also be administered after
exposure to the DNA-damaging agent, in which case, the exposure is
preferably within one day, a week or a month. In one embodiment,
the effective amount of a chloroquine compound is an amount which
reduces DNA damage, reduces DNA mutation or increases survival of
cells exposed to a DNA damaging agent when compared to cells
exposed to the same DNA damaging agent and not receiving a
chloroquine compound.
[0056] 3. Cancer
[0057] The chloroquine compounds are useful in the prophylaxis,
including prevention, and therapeutic treatment of cancers caused
by toxins, carcinogens, DNA damaging radiations, and/or genetic
mutations, among other causes. For example, chloroquine compounds
are useful in the treatment of cancers caused by exposure to toxins
and carcinogens like aromatic hydrocarbons, cigarette smoke, acetyl
amino fluorine, and MTBE. Also, chloroquine compounds are useful in
prophylaxis or therapeutic treatment of cancers caused by DNA
damaging radiations like UV and ionizing radiation. The ionizing
radiation includes both natural and therapeutic radiation
exposures. Examples of ionizing radiations are X-rays for
diagnostics and radiation therapy used for tumors. For example,
chloroquine compounds can be used in the prophylaxis or treatment
of one or more of the following cancers: melanomas, prostate
cancer, breast cancer, colon cancer, lung cancer, non-Hodgkin's
lymphoma, retinoblastoma, neuroblastoma, sarcomas, and ovarian
cancer.
[0058] Chloroquine compounds can be used in prophylaxis of subjects
at risk of cancer. Cancer is the uncontrolled, proliferation
(growth and multiplication) of a single type of cell, occurring in
any tissue of the body. Malignant cancers are those that invade
surrounding tissues and metastasize (spread) to other body sites
via the blood and lymphatic circulations. Metastasized cancers
usually remain the same type of cell as the initial site of cancer
development; for example, if breast cancer metastasizes to a lung,
the cancer in the lung consists of breast cells. Benign cancers do
not invade other tissues or spread, have a slower growth rate than
malignant cancers, and in most cases are not fatal. The methods are
particularly useful for prophylaxis or therapeutic treatment of
malignant cancers.
[0059] Examples of subjects at risk of cancer include subjects
having a primary cancer who are at risk of a second cancer,
subjects having been exposed to a carcinogen (e.g., asbestos,
benzene) or radiation, including X-rays, radioactivity, nuclear
bombs, dirty bombs, excessive exposure to sun rays, or ultraviolet
irradiation, subjects have a genetic variation associated with
cancer, subjects having first degree relatives with a cancer known
to be hereditary, subjects showing a biochemical marker associated
with cancer (e.g., carcinoembryonic antigen), subjects having an
abnormal cellular morphology (precancerous tissue) associated with
development of cancer (e.g., melanocytic naevi), subjects infected
with an oncogenic virus (e.g., certain forms of HPV, HBV, KSHV and
EBV) or helicobacter pylori, and subjects who smoke or engage in
excessive drinking. An exemplary class of subjects at risk of
cancer are subjects who have previously had a melanoma and are risk
of developing a further melanoma. A further exemplary class of
subjects at risk of metastatic melanoma are subjects having a
nonmetastatic melanoma and at least two family members with a
history of melanoma. Another exemplary class of subjects at risk of
melanoma are those having congenital melanocytic naevi. They are
brown or black moles which are present at birth, or which develop
in the first month or so of life.
[0060] Genetic risk of cancer can be associated with either
homozygous or heterozygous variations in genes. The variations can
be present in the germline (and other cells of a patient) or may
appear only in precancerous or cancerous tissue (i.e., somatic
variations). 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. Some variant
alleles in some genes are associated with cancer. Variation can
result in cancer either by increased or altered function (e.g.,
oncogenes) or reduced or lack of function (e.g., tumor suppressor
genes). Variant forms associated with cancer can be recognized by
comparing alleles in populations with and without a cancer (usually
the individuals in the population with cancer have the same type
for purposes of analysis). Alleles occurring significantly more
frequently in the population having cancer are associated with
cancer. 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 cancer
in the transformed cell or animal.
[0061] Many variations in many genes have already been
characterized as being associated with cancer. Examples of genes in
which variation causes increased risk of cancer include RB 1
retinoblastoma, the WT1 gene for Wilms' tumor, the NF 1 and NF2
genes for neuroblastomatosis, types 1 and 2, the VHL gene for renal
cancer tumors association with Von Hippel-Lindau disease, the APC
gene for adenomatous polyposis coli, and the BRCAI and II genes for
breast and ovarian cancer, PTEN and HRAS-1 both associated with
breast cancer (see, e.g., Mulligan et al., Nature Genet 6, 70-74
(1994); Knudson, Cancer Res. 63, 1888-1891 (1989); Miki et al.,
Sciences 266, 66-71 (1994); Bronner, Nature 368, 258-261 (1994),
Leach, Cell 75, 1215, Lieberman, Amer. J. Gastro. 87, 1085-1093
(1992), Easton, Breast Cancer Research 1, 14-17 (1999), Peto et
al., J. Natl. Cancer Inst 91, 943-949 (1999), Kronitiris, N. Engl.
J. Med. 329, 517-523 (1993); Tirkkonen et al. Cancer Research
57(7), 1222-1227 (1997)).
[0062] ATM and/or p53 are further examples of genes having
variations associated with cancer (see, e.g., Garber et al., Cancer
Research 51, 6094-6097 (1991); Olsen et al., J. Nat. Cancer Inst.
93, 121-127 (2001)). As noted these genes are also thought at least
in part to effect 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 treatment with chloroquine compounds.
[0063] For example, retinoblastoma is a cancer affecting young
children in which tumors develop in the retina of one (unilateral,
75% of cases) or both (bilateral, 25% of cases) eyes. In all cases,
retinoblastoma seems to be caused by a mutation in the RB1 gene
located on chromosome 13, which is responsible for controlling
retinal cell division. Although retinoblastoma may occur at any
age, it most often occurs in younger children, with 80% of cases
diagnosed before the age of 5 years. Retinoblastoma presents in
both hereditary (40%) and nonhereditary (60%) forms. The hereditary
form may manifest as unilateral or bilateral disease. Most
unilateral disease is not hereditary, whereas all bilateral disease
is hereditary. For persons carrying the hereditary form of
retinoblastoma, the disease demonstrates with high penetrance
(about 90% will manifest disease). Patients with the hereditary
type of retinoblastoma have a markedly increased frequency of
additional malignancies. The cumulative incidence is about 26+/-10%
in nonirradiated and 58+/-10% in irradiated patients by 50 years
after diagnosis of retinoblastoma. Most of the excess cancers were
osteosarcomas, soft tissue sarcomas, or melanomas. The carcinogenic
effect of radiation increased with dose. In irradiated patients,
two thirds of the second cancers occur within irradiated tissue and
one third outside the radiation field. Treatment of patients having
a genetic variation of the RB1 gene can prevents or reduce the
frequency of both retinoblastomas and/or the additional
malignancies.
[0064] Methods for determining presence of genetic variations
individuals are described in attorney docket 100/1046-20, Ser. No.
______, "Improvements to Analysis Methods for Individual
Genotyping", filed Feb. 24, 2004, U.S. Ser. No. 10/768,788,
"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 EP 0 730 663, each of which is incorporated by
reference.
[0065] The presence of risk of cancer in a subject can be
determined by biochemical analysis, cytological examination to
detect precancerous or cancerous tissues (e.g., Pap smear), genetic
analysis to detect genetic variation, family history of hereditary
cancer, subject history of exposure to a carcinogen or radiation,
or knowledge that the subject will be exposed to a carcinogen or
radiation. If the risk of cancer is due to an event that has not
yet happened (e.g., occupational exposure to radiation or a
carcinogen, or exposure to radiation through treatment of an
existing cancer), chloroquine treatment is preferably administered
before the event creating the risk, and or during the event, and/or
as soon as possible after the event. Administration can then be
continued, preferably at regular intervals, thereafter. If the risk
of cancer is due to an event that has already occurred (e.g., past
exposure to a carcinogen or radiation or genetic mutation),
chloroquine treatment is preferably administered as soon as
possible after the event, and continued at regular intervals
thereafter.
[0066] In some methods, the subject is monitored to determine
response of the subject to administration of the chloroquine
compound. If the subject is at risk of cancer but does not have
cancer when the administration is begun, the subject is monitored
to determine whether a cancer develops. The monitoring can be
performed by similar techniques to those used in assessing risk of
cancer. In addition, cancers or lack thereof can be monitored by
performing scans (e.g., X-rays, CAT scans, MRI) of internal organs
or visuals scans of the skin. If the subject does already have a
cancer when the administration is begun, the subject is monitored
to determine changes in the cancer responsive to the administration
of the chloroquine compound. Preferably, the cancer does not show
further increase in size, shrinks in size or disappears.
Alternatively, administration can inhibit or stop further growth of
the cancer. Monitoring of an existing cancer is performed by visual
observation, scanning (e.g., MRI) or biopsy followed by cytological
or biochemical analysis.
[0067] Prophylactic administration of a chloroquine compound can be
accompanied by administration of another chomopreventive agent
(e.g., tamoxifen (Nolvadex), Sulindac (Clinoril), or aspirin) or
regime designed to decrease the risk of the subject developing
cancer. Likewise, therapeutic administration of a chloroquine
compound can be accompanied by administration of a chemotherapeutic
agent (e.g., busulfan, cisplatin, cyclophosphamide, methotrexate,
daunorubicin, doxorubicin, melphalan, cladribine, vincristine,
vinblastine, and chlorambucil) or radiation or other regime
designed to treat the tumor.
[0068] The chloroquine compounds can be used to prevent secondary
cancers, i.e., cancers that are caused by radiation therapy and/or
chemotherapy used to treat the primary cancer. For example, the
chloroquine compounds are used to prevent the occurrence of breast
cancer in subjects receiving radiation therapy for Hodgkin's
lymphoma (or other cancers). Also, in these subjects the
chloroquine compounds can be used to inhibit the cellular damage
caused by the radiation therapy to normal cells and enhance the
repair process of the normal cells. The chloroquine compounds are
also suitable for prevention of the reoccurrence of cancers in
subjects who have had prior incidences of cancer.
[0069] The chloroquine compounds can be administered prior to,
during, or after treatment with radiation. In this embodiment, the
beneficial effect of the chloroquine compounds is contemplated to
be not solely limited to a beneficial effect on pathological skin
conditions like skin carcinomas and dermatoses. The use of
chloroquine compounds in combination with radiation therapy is
contemplated to protect the normal cells and inhibit the cellular
damage caused by the radiation therapy to normal cells and enhance
the repair process of the normal cells.
[0070] The chloroquine compounds can be used in immunosuppressed
subjects, like transplant subjects, who have an increased risk of
cancer. In immunosuppressed subjects, the chloroquine compounds can
be used to prevent cancers. The chloroquine compounds can be used
to prevent Epstein Barr virus induced lymphoproliferative
syndrome.
[0071] 4. Oxidative Stress
[0072] Normal metabolism produces free radical molecules. Free
radicals are atoms or molecules which have at least one unpaired
electron in the outer orbital. These radicals are the same as
generated by external radiation and include hydrogen peroxide and
superoxide. Mitochondria are the main source of oxygen free
radicals under normal conditions. Free radicals can react with any
biological molecule (proteins, lipids, sugars, DNA) altering its
structure and often also its function. Therefore living organisms
are provided with a rich system of antioxidant defenses whose main
purpose is to prevent the free radicals attack to other molecules.
Antioxidants can also be supplied in the form of nutrition (e.g.,
phytochemicals in fruit and vegetables). Oxidative stress arises
from an imbalance of these radicals and antioxidants as a result of
which unneutralized radicals damage DNA and other
macromolecules.
[0073] Oxidative stress contributes to a number of disorders
including cancer, coronary disease, atherosclerosis, stroke,
cataracts, macular degeneration, depression, neurodegenerative
disease, and premature aging. Oxidative stress occurs to some
extent in everyone. However, levels of oxidative stress that
substantially exceed the mean plus a standard deviation in a
population of disease free individuals can be recognized as a cause
of present or future symptoms, including cancer, coronary disease,
atherosclerosis, stroke, cataracts, macular degeneration,
depression, neurodegenerative disease, and premature aging.
[0074] The level of oxidative stress can be quantified by measuring
the level of a series of prostaglandin-like compounds termed
isoprostanes. These compounds are formed by perodixation of
arachidonic acid. The level of these compounds in the plasma or
urine is a measure of oxidative stress in a subject (see Morrow,
Am. J. Resp. Crit. Care Med. 166, S25-S30 (2002)). Subjects having
an abnormal level of oxidative stress can be recognized by a level
of at least one of isoprostane that exceeds the mean plus a
standard deviation, preferably, at least two standard deviations,
and more preferably at least five standard deviations of the mean
level in a population of individuals free of disease. Usually, a
determination of the level of oxidative stress is performed before
commencing administration of a chloroquine compound. Levels of
oxidative stress can be measured at intervals thereafter and
compared with a baseline measurement before beginning treatment.
Preferably, the level decreases after beginning treatment,
preferably toward a normal level, or at least the level does not
further increase. The subject can also be monitored for symptoms of
oxidative stress, such as macular degeneration, cataracts or
premature aging, cancer, heart disease and improvement therein
responsive to administration of the chloroquine compound.
[0075] The chloroquine compound can be administered in a regime
that includes one or more other 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.
[0076] 5. Ischemia-Reperfusion
[0077] Ischemia refers to a disorder caused by an imbalance between
supply and demand of oxygen to tissue, usually caused by a
reduction in blood flow to the tissue. Organs such as the heart and
brain are most vulnerable to ischemia due to their high extraction
of oxygen. Reperfusion is the process of restoring blood flow to
the tissue. Ischemia and reperfusion result in different damage to
the tissues deprived of oxygen. The reduction of blood flow
decreases the production of high energy phosphates. The energy
failure causes membrane depolarization and uncontrolled release of
excitatory amino acids, such as glutamate, in the extracellular
space (excitotoxicity). Glutamate acts on various types of
receptors, e.g. NMDA and AMPA, eventually causing calcium overload
of neuronal cells. Calcium activates proteolytic enzymes that begin
to degrade both intracellular and extracellular structures, and
other enzymes, i.e. phospholipase A2 and cyclooxigenase, which can
produce free radicals. Neuronal nitric oxide synthase is also
calcium dependent and produces nitric oxide, which is able to react
with superoxide generating the highly reactive radical
peroxynitrite. Secondary to ischemia proinflammatory genes are
expressed and several inflammatory mediators are released, such as
tumor necrosis factor, interleukin 113. Adhesion molecules are also
expressed and therefore neutrophils, monocytes and macrophages
start to bind the endothelium causing microvascular occlusion and
cross the vascular wall penetrating in the brain. These
inflammatory cells can also produce free radicals.
[0078] Restarting blood flow after more than about ten minutes of
ischemia is typically more damaging than the ischemia itself
because the ischemia sets the stage for oxygen to generate
free-radicals. Arachidonic acid conversion to eicosanoids can lead
to production of the superoxide radical when oxygen is available.
Lipid peroxidation chain reactions occur in the membranes of
neurons as well as of astrocytes. Free radical damage to blood
vessels is particularly severe. Ischemia results in large amounts
of ATP being broken-down to xanthine. Reperfusion allows the
endothelial enzyme xanthine oxidase to convert xanthine plus oxygen
to superoxide and uric acid. Liberated iron and zinc ions further
increase free radical damage. Superoxide increases the adhesion of
leucocytes to vessel walls. Normally, nitric oxide can combine with
superoxide to produce peroxynitrite in small amounts, reducing the
adhesion. During ischemia, impaired nitric oxide production
accompanies increased leukocyte adhesion. Reperfusion increases the
amount of nitric oxide produced by the endothelial cells. During
reperfusion, abnormally high amounts of superoxide converts almost
all available nitric oxide to perxoynitrite, which is regarded as
the agent causing most of the damage to brain capillary endothelial
cells.
[0079] Neutrophils which have accumulated in blood vessels due to
the ischemia can also release oxygen-rich free radicals with the
availability of oxygen from reperfusion.
[0080] Administration of a chloroquine compound to a subject having
or at risk of ischemia is effective in prophylaxis or treatment of
tissues damage caused by ischemia and/or reperfusion, particularly
tissue damage or death resulting from the generation of free
radicals following reperfusion.
[0081] Subjects at risk of ischemia include those having previously
had heart disease, those having elevated biochemical markers of the
disease (e.g., protein C), those identified as having blockage of
blood vessels by angioplasty or MRI imaging, 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 No. 1 Vol. 2 (2000);
Alpert, J. Am. College. Cardiol. 36, 959-69 (2000)). 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 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 followed administration of a
chloroquine compound relative to a placebo.
[0082] In some methods, a chloroquine compound is administered to a
subject having or at risk of ischemia in combination with a second
agent effective in prophylaxis 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.
[0083] 6. Stroke
[0084] Stroke is a sudden loss of brain function resulting from
interference with the blood supply to the central nervous system.
Acute stroke can be classified either as ischemic (80% of stroke
cases), which can be further classified to extra-cranial embolism
and intracranial thrombosis, or a hemorrhagic stroke (20% of stroke
cases), which can be further classified to intracerebral hemorrhage
and subarachnoid hemorrhage. Stroke is a leading cause of morbidity
and mortality, particularly in the elderly. Its incidence and
prevalence sharply increase with age: in the United States 72% of
the subjects suffering a stroke are age 65 and older and up to 88%
of stroke deaths occur in this population. Moreover stroke is an
important cause of long-term disability: up to 40% of survivors are
not expected to recover independence with self-care and 25% become
unable to walk independently. Ischemic stroke accounts for 70 to
80% of all strokes and haemorregic stoke accounts for the
remainder. Free radicals play an important role in the pathogenesis
of stroke, especially superoxide interact with NO, leading to
peroxynitrite formation. Although practice of the invention is not
dependent on an understanding of mechanism, it is believed that
administration of a chloroquine compound to a subject having or at
risk of stroke effects prophylaxis or treatment by mitigating the
effects of free radicals, and the response of cells to damage by
the free radicals.
[0085] 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 to 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 treatment.
[0086] Administration of chloroquine can be accompanied by
administration of other agents to treat stroke. These include the
same agents discussed for treating ischemia and oxidative stress as
noted above.
[0087] 7. Atherosclerosis
[0088] Atherosclerosis is a process in which deposits of fatty
substances, cholesterol, cellular waste products, calcium and other
substances build up in the inner lining of an artery. This buildup
is called plaque. It usually affects large and medium-sized
arteries. Some hardening of arteries often occurs when people grow
older. Plaques can grow large enough to significantly reduce the
blood's flow through an artery. But most of the damage occurs when
they become fragile and rupture. Plaques that rupture cause blood
clots to form that can block blood flow or break off and travel to
another part of the body. If either happens and blocks a blood
vessel that feeds the heart, it causes a heart attack. If it blocks
a blood vessel that feeds the brain, it causes a stroke. And if
blood supply to the arms or legs is reduced, it can cause
difficulty walking and eventually gangrene. Thus, atherosclerosis
as well as being a disorder in itself, is a risk factor for
ischemia and stroke. Insofar as atherosclerosis causes ischemia
and/or stroke, it generates free radicals as these specific
diseases and can also be subject to prophylaxis or treatment with a
chloroquine compound
[0089] Risk factors of atherosclerosis are high blood pressure,
high LDL, obesity, high cholesterol levels and smoking.
Atherosclerosis can be detected by MRI or B-mode ultrasound methods
(see Tang, Am J Cardiac Imaging 6:333-339 (1992)). Subjects can be
screened for risk factors before commencing treatment. Tissue
damage resulting from atherosclerosis and its sequelae can be
monitored following treatment as for ischemia or stroke.
[0090] Administration of a chloroquine compound can be combined
with other agents conventionally used in prophylaxis or treatment
of atherosclerosis. These include antiplatelet agents, lipid
lowering agents, bile acid sequestrants, fibrinates, HMG-CoA
reductase inhibitors, nocotinic acid derivatives, and blood
pressure lowering agents.
[0091] 9. Optional Features
[0092] Optionally, the methods of the invention are not practiced
on subjects who have presented with a skin carcinoma or
epithelioma, like basal cell epithelioma and squamous within one
year of beginning treatment with the chloroquine compound. Examples
of cancers that are not localized skin carcinomas or epitheliomas
include, but are not limited to, melanomas, lymphomas, prostate
cancer, breast cancer, colon cancer, lung cancer, retinoblastoma,
neuroblastoma, sarcomas, and ovarian cancer. Optionally, the
subject has never had a localized skin carcinoma or epithelioma.
Optionally, the subject has not suffered from Burkitt's lymphoma,
or skin pathologies caused by harmful radiation before
administering the chloroquine compound. Optionally, the subject
lacks factors predisposing the subject to Burkitt's lymphomas, such
factors being one or more of malarial infection, EBV infection (as
manifested by antibodies to capsid proteins in the serum detectable
by ELISA) and HIV infection. 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 erythematosus, rheumatism,
hypercalcemia, multiple sclerosis and migraine before administering
the chloroquine compound.
[0093] IV. Therapeutic and Prophylactic Benefits
[0094] Chloroquine compounds are used as prophylactic agents. For
prophylactic benefit, the chloroquine compound can be administered
to a subject at risk of developing one of the above disorders but
not presently showing symptoms of the disorder being treated. 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.
[0095] Chloroquine compounds can be used for their therapeutic
benefits in treating the disorders described above. A therapeutic
benefit includes eradication or amelioration of the underlying
disorder being treated. For example, in a cancer subject,
therapeutic benefit includes eradication, or amelioration of the
underlying cancer, or cessation or reduction in its rate of growth.
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. For example, administration of a
chloroquine compound to a subject suffering from cancer provides
therapeutic benefit not only when the subject's tumor marker level
is decreased, but also when an improvement is observed in the
subject with respect to other complications that accompany the
cancer like pain and psychiatric disorders. A therapeutic benefit
also includes elimination or reduction of consequences of the
underlying disorder, such as 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.
[0096] V. Effective Amount
[0097] 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. The effective amounts 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 animals.
[0098] In some methods, the effective 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.
[0099] In some methods, the daily dosage range of chloroquine, can
vary between about 0.1 mg/kg to about 2 gm/kg body weight. The
daily dose of a chloroquine compound can be less than about 2
gm/kg, less than about 1.5 gm/kg, or less than about 1 gm/kg. In
some methods, the daily dose of a chloroquine compound is more than
about 0.5 mg/kg, more than about 500 mg/kg, or more than about 1
gm/kg. Some daily dosage ranges of a chloroquine compound are about
0.5 mg/kg to about 50 mg/kg or about 1.0 mg/kg to about 10 mg/kg
body weight. Some daily doses of chloroquine diphosphate are about
3.5 mg/kg and 7.0 mg/kg.
[0100] The dosage can vary depending on the subject being treated.
For example, a preferred dosage in mice is 3.5 mg/kg once or twice
a day. The equivalent dosages in monkeys and humans are shown in
the Table 1.
1TABLE 1 Man (60 kg) Mouse (20 g) Monkey (3.0 kg) Man (60 kg) CHG
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
[0101] Preferred dosages ranges in human are from 0.05-1 mg/kg,
more preferably 0.1 to 0.8 mg/kg, more preferably 0.2-0.6 mg/kg or
0.2 to 0.4 mg/kg. The dosage can be administered daily, weekly,
monthly or bimonthly (every two months). In patients whose risk to
cancer is occasioned by a distinct event (e.g., exposure to
carcinogen or radiation), the dosage is preferably administered
daily before, during and/or immediately following the event, for an
initial period of at least 1 day, 3 days, a week or a month, and at
larger intervals thereafter (e.g., weekly). For example, if the
risk of exposure is known in advance, an exemplary regime includes
administering the chloroquine compound on the day before, the day
of exposure and the day after exposure. Another exemplary regime
comprises administering the chloroquine compound 24 hours before
the exposure to a known risk of cancer. If the risk of exposure is
not known in advance, an exemplary regime includes administering
the chloroquine compound at least one the day of exposure and the
day following exposure. 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., 0.05-0.2 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.
[0102] In some methods, the effective amount of chloroquine 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
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.
[0103] VI. Kits
[0104] The invention provides a kit comprising a chloroquine
compound 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.
[0105] VII. Routes of Administration and Formulation
[0106] 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.
[0107] The chloroquine compounds can be administered topically or
systemically. Systemic administration is preferred. In some
methods, topical administration also has a systemic effect.
[0108] 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.
[0109] If necessary, the compounds and useful herein can be
administered in combination with other therapeutic agents or
regimes as discussed. The choice of therapeutic agents that can be
co-administered with the compounds of the invention depends, in
part, on the condition being treated.
[0110] 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-accepta- ble 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.
[0111] Examples of materials which can serve as
pharmaceutically-acceptabl- e 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 in 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.
[0112] 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.
[0113] 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.
[0114] 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 aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring. The above listing 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.
[0115] 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.
[0116] 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., dichlorodifluoromethane,
trichlorofluoromethane, 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.
[0117] 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 as 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] The invention is described in greater detail by the
following non-limiting examples.
EXAMPLES
Example 1
Radioprotection Assay
[0122] HeLa cells were treated with 2 .mu.g/ml of chloroquine for
one hour, washed for one hour, and irradiated at 2 or 6 Gy.
Subsequently, 1000 cells were plated and assessed for colony
formation. Table 2 shows that exposure to chloroquine prior to
irradiation increased cell survival by 30%.
2TABLE 2 Treatment Average Number of Colonies* Std. Dev. 2 Gy 444
19.5 Chloroquine + 2 Gy 580 21.2 6 Gy 94.6 10.6 Chloroquine + 6 Gy
129 8.6 *Averages were from five individual samples.
[0123] To test the possibility that chloroquine activation of ATM
may cause radioprotection, C57/BL6 mice were exposed to 8 Gy IR, a
dose which kills approximately 80% of the mice at around two weeks.
Death appears to result from hematopoietic toxicities. The day
before total body irradiation (TBI), mice were either given an i.p.
injection of chloroquine or chloroquine was added to the drinking
water (5 mice--i.p. 1.75 mg/kg chloroquine; 5 mice--i.p. 3.5 mg/kg
chloroquine; 5 mice--1.75 mg/kg chloroquine in drinking water; 5
mice--3.5 mg/kg chloroquine in drinking water). FIG. 1 shows a
Kaplan-Meier survival curve indicating that a dose of chloroquine
prior to the TBI provided significant protection from death.
Significant protection was not observed in ATM deficient
(homozygous) transgenic mice. The experiment was reproduced
numerous times and analyses of tissues indicated that the
protective effect was due to enhanced recovery of hematopoietic
cells (bone marrow, spleen, thymus) following irradiation (FIG. 2).
Injection of chloroquine prior to the TBI had no effect on the
survival of mice lacking ATM genes (FIG. 3), thus indicating that
radioprotection may be dependent on ATM.
[0124] Treatment with chloroquine or hydroxychloroquine also
provided significant protection against loss of coat color in
surviving mice. FIG. 10 shows three pairs of mice subject to 8 Gy
total body irradiation. The two control mice on the left of the
figure show significant loss of coat color. The pair in the middle
which were treated with chloroquine before exposure to total body
irradiation show no significant loss of coat color. The pair on the
right treated with hydroxyquinolone show an intermediate extent of
protection.
Example 2
Cancer Prevention
[0125] Transgenic mice expressing the c-myc oncogene under the
control of the immunoglobulin enhancer (i.e., E1-myc mice) develop
B-cell lymphomas and leukemias with relatively short latencies.
Chloroquine was added to the drinking water of a cohort of
E.mu.-myc mice and the mice were observed for the development of
B-cell malignancies. FIG. 4 demonstrates that 100% of the control
transgenic mice developed malignancies within 100 days of birth
while 0% of the transgenic mice on chloroquine developed tumors.
After .about.120 days, half of the cohort of chloroquine-treated
mice were taken off of chloroquine and the other half were switched
to receiving a dose of chloroquine by i.p. injection once a week.
Within .about.30 days, all of the transgenic mice taken off of the
chloroquine had developed tumors while none of the mice receiving
weekly i.p. injections developed cancer. At 10 months of age, these
mice on weekly chloroquine remained cancer-free and appeared
healthy and normal.
[0126] The carcinogen 3-methylcholanthrene (3-MC) induces soft
tissue sarcomas if injected into muscle and skin carcinomas if
applied to the skin (Smart et al., Carcinogenesis 7:1669-1675
(1986); Noguchi, et al., Proc. Natl. Acad. Sci. U.S.A
93:11798-11801 (1996); Horak et al., Br. J. Cancer 49:637-644
1984)). This model system has been used to demonstrate that
superinduction of p53 after DNA damage (e.g., in a mouse carrying
an extra copy of chromosomal DNA containing the p53 gene) protects
mice from the development of cancers induced by chemical carcinogen
treatments (Garcia-Cao et al., EMBO J. 21:6225-6235 (2002).
Therefore, it was determined whether the protective effect observed
in these studies could likewise be achieved by biochemically
enhancing p53 induction. As demonstrated herein, ATM kinase
activation by chloroquine did not induce strand breaks or induce
phosphorylation of substrates that normally get phosphorylated by
ATM at the sites of DNA break; however, it did lead to induction
and phosphorylation of p53 protein.
[0127] In a further experiment, doses of 3.5 mg/kg of chloroquine
were given by i.p. injection 24 and 4 hours prior to 3-MC injection
in 30 wild type (strain C57B1/6) mice. The occurrence of these
tumors was readily apparent by visual inspection and confirmed by
histologic assessment. Results are shown in FIG. 5. Treatment with
chloroquine significantly increased the percentage of mice
surviving tumor free (p=0.0013).
[0128] In a further experiment 3-MC was injected into the skin on
the leg of a mouse once a week for 4 weeks. Three genetic
backgrounds were used: wild-type, ATM-null, and p53-null. One half
of each cohort of mice received 3.5 mg/kg of chloroquine (CHQ) via
IP injection 24 hours and 4 hours prior to each of the four 3-MC
administration. The development of skin carcinomas was followed
over time. FIG. 7 shows tumor incidence in mice receiving either
placebo or chloroquine prior to 3-MC injection. Chloroquine
markedly protected from tumor development. FIG. 8 shows tumor
incidence in ATM-null mice receiving either placebo or chloroquine
prior to 3-MC injection. Chloroquine does not protect from tumor
development. FIG. 9 shows tumor incidence in p53-null mice
receiving either placebo or chloroquine prior to 3-MC injection.
Again chloroquine did not protect from tumor development. These
results shows that that the prophylactic effect of chloroquine is
mediated at least in part through ATM and p53.
[0129] Multiple exposures to non-lethal doses of ionizing radiation
can induce thymic lymphomas in C57BL/6 mice (Boniver et al., In
Vivo 4(1):41-3 (1990)). Using a classical, tumor-inducing protocol
(Kaplan and Brown, J. Natl. Cancer Inst., 13, 185-206 (1952)),
which consists of four weekly whole-body exposures of 1.75 Gy each,
the effect of chloroquine administration on thymic lymphoma
formation was examined. Chloroquine (3.5 mg/kg) was administered to
4-week old female C57BL/6 mice by i.p. injection 24 hours and 4
hours prior to each of the four doses of radiation described in the
protocol. According to the protocol, tumors were expected to appear
within 4-6 months after the last dose of irradiation in 90% of
control (untreated) mice. FIG. 6 shows the results of this
analysis. Treatment with chloroquine significantly increased the
percentage of tumor free survival (p<0.0001).
[0130] As will be apparent from the foregoing the invention
provides for various uses. In particular, the invention provides
for the use of a chloroquine compound in the manufacture of a
medicament for prophylaxis or treatment of cancer. The invention
further provides for the use of a chloroquine compound in the
manufacture of a medicament for prophylaxis and treatment of tissue
damage resulting from oxidative stress. The invention further
provides for the use of a chloroquine compound in the manufacture
of a medicament for the prophylaxis and treatment of tissue damage
resulting from ischemia and/or reperfusion. The invention further
provides for the use of a chloroquine compound in the manufacture
of a medicament for the prophylaxis and treatment of tissue damage
resulting from stroke. The invention further provides for the use
of chloroquine in the manufacture of a medicament for the
prophylaxis and treatment of tissue damages resulting from
atherosclerosis.
[0131] 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.
[0132] It will be apparent to one of ordinary skill in the art that
many changes and modifications can be made thereto without
departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
1 1 3056 PRT Homo sapiens 1 Met 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
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