U.S. patent application number 10/857167 was filed with the patent office on 2005-01-20 for treatment of radiation disorders.
This patent application is currently assigned to Perlegen Sciences, Inc.. Invention is credited to Bakkenist, Christopher, Kastan, Michael B., McCamish, Mark.
Application Number | 20050014785 10/857167 |
Document ID | / |
Family ID | 23188144 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050014785 |
Kind Code |
A1 |
Kastan, Michael B. ; et
al. |
January 20, 2005 |
Treatment of radiation disorders
Abstract
The present invention provides methods and compositions for the
treatment of DNA damage related disorders. One embodiment is a
method for the inhibition of side effects associated with
chemotherapeutic and radiotherapeutic agents using chloroquine
compounds. Another embodiment is a method for treatment and/or
prevention of lethal or sub-lethal radiation toxicities associated
with terrorist acts or war.
Inventors: |
Kastan, Michael B.;
(Cordova, TN) ; Bakkenist, Christopher; (Cordova,
TN) ; McCamish, Mark; (Cupertino, CA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
943041050
|
Assignee: |
Perlegen Sciences, Inc.
Mountain View
CA
St. Jude Children's Research Hospital
Memphis
TN
|
Family ID: |
23188144 |
Appl. No.: |
10/857167 |
Filed: |
May 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10857167 |
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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10307077 |
Nov 27, 2002 |
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Current U.S.
Class: |
514/313 |
Current CPC
Class: |
A61K 31/4706 20130101;
A61K 31/675 20130101; G01N 33/6842 20130101; C12Q 1/485 20130101;
G01N 33/6812 20130101; G01N 2800/52 20130101; C07K 16/40 20130101;
G01N 33/6893 20130101 |
Class at
Publication: |
514/313 |
International
Class: |
A61K 031/47 |
Goverment Interests
[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.
Claims
1. A method of treatment for a radiation related disorder
comprising administering to an animal subject in need thereof an
effective amount of a chloroquine compound.
2. The method of claim 1 wherein said radiation related disorder is
caused by an exposure to a non-ionizing radiation.
3. The method of claim 1 wherein said radiation related disorder is
caused by an exposure to an ionizing radiation.
4. The method of claim 3 wherein said chloroquine compound treats a
skin damage symptom caused by said ionizing radiation.
5. The method of claim 4 wherein said skin damage symptom is at
least one symptom selected from swelling, itching, redness of skin,
and hair loss.
6. The method of claim 3 wherein said chloroquine compound treats a
non-skin damage symptom caused by said ionizing radiation.
7. The method of claim 6 wherein said non-skin damage symptom is at
least one symptom selected from nausea, vomiting, diarrhea, loss of
appetite, fatigue, fever, diminished organ function, seizures,
cancer, and coma.
8. The method of claim 1 wherein said radiation related disorder is
acute radiation syndrome.
9. The method of claim 1 wherein said chloroquine compound prevents
a death due to radiation exposure.
10. The method of claim 1 wherein said chloroquine compound
protects from sub-lethal and/or unintentional radiation
exposure.
11. The method of claim 1 wherein said chloroquine compound
protects from sub-lethal and/or lethal intentional radiation
exposure.
12. The method of claim 11 wherein said intentional radiation
exposure is due to an act of terrorism.
13. A method of treatment for an adverse effect of a radiation
therapy comprising administering to an animal subject in need
thereof an effective amount of a chloroquine compound.
14. The method of claim 13 wherein said adverse effect is at least
one effect selected from a skin damage, alopecia, or
neutropenia.
15. The method of claim 1 or 13 wherein said chloroquine compound
is at least one compound selected from chloroquine, chloroquine
phosphate, hydroxychloroquine, chloroquine diphosphate, chloroquine
sulphate, hydroxychloroquine sulphate, or enantiomers, derivatives,
analogs, metabolites, pharmaceutically acceptable salts, and
mixtures thereof.
16. The method of claim 15 wherein said compound is at least one
compound selected from chloroquine, chloroquine phosphate,
hydroxychloroquine, chloroquine diphosphate.
17. The method of claim 16 wherein said compound is
chloroquine.
18. The method of claim 16 wherein said compound is
hydroxychloroquine.
19. The method of claim 17 or 18 wherein said compound is an
essentially pure (+) isomer.
20. The method of claim 17 or 18 wherein said compound is an
essentially pure (-) isomer.
21. The method of claim 1 or 13 wherein the amount of the compound
administered is at least about 0.1 mg/kg/day.
22. The method of claim 1 or 13 wherein the amount of the compound
administered is up to about 10 mg/kg/day.
23. The method of claim 1 or 13 wherein the amount of the compound
administered is more than about 0.1 mg/kg/day.
24 The method of claim 1 or 13 wherein the amount of the compound
administered is more than about 1.0 mg/kg/day.
25. The method of claim 1 or 13 wherein the amount of the compound
administered is less than about 50 mg/kg/day.
26. The method of claim 1 or 13 wherein the amount of the compound
administered is less than about 10 mg/kg/day.
27. The method of claim 1 or 13 wherein the chloroquine compound is
administered more than once a week.
28. The method claim 1 or 13 wherein the chloroquine compound is
administered daily, every two weeks, or once a month.
29. The method of claim 1 or 13 wherein the chloroquine compound is
formulated in a sustained release formulation.
30. The method of claim 1 or 13 wherein said amount of said
compound administered is about 0.1 to about 9 mg/kg once a week.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. patent application Ser. No. ______, filed on May 26, 2004,
Attorney Docket No. 29202-710.831, which is a national stage
application under 35 USC 371 of PCT/US03/37838 filed Nov. 26, 2003
which claims the benefit of priority from U.S. patent application
Ser. No. 10/351,733 filed Jan. 24, 2003 and Ser. No. 10/307,077,
filed Nov. 27, 2002 which are incorporated herein by reference in
their entirety.
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.
[0005] Radiation is all around us. Exposure to radiation includes
radiation from natural sources and man-made sources. Acute and/or
chronic exposure to radiation causes several adverse effects,
including occasionally causing death. The current treatments for
treating the adverse effects following exposure to radiation are
ineffective most of the time and also have many side effects.
Hence, there is a demand for more effective treatment of effects
following exposure to radiation.
SUMMARY OF THE INVENTION
[0006] The present invention provides compositions, methods, and
kits for the treatment and/or prevention of DNA damage related
disorders, disorders caused by radiation exposure, and death
associated with radiation exposure. In one embodiment, a
chloroquine compound is administered for the prevention of DNA
damage related disorders, like cancer and radiation related
disorders. In another embodiment, the chloroquine compound does not
prevent a localized skin cancer alone. In yet another embodiment,
the cancers prevented by the chloroquine compounds are a localized
skin cancer and a cancer that is not a localized skin cancer. Also
described herein are methods of inhibiting the side effects of
chemotherapeutic and/or radiotherapeutic agents using chloroquine
compounds. Another embodiment is the prevention and/or treatment of
lethal or sub-lethal radiation toxicities associated with acts of
terrorism or war on response to such acts. The present invention
also provides compositions, methods, and kits for use in the
treatment of radiation related disorders.
BRIEF DESCRIPTION OF THE FIGURES
[0007] 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 i.p.
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.
[0008] 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.
[0009] 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.
[0010] FIG. 4 demonstrates that chloroquine treatment prevents the
development of tumors in E.mu.-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.
[0011] 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.
[0012] 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 in four successive weeks and animals were subsequently
observed for the development of tumors. Statistical significance,
log rank test P=0.0012.
[0013] FIG. 7 shows tumor incidence in wildtype mice receiving
either placebo or CHQ before 3-MC injection. CHQ markedly protects
from tumor development.
[0014] 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.
[0015] 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.
[0016] FIG. 10 demonstrates the efficacy of two chloroquine
compounds in preventing, in varying degree, the change in coat
color in mice treated with 8 GY radiation.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Chloroquine Compounds
[0018] The present invention provides methods, compositions, and
kits for the prevention and/or treatment of DNA damage related
disorders and death due to radiation exposure. Chloroquine
compounds are useful in practicing the invention described herein.
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.
[0019] 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
[0020] 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.
[0021] 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. In
certain preferred embodiments, chloroquine is used in the methods
described herein. In other embodiments hydroxychloroquine is
used.
[0022] 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)quinol
ine (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]aminoquiinoli-
n e phosphate; 3-chloro-4-(4-hydroxy-.alpha.,
.alpha.'-bis(2-methyl-1-pyrr- olidinyl)-2,5-xyl idinoquinoline,
4-[(4-diethylamino)-1-methylbutyl)amino]- -6-methoxyquinoline;
3,4-dihydro-1 (2H)-quinolinecarboxyaldehyde;
1,1'-pentamethylenediquinoleinium diiodide; and 8-quinolinol
sulfate, enantiomers thereof, as well as suitable pharmaceutical
salts thereof.
[0023] 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)-am
ine;
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-- pyrrol
idin-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-4-(benzyl)-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quin-
olin-4-yl)-N.sub.4-(3-chloro-benzyl)-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(2-hydroxy-4-methoxy-benzyl)-
-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(3,-
5-dimethoxy-benzyl)-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinoli-
n-4-yl)-N.sub.4-(4-methylsulphanyl-benzyl)-cyclohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(4-diethylamino-benzyl)-cycl-
ohexane-1,4-diamine;
cis-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(bipheny-
l-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.sub.4-(4-methoxy-benzyl)-cyclohexa-
ne-1,4-diamine;
trans-N.sub.1-(7-chloro-quinolin-4-yl)-N.sub.4-(4-dimethyl-
amino-benzyl)-cyclohexane-1,4-diamine; and
trans-N.sub.1-(7-chloro-quinoli-
n-4-yl)-N.sub.4-(2,6-difluoro-benzyl)-cyclohexane-1,4-diamine.
[0024] 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.
[0025] 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. In other embodiments, the (+) enantiomer of
chloroquine is employed.
[0026] In certain embodiments, essentially pure (+) or (-)
enantiomers are used in the methods described herein. The term
"essentially pure" does not require a 100% enantiomer. The term
refers to about more than 95% pure enatiomer, preferably more than
about 90%, more preferably more than about 85% pure, even more
preferably more than about 80%, and most preferably more than 75%
pure. In certain embodiments mixtures containing various ratios of
the (+) or (-) form in combination with the racemic mixture are
used. In certain embodiments employing the (+) or (-) forms, the
doses of the enantiomer used can be less than if the racemic
mixture were used. The enantiomers of chloroquine and
hydroxychloroquine may be prepared by procedures known to the
art.
[0027] The chloroquine compounds may metabolize to produce active
metabolites. The used of active metabolites is also within the
scope of the present invention.
[0028] Not intending to be limited by one mechanism, it is believed
that the chloroquine compounds and chloroquine-like compounds act
by enhancing the activity of Ataxia-Telangiectasia Mutated (ATM)
kinase and downstream events. The agonistic properties of
chloroquine on ATM kinase have been demonstrated. Hence, it is
intended herein that 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.
[0029] Use of Chloroquine Compounds
[0030] In one aspect, the invention provides methods of treating an
animal subject, including a human. The term "animal subject" as
used herein includes humans as well as other mammals. The methods
described herein generally involve the administration of effective
amounts of chloroquine compounds and/or chloroquine like compounds
for the treatment and/ore prevention of DNA damage related
disorders. The term "DNA damage related disorders" include, but are
not limited to, cancer, aging, disorders caused by damage to DNA
due to exposure to carcinogens, toxins, free radicals, like oxygen
radical, or DNA damaging radiations like ionizing radiation and UV
radiation. The chloroquine compounds are also useful for prevention
of tissue injury resulting from ischemia, such as that which occurs
following myocardial infarction or stroke. The effects of the
chloroquine compounds used in the methods described herein include
systemic, local, and topical effects. It is preferred that the
effects of the chloroquine compounds in the methods described
herein are systemic.
[0031] In one embodiment, the chloroquine compounds are used as
prophylactics to prevent DNA damage related disorders. The
chloroquine compounds are useful in the prevention of cancers
caused by toxins, carcinogens, DNA damaging radiations, and/or
genetic mutations. For example, chloroquine compounds are useful in
the prevention of cancers caused by exposure to toxins and
carcinogens like aromatic hydrocarbons, cigarette smoke, acetyl
amino fluorine, MTBE, etc. Also, chloroquine compounds are useful
in prevention of cancers caused by DNA damaging radiations like UV
and ionizing radiation. The ionizing radiations includes both
natural and therapeutic radiation exposures. Examples of ionizing
radiations are X-rays for diagnostics and radiation therapy used
for tumors and unintended exposure to radiation as an act of
terrorism or war.
[0032] The prophylactic uses for cancer described herein are not
envisioned to encompass the prevention solely of localized skin
carcinomas like basal cell epithelioma and squamous cell carcinoma,
skin carcinomas, Burkitt's lymphoma, or skin pathologies caused by
harmful radiation. When used in patients with actinic keratosis, it
is envisioned the chloroquine compounds do not solely inhibit basal
cell epithelioma and squamous cell carcinoma. In one embodiment,
the chloroquine compounds are used to prevent a localized skin
carcinoma and at least one cancer that is not a localized skin
carcinoma. Examples of cancers that are not localized skin
carcinomas include, but are not limited to, melanomas, lymphomas,
prostate cancer, breast cancer, colon cancer, lung cancer,
retinoblastoma, neuroblastoma, sarcomas, and ovarian cancer.
[0033] In a preferred embodiment, chloroquine compounds are used in
the prevention of one or more of the following cancers--melanomas,
prostate cancer, breast cancer, colon cancer, lung cancer,
non-Hodgkins lymphoma, retinoblastoma, neuroblastoma, sarcomas, and
ovarian cancer.
[0034] The chloroquine compounds can be used to prevent secondary
cancers, i.e., cancers that are caused by radiation therapy and
chemotherapy used to treat the primary cancer. In one embodiment,
the chloroquine compounds are used to prevent the occurrence of
breast cancer in patients receiving radiation therapy for
non-Hodgkin's lymphoma. Also, in these patients 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 patients who have had
prior incidences of cancer.
[0035] In one embodiment, the chloroquine compounds are
administered to decrease or prevent the side-effects of radiation
therapy used to treat cancer. 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.
[0036] In one embodiment, the chloroquine compounds are used in
immunosuppressed patients, like transplant patients. In
immunosuppressed patients, the chloroquine compounds can be used to
prevent cancers. The chloroquine compounds can be used to prevent
Epstein Barr virus induced lymphoproliferative syndrome.
[0037] In another embodiment, chloroquine compounds are used as
prophylactics to inhibit side effects of frequent exposure to
X-rays in athletes. This method would also be useful for other
patient populations that are frequently exposed to DNA damaging
radiations, such as X-ray technicians, pilots, 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 inhibiting the damage to cells
due to damage to DNA.
[0038] The present invention also provides methods for preventing
DNA damage, inhibiting the effects of DNA damage, and stimulating
cellular response to DNA damage by administering an effective
amount of chloroquine compounds. Not intending to be limited by one
mechanism of action, it is contemplated that cellular responses are
enhanced by an agonistic activity on ATM kinase by priming the cell
to respond to agents which cause DNA damage. Further details on ATM
kinase are provided in International PCT application no. US03/38091
filed Nov. 26, 2003, which is incorporated by reference herein in
its entirety.
[0039] 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. 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. In another
embodiment, the effective amount of a chloroquine compound is an
amount which produces anti-oxidant effects.
[0040] The prophylactic use of chloroquine includes the prevention
of tissue injury resulting from ischemia, such as that which occurs
following myocardial infarction or stroke. While not intending to
be limited to one mechanism of action, it is believed that the
chloroquine compounds prevent cellular death due to oxidative
damage during reperfusion and as such can ameliorate tissue injury
resulting from ischemic injury.
[0041] In one embodiment, chloroquine compounds are used in the
treatment of DNA damage related disorders. The chloroquine
compounds are used preferably in combination with chemotherapeutic
or radiotherapeutic agents to prevent the side-effects associated
with the chemotherapeutic agents. It is known that chloroquine
compounds can inhibit multiple drug resistance. Hence, it is not
intended that the methods described herein produce a beneficial
effect on multiple drug resistance alone. In a preferred
embodiment, the beneficial effects of chloroquine compounds, when
used in combination with chemotherapeutic agents, are due to
modulation of ATM kinase activity. It is contemplated that the
chloroquine compounds 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.
[0042] In one embodiment, the chloroquine compounds are used to
treat and/or prevent disorders caused by oxidative damage. The
chloroquine compounds can be administered with anti-oxidants, like
vitamin B12, to stimulate the cellular response to DNA damage and
promote the repair of the cells exposed to the oxidative
agents.
[0043] Treatment of Radiation Therapy Related Adverse Effects
[0044] In one aspect of the invention the chloroquine compounds are
used in the treatment of adverse effects associated with radiation
therapy. The chloroquine compounds may be used to therapeutically
treat the adverse effects of radiation therapy and/or
prophylactically to prevent the occurrence of the adverse effects
associated with radiation therapy. In preferred embodiments, the
use of the chloroquine compounds does not adversely affect the
efficacy and/or potency of the radiation therapy in the disease
being treated.
[0045] Radiation therapy, also known as radiotherapy, x-ray
therapy, or irradiation, is the treatment of disease using
penetrating beams of high-energy or low-energy waves or streams of
particles called radiation. The radiation is used for the treatment
of cancer and is usually administered from special machines or from
radioactive substances. The doses of radiation that damage or
destroy the diseased cells, such as cancer cells, can also injure
or kill normal cells. These effects of radiation on normal cells
cause treatment side effects. In one embodiment, the chloroquine
compounds described herein are used in the treatment of adverse
effects associated with radiation therapy. The chloroquine
compounds can be administered prior to the radiation therapy or
after the radiation therapy is started. Preferably the chloroquine
compounds minimize the effects of the radiation therapy on normal
cells.
[0046] The high energy rays used for radiation therapy can include
for example, x-rays, an electron beam, or cobalt-60 gamma rays.
Also beams of protons or neutrons may be used for radiation
therapy. Internal radiation therapy places the radiation source as
close as possible to the diseased cells. Some of the radioactive
substances used for internal radiation treatment include cesium,
iridium, iodine, phosphorus, and palladium.
[0047] Side effects of treatment with radiation include temporary
or permanent loss of hair in the area being treated, skin
irritation, temporary change in skin color in the treated area, and
tiredness. Some people who receive radiation to the head and neck
experience redness and irritation in the mouth, a dry mouth,
difficulty in swallowing, changes in taste, or nausea. Other
possible side effects include a loss of taste, earaches, and
swelling. Radiation therapy can cause hair loss (alopecia).
Radiation therapy can also cause low white blood cell counts or low
levels of platelets. Neutropenia, which refers to an abnormally low
number of neutrophils in the blood, can also be an adverse effect
associated with radiation therapy.
[0048] In certain embodiments, the chloroquine compounds are used
to treat the adverse effects of radiation therapy on the skin. In
other embodiments, the compounds are used to treat alopecia
associated with radiation therapy. In other preferred embodiments,
the chloroquine compounds are used to treat the side effects
associated with blood, such as neutropenia.
[0049] Treatment of Radiation Related Disorders
[0050] In one aspect of the invention the chloroquine compounds are
used in the treatment of radiation-related disorders.
[0051] Radiation is typically classified as non-ionizing and
ionizing radiation. Examples of sources of non-ionizing radiation
include, but are not limited to, power lines, AM/FM radio and
televsion, microwave oven, heat lamps, and tanning salons. In one
embodiment, chloroquine compounds are used to treat adverse health
effects caused by non-ionizing radiations. The compounds can be
used therapeutically and/or prophylactically.
[0052] The kinds of ionizing radiation include alpha particles,
beta particles, gamma rays, and x-rays. Ionizing radiations have
enough energy to break chemical bonds and typically cause
biological damage by breaking and/or damaging DNA bonds. In
preferred embodiments, the chloroquine compounds are used to treat
adverse health effects caused by ionizing radiations. The compounds
can be used therapeutically and/or prophylactically.
[0053] Typically, due to the biological effects of radiation, cells
either die or due to the damage to the DNA the cells may mutate.
The mutations can be such that the effects of the mutation are seen
immediately or after several days, months, or years. Also, the
mutations could be passed on the affected individuals offsprings or
may show up many generations later. In certain embodiments, the use
of chloroquine has a beneficial effect on the cell death and/or the
mutations of the cells following exposure to radiation. For
example, use of chloroquine prior to exposure to radiation and/or
following to radiation can decrease the number of mutations and
thus cause a decrease in the adverse genetic effects caused by
exposure to radiation.
[0054] Acute radiation syndrome (ARS) is caused by exposure of the
body to high doses of radiation usually over a short period of
time. The symptoms include nausea, vomiting, diarrhea, loss of
appetite, fatigue, fever, diminished organ function, and possibly
even seizures, coma, and death. ARS also typically includes skin
damage, such as swelling, itching, redness of skin, and hair loss.
In some embodiments, chloroquine compounds are used therapeutically
and/or prophylactically for ARS. In the treatment of ARS, in some
embodiments, the beneficial effects are not solely on the skin
damage caused by radiation. Preferably, the chloroquine compounds
have a beneficial effects on both the skin damage and one or more
of the non-skin related symptoms of ARS, such as the nausea,
vomiting, diarrhea, loss of appetite, fatigue, fever, diminished
organ function, seizures and coma.
[0055] Exposure to radiation can also cause cancers, such as
leukemia, breast, bladder, colon, liver, lung, esophagus, ovarian,
multiple myeloma, and stomach cancers. Other cancers that can be
caused by radiation include, prostate, nasal cavity/sinuses,
pharyngeal, and laryngeal, and pancreatic cancer. In some
embodiments, chloroquine compounds are used therapeutically and/or
prophylactically for cancers caused by exposure to radiation.
[0056] In some embodiments, the chloroquine compounds are used by
astronauts, prior to or during space flight, to reduce the adverse
effects caused by exposure to radiation during space flights. In
other embodiments, the methods described herein are employed for
the treatment of hospital personnel who are exposed to X-ray
radiation. In yet other embodiments, personnel who are exposed to
radiation in war situations or have to work in areas with
abnormally high levels of natural radiation are treated with the
chloroquine compounds. Also, the compounds and methods described
herein can be used to treat personnel who are involved in clean-up
operations following an accidental or intentional, e.g., a
terrorist attack, release of radiation.
[0057] In some embodiments, the chloroquine compounds can be used
for treatment in anticipation of or following exposure to a "dirty
bomb." A "dirty bomb" typically refers to a bomb that combines
conventional explosives, such as dynamite, with radioactive
materials in the form of powder or pellets. Also, the chloroquine
compounds can be used in the therapeutic and/or prophylactic
treatment of medical and other personnel who would be involved in
the treatment and clean-up operations following the explosion of a
dirty bomb. In certain embodiments, hospitals, pharmacies, and
non-medical personnel stockpile chloroquine compounds to be used in
the event of an explosion of a dirty bomb. The chloroquine
compounds from such a stockpile can be used by subjects exposed to
the radiation from the dirty bomb and subjects likely to exposed to
the radiation.
[0058] In certain other embodiments, the chloroquine compounds are
used for the prevention of death and/or for the prophylactic
treatment of soldiers and other personnel entering areas with
expected weapons of mass destruction or expected to encounter
radiation exposure due to an act of war or terrorism.
[0059] In certain preferred embodiments, chloroquine and
hydroxychloroquine are used in the treatment of radiation related
disorders. Even more preferred is the use of chloroquine. In other
embodiments, the pure enantiomers of chloroquine or
hydroxychloroquine, either the (+) or (-) form are used.
[0060] Therapeutic and Prophylactic Benefits
[0061] In one embodiment, the chloroquine compounds are used as
prophylactic agents. For prophylactic benefit, the chloroquine
compound may be administered to a patient at risk of developing a
DNA damage related disorder like cancer or to a patient reporting
one or more of the physiological symptoms of a DNA damage related
disorder, even though a diagnosis of such disorder may not have
been made. A prophylactic benefit is achieved when a disorder is
prevented from afflicting a patient. This prevention can include
the affliction of the patient with a milder form of the disorder or
the appearance of fewer or no symptoms of the disorder being
prevented or the absence of the disorder in the patient being
treated.
[0062] In addition to a prophylactic benefit, the chloroquine
compounds can be used for their therapeutic benefits. In one
embodiment, the chloroquine compounds are used to treat DNA damage
related disorders. In a preferred embodiment, the beneficial effect
of the chloroquine compounds is not due to an inhibition of
multiple drug resistance. The term "treating" as used herein
includes achieving a therapeutic benefit and/or a prophylactic
benefit. By therapeutic benefit is meant eradication or
amelioration of the underlying disorder being treated. For example,
in a cancer patient, therapeutic benefit includes eradication or
amelioration of the underlying cancer. Also, a therapeutic benefit
is achieved with 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 patient,
notwithstanding that the patient may still be afflicted with the
underlying disorder. For example, administration of a chloroquine
compound to a patient suffering from cancer provides therapeutic
benefit not only when the patient's tumor marker level is
decreased, but also when an improvement is observed in the patient
with respect to other complications that accompany the cancer like
pain and psychiatric disorders.
[0063] Effective Amount
[0064] A physician or veterinarian having ordinary skill in the art
may readily determine and prescribe the effective amount of the
chloroquine compound required in the methods described herein.
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 will depend on the condition being treated and the
route of administration. Determination of an effective amount is
well within the capabilities of those skilled in the art,
especially in light of the disclosure herein.
[0065] 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.
[0066] In one embodiment, the effective amount can include the dose
ranges, modes of administration, formulations, etc., that have been
recommended or approved by any of the various regulatory or
advisory organizations in the medical or pharmaceutical arts (eg,
FDA, AMA) or by the manufacturer or supplier. Effective amounts of
chloroquine can be found, for example, in the Physicians Desk
Reference.
[0067] The daily dosage range of chloroquine, in one embodiment,
can vary between about 0.1 mg/kg to about 2 gm/kg body weight. The
daily dose of a chloroquine compound may be less than about 2
gm/kg, less than about 1.5 gm/kg, or less than about 1 gm/kg. In
one embodiment, the daily dose of a chloroquine coumpound is more
than about 0.5 mg/kg, more than about 500 mg/kg, or more than about
1 gm/kg. Preferred 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 or about 30 mg/kg to about 50 mg/kg body weight. Preferred
doses of chloroquine diphosphate and hydroxychloroquine are about
3.5 mg/kg and 7.0 mg/kg.
[0068] 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 below.
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
[0069] 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. 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 a total period of at least 1 day, 3 days,
a week or a month. For example, if the risk of exposure is known in
advance, an exemplary regime entails administering the chloroquine
compound on the day before, the day of exposure and the day after
exposure. If the risk of exposure is not known in advance, an
exemplary regime entails 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 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, such as the purified (-) form or the purified (+)
form.
[0070] In some embodiments, the effective amount of chloroquine is
administered once a month, 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 yet another
embodiment, 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.
[0071] In one embodiment, 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.
[0072] Routes of Administration and Formulation
[0073] The compounds useful in the present invention, or
pharmaceutically acceptable salts thereof, can be delivered to the
patient 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.
[0074] The formulations useful herein can administer the
chloroquine compounds topically or systemically. In one embodiment,
the formulation of chloroquine compound is administered
systemically. In another embodiment, the formulation of chloroquine
compound has a systemic effect if administered either topically or
systemically.
[0075] 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 may 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.
[0076] If necessary, the compounds and useful herein may be
administered in combination with other therapeutic agents. The
choice of therapeutic agents that can be co-administered with the
compounds of the invention will depend, in part, on the condition
being treated.
[0077] Agents used in accordance with the methods of the invention
may be conveniently administered in a pharmaceutical composition
containing the active compound in combination with a suitable
carrier. Such pharmaceutical compositions may 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 & Wilkins:
Philadelphia, Pa., 2000. A pharmaceutically-acceptabl- e 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
patient.
[0078] Examples of materials which may 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 harmaceutical
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.
[0079] Agents of use in the invention may 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.
[0080] For oral therapeutic administration, the composition may 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 may 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 may, of course, be varied and may 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 will be
obtained.
[0081] The tablets, troches, pills, capsules, and the like may 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 may contain, in addition to materials of the above
type, a liquid carrier, such as a vegetable oil or a polyethylene
glycol. Various other materials may be present as coatings or to
otherwise modify the physical form of the solid unit dosage form.
For instance, tablets, pills, or capsules may be coated with
gelatin, wax, shellac or sugar and the like.
[0082] For administration orally, the compounds may 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.
[0083] For administration by inhalation, the active compound(s) may
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 may 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 may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch.
[0084] A syrup or elixir may 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 may 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.
[0085] The compounds may 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.
[0086] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation or
transcutaneous delivery (for example subcutaneously or
intramuscularly), intramuscular injection or a transdermal patch.
Thus, for example, the compounds may 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.
[0087] The selected dosage level will depend upon 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 patient being
treated, and like factors well-known in the medical arts.
[0088] The invention is described in greater detail by the
following non-limiting examples.
EXAMPLES
Example 1
Radioprotection Assay
[0089] 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 1 shows that exposure to chloroquine prior to
irradiation increased cell survival by 30%.
2 TABLE 1 Average Standard Treatment Number of Colonies* Deviation
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.
[0090] 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.
[0091] 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
[0092] Transgenic mice expressing the c-myc oncogene under the
control of the immunoglobulin enhancer (i.e., E.mu.-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 .about.10 months of
age, these mice on weekly chloroquine remained cancer-free and
appeared healthy and normal.
[0093] 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. (1986) Carcinogenesis
7:1669-1675; Noguchi, et al. (1996) Proc. Natl. Acad. Sci. U.S.A
93:11798-11801; Horak, et al. (1984) Br. J. Cancer 49:637-644).
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. (2002) EMBO J. 21:6225-6235). 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 breaks, however, it did lead to induction and
phosphorylation of p53 protein. Thus, chloroquine pre-treatment may
prevent/reduce tumor development resulting from 3-MC injections.
Accordingly, 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 mice.
Results are shown in FIG. 5. The occurrence of these tumors was
readily apparent by visual inspection and confirmed by histologic
assessment.
[0094] 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 C57Bl/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).
[0095] 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 chlorquine (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 showsthat that the prophylactic effect of chloroquine is
mediated at least in part through ATM and p53.
[0096] Multiple exposures to non-lethal doses of ionizing radiation
can induce thymic lymphomas in C57BL/6 mice (Boniver, et al. (1990)
Int. J. Radiat. Biol. 57:693-698). Using a classical,
tumor-inducing protocol (Kaplan and Brown (1952) J. Natl. Cancer
Inst. 13:185-208), 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.
[0097] All patents, publications, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual patent, publication, or patent
application was specifically and individually indicated to be
incorporated by reference.
[0098] 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.
* * * * *