U.S. patent application number 10/858450 was filed with the patent office on 2004-12-02 for compositions and methods for inhibiting an isoform of human manganese superoxide dismutase.
Invention is credited to Anziano, Paul Q..
Application Number | 20040242655 10/858450 |
Document ID | / |
Family ID | 33551448 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040242655 |
Kind Code |
A1 |
Anziano, Paul Q. |
December 2, 2004 |
Compositions and methods for inhibiting an isoform of human
manganese superoxide dismutase
Abstract
The present invention is directed to methods of modulating the
activity of an isoform of manganese superoxide dismutase which is
useful for the treatment of diseases such as heart failure.
Inventors: |
Anziano, Paul Q.;
(Philadelphia, PA) |
Correspondence
Address: |
COZEN O'CONNOR, P.C.
1900 MARKET STREET
PHILADELPHIA
PA
19103-3508
US
|
Family ID: |
33551448 |
Appl. No.: |
10/858450 |
Filed: |
May 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60473458 |
May 28, 2003 |
|
|
|
Current U.S.
Class: |
514/357 ;
514/408; 514/485; 514/520; 514/568; 514/585; 514/596; 514/603;
514/617 |
Current CPC
Class: |
A61K 31/165 20130101;
A61K 31/675 20130101; A61K 31/40 20130101; A61K 31/17 20130101;
A61K 31/27 20130101; A61K 31/44 20130101; A61K 31/00 20130101; C12N
9/0089 20130101 |
Class at
Publication: |
514/357 ;
514/408; 514/485; 514/603; 514/617; 514/520; 514/568; 514/585;
514/596 |
International
Class: |
A61K 031/675; A61K
031/44; A61K 031/40; A61K 031/27; A61K 031/165; A61K 031/17 |
Claims
What is claimed is:
1. A method of inhibiting an activity of isoMnSOD in a cell
comprising contacting said cell with a compound of Formula I:
Alk-L.sup.1-L.sup.2-D I or pharmaceutically acceptable salt or
prodrug thereof, wherein: Alk is C.sub.2-100 alkenyl or C.sub.2-100
alkynyl, each optionally substituted by one or more R.sup.1;
L.sup.1 is O, S, CO, C(O)O, C(O)NR.sup.2, SO, S(O).sub.2,
S(O)NR.sup.2, S(O).sub.2NR.sup.2, NR.sup.2, NR.sup.2C(O)NR.sup.3,
or NR C(S)NR.sup.3; L.sup.2 is absent, C.sub.1-6 alkylenyl,
C.sub.2-6 alkenylenyl, or C.sub.2-6 alkynylenyl, each optionally
substituted by one or more R.sup.4; D is aryl or heteroaryl, each
optionally substituted by one or more R.sup.5; R.sup.1 and R.sup.4
are each, independently, halo, cyano, nitro, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
heteroaryl, C.sub.3-7 cycloalkyl, heterocycloalkyl, S(O)R.sup.6,
S(O).sub.2R.sup.6, C(O)R.sup.6, OR.sup.7, SR.sup.7, C(O)OR.sup.7,
NR.sup.8R.sup.9 or NR.sup.8C(O)R.sup.6; R.sup.2 and R.sup.3 are
each, independently, H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, aryl, heteroaryl, C.sub.3-7
cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl;
(C.sub.3-7 cycloalkyl)alkyl or heterocycloalkylalkyl; R.sup.5 is
halo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.1-4 haloalkyl, aryl, cycloalkyl, heteroaryl,
heterocycloalkyl, CN, NO.sub.2, OR.sup.2, SR.sup.12, C(O)R.sup.3,
C(O)NR.sup.14R.sup.15, C(O)OR.sup.12OC(O)R.sup.13,
OC(O)NR.sup.14R.sup.15, NR.sup.14R.sup.15, NR.sup.14 C(O)R.sup.15,
NR.sup.14C(O)OR.sup.12, S(O)R.sup.13, S(O)NR.sup.14R.sup.15,
S(O).sub.2R.sup.13, or S(O).sub.2NR.sup.14R.sup.15; R.sup.6 is H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl, (C.sub.3-7
cycloalkyl)alkyl, heterocycloalkylalkyl, or NR.sup.10R.sup.11;
R.sup.7 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 haloalkyl, alkoxyalkyl, haloalkoxyalkyl,
aryloxyalkyl, heteroaryloxyalkyl, cycloalkyloxyalkyl,
heterocycloalkyloxyalkyl, aryl, heteroaryl, C.sub.3-C.sub.7
cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl;
(C.sub.3-7 cycloalkyl)alkyl or heterocycloalkylalkyl; R.sup.8 and
R.sup.9 are each, independently, H, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, aryl, heteroaryl,
C.sub.3-7 cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl;
(C.sub.3-7 cycloalkyl)alkyl or heterocycloalkylalkyl; or R.sup.8
and R.sup.9 together with the N atom to which they are attached
form a 3-, 4-, 5-, 6-, or 7-membered heterocycloalkyl group;
R.sup.10 and R.sup.11 are each, independently, H, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
C.sub.1-C.sub.6 haloalkyl, aryl, heteroaryl, C.sub.3-C.sub.7
cycloalkyl or heterocycloalkyl; R.sup.12 is H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl or heterocycloalkyl; R.sup.13 is H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, aryl, cycloalkyl, heteroaryl or heterocycloalkyl; and
R.sup.14 and R.sup.15 are each, independently, H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, arylalkyl, or cycloalkylalkyl; or or R.sup.14 and
R.sup.15 together with the N atom to which they are attached form a
4-, 5-, 6- or 7-membered heterocycloalkyl group.
2. The method of claim 1 wherein said activity is pro-oxidant
activity.
3. The method of claim 1, wherein said compound is arachidonoyl
dopamine, arachidonoyl serotonin, eicosapentaenoyl dopamine,
docosahexaenoyl dopamine, or C18:4(.omega.-3)-dopamine.
4. A method of treating drug-induced toxicity in an individual
comprising administering to said individual a therapeutically
effective amount of an isoMnSOD inhibitor.
5. The method of claim 3 wherein said drug is a
chemotherapeutic.
6. The method of claim 5 wherein said chemotherapeutic is a
Topoisomerase II inhibitor.
7. The method of claim 6 wherein said Topoisomerase II inhibitor is
doxorubicin.
8. The method of claim 3 wherein said toxicity is organ
toxicity.
9. The method of claim 8 wherein said organ is heart, pancreas,
liver, kidney, brain, colon, or stomach.
10. The method of claim 9 wherein said organ is heart.
11. A method of treating heart failure in an individual comprising
administering to said individual a therapeutically effective amount
of an isoMnSOD inhibitor.
12. The method of 11 wherein said heart failure is non-ischemic
heart failure.
13. The method of claim 11 wherein said heart failure is drug
induced.
14. The method of claim 13 wherein said heart failure is induced by
an anthracycline.
15. The method of claim 14 wherein said anthracycline is
doxorubicin, epirubicin, daunorubicin, idarubicin, or
anthracenedione (mitoxantrone).
16. The method of claim 13 wherein said heart failure is induced by
chemotherapy.
17. The method of claim 16 wherein said chemotherapy comprises a
DNA damaging chemotherapeutic.
18. The method of claim 17 wherein said DNA damaging
chemotherapeutic is a Topoisomerase II inhibitor.
19. The method of claim 18 wherein said Topoisomerase II inhibitor
is doxorubicin.
20. The method of claim 17 wherein said chemotherapy further
comprises a non-DNA damaging chemotherapeutic.
21. The method of claim 20 wherein said non-DNA damaging
chemotherapeutic is an antibody.
22. The method of claim 21 wherein said antibody recognizes
HER-2.
23. The method of claim 22 wherein said antibody that recognizes
HER-2 is Herceptin.
24. The method of claim 13 wherein said isoMnSOD inhibitor
comprises a compound of Formula I: Alk-L.sup.1-L.sup.2-D I or
pharmaceutically acceptable salt or prodrug thereof, wherein: Alk
is C.sub.2-100 alkenyl or C.sub.2-100 alkynyl, each optionally
substituted by one or more R.sup.1; L.sup.1 is O, S, CO, C(O)O,
C(O)NR.sup.2, SO, S(O).sub.2, S(O)NR.sup.2, S(O).sub.2NR.sup.2,
NR.sup.2, NR.sup.2C(O)NR.sup.3, or NR.sup.2C(S)NR.sup.3; L.sup.2 is
absent, C.sub.1-6 alkylenyl, C.sub.2-6 alkenylenyl, or C.sub.2-6
alkynylenyl, each optionally substituted by one or more R.sup.4, D
is aryl or heteroaryl, each optionally substituted by one or more
R.sup.5; R.sup.1 and R.sup.4 are each, independently, halo, cyano,
nitro, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, S(O)R.sup.6, S(O).sub.2R.sup.6, C(O)R.sup.6,
OR.sup.7, SR.sup.7, C(O)OR.sup.7, NR.sup.8R.sup.9 or
NR.sup.8C(O)R.sup.6; R.sup.2 and R.sup.3 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; R.sup.5 is halo,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-4
haloalkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, CN,
NO.sub.2, OR.sup.2, SR.sup.2, C(O)R.sup.3, C(O)NR.sup.4R.sup.5,
C(O)OR OC(O)R.sup.13, OC(O)NR.sup.14R 5 NR.sup.14 R 5
NR.sup.4C(O)R.sup.5, NR.sup.14C(O)OR.sup.12, S(O)R.sup.13,
S(O)NR.sup.14R.sup.15, S(O).sub.2R.sup.13, or
S(O).sub.2NR.sup.14R.sup.15; R.sup.6 is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, aryl,
heteroaryl, C.sub.3-7 cycloalkyl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, (C.sub.3-7 cycloalkyl)alkyl,
heterocycloalkylalkyl, or NR.sup.10R.sup.11; R.sup.7 is H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryloxyalkyl,
heteroaryloxyalkyl, cycloalkyloxyalkyl, heterocycloalkyloxyalkyl,
aryl, heteroaryl, C.sub.3-C.sub.7 cycloalkyl, heterocycloalkyl,
arylalkyl, heteroarylalkyl; (C.sub.3-7 cycloalkyl)alkyl or
heterocycloalkylalkyl; R.sup.8 and R.sup.9 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; or R.sup.8 and R.sup.9
together with the N atom to which they are attached form a 3-, 4-,
5-, 6-, or 7-membered heterocycloalkyl group; R.sup.10 and R.sup.11
are each, independently, H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 haloalkyl, aryl,
heteroaryl, C.sub.3-C.sub.7 cycloalkyl or heterocycloalkyl;
R.sup.12 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl or
heterocycloalkyl; R.sup.13 is H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl,
heteroaryl or heterocycloalkyl; and R.sup.14 and R.sup.15 are each,
independently, H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, arylalkyl, or
cycloalkylalkyl; or or R.sup.14 and R.sup.15 together with the N
atom to which they are attached form a 4-, 5-, 6- or 7-membered
heterocycloalkyl group.
25. The method of claim 24 wherein said compound is arachidonoyl
dopamine, arachidonoyl serotonin, eicosapentaenoyl dopamine,
docosahexaenoyl dopamine, or C18:4(.omega.-3)-dopamine.
26. A method of treating a mitochondrial related disease or
condition in an individual comprising administering to said an
individual a therapeutically effective amount of an isoMnSOD
inhibitor.
27. The method of claim 26 wherein said inhibitor comprises a
compound of Formula I: Alk-L.sup.1-L.sup.2-D I or pharmaceutically
acceptable salt or prodrug thereof, wherein: Alk is C.sub.2-100
alkenyl or C.sub.2-100 alkynyl, each optionally substituted by one
or more R.sup.1; L.sup.1 is O, S, CO, C(O)O, C(O)NR.sup.2, SO,
S(O).sub.2, S(O)NR.sup.2, S(O).sub.2NR.sup.2, NR.sup.2,
NR.sup.2C(O)NR.sup.3, or NR.sup.2C(S)NR.sup.3; L.sup.2 is absent,
C.sub.1-6 alkylenyl, C.sub.2-6 alkenylenyl, or C.sub.2-6
alkynylenyl, each optionally substituted by one or more R.sup.4; D
is aryl or heteroaryl, each optionally substituted by one or more
R.sup.5; R.sup.1 and R.sup.4 are each, independently, halo, cyano,
nitro, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, S(O)R.sup.6, S(O).sub.2R.sup.6, C(O)R.sup.6,
OR.sup.7, SR.sup.7, C(O)OR.sup.7, NR.sup.8R.sup.9 or
NR.sup.8C(O)R.sup.6; R.sup.2 and R.sup.3 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; R.sup.5 is halo,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-4
haloalkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, CN,
NO.sub.2, OR.sup.12, SR.sup.12, C(O)R.sup.3, C(O)NR.sup.4R.sup.15,
C(O)OR.sup.12, OC(O)R.sup.13, OC(O)NR.sup.14R.sup.15,
NR.sup.14R.sup.15, NR.sup.14C(O)R.sup.15, NR.sup.14C(O)OR.sup.12,
S(O)R.sup.13, S(O)NR.sup.14R.sup.15, S(O).sub.2R.sup.3, or
S(O).sub.2NR.sup.4R.sup.15; R.sup.6 is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, aryl,
heteroaryl, C.sub.3-7 cycloalkyl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, (C.sub.3-7 cycloalkyl)alkyl,
heterocycloalkylalkyl, or NR.sup.10R.sup.11; R.sup.7 is H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryloxyalkyl,
heteroaryloxyalkyl, cycloalkyloxyalkyl, heterocycloalkyloxyalkyl,
aryl, heteroaryl, C.sub.3-C.sub.7 cycloalkyl, heterocycloalkyl,
arylalkyl, heteroarylalkyl; (C.sub.3-7 cycloalkyl)alkyl or
heterocycloalkylalkyl; R.sup.8 and R.sup.9 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; or R.sup.8 and R.sup.9
together with the N atom to which they are attached form a 3-, 4-,
5-, 6-, or 7-membered heterocycloalkyl group; R.sup.10 and R.sup.11
are each, independently, H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 haloalkyl, aryl,
heteroaryl, C.sub.3-C.sub.7 cycloalkyl or heterocycloalkyl;
R.sup.12 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl or
heterocycloalkyl; R.sup.13 is H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl,
heteroaryl or heterocycloalkyl; and R.sup.14 and R.sup.15 are each,
independently, H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, arylalkyl, or
cycloalkylalkyl; or or R.sup.14 and R.sup.15 together with the N
atom to which they are attached form a 4-, 5-, 6- or 7-membered
heterocycloalkyl group.
28. The method of claim 26 wherein said compound is arachidonoyl
dopamine, arachidonoyl serotonin, eicosapentaenoyl dopamine,
docosahexaenoyl dopamine, or C18:4(.omega.-3)-dopamine.
29. A method of inhibiting cell death comprising contacting a cell
with an isoMnSOD inhibitor, wherein said inhibitor does not
modulate expression of a nucleic acid molecule encoding
isoMnSOD.
30. The method of claim 29 wherein said inhibitor comprises a
compound of Formula I: Alk-L.sup.1-L.sup.2-D I or pharmaceutically
acceptable salt or prodrug thereof, wherein: Alk is C.sub.2-100
alkenyl or C.sub.2-100 alkynyl, each optionally substituted by one
or more R.sup.1; L.sup.1 is O, S, CO, C(O)O, C(O)NR.sup.2, SO,
S(O).sub.2, S(O)NR.sup.2, S(O).sub.2NR.sup.2, NR.sup.2,
NR.sup.2C(O)NR.sup.3, or NR.sup.2C(S)NR.sup.1; L.sup.2 is absent,
C.sub.1-6 alkylenyl, C.sub.2-6 alkenylenyl, or C.sub.2-6
alkynylenyl, each optionally substituted by one or more R.sup.4; D
is aryl or heteroaryl, each optionally substituted by one or more
R.sup.5; R.sup.1 and R.sup.4 are each, independently, halo, cyano,
nitro, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, S(O)R.sup.6, S(O).sub.2R.sup.6, C(O)R.sup.6,
OR.sup.7, SR.sup.7, C(O)OR.sup.7, NR.sup.8R.sup.9 or
NR.sup.8C(O)R.sup.6; R.sup.2 and R.sup.3 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; R.sup.5 is halo,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-4
haloalkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, CN,
NO.sub.2, OR.sup.2, SR.sup.2, C(O)R.sup.3, C(O)NR.sup.14R.sup.5,
C(O)OR OC(O)R.sup.13, OC(O)NR.sup.14R.sup.15", R
NR.sup.4C(O)R.sup.5, NR.sup.4C(O)OR.sup.2, S(O)R.sup.3,
S(O)NR.sup.14R.sup.15 S(O).sub.2R.sup.13, or
S(O).sub.2NR.sup.4R.sup.15; R.sup.6 is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, aryl,
heteroaryl, C.sub.3-7 cycloalkyl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, (C.sub.3-7 cycloalkyl)alkyl,
heterocycloalkylalkyl, or NR.sup.10R.sup.11; R.sup.7 is H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryloxyalkyl,
heteroaryloxyalkyl, cycloalkyloxyalkyl, heterocycloalkyloxyalkyl,
aryl, heteroaryl, C.sub.3-C.sub.7 cycloalkyl, heterocycloalkyl,
arylalkyl, heteroarylalkyl; (C.sub.3-7 cycloalkyl)alkyl or
heterocycloalkylalkyl; R.sup.8 and R.sup.9 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; or R.sup.8 and R.sup.9
together with the N atom to which they are attached form a 3-, 4-,
5-, 6-, or 7-membered heterocycloalkyl group; R.sup.10 and R.sup.11
are each, independently, H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 haloalkyl, aryl,
heteroaryl, C.sub.3-C.sub.7 cycloalkyl or heterocycloalkyl;
R.sup.12 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl or
heterocycloalkyl; R.sup.13 is H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl,
heteroaryl or heterocycloalkyl; and R.sup.14 and R.sup.15 are each,
independently, H, C.sub.1-6 alkyl, C-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, arylalkyl, or
cycloalkylalkyl; or or R.sup.14 and R.sup.15 together with the N
atom to which they are attached form a 4-, 5-, 6- or 7-membered
heterocycloalkyl group.
31. The method of claim 29 wherein said compound is arachidonoyl
dopamine, arachidonoyl serotonin, eicosapentaenoyl dopamine,
docosahexaenoyl dopamine, or C18:4(.omega.-3)-dopamine.
32. The method of claim 29 wherein said cell expresses
isoMnSOD.
33. The method of claim 29 wherein said cell is a
cardiomyocyte.
34. The method of claim 29 wherein said cell death is
apoptosis.
35. A method of inhibiting mitochondrial oxidative stress in a cell
comprising contacting said cell with an isoMnSOD inhibitor.
36. The method of claim 35 wherein said inhibitor comprises a
compound of Formula I: Alk-L.sup.1-L.sup.2-D I or pharmaceutically
acceptable salt or prodrug thereof, wherein: Alk is C.sub.2-100
alkenyl or C.sub.2-100 alkynyl, each optionally substituted by one
or more R.sup.1; L.sup.1 is O, S, Co, C(O)O, C(O)NR.sup.2, SO,
S(O).sub.2, S(O)).sub.2NR.sup.2, NR.sup.2, NR.sup.2C(O)NR.sup.3, or
NR C(S)NR.sup.3; L.sup.2 is absent, C.sub.1-6 alkylenyl, C.sub.2-6
alkenylenyl, or C.sub.2-6 alkynylenyl, each optionally substituted
by one or more R.sup.4; D is aryl or heteroaryl, each optionally
substituted by one or more R.sup.5; R.sup.1 and R.sup.4 are each,
independently, halo, cyano, nitro, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, heteroaryl,
C.sub.3-7 cycloalkyl, heterocycloalkyl, S(O)R.sup.6,
S(O).sub.2R.sup.6, C(O)R.sup.6, OR.sup.7, SR.sup.7, C(O)OR.sup.7,
NR.sup.8R.sup.9 or NR.sup.8C(O)R.sup.6; R and R.sup.3 are each,
independently, H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 haloalkyl, aryl, heteroaryl, C.sub.3-7
cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl;
(C.sub.3-7 cycloalkyl)alkyl or heterocycloalkylalkyl; R.sup.5 is
halo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.1-4 haloalkyl, aryl, cycloalkyl, heteroaryl,
heterocycloalkyl, CN, NO.sub.2, OR.sup.12, SR.sup.12, C(O)R.sup.3,
C(O)NR.sup.4R.sup.15, C(O)OR.sup.12, OC(O)R.sup.13,
OC(O)NR.sup.14R.sup.5, NR.sup.14R.sup.15, NR.sup.14C(O)R.sup.15,
NR.sup.14C(O)OR.sup.12, S(O)R.sup.13, S(O)NR.sup.14 R.,
S(O).sub.2R.sup.3, or S(O).sub.2NR.sup.4R.sup.15; R.sup.6 is H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl, (C.sub.3-7
cycloalkyl)alkyl, heterocycloalkylalkyl, or NR.sup.10R.sup.11;
R.sup.7 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 haloalkyl, alkoxyalkyl, haloalkoxyalkyl,
aryloxyalkyl, heteroaryloxyalkyl, cycloalkyloxyalkyl,
heterocycloalkyloxyalkyl, aryl, heteroaryl, C.sub.3-C.sub.7
cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl;
(C.sub.3-7 cycloalkyl)alkyl or heterocycloalkylalkyl; R.sup.8 and
R.sup.9 are each, independently, H, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, aryl, heteroaryl,
C.sub.3-7 cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl;
(C.sub.3-7 cycloalkyl)alkyl or heterocycloalkylalkyl; or R.sup.8
and R.sup.9 together with the N atom to which they are attached
form a 3-, 4-, 5-, 6-, or 7-membered heterocycloalkyl group;
R.sup.10 and R.sup.11 are each, independently, H, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
C.sub.1-C.sub.6 haloalkyl, aryl, heteroaryl, C.sub.3-C.sub.7
cycloalkyl or heterocycloalkyl; R.sup.12 is H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl or heterocycloalkyl; R.sup.13 is H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, aryl, cycloalkyl, heteroaryl or heterocycloalkyl; and
R.sup.14 and R.sup.15 are each, independently, H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, arylalkyl, or cycloalkylalkyl; or or R.sup.14 and
R.sup.15 together with the N atom to which they are attached form a
4-, 5-, 6- or 7-membered heterocycloalkyl group.
37. A method of inhibiting mtDNA damage in a cell comprising
contacting said cell with an isoMnSOD inhibitor.
38. A method of inhibiting production of reactive oxidative species
(ROS) in a cell comprising contacting said cell with an isoMnSOD
inhibitor.
39. A method of inhibiting lipid peroxidation in a cell comprising
contacting said cell with an isoMnSOD inhibitor.
40. A method of treating cancer comprising administering to a
patient a chemotherapeutic and an isoMnSOD inhibitor.
41. The method of claim 40 wherein said isoMnSOD inhibitor
comprises a compound of Formula I: Alk-L.sup.1-L.sup.2-D I or
pharmaceutically acceptable salt or prodrug thereof, wherein: Alk
is C.sub.2-100 alkenyl or C.sub.2-100 alkynyl, each optionally
substituted by one or more R.sup.1; L.sup.1 is O, S, CO, C(O)O,
C(O)NR.sup.2, SO, S(O).sub.2, S(O)NR.sup.2, S(O).sub.2NR.sup.2,
NR.sup.2, NR.sup.2C(O)NR.sup.3, or NR.sup.2C(S)NR.sup.1; L.sup.2 is
absent, C.sub.1-6 alkylenyl, C.sub.2-6 alkenylenyl, or C.sub.2-6
alkynylenyl, each optionally substituted by one or more R.sup.4; D
is aryl or heteroaryl, each optionally substituted by one or more
R.sup.5; R.sup.1 and R.sup.4 are each, independently, halo, cyano,
nitro, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, S(O)R.sup.6, S(O).sub.2R.sup.6, C(O)R.sup.6,
OR.sup.7, SR.sup.7, C(O)OR.sup.7, NR.sup.8R.sup.9 or
NR.sup.8C(O)R.sup.6; R.sup.2 and R.sup.3 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; R.sup.5 is halo,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-4
haloalkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, CN,
NO.sub.2, OR.sup.12, SR.sup.12, C(O)R.sup.3, C(O)NR.sup.14R.sup.15,
C(O)OR.sup.12, OC(O)R.sup.13, OC(O)NR.sup.14R.sup.15,
NR.sup.14R.sup.15, NR.sup.14C(O)R.sup.15, NR.sup.14C(O)OR.sup.12,
S(O)R.sup.13, S(O)NR.sup.14R.sup.15, S(O).sub.2R.sup.13, or
S(O).sub.2NR.sup.14R.sup.15- ; R.sup.6 is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, aryl,
heteroaryl, C.sub.3-7 cycloalkyl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, (C.sub.3-7 cycloalkyl)alkyl,
heterocycloalkylalkyl, or NR.sup.10R.sup.11; R.sup.7 is H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryloxyalkyl,
heteroaryloxyalkyl, cycloalkyloxyalkyl, heterocycloalkyloxyalkyl,
aryl, heteroaryl, C.sub.3-C.sub.7 cycloalkyl, heterocycloalkyl,
arylalkyl, heteroarylalkyl; (C.sub.3-7 cycloalkyl)alkyl or
heterocycloalkylalkyl; R.sup.8 and R.sup.9 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; or R.sup.8 and R.sup.9
together with the N atom to which they are attached form a 3-, 4-,
5-, 6-, or 7-membered heterocycloalkyl group; R.sup.10 and R.sup.11
are each, independently, H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 haloalkyl, aryl,
heteroaryl, C.sub.3-C.sub.7 cycloalkyl or heterocycloalkyl;
R.sup.12 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl or
heterocycloalkyl; R.sup.13 is H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl,
heteroaryl or heterocycloalkyl; and R.sup.14 and R.sup.15 are each,
independently, H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, arylalkyl, or
cycloalkylalkyl; or or R.sup.14 and R.sup.15 together with the N
atom to which they are attached form a 4-, 5-, 6- or 7-membered
heterocycloalkyl group.
42. The method of claim 40 wherein said cancer is hodgkin's
lymphoma, non-hodgkin's lymphoma, breast cancer, ovarian cancer,
testicular cancer, acute leukemia, soft tissue sarcoma, lung
cancer, bladder cancer, gastric cancer, thyroid cancer, hepatoma,
wilm's tumor, or neuroblastoma.
43. The method of claim 40 wherein said chemotherapeutic comprises
doxorubicin.
44. The method of claim 40 wherein said chemotherapeutic comprises
Herceptin.
45. A composition comprising a chemotherapeutic and an isoMnSOD
inhibitor.
46. The composition of claim 45 wherein said chemotherapeutic
comprises doxorubicin.
47. The composition of claim 45 wherein said isoMnSOD inhibitor
comprises a compound of Formula I: Alk-L.sup.1-L.sup.2-D I or
pharmaceutically acceptable salt or prodrug thereof, wherein: Alk
is C.sub.2-100 alkenyl or C.sub.2-100 alkynyl, each optionally
substituted by one or more R.sup.1; L.sup.1 is O, S, CO, C(O)O,
C(O)NR.sup.2, SO, S(O).sub.2, S(O)NR.sup.2, S(O).sub.2NR.sup.2,
NR.sup.2, NR.sup.2C(O)NR.sup.3, or NR.sup.2C(S)NR.sup.3; L.sup.2 is
absent, C.sub.1-6 alkylenyl, C.sub.2-6 alkenylenyl, or C.sub.2-6
alkynylenyl, each optionally substituted by one or more R.sup.4; D
is aryl or heteroaryl, each optionally substituted by one or more
R.sup.5; R.sup.1 and R.sup.4 are each, independently, halo, cyano,
nitro, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, S(O)R.sup.6, S(O).sub.2R.sup.6, C(O)R.sup.6,
OR.sup.7, SR.sup.7, C(O)OR.sup.7, NR.sup.8R.sup.9 or
NR.sup.8C(O)R.sup.6; R.sup.2 and R.sup.3 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; R.sup.5 is halo,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-4
haloalkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, CN,
NO.sub.2, OR.sup.2, SR.sup.2, C(O)R.sup.3, C(O)NR.sup.4R.sup.15,
C(O)OR.sup.12, OC(O)R.sup.13, OC(O)NR.sup.14R.sup.15,
NR.sup.14R.sup.15, NR.sup.14C(O)R.sup.5, NR.sup.14C(O)OR.sup.12,
S(O)R.sup.13, S(O)NR.sup.14R.sup.15, S(O).sub.2R.sup.13, or
S(O).sub.2NR.sup.14R.sup.15- ; R.sup.6 is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, aryl,
heteroaryl, C.sub.3-7 cycloalkyl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, (C.sub.3-7 cycloalkyl)alkyl,
heterocycloalkylalkyl, or NR.sup.10R.sup.11; R.sup.7 is H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryloxyalkyl,
heteroaryloxyalkyl, cycloalkyloxyalkyl, heterocycloalkyloxyalkyl,
aryl, heteroaryl, C.sub.3-C.sub.7 cycloalkyl, heterocycloalkyl,
arylalkyl, heteroarylalkyl; (C.sub.3-7 cycloalkyl)alkyl or
heterocycloalkylalkyl; R.sup.8 and R.sup.9 are each, independently,
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl; (C.sub.3-7
cycloalkyl)alkyl or heterocycloalkylalkyl; or R.sup.8 and R.sup.9
together with the N atom to which they are attached form a 3-, 4-,
5-, 6-, or 7-membered heterocycloalkyl group; R.sup.10 and R.sup.11
are each, independently, H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 haloalkyl, aryl,
heteroaryl, C.sub.3-C.sub.7 cycloalkyl or heterocycloalkyl;
R.sup.12 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl or
heterocycloalkyl; R.sup.13 is H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl,
heteroaryl or heterocycloalkyl; and R.sup.14 and R.sup.15 are each,
independently, H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, arylalkyl, or
cycloalkylalkyl; or or R.sup.14 and R.sup.15 together with the N
atom to which they are attached form a 4-, 5-, 6- or 7-membered
heterocycloalkyl group.
48. A method of identifying an isoMnSOD inhibitor comprising: a)
contacting a cell expressing isoMnSOD with a test compound; b)
measuring an isoMnSOD mediated event in said cell; and c) comparing
said isoMnSOD mediated event in said cell to a cell that has not
been contacted with said test compound; wherein a decrease in said
isoMnSOD mediated event indicates that said test compound is an
isoMnSOD inhibitor.
49. The method of claim 48 wherein said cell expressing isoMnSOD is
transfected with a vector comprising a nucleic acid molecule
encoding for isoMnSOD.
50. The method of claim 49 wherein said vector is a plasmid or a
virus.
51. The method of claim 48 wherein said isoMnSOD mediated event is
cell death, lipid peroxidation, isoprostane production, HNE
production, isoprostane protein modification, or HNE protein
modification.
52. A method of measuring toxicity of a compound comprising
contacting said compound to a cell, wherein said cell comprises a
nucleic acid molecule comprising a genomic fragment of MnSOD gene
spanning Exon 2 to Exon 4 of said gene, wherein said fragment
comprises a frameshift mutation in Exon 3, and is operably linked
to a reporter gene, wherein expression of said reporter gene is
indicative of said compound being toxic.
53. The method of claim 52 wherein said reporter gene is a
luciferase gene, B-galactosidase gene, or secreted alkaline
phosphatase gene, or a fluorescent protein.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/473,458, filed May 28, 2003, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to methods of modulating
oxidative damage to intracellular components such as mitochondrial
DNA (mtDNA), mitochondrial lipids, and mitochondrial proteins. The
present invention is also directed to modulating the activity of a
spliced isoform of manganese superoxide dismutase.
BACKGROUND OF THE INVENTION
[0003] Manganese Superoxide Dismutase (MnSOD) is a component of the
cellular antioxidant defense mechanism that is necessary for
mitochondrial function, cellular energy production and cell
viability. Native MnSOD is a mitochondrial protein that is imported
from the cytoplasm and localized to the mitochondrial matrix, where
it scavenges superoxide free radicals or anions and converts these
reactive oxygen species into the benign oxidant, hydrogen peroxide
(H.sub.2O.sub.2), and oxygen. MnSOD is expressed in all cell types
and provides an essential function. MnSOD importance was clearly
illustrated in mice neonatal offspring containing a homozygous
disruption (knockout) of MnSOD gene. These mice develop
multisystem, mitochondrial energy-loss pathologies that include
cardiomyopathy, neurological and liver dysfunction and exhibit
perinatal lethality. The homozygous MnSOD knock-out serves as a
proof-of-principle for complete loss of MnSOD function.
[0004] The multisystem energy-loss phenotype in the MnSOD knockout
mouse may be due to an adverse accumulation of superoxide free
radicals within the mitochondria upon the onset of an aerobic
environment, causing loss of the ATP-synthesizing capacity of
mitochondria and either initiating premature cell death by necrosis
or by initiating the mitochondrial membrane permeability
transition, causing release of mitochondrial pro-apoptotic proteins
from the mitochondria due to depolarization of the mitochondrial
membrane. Adverse accumulation of superoxide will result in
spontaneous dismutation of superoxide into hydrogen peroxide, which
is normally a benign oxidant unless in the presence of a transition
metal, which then initiates Fenton-type chemistry to generate
reactive oxygen species similar to hydroxyl free radicals.
[0005] Research has shown that the MnSOD gene can express a splice
isoform (isoMnSOD) during stress conditions that expresses a
pro-oxidant form instead of the normal antioxidant activity of the
normal MnSOD (Anziano, et al., Pediatrics Research, 47; 2000;).
IsoMnSOD is also described in WO 99/43697. MnSOD alternative
splicing is inducible and depends on the deregulation of the normal
MnSOD splicing pathway. Alternative splicing of the MnSOD RNA
removes coding Exon 3, and fuses in-frame flanking Exons 2 and 4.
The isoMnSOD protein is internally deleted for key alpha helical
domain that serves in the parent MnSOD as a portal for the
selective entry of superoxide anions into the MnSOD metal pocket.
IsoMnSOD does not exhibit antioxidant, dismutase activity as the
parent MnSOD, but exhibits in vitro a gain-of-function peroxidative
activity that generates reactive oxygen free radicals from hydrogen
peroxide (H.sub.2O.sub.2). In vivo, isoMnSOD initiates lipid
peroxidation within the mitochondrial membrane and it causes
modification of target proteins by oxidative stress markers such as
the reactive lipid byproduct, 4-hydroxynonenal (HNE).
[0006] In addition, stress from internal factors (e.g. diseases) or
by exogenous or external influences such as, for example, drugs,
can impair a cell's and organism's viability. Although the
pharmacological properties of drugs or potential drugs are well
understood, companies still spend billions of dollars a year on
candidates that fail during preclinical and clinical trials due to
unforeseen drug toxicity. Current methods for predicting whether a
drug will be toxic in an individual have not been particularly
effective because there are few useful markers of drug-induced
toxicity that would indicate whether a drug is worthwhile
pursuing.
[0007] Additionally, there are numerous drugs that are used today
whose effectiveness is diminished because of toxicity that is
caused by inherent toxicity of the drugs, thereby limiting the
useful dosage. Some of the toxicity that is caused by the drugs can
be related to damage to mitochondrial contents due to altering and
diminishing its antioxidant environment and, therefore, if the
ancillary toxic event can be prevented it should enhance the
effectiveness of the compounds.
[0008] In view of the above evidence, there is a need to identify
modulators of isoMnSOD activity so that one can control the effects
of isoMnSOD expression. There is also a need to identify compounds
that can be used to reduce or prevent drug-induced toxicity. There
are further needs for assays and methods that can be used to
predict if a composition will cause drug-induced toxicity in an
individual or a cell. The present invention helps to fulfill these
needs as well as others.
SUMMARY OF THE INVENTION
[0009] The present invention provides methods of inhibiting an
activity of isoMnSOD in a cell comprising contacting said cell with
a compound of Formula I: Alk-L.sup.1-L.sup.2-D, wherein constituent
members are defined herein.
[0010] In some embodiments, the present invention provides methods
of treating drug-induced toxicity in an individual comprising
administering to said individual a therapeutically effective amount
of an isoMnSOD inhibitor.
[0011] The present invention also provides methods of treating
heart failure in an individual comprising administering to said
individual a therapeutically effective amount of an isoMnSOD
inhibitor.
[0012] In some embodiments, the present invention provides methods
of treating a mitochondrial related disease or condition in an
individual comprising administering to said an individual a
therapeutically effective amount of an isoMnSOD inhibitor.
[0013] In some embodiments, the present invention provides methods
of inhibiting cell death comprising contacting a cell with an
isoMnSOD inhibitor, wherein said inhibitor does not modulate
expression of a nucleic acid molecule encoding isoMnSOD.
[0014] The present invention also provides methods of inhibiting
mitochondrial oxidative stress in a cell comprising contacting said
cell with an isoMnSOD inhibitor.
[0015] In some embodiments, the present invention provides methods
of inhibiting mtDNA damage in a cell comprising contacting said
cell with an isoMnSOD inhibitor.
[0016] In some embodiments, the present invention provides methods
of inhibiting production of reactive oxidative species (ROS) in a
cell comprising contacting said cell with an isoMnSOD
inhibitor.
[0017] The present invention also provides, methods of inhibiting
lipid peroxidation in a cell comprising contacting said cell with
an isoMnSOD inhibitor.
[0018] In some embodiments, the present invention provides methods
of treating cancer comprising administering to a patient a
chemotherapeutic and an isoMnSOD inhibitor.
[0019] The present invention also provides compositions comprising
a chemotherapeutic and an isoMnSOD inhibitor.
[0020] In some embodiments, the present invention provides methods
of identifying an isoMnSOD inhibitor comprising:
[0021] a) contacting a cell expressing isoMnSOD with a test
compound;
[0022] b) measuring an isoMnSOD mediated event in said cell;
and
[0023] c) comparing said isoMnSOD mediated event in said cell to a
cell that has not been contacted with said test compound;
[0024] wherein a decrease in said isoMnSOD mediated event indicates
that said test compound is an isoMnSOD inhibitor.
[0025] The present invention also provides methods of measuring
toxicity of a compound comprising contacting said compound to a
cell, wherein said cell comprises a nucleic acid molecule
comprising a genomic fragment of MnSOD gene spanning Exon 2 to Exon
4 of said gene, wherein said fragment comprises a frameshift
mutation in Exon 3, and is operably linked to a reporter gene,
wherein expression of said reporter gene is indicative of compound
being toxic.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1: Peroxidative assay of native mitochondrial extracts
analyzed by the ROS-activity blot according to Example 13. Lane 1:
no treatments; Lane 2: arachidonoyl-dopamine; Lane 3:
eicosapentaenoyl (EPA)-dopamine; Lane 4: docosahexaenoyl
(DHA)-dopamine; Lane 5: C18:4.omega.-3-dopamine; Lane 6:
arachidonoyl-serotonin; Lane 7: no treatment.
[0027] FIG. 2: Covalent modification of the MnSOD-Exon3-deleted
isoform by isoMnSOD inhibitors according to Example 14. A.
Detection of dopamine-modification--Lane 1: no treatment; Lane 2:
arachidonoyl-dopamine; Lane 3: eicosapentaenoyl (EPA)-dopamine;
Lane 4 docosahexaenoyl (DHA)-dopamine; Lane 5:
C18:4(.omega.-3)-dopamine. B. Detection of
serotonin-modification--Lane 1: no treatment; Lane 2:
arachidonoyl-serotonin.
[0028] FIG. 3: Doxorubicin induces the expression of isoMnSOD
according to Example 10. Lane 1: no treatment; Lane 2: 0.1 .mu.M
doxorubicin; Lane 3: 1.0 .mu.M doxorubicin. Membrane was probed for
isoMnSOD protein by Western Blot.
[0029] FIG. 4: In vitro analysis of the effect of test compounds on
the peroxidative activity of the MnSOD-Exon3-deleted isoform
according to Example 4. Lane 1: no treatment; Lane 2:
arachidonoyl-glycine; Lane 3:
arachidonoyl-3-hydroxy-.gamma.-aminobutyric acid (GABA); Lane 4:
arachidonoyl-dopamine; Lane 5: eicosapentaenoyl (EPA)-dopamine;
Lane 6: docosahexaenoyl (DHA)-dopamine; Lane 7:
C18:4(.omega.-3)-dopamine; Lane 8: arachidonoyl-ethanolamide.
[0030] FIG. 5: Characterization of isoMnSOD peroxidative activity.
A. Reactive Oxygen Species generating activity gel (left panel) and
western immunoblot (right panel) analysis of recombinant isoMnSOD,
produced in bacterial BL21 cells. B. Characterization of the
isoMnSOD protein in pig pancreatic mitochondria. Four lanes each of
25 .mu.g of pig pancreatic mitochondrial proteins were separated by
a 7% PAGE without SDS. Measurement of the antioxidant, native MnSOD
superoxide dismutase activity was performed in-gel (lane 1). The
additional three gel-lanes were electrotransferred to
nitrocellulose and analyzed for peroxidative analysis using the
"ROS-generating activity blot" (lane 2) and protein assignment by
Western immunoblot analysis using the anti-MnSOD (lane 3) and
anti-MnSOD E2/E4 peptide (lane 4) polyclonal antibodies. C. The
peroxidative activity of pig isoMnSOD (left lane) is supershifted
by pre-incubation with 1 .mu.g of the anti-MnSOD polyclonal
antibody prior to native gel electrophoresis (right lane).
DETAILED DESCRIPTION
[0031] Superoxide radicals and other highly reactive oxygen species
are harmful by-products in every respiring cell, causing oxidative
damage to a wide variety of macromolecules and cellular components.
A group of metalloproteins known as superoxide dismutases (SOD)
catalyzes the oxidation-reduction reaction that converts
free-radical oxygen (O.sub.2.sup.-) to hydrogen peroxide
(H.sub.2O.sub.2) and oxygen (O.sub.2) and thus provides a defense
mechanism against oxygen toxicity. SODs may contain manganese (Mn)
or iron (Fe) or a combination of copper (Cu) and Zinc (Zn). SODs
are described in U.S. Pat. No. 5,540,911.
[0032] Mitochondria are essential for meeting the body's acute and
chronic energy demands because all cells of the body synthesize
their fuel within the mitochondria. Mitochondrial DNA (mtDNA)
contains thirteen genes encoding protein products that are
necessary to synthesize the cell's fuel, adenosine triphosphate
(ATP).
[0033] Diseases, drugs, or other factors that cause mitochondrial
oxidative stress have often been implicated as an initiator of
mitochondrial damage. If oxygen is not metabolized efficiently,
oxygen free radicals can accumulate in the cell. Free radicals can
cause damage to nucleic acid molecules, proteins or lipids. Since
the mitochondria consume the most of the cell's oxygen, the
mitochondria can be the most exposed organelle to free radical
damage, especially mtDNA. If oxidative damage to mtDNA is left
unrepaired, the point-mutation and deletion rate of the mtDNA
increases, which may eventually lead to permanent organ
fatigue.
[0034] An increase in mitochondrial mutations or an increase in
free oxygen radicals can lead to an energy-loss syndrome which
could effect the heart (cardiomyopathy or conduction disorders,
e.g. heart failure), the nervous system, the pancreas (e.g.,
non-insulin dependent diabetes), the gastrointestinal tract (e.g.,
dysmotility or pseudo-obstruction), inner ear (e.g., sensorineural
hearing loss), kidney (e.g. glomerulopathy) and/or the skeletal
muscle (e.g., myopathy).
[0035] Because oxidative damage in cells can lead to various
diseases, disorders, or conditions, there is an unfulfilled need to
identify methods and compounds for treating diseases related to an
increase in oxidative damage. The present invention helps to
fulfill this need by identifying compounds that can inhibit or
reduce isoMnSOD and, therefore, inhibit or reduce oxidative
damage.
[0036] The recent discovery of an alternative splice form of MnSOD
that has pro-oxidant activity and pro-apoptotic activity as
compared to the antioxidant activity of MnSOD has led to the
invention that oxidative damage caused by these species can be
inhibited or reduced by compounds that inhibit or reduce an
activity of isoMnSOD.
[0037] The present invention relates to the discovery that an
alternative splice form of Manganese Superoxide Dismutase,
hereinafter "isoMnSOD", is a pro-oxidant version of the normal
splice form of Manganese Superoxide Dismutase, hereinafter "MnSOD".
IsoMnSOD can be induced in all cell types, in all individuals, and
is believed to be expressed in response to oxidative and
mitochondrial damage. IsoMnSOD acts as a dominant, gain-of-function
protein that when expressed at significant levels, induces
mitochondrial dysfunction and mitochondrial energy-loss
pathologies, including mitochondrial-mediated apoptotic cell death
and acts as a modifier of an underlying genetic condition. IsoMnSOD
generates oxygen free radicals other than superoxide in the
presence of hydrogen peroxide and are not scavenged by native
MnSOD. IsoMnSOD has been previously described in U.S. Pat. No.
6,737,506. The expression of isoMnSOD can lead to cell death and
cellular toxicity. IsoMnSOD expression can be induced by drugs or
compositions that have been shown to cause drug-related toxicity
such as, for example, doxorubicin. The present invention describes
methods and compositions that can be used, for example, to inhibit
or reduce the activity of isoMnSOD, inhibit or reduce drug induced
toxicity, and/or to inhibit or reduce cell death. The activity of
isoMnSOD can also be modulated for the treatment of various
mitochondrial related, energy-loss diseases, which are prone to
oxidative damage of their remaining energy capacity.
[0038] Upon oxidative stress or mitochondrial damage isoMnSOD
expression is increased by alternative splicing and has been found
to not only create additional reactive oxidative species, but also
to increase isoprostanes and HNE modifications of other proteins
and itself. Isoprostanes are produced by the peroxidation of
lipoproteins, which can be regulated by isoMnSOD. Elevated
isoprostane levels have been associated with, for example,
hepatorenal syndrome, rheumatoid arthritis, inflammation,
atherosclerosis, inflammatory vascular diseases, and carcinogenesis
(Jansenn, Chemistry and Physics of Lipids, 128 (2004) 101-116;
Cracowski et al., Journal of Vascular Research 2001;38:93-103;
Eaton et al., Am J. Physiol. 1999 Mar;276(3 Pt 2):H935-43).
Therefore, an increase in isoMnSOD activity can increase the risk
of the patient of developing these and other conditions. The
production of HNE can also lead to diseases or disorders by
inactivating other proteins in the cell. Protein inactivation by
HNE occurs because HNE covalently modifies enzymes in a cell which
can assist in targeting proteins for degradation by the proteosome
(Grune et al. Mol Aspects Med. 2003 Aug-Oct;24(4-5):195-204).
However, if when HNE is overproduced and causes the modification of
a large number of proteins, clogging of the proteosome can result,
which can inhibit the function of a cell and lead to cell death.
Under normal circumstances HNE is metabolized and is prevented from
damaging the cell. However, when HNE accumulates, in addition to
clogging the proteosome, HNE can lead to atherogenesis (Hoff et al.
Klin Wochenschr. 1991 Dec. 15;69(21-23):1032-8). Therefore,
inhibiting HNE production by inhibiting isoMnSOD according to the
present invention can be beneficial to the cell.
[0039] Accordingly, in one aspect, the present invention provides
methods of inhibiting or reducing an activity of isoMnSOD in a cell
comprising administering an isoMnSOD inhibitor.
[0040] As used herein, the term "MnSOD" refers to the gene
Mangenese Superoxide Dismutase. "MnSOD" can also refer to either
the cDNA, RNA, mRNA, genomic DNA, or gene product (e.g. protein) of
the MnSOD gene. In some embodiments, "MnSOD" refers to the
antioxidant form of MnSOD. "MnSOD" also refers to a cDNA, mRNA, or
protein that comprises the third exon of the MnSOD gene.
[0041] As used herein, the term "isoMnSOD" or "alternative splice
form of MnSOD" refers to a splice form the MnSOD gene that lacks
exon 3. "IsoMnSOD" can also be referred to as
"MnSOD-exon3-deleted", "exon 3 deleted MnSOD" or "altisoMnSOD". The
genomic structure of MnSOD, nucleic acid sequence of isoMnSOD, and
the amino acid sequence of isoMnSOD are described in U.S. Pat. No.
6,737,506. An exon 3 deleted MnSOD lacks the active site histidine
at position 74 (74H; numbering based upon the mature form of native
MnSOD, where the mitochondrial targeting signal in cleaved at K25
for both native MnSOD and isoMnSOD) that is required selective
entry of superoxide into the native MnSOD active site and for
manganese binding as well as the peptide sequences required for
forming the final homotetrameric complex of antioxidant-MnSOD
associated with superoxide capture, but molecular modeling of
isoMnSOD has shown that histidine at position 31 (H31) is
reoriented and now substitutes for 74H, with otherwise complete
conservation of the positioning of active site amino acids in
native MnSOD. The active site for isoMnSOD is more accessible to
molecules larger than superoxide, such as H.sub.2O.sub.2. In some
embodiments, "isoMnSOD" refers to polynucleotides that encode
polypeptides or the polypeptides themselves that are defined by the
above described structure/function parameters. In some embodiments,
"isoMnSOD" refers to a polypeptide that has pro-oxidant
activity.
[0042] An "activity of isoMnSOD" refers to the protein activity of
the polypeptide encoded by the isoMnSOD transcript. In some
embodiments, the activity is pro-oxidant activity of the isoMnSOD
protein. Pro-oxidant activities include any activity that can lead
to oxidation of cellular components and includes, for example,
generating and/or facilitating the accumulation of reactive oxygen
species as well as damaging nucleic acids, proteins, lipids, and
the like by oxidation. The activity can also refer to isoMnSOD
pro-apoptotic activity. The activity can also refer to the binding
activity of a polypeptide comprising isoMnSOD. For example, the
binding activity can refer to the ability of the protein to bind
other polypeptides, reactive oxygen species (ROS), metals,
transition metals, compounds, inhibitors, activators, or other
co-factors.
[0043] An "isoMnSOD inhibitor" refers to a pharmaceutical agent
such as, for example, a protein (e.g., antibody or other binding
protein) or small molecule (compound) that is able to inhibit or
reduce an activity of the isoMnSOD gene product. In some
embodiments, the isoMnSOD inhibitor inhibits or reduces the
pro-oxidant activity, the binding activity, both types of activity
or other activity. The isoMnSOD inhibitor can also inhibit or
reduce the binding of isoMnSOD to other polypeptides, to reactive
oxygen species, to metals, to transition metals, co-factors,
activators, inhibitors, or combinations thereof.
[0044] The isoMnSOD inhibitor or composition, such as a
pharmaceutical composition, thereof can be administered to any cell
expressing isoMnSOD either in vitro, in vivo, ex vivo, or
combinations thereof. Methods of administration are well known to
the skilled artisan and described herein. A composition comprising
the isoMnSOD inhibitor and an acceptable carrier (such as a
pharmaceutically acceptable carrier) or diluent may be formulated
by one having ordinary skill in the art depending upon the chosen
mode of administration. Suitable carriers are described in the most
recent edition of Remington's Pharmaceutical Sciences, A. Osol, a
standard reference text in this field.
[0045] The compositions according to the present invention may be
administered as a single dose or in multiple doses. The
compositions of the present invention may be administered either as
individual therapeutic agents or in combination with other
therapeutic agents.
[0046] The compositions or inhibitors of the present invention may
be combined with conventional therapies, which may be administered
sequentially or simultaneously. One or more additional agents such
as, for example, anti-viral agents, antibodies, anti-inflammatory
agents, chemotherapeutics, antibiotics, and/or immunosuppressants
can be used in combination with the compounds of the present
invention for treatment of drug induced toxicity or other
conditions relating to an oxidative damage disorder. The agents can
be combined with the present compounds in a single dosage form, or
the agents can be administered simultaneously or sequentially as
separate dosage forms.
[0047] The compositions comprising an isoMnSOD inhibitor may be
administered by any means that enables the active agent to reach
the agent's site of action in the cell or in the body of an
individual. Because some compounds are subject to being digested,
hydrolyzed or otherwise broken down when administered orally,
parenteral administration (e.g., intravenous, subcutaneous,
intramuscular) can be used to optimize absorption. In addition, the
compositions of the present invention may be injected at a site at
or near the oxidative damage. For example, administration may be by
direct injection into tissue directly adjacent to the oxidative
damage. If the individual to be treated is suffering from oxidative
damage on the skin or drug-induced toxicity on the skin, the
isoMnSOD inhibitor may be formulated with a topical carrier (such
as a pharmaceutically acceptable topical carrier) and the
formulation may be administered topically as, for example, a cream,
a lotion, or an ointment.
[0048] The dosage administered varies depending upon factors such
as: pharmacodynamic characteristics; its mode and route of
administration; age, health, and weight of the recipient; nature
and extent of symptoms; kind of concurrent treatment; and frequency
of treatment. Usually, a daily dosage of isoMnSOD inhibitor can be
about 1 .mu.g to 100 milligrams per kilogram of body weight. In
some embodiments, about 0.5 to about 500, about 1 to about 400,
about 1 to about 300, about 1 to about 200, about 1 to about 100,
about 1 to about 50, about 1 to about 10 milligrams per kilogram
per day is given in divided doses 1 to 6 times a day or in
sustained release form in an effective to obtain desired
results.
[0049] In some embodiments, the isoMnSOD inhibitor is administered
in a therapeutically effective amount. As used herein, the term
"therapeutically effective amount" is meant to refer to an amount
of an isoMnSOD inhibitor which produces a clinical effect observed
as reduction or reverse in drug related toxicity, clinical
endpoints, or oxidative damage in an individual when a
therapeutically effective amount of an isoMnSOD inhibitor is
administered to an individual or to a cell. Therapeutically
effective amounts are typically determined by the effect they have
compared to the effect observed when a composition which includes
no active ingredient is administered to a similarly situated
individual. The precise effective amount for a subject will depend
upon the subject's size and health, the nature and extent of the
condition, and the therapeutics or combination of therapeutics
selected for administration. However, the effective amount for a
given situation is determined by routine experimentation and is
within the judgment of the clinician.
[0050] As used herein, the term "contacting" refers to the bringing
together of indicated moieties in an in vitro system or an in vivo
system. For example, "contacting" a cell with an isoMnSOD inhibitor
or another compound of the invention includes the administration of
the isoMnSOD inhibitor to an individual or patient, such as a
human, having an isoMnSOD protein, as well as, for example,
introducing the isoMnSOD inhibitor into a sample containing a
cellular or purified preparation containing the isoMnSOD
protein.
[0051] As used herein, the term "individual" or "patient," used
interchangeably, refers to any animal, including mammals,
preferably mice, rats, other rodents, rabbits, dogs, cats, swine,
cattle, sheep, horses, or primates, and most preferably humans.
[0052] As used herein, the term "cell" refers to a cell that is
either in vivo, in vitro, or ex vivo. The isoMnSOD inhibitor can be
contacted with any type of cell including, for example, dividing
cells, non-dividing cells, multinucleated cells, cancer cells,
neural cells, muscle cells, heart cells, brain cells, digestive
tract cells (e.g. stomach, small intestine, large intestine,
esophagus, and the like), pancreas cells, and the like. In some
embodiments, the cell is a non-dividing cell. In some embodiments,
the cell is a heart cell.
[0053] The present invention also provides methods of inhibiting or
reducing the production of free isoprostanes, HNE, and modification
of proteins by HNE and/or isoprostanes. As discussed above, the
accumulation of HNE and isoprostanes can be deleterious to a cell
and an individual or be indicative cellular damage and disease. The
expression and activity of isoMnSOD can lead to the accumulation of
these two product or related products and, therefore, directly
contribute to the modification of proteins which can result in the
degradation of essential proteins that would otherwise not be
degraded, but also can lead to the clogging of the proteosome and
eventually to the death or dysfunction of a cell.
[0054] In some embodiments, the present invention provides methods
of inhibiting or reducing isoprostane production in a cell
comprising contacting the cell with an isoMnSOD inhibitor. In some
embodiments, the isoprostane is, for example,
8-epi-PGF.sub.2.alpha., or isolevuglandin (e.g. .alpha., .nu., and,
.delta.). In some embodiments, the inhibitor inhibits or reduces
isoprostane protein modification.
[0055] As used herein, the term "isoprostane protein modification"
refers to a covalent modification of a protein that occurs when the
protein comes in contact with one or more isoprostane molecules. In
some embodiments, the isoprostane modification inhibits or reduces
an activity of a protein. The "activity of a protein" can be
enzymatic or non-enzymatic (e.g. binding to another molecule,
signal transduction, targeting of itself or another protein to a
specific location or organelle within or outside a cell). In some
embodiments, the protein is modified with an oxidized lipid or with
an oxidized isolevuglandin, such as isolevuglandin-lysine adducts
on apolipoprotein B. (Identification of Extremely Reactive
.gamma.-Ketoaldehydes (Isolevuglandins) as Products of the
Isoprostane Pathway and Characterization of Their Lysyl Protein
Adducts. JBC Vol. 274, pp. 13139-13146, 1999. Cynthia J. Brame,
Robert G. Salomon, Jason D. Morrow, and L. Jackson Roberts.).
[0056] In further embodiments, the present invention can be used to
inhibit or reduce HNE production and/or HNE protein modification in
a cell, for example, by contacting the cell with an isoMnSOD
inhibitor. As used herein, the term "HNE protein modification"
refers to a covalent modification of a protein that occurs when the
protein comes in contact with one or more HNE molecules. In some
embodiments the protein is modified by 4-hydroxy-2-nonenal. In some
embodiments, the protein being modified is cytochrome c or an
oxidase subunit of cytochrome c.
[0057] Since HNE and/or isoprostane production can lead to
hepatorenal syndrome, rheumatoid arthritis, atherosclerosis,
carcinogenesis, and other diseases, the present invention provides
methods of treating diseases related to HNE and/or isoprostane
production comprising administering to an individual a
therapeutically effective amount of an isoMnSOD inhibitor. In some
embodiments, the disease to be treated is hepatorenal syndrome,
rheumatoid arthritis, atherosclerosis, or carcinogenesis. In some
embodiments, the isoMnSOD inhibitor is combined with one or more
compounds used to treat hepatorenal syndrome, rheumatoid arthritis,
atherosclerosis, or carcinogenesis. In some embodiments, an
isoMnSOD inhibitor is co-administered with statins (e.g.
Lipitor.TM., Zocor.TM., Crestor.TM., Mevacor.TM., Pravachol.TM.,
and the like), ezetimibe (Zetia.TM.), non-steroidal
anti-inflammatory agents (NSAIDs; e.g. ibuprofen, COX-2 inhibitors,
aspirin, acetaminophen, disease modifying anti-rheumatic drugs
(DMARDs; e.g. methotrexate, leflunomide (Arava.TM.), etanercept
(Enbrel.TM.), infliximab (Remicade.TM.), adalimumab (Humira.TM.),
anakinra (Kineret.TM.)), antimalarials, gold salts, sulfasalazine,
d-penicillamine, cyclosporin A, cyclophosphamide, azathioprine,
corticosteroids, dopamine, Misoprostol, renal vasoconstrictor
antagonists, systemic vasoconstrictors, N-acetylcysteine, and the
like.
[0058] Drugs such as doxorubicin and Herceptin.TM. have been shown
to cause toxicity in individuals through a mechanism that would
implicate isoMnSOD. As discussed, herein, many compounds cause
adverse events in patients that are due to oxidative damage that
can be facilitated through the expression and activity of isoMnSOD.
Doxorubicin has been shown to induce and/or increase the expression
of isoMnSOD (described herein) in cells such as, for example, heart
cells. Therefore, the present invention provides methods of
treating drug-induced toxicity in an individual comprising
administering to the individual a therapeutically effective amount
of an isoMnSOD inhibitor. In some embodiments, the toxicity is
organ toxicity. Examples of organs that can be effected by drug
induced toxicity include, but are not limited to, heart, pancreas,
liver, kidney, brain, colon, stomach, and bone marrow. Drug
toxicity can also affect the skin (e.g., leading to hair loss), the
eye (e.g., leading to macular degeneration), and the nervous system
(e.g., drug induced neural degeneration). In some embodiments, the
drug-induced toxicity leads to heart failure.
[0059] According to another aspect of the invention, it provides
methods of treating heart failure in an individual comprising
administering to the individual a therapeutically effective amount
of an isoMnSOD inhibitor. In some embodiments, the heart failure is
ischemic or non-ischemic heart failure. In some embodiments, the
heart failure is non-ischemic heart failure. In some embodiments,
the heart failure is drug induced.
[0060] As used herein, the term "heart failure" refers to a
condition initiated by impairment of the heart's function as a
pump. Heart failure is a progressive disorder in which damage to
the heart causes weakening of the cardiovascular system. It is
clinically manifested by fluid congestion or inadequate blood flow
to tissues. Heart failure progresses by inappropriate responses of
the body to heart injury. Heart failure may be the sum of one or
many causes. "Ischemia" is characterized by a decrease in the blood
supply to a bodily organ, tissue, or part caused by constriction or
obstruction of the blood vessels. Thus, "ischemic heart failure"
refers to heart failure facilitated by a chronic or acute decrease
in blood supply to the heart leading to necrotic cell death. In
contrast, "non-ischemic heart failure" refers to damage to the
heart that is not characterized by a decrease in the blood supply
to the heart or caused by constriction or obstruction of the blood
vessels. An example of non-ischemic heart failure is, but not
limited to, oxidative damage to the heart. In some embodiments,
non-ischemic heart failure is drug induced. An example drug that
can lead to drug induced, non-ischemic heart failure is an
anthracycline compound. Examples of anthracycline compounds
include, but are not limited to, doxorubicin, epirubicin,
daunorubicine, idarubicin, and anthracenedione (mitoxantrone).
Non-ischemic heart failure can also be induced by chemotherapy,
such as, for example, by treatment with nucleic acid damaging
agents (e.g., DNA damaging agents) such as Topoisomerase II
inhibitors, as well as non-nucleic acid damaging agents.
Non-ischemic heart failure can also be induced by radiation (such
as in combination with chemotherapy) and is an example of a nucleic
acid damaging agent. A "nucleic acid damaging agent" refers to a
compound or agent that causes damage to DNA or RNA. The compound or
agent can cause damage to the nucleic acid molecule directly or
indirectly. By "direct" damage to the nucleic acid molecule it is
meant that the compound or agent modifies the nucleic acid molecule
itself, rather than effecting another molecule which results in the
damage of the nucleic acid molecule. A compound or agent that
effects another molecule that results in damaging a nucleic acid
molecule in the cell is said to "indirectly damage" the nucleic
acid molecule. Examples of "agents" that can damage DNA are, but
not limited to, radiation (e.g. gamma or ultraviolet), cytotoxins
(e.g., chemotherapeutics), and the like.
[0061] Antibodies that recognize and inhibit proteins involved in
tumorigenesis have been used to treat cancer, but can be toxic and
in some instances lead to heart failure. Therefore, the present
invention also provides for methods of treating heart failure (e.g.
non-ischemic) induced by antibodies that are used to treat cancer.
The type of cancer that is being treated can be any cancer
including, for example, breast cancer, lung cancer (e.g., non-small
cell), pancreatic cancer, colon cancer, prostate cancer, ovarian
cancer, glioma and the like. An example of an antibody that can
facilitate, lead to, or cause heart failure is an antibody that
recognizes the protein HER-2 (e.g. Herceptin.TM.). The HER-2
proto-oncogene (also known as erbB-2, c-neu and HER-2/neu) and its
association with various cancers is known to one of ordinary skill
in the art. The risk of heart failure in an individual treated with
antibodies used to treat cancer in an individual is further
increased if the antibody treatment is combined with a nucleic acid
damaging agent such as, for example, a Topoisomerase II inhibitor
(J. Sparano. Semin Oncol 28: 20-27 (2001)). Therefore, in some
embodiments, methods of treating cancer include administration of
either or both of a chemotherapeutic or antibody in combination
with an isoMnSOD inhibitor.
[0062] Although the exact mechanism of how doxorubicin (Dox)
increases the risk of heart failure is not clearly understood, it
is believed that doxorubicin generates superoxide which is then
converted into hydrogen peroxide in the presence or absence of
MnSOD. At low concentrations of doxorubicin, little of the
H.sub.2O.sub.2 accumulates in the mitochondria. While not wishing
to be bound by theory, it is possible that native MnSOD scavenges
all or most of the superoxide. Although the isoMnSOD is induced by
doxorubicin and is present in the mitochondria, relatively little
lipid peroxidation occurs. At higher concentrations of Dox, the
Dox-generated superoxide is believed to overwhelm the endogenous
native MnSOD (antioxidant) protein, allowing H.sub.2O.sub.2 to
accumulate due to spontaneous dismutation of superoxide into
H.sub.2O.sub.2. At these higher concentrations, isoMnSOD is
believed to accept hydrogen peroxide and convert it into reactive
oxygen species, which can result in oxidative damage including, but
not limited to, lipid peroxidation, generation of HNE, generation
of isoprostanes, and/or mitochondrial damage. In some instances, it
is believed that the generated HNE can modify isoMnSOD. Thus, the
HNE-modified isoMnSOD can be used as a marker of lipid peroxidation
and presence of HNE, as well as antibodies directed against amino
acid sequences of isoMnSOD, not restricted to the MnSOD E2/E4
junction, that can either detect reactive lipid byproducts and
drugs modifications or have their binding reduced or blocked by
amino acid modifications, including those at positions H26, H29,
and K51. Accordingly, the inhibition of isoMnSOD can lead to a
reduction in lipid peroxidation and reduce the toxicity of
doxorubicin and other drugs that induce or increase the expression
of isoMnSOD. Thus, in some embodiments, the present invention
provides a method of inhibiting or reducing doxorubicin induced
toxicity comprising administering to an individual a
therapeutically effective amount of an isoMnSOD inhibitor.
[0063] The present invention also provides methods of treating
mitochondrial related diseases or conditions in an individual
comprising administering to said individual a therapeutically
effective amount of an isoMnSOD inhibitor.
[0064] As used herein, the term "mitochondrial related disease"
refers to a disease, condition, or disorder where the function of
the mitochondria is disrupted. The function can be disrupted by
mitochondrial DNA (mtDNA) damage, proteins functioning abnormally
within the mitochondria, membrane depolarization, and the like. A
"mitochondrial related disease" can also be referred to as an
energy-loss disease because the mitochondria supplies the energy
for the cell. A "mitochondrial related disease" can also be
referred to as an premature cell death disease because loss of the
mitochondrial energy supply can initiate necrosis or control the
release of pro-apoptotic proteins into the cytoplasm. The
expression of isoMnSOD disrupts the function of the mitochondria,
such as by facilitating oxidative damage to the mitochondria, and
can be therefore detrimental to the viability of a cell, tissue or
individual. Thus, inhibiting or reducing the activity of isoMnSOD
can improve the condition of a cell, tissue, or individual
suffering from a mitochondrial related disease. In some
embodiments, the mitochondrial related disease is characterized by
abnormal levels of oxidative damage to the mtRNA. An example of a
mitochondrial related disease is cardiomyopathy (e.g., non-ischemic
heart failure) Additional examples are well known to those skilled
in the art.
[0065] Another aspect of the present invention includes methods of
inhibiting or reducing mitochondrial oxidative stress in a cell
comprising contacting said cell with an isoMnSOD inhibitor. As used
herein, the term "mitochondrial oxidative stress" refers to a
condition in the cell in which the mitochondria is damaged by
oxidative agents or a condition that produces oxidative agents that
can cause damage to the mitochondria or to a cell. Mitochondrial
oxidative stress includes, but is not limited to, conditions where
the mitochondria or cell's antioxidant environment has been
substantially altered or overwhelmed and contains abnormal amounts
of reactive oxygen species. An example of a disease characterized
by mitochondrial oxidative stress is drug-induced organ failure
(such as drug induced heart failure). One of skill in the art can
determine if a cell contains abnormal amounts of reactive oxygen
species by comparing the cell or cells in question to a cell or
cells that is known to be normal. Methods of measuring the presence
or level of reactive oxygen species in a cell are known in the
art.
[0066] In some embodiments, the present invention provides methods
of inhibiting or reducing mtDNA damage in a cell comprising
contacting the cell with an isoMnSOD inhibitor. "mtDNA damage"
refers to damage to the DNA or nucleic acids present inside the
mitochondria. Examples of mtDNA damage include, but are not limited
to, mutations, deletions, insertions, cross-linkages, or other
modifications that are not found in undamaged mtDNA. In some
embodiments, mtDNA damage includes oxidative damage caused directly
or indirectly by reactive oxygen species.
[0067] The present invention also provides methods of inhibiting or
reducing cell death comprising contacting a cell with an isoMnSOD
inhibitor. In some embodiments, the inhibitor does not include an
inhibitor that modulates expression of a nucleic acid molecule
encoding a polypeptide comprising isoMnSOD. In some embodiments,
the cell death is necrotic or apoptotic cell death. In some
embodiments, the cell death is caused by elevated levels of
reactive oxygen species, lipid peroxidation, HNE, isoprostanes or
the like.
[0068] In some embodiments, the isoMnSOD inhibitors of the present
invention are administered to any cell that expresses isoMnSOD or
may be likely to express isoMnSOD (such as, for instance, the cell
will be contacted with chemotherapeutic). In some embodiments, the
cell is a non-dividing cell, dividing cell, or a cell that is
undergoing senescence. A "non-dividing cell" refers to cell that is
no longer undergoing cell division (e.g. mitosis or meiosis).
Examples of non-dividing cells include, but are not limited to,
cardiomyocytes, hepatocytes, pancreatic cells, and the like.
[0069] As used herein the term "cell death" or "premature cell
death" refers to the death of a cell through apoptosis (e.g.
programmed cell death) or necrosis. The apoptosis can be regulated
through a p53 dependent pathway or a p53 independent pathway.
[0070] In some embodiments, the present invention provides methods
of inhibiting or reducing the production of reactive oxygen species
(ROS) in a cell comprising contacting the cell with an isoMnSOD
inhibitor. "Reactive oxygen species" refers to compounds or
molecules that have a reactive oxygen moiety. When oxygen abstracts
electrons from other molecules in the cell, reactive oxygen species
(ROS) can be formed. This electron abstraction leaves donor
molecules with unpaired electrons, causing them to become highly
reactive radicals. Reactive Oxygen Species (ROS) is a term that
collectively describes radicals and other non-radical reactive
oxygen derivatives. These intermediates can participate in
reactions giving rise to free radicals or that are damaging to
organic substrates. Examples of radical containing reactive oxygen
species include, but are not limited to, hydroxyl radicals (e.g.,
OH), superoxide (e.g., 2--), nitric oxide (e.g., NO), thyl (e.g.,
RS), peroxyl (e.g., RO.sub.2), lipid peroxyl (e.g., LOO), and the
like. Examples of non-radical containing reactive oxygen species
include, but are not limited to, hydrogen peroxide (e.g.,
H.sub.2O.sub.2), hypochloric acid (e.g., HOCl), singlet oxygen
(e.g., .sup.-1O.sub.2), ozone (e.g., O.sub.3), lipid peroxide
(e.g., LOOH), and the like (where R is H or alkyl, L is a lipid
moiety). In some embodiments, the isoMnSOD inhibitor inhibits the
production of radical containing reactive oxygen species,
non-radical containing reactive oxygen species, or both.
[0071] Cell membranes, organelle membranes and other structures
within the cell contain lipids that are essential for the viability
of the cell. The lipids present in the cellular membranes can be
damaged by oxidative species and, therefore, negatively impact the
cell's viability. Lipid peroxidation of a cell can occur by the
interaction of a lipid with a reactive oxygen species, which, in
some embodiments, can be facilitated by a protein within the cell,
such as, for example, isoMnSOD. Accordingly, another aspect of the
present invention involves methods of inhibiting or reducing lipid
peroxidation in a cell comprising contacting the cell with an
isoMnSOD inhibitor. "Lipid peroxidation", as used herein, refers to
the oxidative deterioration of one or more lipids containing any
number of carbon-carbon double bonds.
[0072] As discussed above, isoMnSOD inhibitors can be combined with
other pharmaceutical compositions, agents, or compounds when being
administered to a cell or an individual. One class of compounds
that can be co-administered with an isoMnSOD inhibitor are
chemotherapeutics. Antibodies that recognize HER-2 (e.g.
Herceptin.TM.) and nucleic acid damaging agents, are compounds that
can be co-administered with an isoMnSOD inhibitor. Herceptin.TM.
has been a useful tool in the fight against breast cancer and in
some cases is used in conjunction with chemotherapeutics including,
but not limited to, Topoisomerase II inhibitors (e.g. doxorubicin,
epirubicin, and the like). However, a HER-2 antibody or the
combination of a HER-2 antibody and a Topoisomerase II inhibitor
has been reported to increase the risk of heart failure (see, for
example, Cancer. 2002 Oct. 1;95(7):1592-600, which is herein
incorporated by reference). This increase is related to an increase
in oxidative damage in the heart. Accordingly, the present
invention provides methods of treating cancer comprising
administering to a patient a therapeutically effective amount of
one or more chemotherapeutics and an isoMnSOD inhibitor. The
present invention further provides methods of treating cancer
comprising administering to a patient a therapeutically effective
amount of an antibody and an isoMnSOD inhibitor. The present
invention further provides methods of treating cancer comprising
administering to a patient a therapeutically effective amount of a
chemotherapeutic, an antibody, and an isoMnSOD inhibitor. The
administration of an isoMnSOD inhibitor reduces the oxidative
damage to the individuals cells exposed to the chemotherapeutic
and/or antibody and, therefore, allow the anti-cancer drugs to be
given with a reduced risk of heart failure or other damage that is
caused by the drugs.
[0073] In some embodiments, an inhibitor of isoMnSOD is
administered to an individual who has been identified in need of an
isoMnSOD inhibitor (e.g. an individual susceptible to heart
failure). In some embodiments, an individual is in need of an
isoMnSOD inhibitor if the individual has been identified as at-risk
for heart failure. In some embodiments, the heart failure is drug
induced or caused by a cancer treatment (e.g. Herceptin.TM. with or
without an anthracycline). An individual can also be in need of an
isoMnSOD inhibitor if the individual has been identified as
suffering from an oxidative disease, mitochondrial-related disease,
mtDNA damage, HNE and/or isoprostane protein modification related
disease (e.g artherosclerosis), too much lipid peroxidation, and
the like.
[0074] As discussed previously, isoMnSOD inhibitors can be combined
with any chemotherapeutic or anti-cancer drug. The term
"chemotherapeutic" refers to any chemical compound such as a
cytotoxin used to treat the disease generally known as cancer. In
some embodiments, at least one isoMnSOD inhibitor is
co-administered with at least one, at least two, at least three, at
least four, at least five, at least six, at least seven, or at
least ten chemotherapeutics. In some embodiments, one or more
isoMnSOD inhibitors are co-administered with an antibody that
recognizes HER-2 (e.g. Herceptin.TM.), a nucleic acid damaging
agent (e.g. Topoisomerase II inhibitor, doxorubicin, epirubicin,
daunorubicin, idarubicin, or anthracenedione), or combinations
thereof. The cancer to be treated can be any cancer including, but
not limited to Hodgkin's lymphoma, non-Hodgkin's lymphoma, breast
cancer, ovarian cancer, testicular cancer, acute leukemia, soft
tissue sarcoma, lung cancer (e.g., non-small cell), bladder cancer,
pancreatic cancer, gastric cancer, thyroid cancer, hepatoma, wilm's
tumor, glioma, or neuroblastoma. In some embodiments, the cancer is
breast cancer.
[0075] In some embodiments, the present invention provides a
composition comprising one or more chemotherapeutics and one or
more isoMnSOD inhibitors. In some embodiments, the present
invention provides a composition comprising one or more antibodies
and one or more isoMnSOD inhibitors. In some embodiments, the
present invention provides a composition comprising one or more
chemotherapeutics, one or more antibodies, and one or more isoMnSOD
inhibitors. In some embodiments, the composition is a
pharmaceutical composition. The enzyme fatty acid amide hydrolase
(FAAH) is known to recognize and break down fatty acids and their
derivatives. Therefore, in some embodiments, an isoMnSOD inhibitor,
such as an inhibitor that may be subject to cleavage by FAAH, is
co-administered with a FAAH inhibitor, which would prevent or
inhibit the degradation of the isoMnSOD inhibitors by FAAH.
Examples of FAAH inhibitors include, but are not limited to,
4-benzyloxyphenyl-n-butylcarb- amate, CAY10400.TM., and the
inhibitors described in Boger et al. (PNAS, 97(10):5044-5049, which
is herein incorporated by reference in its entirety).
[0076] Example isoMnSOD inhibitors include, for example, compounds
of Formula I:
Alk-L.sup.1-L.sup.2-D I
[0077] or pharmaceutically acceptable salts or prodrugs thereof,
wherein:
[0078] Alk is C.sub.2-100 alkenyl or C.sub.2-100 alkynyl, each
optionally substituted by one or more R.sup.1;
[0079] L.sup.1 is O, S, CO, C(O)O, C(O)NR.sup.2, SO, S(O).sub.2,
S(O)NR.sup.2, S(O).sub.2NR.sup.2, NR.sup.2, NR.sup.2C(O)NR.sup.3,
or NR C(S)NR.sup.3;
[0080] L.sup.2 is absent, C.sub.1-6 alkylenyl, C.sub.2-6
alkenylenyl, or C.sub.2-6 alkynylenyl, each optionally substituted
by one or more R.sup.4;
[0081] D is aryl or heteroaryl, each optionally substituted by one
or more R.sup.5;
[0082] R.sup.1 and R.sup.4 are each, independently, halo, cyano,
nitro, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, heteroaryl, C.sub.3-7 cycloalkyl,
heterocycloalkyl, S(O)R.sup.6, S(O).sub.2R.sup.6, C(O)R.sup.6,
OR.sup.7, SR.sup.7, C(O)OR.sup.7, NR.sup.8R.sup.9 or
NR.sup.8C(O)R.sup.6;
[0083] R.sup.2 and R.sup.3 are each, independently, H, C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl,
aryl, heteroaryl, C.sub.3-7 cycloalkyl, heterocycloalkyl,
arylalkyl, heteroarylalkyl; (C.sub.3-7 cycloalkyl)alkyl or
heterocycloalkylalkyl;
[0084] R.sup.5 is halo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-4 haloalkyl, aryl, cycloalkyl,
heteroaryl, heterocycloalkyl, CN, NO.sub.2, OR.sup.12, SR.sup.12,
C(O)R.sup.13, C(O)NR.sup.14R.sup.15, C(O)OR.sup.12, OC(O)R.sup.13,
OC(O)NR.sup.14R.sup.15, NR.sup.14R.sup.15, NR.sup.14C(O)R.sup.15,
NR.sup.14C(O)OR.sup.12, S(O)R.sup.13, S(O)NR.sup.14R.sup.15,
S(O).sub.2R.sup.13, or S(O).sub.2NR.sup.14R.sup.15;
[0085] R.sup.6 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 haloalkyl, aryl, heteroaryl, C.sub.3-7
cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl,
(C.sub.3-7 cycloalkyl)alkyl, heterocycloalkylalkyl, or
NR.sup.10R.sup.11;
[0086] R.sup.7 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 haloalkyl, alkoxyalkyl, haloalkoxyalkyl,
aryloxyalkyl, heteroaryloxyalkyl, cycloalkyloxyalkyl,
heterocycloalkyloxyalkyl, aryl, heteroaryl, C.sub.3-C.sub.7
cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl;
(C.sub.3-7 cycloalkyl)alkyl or heterocycloalkylalkyl;
[0087] R.sup.8 and R.sup.9 are each, independently, H, C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl,
aryl, heteroaryl, C.sub.3-7 cycloalkyl, heterocycloalkyl,
arylalkyl, heteroarylalkyl; (C.sub.3-7 cycloalkyl)alkyl or
heterocycloalkylalkyl;
[0088] or R.sup.8 and R.sup.9 together with the N atom to which
they are attached form a 3-, 4-, 5-, 6-, or 7-membered
heterocycloalkyl group;
[0089] R.sup.10 and R.sup.11 are each, independently, H,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.1-C.sub.6 haloalkyl, aryl, heteroaryl,
C.sub.3-C.sub.7 cycloalkyl or heterocycloalkyl;
[0090] R.sup.12 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl
or heterocycloalkyl;
[0091] R.sup.13 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl
or heterocycloalkyl; and
[0092] R.sup.14 and R.sup.15 are each, independently, H, C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
aryl, cycloalkyl, arylalkyl, or cycloalkylalkyl; or or R.sup.14 and
R.sup.15 together with the N atom to which they are attached form a
4-, 5-, 6- or 7-membered heterocycloalkyl group.
[0093] In some embodiments, Alk is C.sub.2-50 alkenyl.
[0094] In some embodiments, Alk is C.sub.4-30 alkenyl.
[0095] In some embodiments, Alk is C.sub.16-20 alkenyl.
[0096] In some embodiments, Alk is C.sub.17, C.sub.18 or C.sub.19
alkenyl.
[0097] In some embodiments, Alk is C.sub.2-50 alkenyl comprising at
least two double bonds.
[0098] In some embodiments, Alk is C.sub.2-50 alkenyl comprising at
least four double bonds.
[0099] In some embodiments, Alk comprises at least one bis-allylic
methylene group.
[0100] In some embodiments, Alk comprises 1 to about 10 bis-allylic
methylene groups.
[0101] In some embodiments, Alk comprises 2 to 5 bis-allylic
methylene groups.
[0102] In some embodiments, Alk corresponds to the alkenyl group of
a polyunsaturated fatty acid.
[0103] In some embodiments, L.sup.1 is O, CO, C(O)O, C(O)NR.sup.2,
NR.sup.2 or NR.sup.2C(O)NR.sup.3.
[0104] In some embodiments, L.sup.1 is CO, C(O)O, C(O)NR.sup.2 or
NR.sup.2.
[0105] In some embodiments, L.sup.1 is C(O)NR.sup.2.
[0106] In some embodiments, L.sup.2 is absent or C.sub.1-6
alkylenyl.
[0107] In some embodiments, L.sup.2 is Ca 3 alkylenyl.
[0108] In some embodiments, D is phenyl, naphthyl, anthracenyl,
phenanthrenyl, indanyl, indenyl, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl,
imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl,
benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl,
tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl,
benzothienyl, purinyl, carbazolyl, benzimidazolyl, or indolinyl,
each optionally substituted by one or more R.sup.5.
[0109] In some embodiments, R.sup.5 is halo, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-4 haloalkyl, aryl,
cycloalkyl, heteroaryl, heterocycloalkyl, CN, NO.sub.2, OR.sup.12
or NR.sup.4R.sup.15.
[0110] In some embodiments, R.sup.5 is halo, C.sub.1-6 alkyl,
C.sub.1-4 haloalkyl, CN, NO.sub.2, OR 2 or NR.sup.14R.sup.15.
[0111] In some embodiments, R.sup.5 is OH.
[0112] In some embodiments, R.sup.5 is methoxy.
[0113] Further examples isoMnSOD inhibitors include arachidonoyl
dopamine (AA-DA; Cayman Chemical, MI), arachidonoyl serotonin
(AA-serotonin; Cayman Chemical, MI), eicosapentaenoyl dopamine
(EPA-DA; Cayman Chemical, MI), docosahexaenoyl dopamine (DPH-DA;
Cayman Chemical, MI), and C18:4(.omega.-3)-dopamine (International
Technology Transfer Concepts), and the like, each of which was
found to inhibit pro-oxidant activity of isoMnSOD according to at
least one of the assays provided in the Examples.
[0114] Further examples of isoMnSOD inhibitors include
arachidonoyl-5-methoxytryptamine (AA-MOT; Cayman Chemical, MI),
eicosapentaenoyl-5-methoxytryptamine (EPA-MOT; Cayman Chemical,
MI), N-(4-hydroxyphenyl)arachidonoylamide (AM404; Cayman Chemical,
MI); eicosapentaenoyl serotonin; docosahexaenoyl serotonin;
arachidonoyl tyramine; arachidonoyl phenethylamine,
C18:3(.omega.-3)-dopamine; C20:3(.omega.-6)-dopamine;
C22:5(.omega.-3)-dopamine, and the like.
[0115] At various places in the present specification, substituents
of compounds of the invention are disclosed in groups or in ranges.
It is specifically intended that the invention include each and
every individual subcombination of the members of such groups and
ranges. For example, the term "C.sub.1-6 alkyl" is specifically
intended to individually disclose methyl, ethyl, C.sub.3 alkyl,
C.sub.4 alkyl, C.sub.5 alkyl, and C.sub.6 alkyl.
[0116] For compounds of the invention in which a variable appears
more than once, each variable can be a different moiety selected
from the Markush group defining the variable. For example, where a
structure is described having two R groups that are simultaneously
present on the same compound; the two R groups can represent
different moieties selected from the Markush group defined for
R.
[0117] It is further appreciated that certain features of the
invention, which are, for clarity, described in the context of
separate embodiments, can also be provided in combination in a
single embodiment. Conversely, various features of the invention
which are, for brevity, described in the context of a single
embodiment, can also be provided separately or in any suitable
subcombination.
[0118] As used herein, the term "alkyl" is meant to refer to a
saturated hydrocarbon group which is straight-chained or branched.
Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g.,
n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl),
pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An
alkyl group can contain from 1 to about 20, from 2 to about 20,
from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to
about 4, or from 1 to about 3 carbon atoms.
[0119] As used herein, "alkenyl" refers to an alkyl group having
one or more double carbon-carbon bonds. Example alkenyl groups
include ethenyl, propenyl, and the like.
[0120] As used herein, "alkynyl" refers to an alkyl group having
one or more triple carbon-carbon bonds. Example alkynyl groups
include ethynyl, propynyl, and the like.
[0121] As used herein, "alkylenyl" refers to a divalent alkyl
linking group.
[0122] As used herein, "alkenylenyl" refers to a divalent alkenyl
linking group.
[0123] As used herein, "alkynylenyl" refers to a divalent alkynyl
linking group.
[0124] As used herein, "haloalkyl" refers to an alkyl group having
one or more halogen substituents. Example haloalkyl groups include
CF.sub.3, C.sub.2F.sub.5, CHF.sub.2, CCl.sub.3, CHCl.sub.2,
C.sub.2Cl.sub.5, and the like.
[0125] As used herein, "aryl" refers to monocyclic or polycyclic
(e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as,
for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl,
indenyl, and the like. In some embodiments, aryl groups have from 6
to about 20 carbon atoms.
[0126] As used herein, "cycloalkyl" refers to non-aromatic cyclic
hydrocarbons including cyclized alkyl, alkenyl, and alkynyl groups.
Cycloalkyl groups can include mono- or polycyclic (e.g., having 2,
3 or 4 fused rings) ring systems as well as spiro ring systems.
Example cycloalkyl groups include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl,
cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcamyl,
adamantyl, and the like. Also included in the definition of
cycloalkyl are moieties that have one or more aromatic rings fused
(i.e., having a bond in common with) to the cycloalkyl ring, for
example, benzo derivatives of pentane, pentene, hexane, and the
like.
[0127] As used herein, a "heteroaryl" group refers to an aromatic
heterocycle having at least one heteroatom ring member such as
sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic
and polycyclic (e.g., having 2, 3 or 4 fused rings) systems.
Examples of heteroaryl groups include without limitation, pyridyl,
pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl,
isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl,
oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl,
pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl,
isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl,
indolinyl, and the like. In some embodiments, the heteroaryl group
has from 1 to about 20 carbon atoms, and in further embodiments
from about 3 to about 20 carbon atoms. In some embodiments, the
heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6
ring-forming atoms. In some embodiments, the heteroaryl group has 1
to about 4, 1 to about 3, or 1 to 2 heteroatoms.
[0128] As used herein, "heterocycloalkyl" refers to non-aromatic
heterocycles including cyclized alkyl, alkenyl, and alkynyl groups
where one or more of the ring-forming carbon atoms is replaced by a
heteroatom such as an O, N, or S atom. Example "heterocycloalkyl"
groups include morpholino, thiomorpholino, piperazinyl,
tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl,
1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl, pyrrolidinyl,
isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl,
thiazolidinyl, imidazolidinyl, and the like. Also included in the
definition of heterocycloalkyl are moieties that have one or more
aromatic rings fused (i.e., having a bond in common with) to the
nonaromatic heterocyclic ring, for example phthalimidyl,
naphthalimidyl, and benzo derivatives of heterocycles such as
indolene and isoindolene groups. In some embodiments, the
heterocycloalkyl group has from 1 to about 20 carbon atoms, and in
further embodiments from about 3 to about 20 carbon atoms. In some
embodiments, the heterocycloalkyl group contains 3 to about 14, 3
to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the
heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2
heteroatoms. In some embodiments, the heterocycloalkyl group
contains 0 to 3 double bonds. In some embodiments, the
heterocycloalkyl group contains 0 to 2 triple bonds.
[0129] As used herein, "arylalkyl" refers to an alkyl group
substituted by an aryl group.
[0130] As used herein, "cycloalkylalkyl" refers to an alkyl group
substituted by a cycloalkyl group.
[0131] As used herein, "heteroarylalkyl" refers to an alkyl group
substituted by a heteroaryl group.
[0132] As used herein, "heterocycloalkylalkyl" refers to an alkyl
group substituted by a heterocycloalkyl group.
[0133] As used herein, "halo" or "halogen" includes fluoro, chloro,
bromo, and iodo.
[0134] As used herein, "alkoxy" refers to an --O-alkyl group.
Example alkoxy groups include methoxy, ethoxy, propoxy (e.g.,
n-propoxy and isopropoxy), t-butoxy, and the like.
[0135] As used herein, "haloalkoxy" refers to an -O-haloalkyl
group. An example haloalkoxy group is OCF.sub.3.
[0136] As used herein, "aryloxy" refers to --O-aryl.
[0137] As used herein, "heteroaryloxy" refers to
--O-heteroaryl.
[0138] As used herein, "cycloalkyloxy" refers to
--O-cycloalkyl.
[0139] As used herein, "heterocycloalkyloxy" refers to
--O-heterocycloalkyl.
[0140] As used herein, "alkoxyalkyl" refers to alkyl substituted by
alkoxy.
[0141] As used herein, "haloalkoxyalkyl" refers to alkyl
substituted by haloalkoxy.
[0142] As used herein, "aryloxyalkyl" refers to alkyl substituted
by aryloxy.
[0143] As used herein, "heteroaryloxyalkyl" refers to alkyl
substituted by heteroaryloxy.
[0144] As used herein, "cycloalkyloxyalkyl" refers to alkyl
substituted by cycloalkyloxy.
[0145] As used herein, "heterocycloalkyloxyalkyl" refers to alkyl
substituted by heterocycloalkyloxy.
[0146] As used herein, the term "bis-allylic" is used as known in
the art and refers to a metheylene group (CH.sub.2) flanked by
double bonds. For example, the CH.sub.2 moiety of
--CH.dbd.CH--CH.sub.2--CH.dbd.CH-- is a bis-allylic methylene
group.
[0147] The compounds described herein can be asymmetric (e.g.,
having one or more stereocenters). All stereoisomers, such as
enantiomers and diastereomers, are intended unless otherwise
indicated. Compounds of the present invention that contain
asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically active starting materials are
known in the art, such as by resolution of racemic mixtures or by
stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present invention. C is and trans geometric
isomers of the compounds of the present invention are described and
may be isolated as a mixture of isomers or as separated isomeric
forms.
[0148] Resolution of racemic mixtures of compounds can be carried
out by any of numerous methods known in the art. An example method
includes fractional recrystallizaion using a "chiral resolving
acid" which is an optically active, salt-forming organic acid.
Suitable resolving agents for fractional recrystallization methods
are, for example, optically active acids, such as the D and L forms
of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid,
mandelic acid, malic acid, lactic acid or the various optically
active camphorsulfonic acids such as a-camphorsulfonic acid. Other
resolving agents suitable for fractional crystallization methods
include stereoisomerically pure forms of P-methylbenzylamine (e.g.,
S and R forms, or diastereomerically pure forms), 2-phenylglycinol,
norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine,
1,2-diaminocyclohexane, and the like.
[0149] Resolution of racemic mixtures can also be carried out by
elution on a column packed with an optically active resolving agent
(e.g., dinitrobenzoylphenylglycine). Suitable elution solvent
composition can be determined by one skilled in the art.
[0150] Compounds of the invention can also include tautomeric
forms, such as keto-enol tautomers. Tautomeric forms can be in
equilibrium or sterically locked into one form by appropriate
substitution.
[0151] Compounds of the invention can also include all isotopes of
atoms occurring in the intermediates or final compounds. Isotopes
include those atoms having the same atomic number but different
mass numbers. For example, isotopes of hydrogen include tritium and
deuterium.
[0152] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0153] The present invention also includes pharmaceutically
acceptable salts of the compounds described herein. As used herein,
"pharmaceutically acceptable salts" refers to derivatives of the
disclosed compounds wherein the parent compound is modified by
converting an existing acid or base moiety to its salt form.
Examples of pharmaceutically acceptable salts include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines; alkali or organic salts of acidic residues such as
carboxylic acids; and the like. The pharmaceutically acceptable
salts of the present invention include the conventional non-toxic
salts or the quaternary ammonium salts of the parent compound
formed, for example, from non-toxic inorganic or organic acids. The
pharmaceutically acceptable salts of the present invention can be
synthesized from the parent compound which contains a basic or
acidic moiety by conventional chemical methods. Generally, such
salts can be prepared by reacting the free acid or base forms of
these compounds with a stoichiometric amount of the appropriate
base or acid in water or in an organic solvent, or in a mixture of
the two; generally, nonaqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences,
17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and
Journal of Pharmaceutical Science, 66, 2 (1977), each of which is
incorporated herein by reference in its entirety.
[0154] The present invention also includes prodrugs of the
compounds described herein. As used herein, "prodrugs" refer to any
covalently bonded carriers which release the active parent drug
when administered to a mammalian subject. Prodrugs can be prepared
by modifying functional groups present in the compounds in such a
way that the modifications are cleaved, either in routine
manipulation or in vivo, to the parent compounds. Prodrugs include
compounds wherein hydroxyl, amino, sulfhydryl, or carboxyl groups
are bonded to any group that, when administered to a mammalian
subject, cleaves to form a free hydroxyl, amino, sulfhydryl, or
carboxyl group respectively. Examples of prodrugs include, but are
not limited to, acetate, formate and benzoate derivatives of
alcohol and amine functional groups in the compounds of the
invention. Preparation and use of prodrugs is discussed in T.
Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol.
14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in
Drug Design, ed. Edward B. Roche, American Pharmaceutical
Association and Pergamon Press, 1987, both of which are hereby
incorporated by reference in their entirety.
[0155] Synthesis
[0156] IsoMnSOD inhibitors of Formula I, including salts, hydrates,
and solvates thereof, can be prepared using known organic synthesis
techniques and can be synthesized according to any of numerous
possible synthetic routes.
[0157] The reactions for preparing compounds of Formula I can be
carried out in suitable solvents which can be readily selected by
one of skill in the art of organic synthesis. Suitable solvents can
be substantially non-reactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, e.g., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected.
[0158] Preparation of compounds of Formula I can involve the
protection and deprotection of various chemical groups. The need
for protection and deprotection, and the selection of appropriate
protecting groups can be readily determined by one skilled in the
art. The chemistry of protecting groups can be found, for example,
in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic
Synthesis, 3rd. Ed., Wiley & Sons, Inc., New York (1999), which
is incorporated herein by reference in its entirety.
[0159] Reactions can be monitored according to any suitable method
known in the art. For example, product formation can be monitored
by spectroscopic means, such as nuclear magnetic resonance
spectroscopy (e.g., .sup.1H or .sup.13C) infrared spectroscopy,
spectrophotometry (e.g., UV-visible), or mass spectrometry, or by
chromatography such as high performance liquid chromatography
(HPLC) or thin layer chromatography.
[0160] Compounds of Formula I are commercially available (e.g.,
Cayman Chemical, MI) or can be prepared according to numerous
preparatory routes known in the literature. For example,
polyunsatuarated fatty acids (e.g., arachidonic acid, linoleic
acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid,
C18:4(.omega.-3) fatty acid, C20:4(o-3) fatty acid,
C20:3(.omega.-6) fatty acid, C20:3(.omega.-3) fatty acid,
C22:5(.omega.-3) fatty acid, C22:4(.omega.-3) fatty acid,
C22:4(.omega.-6) fatty acid, C22:3(o-3) fatty acid, etc.) can be
combined with arylamines, heteroarylamines, arylalkylamines,
heteroarylalkylamines, etc. optionally in the presence of an acid
or base to form the amide-linked (L.sup.1=CONR.sup.2) product of
Formula I. The resulting amide bond can be further reduced in the
presence of a suitable reducing agent such as hydrogen over Pd
catalyst to form the corresponding amine-linked (L.sup.1=NR.sup.2)
product of Formula I. Additionally, the fatty acids can be combined
with arylhydroxides (e.g. phenols) optionally in the presence of an
acid or base to form ester-linked (L.sup.1=COO) compounds of
Formula I which can, in turn, be reduced to form ether-linked
(L.sup.1=O) compounds of Formula I. Other coupling reactions
between fatty acids or derivatives thereof and aryl or heteroaryl
reagents are well known in the art and can form carbonyl, ureido,
sulfonyl, sulfinyl, sulfonamide and other linkages useful in the
preparation of compounds of Formula I. Example coupling reactions
can be found in March, Advanced Organic Chemistry, 4.sup.th ed.
John Wiley & Sons, NY, 1992 and Kemp et al., Organic Chemistry,
Worth Publishers, Inc. NY, 1980; each of which is incorporated
herein by reference in its entirety.
[0161] In addition to the isoMnSOD inhibitors described herein, any
isoMnSOD inhibitor can be used. The present invention provides
methods for identifying isoMnSOD inhibitors using methods that are
described herein and the Examples. One of skill in the art can
identify an isoMnSOD inhibitor by isolating an isoMnSOD polypeptide
and measuring the activity with and without a candidate compound.
In some embodiments, the candidate compound will inhibit the
pro-oxidant activity of isoMnSOD and would, therefore, be
considered an inhibitor of isoMnSOD activity. In some embodiments,
the compound reduces the pro-oxidant activity of isoMnSOD by at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100%. This assay can also be used to identify activators of
isoMnSOD activity. Methods of measuring pro-oxidant activity are
known in the art and are also described herein.
[0162] In some embodiments, an isoMnSOD inhibitor is identified by
a method comprising contacting a cell expressing isoMnSOD with a
test compound; measuring a isoMnSOD mediated event in the cell; and
comparing the isoMnSOD mediated event in the cell to a cell that
has not been contacted with the test compound; wherein a decrease
in the isoMnSOD mediated event indicates that the test compound is
an isoMnSOD inhibitor. An "isoMnSOD mediated event" refers to a
process or phenotypic event that is regulated or facilitated by
isoMnSOD. In some embodiments, an "isoMnSOD mediated event" is cell
death, oxidation, lipid peroxidation, isoprostane production, HNE
production, isoprostane protein modification, HNE protein
modification, or the like. In some embodiments, the "isoMnSOD
mediated event is not oxidation. In some embodiments, the test
compound is an antibody, peptide, polypeptide, nucleic molecule,
small molecule, and the like. In some embodiments the test compound
is not a nucleic acid molecule. In some embodiments, the test
compound is not a small molecule compound.
[0163] IsoMnSOD activity can also be measured in vivo or in vitro
by its pro-apoptotic activity. Induction of the isoMnSOD protein
can lead to the premature cell death by apoptosis or by another
mechanism. Therefore, by using a cell that normally does not
express isoMnSOD, one of skill in the art can identify inhibitors
of isoMnSOD activity by inhibiting the premature cell death of a
cell.
[0164] As a non-limiting example, one of skill in the art can
transform or transfect a cell line that does not express isoMnSOD
or expresses isoMnSOD at a low level with a nucleic acid molecule
that expresses the isoMnSOD gene and protein. The expression of
isoMnSOD in the cell line can lead to a phenotypic change that is
measurable such as, for example, cell viability, cell death markers
(e.g. caspase cleavage and expression), gene expression, protein
expression, protein cleavage, and the like. A compound can be
tested as an inhibitor of isoMnSOD activity to determine if the
compound has an effect on the phenotype that is observed due to the
expression of isoMnSOD. If the compound reduces the effect that is
observed, the compound is said to be an inhibitor. In contrast,
this assay can also be used to identify activators of isoMnSOD
activity by determining whether the compound increases the effects
of isoMnSOD as measured by a phenotype. In some embodiments, the
compound will be tested in a cell line that does not express
isoMnSOD and compared to a cell line that expresses isoMnSOD to
determine if the change in phenotype is specific for the expression
of isoMnSOD.
[0165] In some embodiments, a compound is considered to be an
isoMnSOD inhibitor if the compound reduces the effect of isoMnSOD
by at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100%. In some embodiments, the compound reduces the pro-oxidant
activity or pro-apoptotic activity of isoMnSOD by at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100%.
[0166] isoMnSOD inhibitors can also be identified using assays that
measure isoMnSOD mediated HNE production, isoprostane production,
or lipid peroxidation. A compound would be considered to be an
isoMnSOD inhibitor if the compound inhibits the amount of HNE or
isoprostanes produced in a cell or if the compound inhibits lipid
peroxidation. In some embodiments, these assays are performed in a
cell where isoMnSOD is exogenously produced.
[0167] As used herein, the term "exogenously produced" refers to
protein, peptide, or nucleic acid molecule that is not normally
expressed in a cell or was not originally part of the genome of a
cell. A gene or protein can be exogenously produced by using a
nucleic acid molecule such as, for example, a plasmid or a virus
(e.g. retrovirus, DNA virus, adenovirus, adeno-associated virus,
and the like). In some embodiments, the nucleic acid molecule is
free of infectious particles.
[0168] IsoMnSOD inhibitors can also be identified using animal
models that have drug induced toxicity, such as non-ischemic heart
failure. For example, a mammal can be treated with one or more
chemotherapeutics that causes toxicity in the mammal. A test
compound can be administered in conjunction with the
chemotherapeutic to determine if it reduces the drug-induced
toxicity. In some embodiments, the drug-induced toxicity is
non-ischemic heart failure. Inducing toxicity using doxorubicin in
animal models has been previously performed, see, for example,
Arola et al. Cancer Research, 60:1789-1792. Therefore, an
individual or non-human animal that has been treated with
doxorubicin can be treated with a compound to determine if it is an
isoMnSOD inhibitor. A compound is said to be an isoMnSOD inhibitor
if it reduces the toxicity of the drug by at least 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or 100%.
[0169] A compound can also be identified as an isoMnSOD inhibitor
by determining if the compound can bind to the isoMnSOD protein.
Methods for determining the binding of a compound to a protein are
known to one of ordinary skill in the art and any routine method
can be used. As non-limiting examples, the compounds can be labeled
to see if the compound interacts with the polypeptide. Once the
compound is contacted with an individual, cell or protein, a
cross-linking agent can be used to determine if the compound binds
to the protein. If a compound is found to bind to an isoMnSOD
protein either in vivo or in vitro and it inhibits an activity of
isoMnSOD it is considered to be an isoMnSOD inhibitor. If a
compound is found to bind to an isoMnSOD protein either in vivo or
in vitro and it activates an activity of isoMnSOD it is considered
to be an isoMnSOD activator.
[0170] The present invention has also identified that isoMnSOD is
induced under specific circumstances, which includes, but is not
limited to, drug-induced toxicity. Therefore, the expression of
isoMnSOD can be used as a marker of drug induced toxicity. The
ability to identify compounds that cause toxicity would save time
and money in the development of drugs by discarding drugs that are
prohibitively toxic and pursuing compounds with little toxicity or
acceptable levels of toxicity. Therefore, the present invention
provides methods of measuring toxicity of one or more compounds by
contacting the one or more compounds with a cell, wherein the cell
contains a nucleic acid molecule having a genomic fragment of MnSOD
gene spanning Exon 2 to Exon 4 of the MnSOD gene, wherein the
genomic fragment comprises a frameshift mutation in Exon 3, and is
operably linked to a reporter gene, wherein expression of the
reporter gene is indicative of toxicity. The reporter gene will
only be expressed if the alternative transcript form of isoMnSOD
gene is expressed or in the case of a genomic fragment if the
alternative splice event occurs. Under non-toxic conditions (e.g.
the drug does not cause toxicity) the reporter gene will not be
expressed due to the presence of the frameshift mutation located in
Exon 3 or equivalent sequence. Under drug-inducing toxic conditions
the cells machinery removes Exon 3 by an alternative splicing
mechanism, thereby allowing the expression of the reporter gene.
The detection of the reporter gene either by RNA expression (e.g.
mRNA or RNA), protein expression or the protein's activity
indicates that the drug is toxic. If the reporter gene is not
expressed or the reporter gene's activity is not detected, the
compound is considered to be non-toxic. Methods of detecting gene
expression are known to one of ordinary skill in the art and
include, but are not limited to, RT-PCR, northern blot, western
blot, immunofluorescence, immunoprecipitation, and the like.
[0171] A "reporter gene" refers to any gene that is used as
indication that the splicing event has occurred. In some
embodiments, the reporter gene is a luciferase gene,
.beta.-galactosidase gene, a secreted alkaline phosphatase gene, or
a fluorescent protein (e.g. green fluorescent protein (GFP), red
fluorescent protein, cyan fluorescent protein, or yellow
fluorescent protein), and the like. In some embodiments, the
reporter gene can be a gene that is not expressed in the cell or is
expressed at low levels, so that an increase in expression is
detected. The reporter gene can be detected using any method
including, but not limited to, Western blot (to measure the protein
expression of the reporter gene), enzymatic activity of the
reporter gene product, fluorescent microscopy (e.g.
immunofluorescence), ultraviolet absorption, RT-PCR (to detect the
presence of the reporter gene's RNA or mRNA), and the like. This
method can also allow the screening of more than one compound to
determine if compositions or compounds in combination with one
another would produce toxic effects. In some embodiments the
composition comprises at least one compound, at least two
compounds, at least three compounds, at least four compounds, or at
least five compounds. In some embodiments, the compound is a
pharmaceutical composition comprising a drug. In some embodiments,
the drug is a small molecule, antibody, nucleic acid molecule (e.g.
DNA, RNA, or virus), and the like.
[0172] In addition to the reporter gene being present in a cell
line and a compound is tested in vitro (e.g. in cell culture or
test tube), a transgenic non-human animal can be created
incorporating a nucleic acid molecule comprising a genomic fragment
of MnSOD gene spanning Exon 2 to Exon 4 of said gene, wherein the
fragment comprises a frameshift mutation in Exon 3, and is operably
linked to a reporter gene. The production of non-human transgenic
animal is well known to one of ordinary skill in the art and only
requires routine experimentation (see, for example, Transgenic
Animal Technology: A Laboratory Handbook 2nd ed., Carl Pinkert,
Academic Press (2002); Mouse Genetics and Transgenics: A Practical
Approach, Oxford University Press (2000); and Gene Targeting: A
Practical Approach 2.sup.nd ed., Oxford University Press (2000).
Upon the generation of a transgenic animal (e.g. mouse or rat) one
or more compounds can be tested to determine if the compound
induces the expression of the reporter gene. The expression of the
reporter gene would be indicative of drug-induced toxicity.
[0173] In either the in vivo or in vitro systems the reporter gene
may be expressed without being induced by the test compound.
However, if a test compound were to increase the expression of the
reporter gene, it would then be considered to be toxic. In some
embodiments, a test compound is considered to be toxic if the test
compound increases the expression of the reporter gene over the
basal levels by at least 10%, by at least 20%, by at least 30%, by
at least 40%, by at least 50%, by at least 60%, by at least 70%, by
at least 80%, by at least 90%, by at least 100%, by at least 200%,
by at least 300%. As used herein, the term "basal level" refers to
the level of expression that occurs in a cell or animal that occurs
without being contacted with a test compound.
[0174] In addition, to using a reporter gene, one of skill in the
art could measure the expression of isoMnSOD mRNA, protein,
activity of isoMnSOD, or combinations thereof to determine if a
compound is toxic. The expression or activity of isoMnSOD can be
compared in the absence and the presence of one or more test
compounds. If the expression or activity of isoMnSOD is increased
in the presence of one or more test compounds as compared to the
expression of isoMnSOD in the absence of the one or more test
compounds, the increase in expression is indicative of the one or
more compounds being toxic. If the expression or activity of
isoMnSOD is not increased in the presence of one or more test
compounds, then the lack of increase in expression is indicative of
the compound not being toxic.
[0175] In some embodiments, a change in expression or activity of
isoMnSOD or of the above-mentioned reporter gene is indicative of
the one or more test compounds being toxic if the expression or
activity of isoMnSOD or the reporter gene is increased by at least
5%, at least 10%, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
at least 100%, at least 200%, at least 300% as compared to the
expression or activity of the genes or gene products in the absence
of the one or more test compounds. Methods to measure changes in
expression of a gene or a gene product are known to one of ordinary
skill in the art.
[0176] Pharmaceutical Formulations and Dosage Forms
[0177] When employed as pharmaceuticals, the inhibitors of the
present invention can be administered in the form of pharmaceutical
compositions. These compositions can be administered by a variety
of routes including oral, rectal, transdermal, subcutaneous,
intravenous, intramuscular, and intranasal, and can be prepared in
a manner well known in the pharmaceutical art.
[0178] This invention also includes pharmaceutical compositions
which contain, as the active ingredient, one or more of the
compounds of Formula I or any other isoMnSOD inhibitor as described
above in combination with one or more pharmaceutically acceptable
carriers. In making the compositions of the invention, the active
ingredient is typically mixed with an excipient, diluted by an
excipient or enclosed within such a carrier in the form of, for
example, a capsule, sachet, paper, or other container. When the
excipient serves as a diluent, it can be a solid, semi-solid, or
liquid material, which acts as a vehicle, carrier or medium for the
active ingredient. Thus, the compositions can be in the form of
tablets, pills, powders, lozenges, sachets, cachets, elixirs,
suspensions, emulsions, solutions, syrups, aerosols (as a solid or
in a liquid medium), ointments containing, for example, up to 10%
by weight of the active compound, soft and hard gelatin capsules,
suppositories, sterile injectable solutions, and sterile packaged
powders.
[0179] In preparing a formulation, the active compound can be
milled to provide the appropriate particle size prior to combining
with the other ingredients. If the active compound is substantially
insoluble, it can be milled to a particle size of less than 200
mesh. If the active compound is substantially water soluble, the
particle size can be adjusted by milling to provide a substantially
uniform distribution in the formulation, e.g. about 40 mesh.
[0180] Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrup, and methyl cellulose. The formulations can
additionally include: lubricating agents such as talc, magnesium
stearate, and mineral oil; wetting agents; emulsifying and
suspending agents; preserving agents such as methyl- and
propylhydroxy-benzoates; sweetening agents; and flavoring agents.
The compositions of the invention can be formulated so as to
provide quick, sustained or delayed release of the active
ingredient after administration to the patient by employing
procedures known in the art.
[0181] The compositions can be formulated in a unit dosage form,
each dosage containing from about 5 to about 100 mg, or about 10 to
about 30 mg, of the active ingredient. The term "unit dosage forms"
refers to physically discrete units suitable as unitary dosages for
human subjects and other mammals, each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect, in association with a suitable
pharmaceutical excipient.
[0182] The active compound can be effective over a wide dosage
range and is generally administered in a pharmaceutically effective
amount. It will be understood, however, that the amount of the
compound actually administered will usually be determined by a
physician, according to the relevant circumstances, including the
condition to be treated, the chosen route of administration, the
actual compound administered, the age, weight, and response of the
individual patient, the severity of the patient's symptoms, and the
like.
[0183] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical
excipient to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention. When
referring to these preformulation compositions as homogeneous, the
active ingredient is typically dispersed evenly throughout the
composition so that the composition can be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation is then subdivided into unit
dosage forms of the type described above containing from, for
example, 0.1 to about 500 mg of the active ingredient of the
present invention.
[0184] The tablets or pills of the present invention can be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0185] The liquid forms in which the compounds and compositions of
the present invention can be incorporated for administration orally
or by injection include aqueous solutions, suitably flavored
syrups, aqueous or oil suspensions, and flavored emulsions with
edible oils such as cottonseed oil, sesame oil, coconut oil, or
peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0186] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as described supra. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in can be
nebulized by use of inert gases. Nebulized solutions may be
breathed directly from the nebulizing device or the nebulizing
device can be attached to a face masks tent, or intermittent
positive pressure breathing machine. Solution, suspension, or
powder compositions can be administered orally or nasally from
devices which deliver the formulation in an appropriate manner.
[0187] The amount of compound or composition administered to a
patient will vary depending upon what is being administered, the
purpose of the administration, such as prophylaxis or therapy, the
state of the patient, the manner of administration, and the like.
In therapeutic applications, compositions can be administered to a
patient already suffering from a disease in an amount sufficient to
cure or at least partially arrest the symptoms of the disease and
its complications. Effective doses will depend on the disease
condition being treated as well as by the judgment of the attending
clinician depending upon factors such as the severity of the
disease, the age, weight and general condition of the patient, and
the like.
[0188] The compositions administered to a patient can be in the
form of pharmaceutical compositions described above. These
compositions can be sterilized by conventional sterilization
techniques, or may be sterile filtered. Aqueous solutions can be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile aqueous carrier prior to
administration. The pH of the compound preparations typically will
be between 3 and 11, more preferably from 5 to 9 and most
preferably from 7 to 8. It will be understood that use of certain
of the foregoing excipients, carriers, or stabilizers will result
in the formation of pharmaceutical salts.
[0189] The therapeutic dosage of the compounds of the present
invention can vary according to, for example, the particular use
for which the treatment is made, the manner of administration of
the compound, the health and condition of the patient, and the
judgment of the prescribing physician. The proportion or
concentration of a compound of the invention in a pharmaceutical
composition can vary depending upon a number of factors including
dosage, chemical characteristics (e.g., hydrophobicity), and the
route of administration. For example, the compounds of the
invention can be provided in an aqueous physiological buffer
solution containing about 0.1 to about 10% w/v of the compound for
parenteral administration. Some typical dose ranges are from about
1 .mu.g/kg to about 1 g/kg of body weight per day. In some
embodiments, the dose range is from about 0.01 mg/kg to about 100
mg/kg of body weight per day. The dosage is likely to depend on
such variables as the type and extent of progression of the disease
or disorder, the overall health status of the particular patient,
the relative biological efficacy of the compound selected,
formulation of the excipient, and its route of administration.
Effective doses can be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0190] The compounds of the invention can also be formulated in
combination with one or more additional active ingredients which
can include any pharmaceutical agent such as a chemotherapeutic,
anti-viral agents, antibiotics, antibodies, immune suppressants,
anti-inflammatory agents, FAAH inhibitors, and the like. In some
embodiments, the compounds of the invention are formulated in
combination with one or more chemotherapeutics and other agents
used for treating cancer or heart failure.
[0191] In order that the invention disclosed herein may be more
efficiently understood, examples are provided below. It should be
understood that these examples are for illustrative purposes only
and are not to be construed as limiting the invention in any
manner.
EXAMPLES
Example 1
E. coli Transformation and Recombinant MnSOD Exon3-Deleted Isoform
Protein Extract
[0192] The pRSET-B/MnSOD Exon3-deleted isoform recombinant DNA
contained as an insert a 600 nucleotide DNA fragment from human
MnSOD Exon3-deleted isoform cDNA from coding nucleotide 95 to 695
(Genbank Accession #: X07834) that was amplified by PCR and cloned
into the Nde1-Hind3 site of pRSET-B (Invitrogen). BL21/pLysS cells
(Invitrogen) were transformed with pRSET-B/MnSOD Exon3-deleted
isoform and the expression of the MnSOD Exon3-deleted isoform cDNA
was induced in a 20 ml culture by addition of 1 mM IPTG at
OD.sub.600 0.6 for 2.5 hrs. Pelleted cells were washed 1.times. in
buffer A (70 mM Mannitol, 200 mM sucrose, 5.0 mM KCL, 1.0 mM EDTA,
0.1 mM DTPA, 1.0 mM EGTA pH 7.4) and stored frozen. To facilitate
lysis, cell pellets were thawed for 10 min, refrozen, and lysed for
20 min at room temperature in 1 ml of B-Per detergent (Pierce)
containing 1/10 vol of 5M NaCl and bacterial protease inhibitors
(Sigma). The cell extract is centrifuged at 7 K rpm for 5 min, the
supernatant removed, and the pellet was re-extracted with 500 .mu.L
of B-PER and centrifuged. B-Per extractions of the bacterial pellet
were continued to fully solubilize the recombinant MnSOD
Exon3-deleted isoform.
Example 2
Transient Transfections
[0193] For cDNA transient transfections into mammalian cell lines,
cells were grown in MCDB131 medium containing 10% FBS. For
transfection, 4.times.10.sup.6 cells were released from the tissue
culture plate using 0.05% Trypsin/EDTA (GIBCO/BRL) and resuspended
in 1 ml of MCDB medium containing 10% FBS, which was then gently
mixed with 4 ml of MCDB medium without serum containing 5 .mu.g of
recombinant DNA complexed with DMRIE-C (20 .mu.L), according to
manufacturer's instructions (BRL). After an incubation of cells
with the DNA/DMRIE-C complex for 20 min, the cells were pelleted at
1000 g for 5 min, washed once with MCDB 10% FBS and replated in
MCDB containing 10% FBS. This transfection procedure resulted in
high viability of cells without potential toxicity by DMRIE-C. At
different time points (24, 48 and 72 hrs), cells were recovered
with 0.25% trypsin, 1 mM EDTA, counted and aliquoted in tubes at
.about.1.times.10.sup.6 cells for western immunoblot analysis.
Example 3
Western Immunoblot Analysis
[0194] Samples were lysed in 2.times.Laemmli SDS lysis buffer (4.0
ml 10% SDS, 2.0 ml glycerol, 1.2 ml Tris 6.8, 2.8 ml H.sub.2O; add
1 .mu.l of .beta.-mercaptoethanol (14.4M) per 1 ml of lysis buffer)
and boiled for 5 min. The proteins were separated on an 12%
polyacrylamide SDS denaturing gel, and electroblotted onto a
nitrocellulose PVDF membrane overnight at 20 volts. Membranes were
blocked for 1 hr in TBS with 0.1% Tween 20 and 1% BSA; this
blocking buffer was also used throughout the immunoblotting
procedure. Proteins were detected using the affinity pure, rabbit
polyclonal antibody, anti-MnSOD Exon3-delted isoform, directed to
12 amino acids flanking the human MnSOD E2/E4 splice junction
peptide rabbit polyclonal antibody, anti-MnSOD Exon3-deleted
isoform, a pan-MnSOD pAb (Upstate Biotechnology), mouse anti-HNE
(Genox), or goat anti-8-epi-PGF2a(isoprostane) (Oxford Biomed). The
secondary antibodies were HRP conjugated (Santa Cruz Biotechnology)
and were detected using Femto Western blotting procedure (Pierce)
following manufacturer's instructions.
Example 4
Drug Inhibition Method
[0195] Each test compound was prepared by mixing 5 .mu.L of a 1 mM
test compound stock solution in ethanol with 5 .mu.L of 0.1 M KOH,
followed by the addition of 5 ml of TBS for a final concentration
of 1 .mu.M test compound. Equal amounts of native protein extracts
from pancreatic mitochondria (obtained according to the protocol of
Example 5) were loaded in separate wells and resolved in a 7% PAGE
gel without SDS, electrotransferred to PVDF and divided into
strips. Each PVDF strip was incubated with 1 .mu.M of test compound
in TBS for 30 minutes, washed twice in TBS for 20 minutes, and the
peroxidative activity of isoMnSOD was measured by incubation of
each strip with 1 mM H.sub.2O.sub.2 in 0.4 ml of the luminol
solution contained within the Femtomole chemiluminescence assay kit
(Pierce) and exposed to x-ray film (FIG. 4). Compounds in lanes 4-7
(arachidonoyl-dopamine; eicosapentaenoyl (EPA))-dopamine;
docosahexaenoyl (DHA)-dopamine; C18:4(.omega.-3)-dopamin- e;
respectively) were found to inhibit peroxidative activity more
significantly than the other compounds.
Example 5
Isolation and Lysis of Pig Pancreatic Mitochondria:
[0196] All procedures were carried out at 4.degree. C. Mitochondria
were obtained from a liquid nitrogen powder of one pig pancreas
(.about.8 g), which was resuspended in 15 ml of buffer A containing
following protease inhibitors: 1 .mu.g/ml aprotinin, 1 .mu.g/ml
pepstatin A, 1 .mu.g/ml chymostatin A, 2 .mu.g/ml leupeptin, 2
.mu.M benzamide hydrochloride, and 1 .mu.M phenylmethylsulfonyl
fluoride. Tissue was broken with a Potter homogenizer and the
homogenate was centrifuged for 10 min at 800 g to remove cell
debris. The supernatant was centrifuged at 10,000 g from 15 min to
recover mitochondria. The mitochondrial pellet was washed twice
with a hand held Dounce homogenizer and mitochondria were stored
frozen. Mitochondria were lysed by slowly adding 1 ml of M-Per
(Pierce) containing 1/10 vol 5M NaCl and 1/10 vol Buffer A. The
mitochondrial lysate was subsequently clarified by centrifugation
for 10 min at 2000 g.
Example 6
Peroxidase Activity
[0197] Peroxidase activity was monitored using luminol-dependent
chemiluminescence. Luminol stock solution (32.2 mM) was prepared by
dissolving 5.7 mg/mL of luminol in 1 N NaOH. A working stock was
prepared daily by adding 0.1 mL of luminol stock to 19.9 mL of 1 M
sodium phosphate buffer, pH 7.0; 0.1 mL of working solution was
added to 0.5-mL aliquots of the sample to be assayed for a final
concentration of 23 .mu.M luminol. H.sub.2O.sub.2 (100 mM in 0.2 M
sodium phosphate, pH 7) was prepared from concentrated (30%)
H.sub.2O.sub.2 daily; 100 .mu.L was added to 0.5-mL aliquots to be
assayed for a final concentration of approximately 1 mM. For
peroxidase assays the luminol and H.sub.2O.sub.2 were added to
samples simultaneously and the chemiluminescence was measured in an
automated luminometer (Stratagene). The maximum chemiluminescence
reading (mV) during a 30-s period was recorded. Changes in
peroxidase activity could easily be detected using a luminometer,
but it can have the drawback of being susceptible of trace amount
of contaminating transition metals. Thus a method was developed to
measure the peroxidative activity of MnSOD Exon3-deleted isoform
directly on nitrocellulose filters.
[0198] Recombinant and mammalian detergent extracts of native
mitochondrial proteins were separated in a 7% PAGE gel without SDS
containing 375 mM Tris pH 8.2 at 100 volts for 1.5 hr. After
electrotransfer of proteins to nitrocellulose using transfer buffer
without SDS, peroxidative activity was measured directly upon the
filter by their incubation with 0.5 ml of the Femtomole luminol
solution (Pierce) containing 1 mM H.sub.2O.sub.2 and exposing to
x-ray film or other means of detecting chemiluminescence.
[0199] The peroxidative activity of MnSOD Exon3-deleted isoform was
also measured using a standard dot blot assay. MnSOD proteins were
immunoprecipitated from either a bacterial or mammalian protein
extract solubilized by 1 ml of B-Per containing 1/10 vol 5 M NaCl
and 1/10 vol Buffer A using 5 .mu.g of the polyclonal rabbit
anti-MnSOD antibody (UpState Biotechnology). Immune complexes were
allowed to form overnight at 4.degree. C. Twenty microliters of
Protein G Plus-Agarose conjugate (Santa Cruz Biotechnology) was
added, and after a 1-h incubation period, the complex was washed
four times with RIPA buffer (PBS with 1% Nonidet P-40/0.5% sodium
deoxycholate/0.1% SDS). The solution was mixed with 100 .mu.l of
TBS, pipeted onto HybondC nitrocellulose in a dot blot manifold,
vacuumed dried, and 0.5 ml of 0.1 M citrate buffer pH 3.5 was added
to the agarose beads to release and transfer the MnSOD
Exon3-deleted isoform onto nitrocellulose. The filter is washed
with TBS and analyzed by chemiluminescence as described above.
Example 7
Induction of MnSOD alternative splicing and MnSOD Exon3-deleted
isoform synthesis by Anti-Fas antibody.
[0200] Induction of MnSOD Exon3-Deleted isoform In Cells By the
Addition of Anti FAS CH-11 Antibody:
[0201] The human prostatic cell line, PC3, was grown in complete
MCDB 131 medium containing 10% FBS. Cells were passed 24 hours
prior to treatment. Two hours prior to treatment, the medium was
replaced with MCDB medium containing 1% FBS. 1.times.10 (5) PC-3
cells treated with anti-Fas mAb (IPO-4, 1 mg/ml) for different
times, and cellular SDS extracts were prepared as described herein.
The anti-Fas mAb induced expression of isoMnSOD.
Example 8
Antisense Targeting of isoMnSOD Induction During Fas-Induced
Apoptosis in Prostate Cancer PC3 Cells Blocks Caspase 9
Activation
[0202] The Fas (CD95) receptor is a type I transmembrane protein
that mediates cell death and can be engaged by the agonist anti-Fas
antibody, CH-11. PC-3 cells are deficient for the recessive
oncogene, p53, and thus, Fas-mediated apoptosis in PC-3 cells is
p53-independent. Fas-mediated apoptosis in PC3 cells is not robust,
but does exhibit release of mitochondrial pro-apoptotic factors
within two hours after Fas-ligation, such as cytochrome c, which is
necessary for protease-activation cleavage of caspase-9 and nuclear
DNA degradation (Gewies, A. et al., (2000). Cancer Res 60,
2163-8.).
[0203] We found that isoMnSOD, as well as normal MnSOD, were
induced within one hour after ligation and engagement of the Fas
receptor by the agonist Fas antibody, mAb CH-- Il in PC-3 (data not
shown). Induction of isoMnSOD expression exhibits a similar time
course as the release of pro-apoptotic cytochrome c from
mitochondria and activation of caspase 9 (Gewies, A. et al.,
(2000). Cancer Res 60, 2163-8.). In order to determine if isoMnSOD
is necessary for caspase 9 activation, we targeted the isoMnSOD
mRNA at the MnSOD E2/E4 splice junction using antisense and as a
control, reverse sense MnSOD oligodeoxynucleotide (ODN). There was
a loss of both isoMnSOD and caspase 9 activation in cells treated
with MnSOD E2/E4 antisense ODN.
Example 9
Doxorubicin Induces isoMnSOD Expression
[0204] Mitochondrial oxidative stress has been implicated as
causative for the energy-loss and increased level of premature cell
death (e.g. apoptosis and/or necrosis) that is observed in
Doxorubicin(DOX)-induced cardiac dysfunction. Although the
antineoplastic action of DOX in dividing cells is due to its
inhibition of Topoisomerase II, Doxorubicin accumulates within the
mitochondria of differentiated cardiomyocytes and leads to
excessive production of the superoxide free radicals or anions.
Doxorubicin itself is not pro-oxidant, but requires
redox-activation of its quinone moiety to a semiquinome. The
semiquinome of DOX reacts with oxygen, which in turn generates
superoxide radicals, while at the same time, the DOX quinome moiety
is regenerated. This process is called redox cycling and leads to
toxic levels of superoxide free radicals and overwhelms the
capacity of the endogenous MnSOD antioxidant defense system.
Consistent with this, a transgenic animal overexpressing MnSOD can
suppress Doxorubicin-induced cardiotoxicity.
[0205] Preparation of Adult Cardiomyocyte Explants.
[0206] Adult cardiomyocytes were isolated and purified following
the combination of perfusion techniques (Borg et al. Recognition of
extracellular matrix components by neonatal and adult cardiac
myocytes. Dev Biol. 1984;104:86-96.) and an attachment procedure
(Bugaisky L B, Zak R. Differentiation of adult rat cardiac myocytes
in cell culture. Circ Res. 1989;64:493-500.) as described by Sil et
al. (Sil et al. Myotrophin in human cardiomyopathic heart. Circ
Res. 1993;73:98-108.). Briefly, after rats (5 weeks old) were
killed by decapitation, the hearts were aseptically excised and
residual blood was removed. The heart was perfused in Joklik's
medium (containing Joklik's minimal essential medium, 25 mmol/L
glutamic acid, 30 mmol/L taurine, and 1 mmol/L adenosine) without
recirculation on a modified Langendorff apparatus for approximately
10 minutes at 37.degree. C. The perfusion was then continued for 30
minutes at the same temperature, with recirculation in Joklik's
medium containing collagenase type II (100 U/mL). After perfusion,
the ventricles were cut into small pieces and tritulated in 0.05%
Trypsin/EDTA for 10 min. Cardiac explants were cultured overnight
on laminin-coated (20 .mu.g per well) 35-mm six-well plates in
medium 199 containing 5% FBS, 5 mM creatine, 2 mM L-carnitine, and
5 mM taurine. Explants were treated with doxorubicin (Sigma)
shortly after plating for 24 hrs at 1 mM.
[0207] Cardiomyocytes only express the normal MnSOD mRNA and
protein, but incubation of adult heart explants for 3 days with 0.1
nM to 1.0 .mu.M Doxorubicin in AdvDMEM 2% FBS induced isoMnSOD
expression (FIG. 3). isoMnSOD expression was analyzed by Western
Blot. isoMnSOD expression was positively correlated with apoptotic
activation of caspase 3.
Example 10
Expression of isoMnSOD Leads to Isoprostane and/or HNE Protein
Modification
[0208] BL21 cells were transformed with pRSET-B/isoMnSOD. Proteins
were analyzed for 8-epi-PGF2.alpha.x-modified proteins using an
antibody that recognizes 8-epi-PGF2.alpha.. The expression of
isoMnSOD induced the modification of proteins by the isoprostane,
whereas the empty vector failed to modify the proteins with
8-epi-PGF2.alpha. (data not shown).
[0209] PC12 were transfected with either empty vector (pcDNA3.1) or
a vector carrying the cDNA of isoMnSOD (pcDNA3.1/isoMnSOD) with its
mitochondrial targeting signal. The DNA was transfected with
DMRIE-C (GIBCO/BRL) for 30 hours. Proteins were analyzed by Western
blot for isoprostane modification. Isoprostane protein modification
was induced when isoMnSOD was expressed, but not when only the
empty vector was used (data not shown).
[0210] The human prostate cell line, PC3, were transfected with
either empty vector or pcDNA3.1/isoMnSOD. Proteins were analyzed
for HNE modification using an antibody that recognizes HNE modified
proteins. HNE modified proteins were increased in response to the
expression of isoMnSOD as compared to the negative control (empty
vector; data not shown).
[0211] PC12 cells were transiently transfected for 30 hours with
either empty vector or a vector carrying the isoMnSOD cDNA. HNE
modification of proteins was detected via Western Blot. Proteins
were modified by HNB in response to the transfection of isoMnSOD,
but not in response to the empty vector (data not shown).
Example 11
Inhibition of Drug-Induced Toxicity
[0212] Doxorubicin is used to induce cardiomyopathy and
cardiotoxicity as described in Arola et al. (Cancer Research,
60:1789-1792). Rats, .about.300 g, are treated once with
doxorubicin by IP injection using 2.5, 5, 10, and 15 mg/kg
doxorubicin. Animals are sacrificed two days afterwards and tissue
is stored frozen at -70.degree. C. The dosage of
arachidonoyl-dopamine to treat rats is at 7.5 mg/kg and 15 mg/kg
and injected IP at the time=0, 6 hrs, and 24 hrs after doxorubicin
injection. A working solution of arachidonoyl-dopamine is prepared
by adding an equal amount (vol/vol) of the stock solution (40 mM
arachidonoyl-dopamine in ethanol) to 0.1 M KOH and then bringing
the final volume of 0.2 ml with PBS. The rats are analyzed for
changes in cardiotoxicity and physiology following treatment of
isoMnSOD inhibitor. MnSOD expression and isoMnSOD expression is
analyzed in the treated rats by Western Blot and
Immunostaining.
[0213] Mitochondrial proteins from heart, kidney, liver and
pancreas are examined by measuring the in vitro peroxidative
activity of the MnSOD Exon3-deleted isoform using the ROS-activity
blots in parallel with the combined immunoprecipitation/dot blot
analysis of the peroxidative activity. Both procedures are based
upon the chemiluminescence detection of ROS by the oxidation of
luminol. Secondly, western immunoblot analysis of mitochondrial
proteins are analyzed for both native MnSOD and MnSOD Exon3-deleted
isoform proteins, as well as the level of covalent modification of
proteins by reactive lipid peroxidation byproducts, such as HNE and
8-epi-PGF2.alpha.c. Total cellular proteins are also be examined by
western immunoblot, probing for markers for premature cell death
including activation of caspase-9 and -3 cleavage, downstream
protein targets of activated caspase proteases, such as cleavage of
PARP and degradation of cellular proteins linked to necrotic
premature cell death, including cardiac connexin-43. The inhibitor
reduces the markers of cell death as compared to a negative
control.
[0214] In order to assess heart function, a Langendorf isolated
perfused heart preparation and left ventricular developed pressure
(systolic-diastolic), the maximal rate of left ventricular pressure
development (dP/dt max) and the minimal (relaxation) rate of left
ventricular pressure development (dP/dt min), or the coronary flow
are measured. An inhibitor of drug-induced toxicity reduces the
heart toxicity.
Example 12
isoMnSOD Inhibitors Inhibit Peroxidase Activity
[0215] F10 medium was used throughout the procedure. Pancreas (8
grams) from a 3 kilo pig was minced, washed twice, and 1 g of
tissue was resuspended in 5 ml of F10 medium. An equal volume of
either 2 .mu.M arachidonoyl-dopamine, eicosapentaenoyl
(EPA)-dopamine, docosahexaenoyl (DHA)-dopamine, C 18:4-dopamine or
arachidonoyl-serotonin was added and each pancreatic explant was
cultured for 1 hr at 37.degree. C. in 5% CO.sub.2 tissue culture
incubator. Explants were stored frozen at -70.degree. C. Tissue was
homogenized in Buffer A at 4.degree. C. and mitochondria was
isolated by differential centrifugation and stored frozen at
-70.degree. C. Peroxidative assay of native mitochondrial extracts
analyzed by the ROS-activity blot. Results are shown in FIG. 1.
Docosahexaenoyl (DHA)-dopamine (lane 4), C18:4.omega.-3-dopamine
(lane 5), and arachidonoyl-serotonin (lane 6) inhibited the
mitochondiral peroxidase activity. Arachidonoyl-dopamine (lane 2),
eicosapentaenoyl (EPA)-dopamine (lane 3) did not inhibit peroxidase
activity to the same effect.
Example 13
Detection of Inhibitors Bound to isoMnSOD
[0216] Covalent modification of the MnSOD-Exon3-deleted isoform was
measured by a combined immuneprecipitation/western blot analysis.
Immunoblots for detecting AA-DA, EPA-DA, DHA-DA,
C18:4(.omega.-3)-DA adducts were performed usng anti-dopamine
rabbit polyclonal (Chemicon) and anti-serotonin goat polyclonal
antibodies (ImmunoStar), respectively. After immunoprecipitating
using an antibody against the inhibitor, isoMnSOD was detected as
described above. Arachidonoyl-dopamine (AA-DA)
docosahexaenoyl-dopamine (DHA-DA), C18:4-dopamine, and
arachidonoyl-serotonin (AA-serotonin) were found to be able to bind
to isoMnSOD (FIG. 2A and FIG. 2B).
Example 14
Identification of isoMnSOD Inhibitors Using Isoprostane Production
as a Marker
[0217] A cell is transfected with an empty vector or a vector
encoding for isoMnSOD. A test compound is added to the cell in vary
concentrations ranging from 1 nm to 1M. The levels of isoprostanes
production are measured using an enzyme immunoassay for
isoprostanes (Oxford Biomedical Research, Product No. EA 84). The
amount of free isoprostanes are compared between a cell that has
and has not been contacted with a test compound. A decrease in free
isoprostanes is found indicating that the test compound is an
isoMnSOD inhibitor.
Example 15
Identification of isoMnSOD Inhibitors Using NHE Production as a
Marker
[0218] A cell is transfected with an empty vector or a vector
encoding for isoMnSOD. A test compound is added to the cell in vary
concentrations ranging from 1 nm to 1M. The levels of HNE
production or HNE protein modification are measured using an enzyme
immunoassay or antibody for HNE. The amount of HNE or HNE protein
modifications are compared between a cell that has and has not been
contacted with a test compound. A decrease in HNE or HNE protein
modifications is found indicating that the test compound is an
isoMnSOD inhibitor.
Example 16
Characterization of Reactive Oxygen Species by isoMnSOD
[0219] The activity of isoMnSOD was analyzed using recombinant
produced isoMnSOD and. isoMnSOD produced in bacterial BL21 cells
was isolated and analyzed via Western Blot and also for reactive
oxygen species (ROS)-generating activity. "ROS-generating activity
gel" was measured (FIG. 5A, left panel) and western immunoblot
(FIG. 5A right panel) analyses of recombinant MnSOD E3-deleted
isoform was analyzed. The results demonstrates the congruence of
the peroxidative activity and immunoreactivity of the recombinant
MnSOD E3-deleted isoform.
[0220] Characterization of the MnSOD E3-Deleted Isoform in Pig
Pancreatic Mitochondria.
[0221] Four lanes each of 25 .mu.g of pig pancreatic mitochondrial
proteins were separated by a 7% PAGE without SDS. Measurement of
the antioxidant, native MnSOD superoxide dismutase activity was
performed in-gel (FIG. 5B, lane 1). The additional three gel-lanes
were electrotransferred to nitrocellulose and analyzed for
peroxidative analysis using the "ROS-generating activity blot"
(FIG. 5B, lane 2) and protein assignment by Western immunoblot
analysis using the anti-MnSOD (FIG. 5B, lane 3) and anti-MnSOD
E2/E4 peptide (lane 4) polyclonal antibodies. Comparing lanes 1 and
2 shows that the dismutase, antioxidant activity strictly
corresponds to native MnSOD (FIG. 5B, lane 3; upper complex),
whereas the peroxidative activity is confined to the MnSOD
E3-deleted isoform (FIG. 5, lanes 3 and 4; lower band). To further
confirm that the peroxidative activity is due to the activity of
isoMnSOD, the pig extract was preincubated with 1 .mu.g anti-MnSOD
polyclonal antibody prior to native gel electrophoresis (FIG. 5C,
right lane). The antibody shifted the ROS-generating band produced
by isoMnSOD as compared to the control (FIG. 5C, left lane), which
indicates that the ROS-generating activity is due to the presence
and activity of isoMnSOD. Thus, the results demonstrated that
isoMnSOD is able to generate reactive oxygen species, which is in
contrast to MnSOD, which has antioxidant activity.
Example 17
Reporter Gene System to Analyze Compounds for Toxicity
[0222] The test reporter system utilizes a recombinant DNA
construct that contains an ATG start codon in MnSOD Exon 2, an in
frame fusion of a genomic fragment spanning MnSOD Exon 2 to Exon 4,
but which also harbors a mutant MnSOD Exon 3, (i.e. frameshift
mutation), to a B-galactosidase gene, or Green Fluorescence Protein
("altMnSODreport"). However, alternative splicing and skipping of
MnSOD (coding) Exon 3 will result in a fusion protein comprising a
short stretch of MnSOD E2 and E4 coding sequences N-terminal to a
reporter protein; the MnSODE2E4 amino acid sequence is 20 amino
acids and should not interfere with the expression of the reporter.
Normal splicing of the MnSOD transcript, with retention of MnSOD
E3, generates a mRNA that contains a stop codon in E3 sequences
upstream of the reporter and will therefore prematurely terminate
translation, and fail to generate a fusion protein a signal, and
result in no reporter signal.
[0223] The genomic fragments are isolated using PCR. The source of
the MnSOD genomic DNA is human BAC RP11-280121 or mouse BAC RP23
64P12. The genomic structure of MnSOD, nucleic acid sequence of
isoMnSOD, and the amino acid sequence of isoMnSOD are described in
U.S. Pat. No. 6,737,506, which is herein incorporated by reference
in its entirety.
[0224] The reporter is tested in prostate cancer cells, PC3, that
are treated with Fas CH-11 mAb because Fas induces dysregulation of
the normal MnSOD splicing pathway and production of isoMnSOD and
this model system is clinically relevant. PC3 cells are transiently
transfected with the reporter construct. Greater than 80% of cells
are transfected using 5 .mu.g of recombinant DNA complexed to
DMRIE-C lipofectant (BRL) and added to trypsinized cells. Once
cells have reattached overnight, transfected cells are treated with
Fas CH-11 mAb. Using Green Fluorescence Protein as the reporter,
the increase in fluorescence is monitored over real-time due to
dysregulation of the normal MnSOD splicing pathway and production
of the MnSODE2E4reporter fusion protein.
[0225] The Toxicology Screen is validated with two tests to assure
that the reporter signal is due to a fusion of MnSOD E2/E4 sequence
to the reporter and not MnSOD E2/E3/E4 sequences. First,
transfected cells are treated with antisense ODN targeted directed
to the MnSOD E2/E4 junction; sense and reverse sense ODNs serve as
controls. Inactivation of the MnSOD E2/E4/reporter mRNA ODN by
MnSOD E2/E4 antisense ODN results in the loss of a reporter signal
or loss of GFP fluorescence, while control ODNs has no effect. The
second control assay is to perform a western immunoblot analysis,
using an antibody directed against the MnSOD E2/E4 peptide
junction. In addition, the expression of the reporter gene is
examined using antibodies against the reporter protein; an antibody
against MnSOD protein (Stressgene) will not detect the short MnSOD
E2/E4 amino acid sequence in the fusion protein. The correct fusion
protein is detected by the MnSODE2/E4 peptide antibody and
confirmed by an antibody against the reporter protein.
[0226] The endogenous expression of the MnSOD and isoMnSOD proteins
is also examined. This verifies that there is induction of isoMnSOD
upon treatment with Fas mAb, CH-11. The steady state levels of
isoMnSOD is quantitatively measured relative to the normal MnSOD
and as a control, subunit II of the Cytochrome C Oxidase, the
terminal electron acceptor in the mitochondrial electron transport
system. The induction of apoptosis is characterized using
antibodies to caspase 9 and 3.
[0227] Testing of Drugs for Toxicity
[0228] Drugs are tested alone or in combination in cancerous or
diseased cells and in healthy cells. The compounds are added to
cells comprising the reporter gene system. The induction of the
reporter is characterized in the absence and the presence of the
drug(s).
[0229] Breast cancer cell line, MDA-MB-435, Her-2/neu
over-expressing (ATCC), fibroblast cells lines NIH3T3 and B104,
which are NIH3T3 cells transfected with the rat ErbB2/Her2
oncogene, and normal primary cells (Clonetics; rat cardiomyocytes,
donated by Kathryn Maschhoff, MD PhD, Neonatology, University of
Pennsylvania) are treated with doxorubicin alone and in combination
with the rat ErbB2/Her2 17.6.4 monoclonal antibody, which
downregulates the human and rat Her2 receptor (Zhang et al.,
1999)(Oncogene), and Herceptin.TM. (Genentech). It is also
determined if the Her2 mAbs induces isoMnSOD expression in
untransfected cells and cells transfected with altMnSODreport. A
dose-response curve is established, using western immunoblot
analysis of the isoMnSOD protein and the expression of the reporter
construct. A similar analysis is performed for doxorubicin
treatment of cells. Both treatments are analyzed for their effects
on mtDNA stability by Southern hybridization analysis, induction of
lipid peroxidation by immunodetection of HNE-modified proteins, and
induction of apoptosis by activation of caspases 9 and 3.
Doxorubicin causes an increase in expression of altMnSOD
report.
[0230] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. Each reference cited
in the present application is incorporated herein by reference in
its entirety. U.S. provisional application Ser. No. 60/473,458,
filed May 28, 2003 is incorporated herein by reference in its
entirety.
* * * * *