U.S. patent application number 09/947757 was filed with the patent office on 2002-02-14 for p53 inhibitors and therapeutic use of the same.
This patent application is currently assigned to The Board of Trustees of the University of Illinois. Invention is credited to Gudkov, Andrei V., Komarov, Pavel G., Komarova, Elena A..
Application Number | 20020019425 09/947757 |
Document ID | / |
Family ID | 22375329 |
Filed Date | 2002-02-14 |
United States Patent
Application |
20020019425 |
Kind Code |
A1 |
Gudkov, Andrei V. ; et
al. |
February 14, 2002 |
p53 Inhibitors and therapeutic use of the same
Abstract
The therapeutic use of temporary p53 inhibitors in the treatment
of p53-mediated diseases, conditions, and injuries is
disclosed.
Inventors: |
Gudkov, Andrei V.; (Glencoe,
IL) ; Komarov, Pavel G.; (Oak Park, IL) ;
Komarova, Elena A.; (Oak Park, IL) |
Correspondence
Address: |
MARSHALL, O'TOOLE, GERSTEIN, MURRAY & BORUN
6300 SEARS TOWER
233 SOUTH WACKER DRIVE
CHICAGO
IL
60606-6402
US
|
Assignee: |
The Board of Trustees of the
University of Illinois
|
Family ID: |
22375329 |
Appl. No.: |
09/947757 |
Filed: |
September 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09947757 |
Sep 6, 2001 |
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09493527 |
Jan 28, 2000 |
|
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60117881 |
Jan 29, 1999 |
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Current U.S.
Class: |
514/366 ;
514/375; 514/386; 514/393 |
Current CPC
Class: |
A61P 25/00 20180101;
A61K 45/06 20130101; A61K 31/00 20130101; A61P 17/02 20180101; A61P
35/00 20180101; A61K 31/427 20130101; A61P 43/00 20180101; A61K
31/428 20130101; A61K 31/424 20130101; A61K 31/429 20130101; A61K
31/4188 20130101; A61P 29/00 20180101 |
Class at
Publication: |
514/366 ;
514/375; 514/393; 514/386 |
International
Class: |
A61K 031/429; A61K
031/424; A61K 031/4188 |
Claims
What is claimed is:
1. A method of treating a disease or condition wherein inhibition
of p53 activity provides a benefit comprising administering a
therapeutically effective amount of a temporary p53 inhibitor to an
individual suffering from the disease or condition.
2. The method of claim 1 wherein the disease or condition comprises
a p53-deficient cancerous tumor.
3. The method of claim 1 wherein the disease or condition comprises
hyperthermia.
4. The method of claim 1 wherein the disease or condition comprises
hypoxia, a burn, a trauma to the central nervous system, a seizure,
or an acute inflammation.
5. The method of claim 1 wherein the disease or condition comprises
senescence of fibroblasts.
6. The method of claim 1 wherein the temporary p53 inhibitor
comprises a compound having the structural formula 11and mixtures
thereof, wherein X is O, S, or NH, m is 0 or 1, n is 1 to 4,
R.sup.1 and R.sup.2, independently, are selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
alkaryl, haloalkyl, haloaryl, a heterocyclic, heteroaryl,
heteroaralkyl, alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl,
aralkoxyalkyl, halo, (alkylthio)alkyl, (arylthio)alkyl, and
(aralkylthio)alkyl, or R.sup.1 and R.sup.2 are taken together to
form an aliphatic or aromatic, 5- to 8-membered ring, either
carbocyclic or heterocyclic; R.sup.3 is selected from the group
consisting of hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, aryl,
aralkyl, haloaryl, heteroaralkyl, a heterocycle, alkoxy, aryloxy,
halo, NR.sup.4R.sup.5, NHSO.sub.2NR.sup.4R.sup.5,
NHSO.sub.2R.sup.4, and SO.sub.2NR.sup.4R.sup.5- ; and R.sup.4 and
R.sup.5, independently, are selected from the group consisting of
hydrogen, alkyl, aryl, heteroaryl, and a heterocycle, or R .sup.4
and R.sup.5 are taken together to form an aliphatic or aromatic, 5-
to 8-membered ring, either carbocyclic or heterocyclic; and
pharmaceutically acceptable salts and hydrates thereof.
7. The method of claim 6 wherein the R.sup.1 through R.sup.5
groups, independently, are optionally substituted with one or more
substituents selected from the group consisting of alkyl, aryl, OH,
NR.sup.4R.sup.5, CN, C(=O)NR.sup.4R.sup.5, SR.sup.4,
SO.sub.2R.sup.4, CO.sub.2R.sup.6, OC(=O)R.sup.6, OR.sup.6,
CF.sub.3, halo, and NO.sub.2 wherein R.sup.6 is hydrogen or
alkyl.
8. The method of claim 6 wherein X is S or NH; m and n each are 1;
R.sup.1 and R.sup.2, independently, are selected from the group
consisting of hydrogen, alkyl, aryl, aralkyl, alkaryl, haloalkyl,
and haloaryl, or are taken together to form a 5- or 6-membered,
carbocyclic or heterocyclic ring; and R.sup.3 is selected from the
group consisting of alkyl, haloalkyl, aryl, alkaryl, aralkyl,
haloaryl, and a heterocycle.
9. The method of claim 6 wherein X is S; m and n each are 1;
R.sup.1 and R.sup.2 are taken together to form a 5- or 6-membered
aliphatic carbocyclic ring; and R.sup.3 is selected from the group
consisting of alkyl, haloaryl, aryl, alkaryl, aralkyl, and a
heterocycle.
10. The method of claim 6 wherein the p53 inhibitor has the
structure 12
11. The method of claim 10 wherein R.sup.1 and R.sup.2,
independently, are selected from the group consisting of hydrogen,
alkyl, aryl, haloalkyl, haloaryl, aralkyl, and alkaryl, or R.sup.1
and R.sup.2 are taken together to form a 5- or 6-membered ring,
carbocyclic or heterocyclic; and R.sup.3 is selected from the group
consisting of alkyl, haloalkyl, aryl, alkaryl, aralkyl, and a
heterocycle.
12. The method of claim 11 wherein R.sup.3 is aryl, optionally
substituted with one to three substituents selected from the group
consisting of halo, CF.sub.3, phenyl, alkyl, nitro, and 13
13. The method of claim 6 wherein the p53 inhibitor has the
structure 14
14. The method of claim 13 wherein R.sup.1 and R.sup.2,
independently, are selected from the group consisting of hydrogen,
alkyl, aryl, haloalkyl, haloaryl, aralkyl, and alkaryl, or R.sup.1
and R.sup.2 are taken together to form a 5- or 6-membered ring,
carbocyclic or heterocyclic; and R.sup.3 is selected from the group
consisting of alkyl, haloalkyl, aryl, alkaryl, aralkyl, and a
heterocycle.
15. The method of claim 14 wherein R.sup.1 and R.sup.2,
independently, are selected from the group consisting of hydrogen,
alkyl, haloalkyl, haloaryl, and aryl, or R.sup.1 and R.sup.2 are
taken together to form a 5- or 6-membered carbocyclic ring; and
R.sup.3 is selected from the group consisting of aryl, haloalkyl,
and alkaryl.
16. The method of claim 15 wherein R.sup.3 is aryl, optionally
substituted with one to three substituents selected from the group
consisting of halo, alkyl, CF.sub.3, phenyl, nitro, 15
17. The method of claim 13 wherein R.sup.3 is 16wherein w is 0
through 5, and R.sup.10 is selected from the group consisting of
alkoxy, CF.sub.3, alkylthio, alkyl, aralkyl, and aryl.
18. The method of claim 6 wherein the p53 inhibitor has the
structure 17wherein R.sup.9 is alkyl, aryl, or halo.
19. The compound of claim 18 wherein R.sup.9 is methyl, phenyl, or
iodo.
20. The method of claim 6 wherein the p53 inhibitor has the
structure 18wherein R.sup.3 is selected from the group consisting
of phenyl, 4-chlorophenyl, 4-nitrophenyl, 3-nitrophenyl,
4-methylphenyl, 4-phenylphenyl, and 4-bromophenyl; R.sup.6 and
R.sup.7, independently, are hydrogen or alkyl; and R.sup.8 is
CO.sub.2R.sup.6 or hydrogen.
21. The method of claim 1 wherein the p53 inhibitor comprises
2-[2-imino-4,5,6,7-tetrahydro-1,
3-benzothiazol-3(2H)-yl]-1-(4-methylphen- yl)-1-ethanone;
2-(4-methylphenyl)-5,6,7,8-tetrahydrobenzo[d]-imidazo[2,1--
b]thiazole;
2-[2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3(2H)-yl]-1-(4--
iodophenyl)-1-ethanone;
2-[2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3(2-
H)-yl]-1-(biphenyl)-1-ethanone;
2-phenyl-5,6,7,8-tetrahydrobenzo[d]imidazo- [2,1-b]-thiazole;
3-methyl-6-phenylimidazo[2,1-b]thiazole;
2,3-dimethyl-6-phenylimidazo[2,1-b]thiazole;
2-(4-trifluoromethylphenyl)--
5,6,7,8-tetrahydrobenzo-[d]imidazo[2,1-b]thiazole;
2-(4-flourophenyl)-5,6,-
7,8-tetrahydrobenzo[d]imidazo[2,1-b]thiazole;
2-(4-nitrophenyl)-5,6,7,8-te-
trahydrobenzo[d]imidazo[2,1-b]thiazole;
2-(3-nitrophenyl)-5,6,7,8-tetrahyd-
robenzo[d]imidazo[2,1-b]thiazole; or a mixture thereof, and
pharmaceutically acceptable salts and hydrates thereof.
22. A method of reducing or eliminating normal cell death
attributable to a treatment of a disease or condition comprising
administering a therapeutically effective amount of a temporary p53
inhibitor to a mammal to reversibly inhibit p53 activity.
23. The method of claim 22 wherein the disease or condition is a
cancer, hyperthermia, hypoxia, stroke, ischemia, acute
inflammation, a burn, or cell aging.
24. The method of claim 23 wherein the disease is a cancer
comprising a tumor that lacks functional p53.
25. A method of reducing or eliminating normal cell death
attributable to contraction of a disease comprising administering a
therapeutically effective amount of a temporary p53 inhibitor to a
mammal to reversibly inhibit p53 activity.
26. A method of reducing or eliminating damage to normal tissue
attributable to a treatment for cancer comprising administering a
therapeutically effective of a temporary p53 inhibitor to a mammal
to reversibly inhibit p53 activity.
27. The method of claim 26 wherein the cancer treatment comprises
chemotherapy.
28. The method of claim 26 wherein the cancer treatment comprises
radiation therapy.
29. A cancer treatment composition comprising: (a) a
chemotherapeutic drug; and (b) a temporary p53 inhibitor.
30. An improved method of treating cancer comprising administration
of a therapeutically effective radiation dose to a mammal to treat
a cancer, and administration of a therapeutically effective amount
of a temporary p53 inhibitor to the mammal to reversibly inhibit
p53 activity.
31. The method of claim 30 wherein the radiation dose and p53
inhibitor are administered simultaneously.
32. The method of claim 30 wherein the p53 inhibitor is
administered prior to administration of the radiation dose.
33. A method of preventing cell death attributable to a
stress-inducing event affecting the cell, said method comprising
treating the cell with therapeutically effective of a compound of a
temporary p53 inhibitor to reversibly inhibit p53 activity.
34. The method of claim 33 wherein the stress-inducing event
comprises a cancer treatment, a trauma, hyperthermia, hypoxia,
ischemia, stroke, a burn, a seizure, a tissue or organ prior to
transplanting, preparing a host for a bone marrow transplant, or
DNA damage.
35. The method of claim 33 wherein p53 activity is inhibited for a
sufficient time for the cell to recover from the stress-inducing
event.
36. A pharmaceutical composition for treating a disease comprising
(a) a drug capable of treating the disease, and (b) a temporary p53
inhibitor.
37. A pharmaceutical composition comprising (a) a temporary p53
inhibitor, and (b) a carrier.
38. A method of modulating tissue aging comprising treating the
tissue with a therapeutically effective amount of a temporary p53
inhibitor to reversibly inhibit p53 activity.
39. A method of sensitizing p53-deficient cells to a cancer therapy
comprising administering, in conjunction with the cancer therapy, a
sufficient amount of a temporary p53 inhibitor to a mammal to
destroy p53-deficient cells that survive in an absence of the p53
inhibitor.
40. An improved method of treating cancer comprising administration
of a therapeutically effective dose of a chemotherapeutic agent to
a mammal to treat a cancer, and administration of a sufficient
amount of a temporary p53 inhibitor to the mammal to reversibly
inhibit p53 activity, wherein the dose of the chemotherapeutic
agent is greater than a dose of the identical chemotherapeutic
agent required to treat the cancer in the absence of administration
of the p53 inhibitor.
41. The method of claim 40 wherein the mammal is free of a cancer
induced by temporary p53 suppression.
42. A method of reducing or eliminating p53-mediated side effects
associated with a cancer therapy comprising administering a
therapeutically effective dose of a temporary p53 inhibitor to a
mammal in conjunction with the cancer therapy.
43. The method of claim 42 wherein the cancer therapy comprises
radiation therapy.
44. The method of claim 42 wherein the cancer therapy comprises
chemotherapy.
45. The method of claim 42 wherein the p53-mediated side effect
comprises one or more of hair loss, testicular cell damage,
intestinal epithelia cell damage, lymphoid system damage, or
hemapoietic system damage.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of provisional
application Ser. No. 60/117,881, filed Jan. 29, 1999.
FIELD OF THE INVENTION
[0002] The present invention relates to temporary p53 inhibitors
and their use in therapy, for example, in cancer treatment, in
modifying tissue response to a stress, and in modifying cell aging.
More particularly, the present invention relates to compounds
having the ability to effectively and temporarily inhibit p53
activity, and that can be used therapeutically, alone or in
conjunction with a therapy, like chemotherapy or radiation therapy
during cancer treatment, to treat a disease or condition where
temporary inhibition of p53 activity provides a benefit. Examples
of compounds that temporarily inhibit p53 activity and can be used
therapeutically have the following general structural formulae (I)
through (IV): 1
[0003] and pharmaceutically acceptable salts and hydrates
thereof.
BACKGROUND OF THE INVENTION
[0004] The p53 gene is one of the most studied and well-known
genes. p53 plays a key role in cellular stress response mechanisms
by converting a variety of different stimuli, for example, DNA
damage, deregulation of transcription or replication, and oncogene
transformation, into cell growth arrest or apoptosis (T. M.
Gottlieb et al., Biochem. Biophys. Acta, 1287, p. 77 (1996)).
[0005] p53 has a short half-life, and, accordingly, is continuously
synthesized and degraded in the cell. However, when a cell is
subjected to stress, p53 is stabilized. Examples of cell stress
that induce p53 stabilization are:
[0006] a) DNA damage, such as damage caused by UV (ultraviolet)
radiation, cell mutations, chemotherapy, and radiation therapy;
[0007] b) hyperthermia; and
[0008] c) deregulation of microtubules caused by some
chemotherapeutic drugs, e.g., treatment using taxol or Vinca
alkaloids.
[0009] When activated, p53 causes cell growth arrest or a
programmed, suicidal cell death, which in turn acts as an important
control mechanism for genomic stability. In particular, p53
controls genomic stability by eliminating genetically damaged cells
from the cell population, and one of its major functions is to
prevent tumor formation.
[0010] p53 is inactivated in a majority of human cancers (A. J.
Levine et al., Br. J. Cancer, 69, p. 409 (1994) and A. M. Thompson
et al., Br. J. Surg., 85, p. 1460 (1998)). When p53 is inactivated,
abnormal tumor cells are not eliminated from the cell population,
and are able to proliferate. For example, it has been observed that
p53-deficient mice almost universally contract cancer because such
mice lack a gene capable of maintaining genomic stability (L. A.
Donehower et al., Nature, 356, p. 215 (1990) and T. Jacks et al.,
Curr. Biol, 4, p. 1 (1994)). A loss or inactivation of p53,
therefore, is associated with a high rate of tumor progression and
a resistance to cancer therapy.
[0011] p53 also imparts a high sensitivity to several types of
normal tissue subjected to genotoxic stress. Specifically,
radiation therapy and chemotherapy exhibit severe side effects,
such as severe damage to the lymphoid and hematopoietic system and
intestinal epithelia, which limit the effectiveness of these
therapies. Other side effects, like hair loss, also are p53
mediated and further detract from cancer therapies. These side
effects are caused by p53-mediated apoptosis, which maps tissues
suffering from side effects of cancer therapies. Therefore, to
eliminate or reduce adverse side effects associated with cancer
treatment, it would be beneficial to inhibit p53 activity in normal
tissue during treatment of p53-deficient tumors, and thereby
protect normal tissue.
[0012] However, loss of p53 activity in tumors is associated with
faster tumor progression and resistance to cancer treatment.
Therefore, conventional theories dictate that suppression of p53
would lead to disease progression and protection of the tumor from
a cancer therapy. Consequently, prior investigators attempted to
restore or imitate the function of p53 in the prevention or
treatment of a cancer.
[0013] Inactivation of p53 has been considered an undesirable and
unwanted event, and considerable effort has been expended to
facilitate cancer treatment by restoring p53 function. However, p53
restoration or imitation causes the above-described problems with
respect to damaging normal tissue cells during chemotherapy or
radiation therapy. These normal cells are subjected to stress
during cancer therapy, which leads the p53 in the cell to cause a
programmed death. The cancer treatment then kills both the tumor
cells and the normal cells. A discussion with respect to
suppression of p53 in various therapies is set forth in the
publication, E. A. Komarova and A. V. Gudkov, "Could p53 be a
target for therapeutic suppression?," Seminars in Cancer Biology,
Vol. 8(5), pages 389-400 (1998), incorporated herein by
reference.
[0014] In summary, p53 has a dual role in cancer therapy. On one
hand, p53 acts as a tumor suppressor by mediating apoptosis and
growth arrest in response to a variety of stresses and controlling
cellular senescence. On the other hand, p53 is responsible for
severe damage to normal tissues during cancer therapies. As
disclosed herein, the damage caused by p53 to normal tissue made
p53 a potential target for therapeutic suppression. In addition,
because more than 50% of human tumors lack functional p53,
suppression of p53 would not affect the efficacy of a treatment for
such tumors, and would protect normal p53-containing tissues.
[0015] The adverse effects of p53 activity on an organism are not
limited to cancer therapies. p53 is activated as a consequence of a
variety of stresses associated with injuries (e.g., burns)
naturally occurring diseases (e.g., hyperthermia associated with
fever, and conditions of local hypoxia associated with a blocked
blood supply, stroke, and ischemia) and cell aging (e.g.,
senescence of fibroblasts), as well as a cancer therapy. Temporary
p53 inhibition, therefore, also can be therapeutically effective
in: (a) reducing or eliminating p53-dependent neuronal death in the
central nervous system, i.e., brain and spinal cord injury, (b) the
preservation of tissues and organs prior to transplanting, (c)
preparation of a host for a bone marrow transplant, and (d)
reducing or eliminating neuronal damage during seizures, for
example.
[0016] Activated p53 induces growth arrest, which often is
irreversible, or apoptosis, thus mediating damage of normal tissues
in response to the applied stress. Such damage could be reduced if
p53 activity is temporarily suppressed shortly before, during, or
shortly after, a p53-activating event. These and other
p53-dependent diseases and conditions, therefore, provide an
additional area for the therapeutic administration of temporary p53
inhibitors. p53 also plays a role in cell aging, and, accordingly,
aging of an organism. In particular, morphological and
physiological alterations of normal tissues associated with aging
may be related to p53 activity. Senescent cells that accumulate in
tissues over time are known to maintain very high levels of
p53-dependent transcription. p53-dependent secretion of growth
inhibitors by senescent cells accumulate in aging tissue. This
accumulation can affect proliferating cells and lead to a gradual
decrease in overall proliferative capacity of tissues associated
with age. Suppression of p53 activity, therefore, is envisioned as
a method of suppressing tissue aging.
[0017] However, there are several important objectives that should
be satisfied before a therapy involving suppression of p53 is
implemented, for example:
[0018] (i) providing a p53 inhibitor that is sufficiently
efficacious in vivo for practical administration as a therapeutic
drug (i.e., inhibits p53 activity in a micromolar (.mu.m) range of
concentrations);
[0019] (ii) providing a p53 inhibitor that has a sufficiently low
toxicity for use in therapy, and also does not cause undesirable
side effects at concentrations sufficient to inhibit p53
activity;
[0020] (iii) exhibiting a p53 inhibition that is reversible because
long-term p53 inactivation can significantly increase the risk of
cancer;
[0021] (iv) during temporary p53 inhibition, the cells should
recover from the applied stress and the p53-activating signal
should be eliminated or reduced, otherwise restoration of p 53
activity while the p53-activating signal is active could result in
cell damage; and
[0022] (v) the p53 suppression therapy is not associated with a
dramatic increase in the frequency of cancer development, i.e., the
therapeutic inhibitors target p53-mediated control of cellular
response to stress, but do not affect p53-mediated control of
oncogene transformation.
[0023] Until the present invention, p53 inhibitors useful in
therapeutic applications have not been disclosed. A potential
therapeutic inhibitor of p53 is a compound that acts at any stage
of the p53 signaling pathway, and leads to functional inactivation
of a p53-mediated response (i.e., blocking of p53-dependent growth
arrest, apoptosis, or both). Prior investigators did not consider
therapeutic p53 inhibitors because therapeutic p53 suppression was
considered a disadvantage leading to the onset and proliferation of
cancerous tumors. The present invention, therefore, is directed to
the therapeutic and temporary inhibition of p53 activity, and to
compounds capable of such inhibition.
SUMMARY OF THE INVENTION
[0024] The present invention is directed to the inhibition of p53
activity in therapeutic applications. The present invention also is
directed to compounds that effectively and temporarily inhibit p53
activity, and to the therapeutic use of such temporary
p53-inhibiting compounds.
[0025] Therefore, one aspect of the present invention is to provide
p53 inhibitors that reversibly inhibit p53 activity and can be used
therapeutically, for example, a compound having the general
structural formulae (I) through (IV): 2
[0026] wherein X is O, S, or NH,
[0027] m is 0 or 1,
[0028] n is 1 to 4,
[0029] R.sup.1 and R.sup.2, independently, are selected from the
group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,
aralkyl, alkaryl, a heterocycle, heteroaryl, heteroaralkyl,
haloalkyl, haloaryl, alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl,
aral-koxyalkyl, halo, (alkylthio)alkyl, (arylthio)alkyl, and
(aralkylthio)alkyl,
[0030] or R.sup.1 and R.sup.2 are taken together to form an
aliphatic or aromatic, 5 to 8-membered ring, either carbocyclic or
heterocyclic;
[0031] R.sup.3 is selected from the group consisting of hydrogen,
alkyl, haloalkyl, alkenyl, alkynyl, aryl, aralkyl, haloaryl,
heteroaralkyl, a heterocycle, alkoxy, aryloxy, halo,
NR.sup.4R.sup.5, NHSO.sub.2NR.sup.4R5, NHSO.sub.2R.sup.4, and
SO.sub.2NR.sup.4R.sup.5; and
[0032] R.sup.4 and R.sup.5, independently, are selected from the
group consisting of hydrogen, alkyl, aryl, heteroaryl, and a
heterocycle,
[0033] or R.sup.4 and R.sup.5 are taken together to form an
aliphatic or aromatic, 5- to 8-membered ring, either carbocyclic or
heterocyclic; and
[0034] pharmaceutically acceptable salts and hydrates thereof.
[0035] Another aspect of the present invention is to provide a
method of reducing or eliminating death of normal cells
attributable to treatment of a disease or condition comprising
administering a therapeutically effective amount of a temporary p53
inhibitor to a mammal to reversibly inhibit p53 activity.
[0036] Yet another aspect of the present invention is to provide a
method of reducing or eliminating normal cell death attributable to
a trauma or contraction of a disease comprising administering a
therapeutically effective amount of a temporary p53 inhibitor to a
mammal to reversibly inhibit p53 activity.
[0037] Another aspect of the present invention is to provide a
method of reducing or eliminating damage to normal tissue
attributable to a treatment for a p53-deficient cancer comprising
administering a therapeutically effective amount of a temporary p53
inhibitor to a mammal to reversibly inhibit p53 activity.
[0038] Still another aspect of the present invention is to provide
an improved cancer treatment composition comprising:
[0039] (a) a chemotherapeutic drug, and
[0040] (b) a temporary p53 inhibitor.
[0041] Another aspect of the present invention is to provide an
improved method of treating cancer comprising administration of a
sufficient radiation dose to a mammal to treat a cancer, and
administration of a therapeutically effective amount of a temporary
p53 inhibitor to the mammal to reversibly inhibit p53 activity.
[0042] Another aspect of the present invention is to provide a
method of preventing cell death attributable to a stress-inducing
event effecting the cell, said method comprising treating the cell
with a therapeutically effective amount of a compound capable of
reversibly inhibiting p53 activity in the cell.
[0043] Another aspect of the present invention is to provide a
pharmaceutical composition for treating a disease comprising
[0044] (a) a drug capable of treating the disease, and
[0045] (b) a temporary p53 inhibitor.
[0046] Another aspect of the present invention is to provide a
pharmaceutical composition comprising
[0047] (a) a temporary p53 inhibitor, and
[0048] (b) a carrier.
[0049] Another aspect of the present invention is to provide a
method of modulating tissue aging comprising treating the tissue
with a therapeutically effective amount of a compound capable of
reversibly inhibiting p53 activity.
[0050] Another aspect of the present invention is to provide a
method of treating a mammal subjected to a dose of radiation
comprising administering to the mammal of a therapeutically
effective amount of a compound capable of reversibly inhibiting p53
activity to protect radiated mammal.
[0051] Yet another aspect of the present invention is to provide a
method of sensitizing p53-deficient cells to a cancer therapy
comprising administering a therapeutically effective amount of a
compound capable of reversibly inhibiting p53 activity to a mammal,
in conjunction with the cancer therapy, to destroy cells that
otherwise are unaffected by the cancer therapy.
[0052] Another aspect of the present invention is to provide an
improved method of treating cancer comprising administration of a
therapeutically effective amount of a chemotherapeutic agent to a
mammal to treat a cancer, and administration of a therapeutically
effective amount of a temporary p53 inhibitor to the mammal to
reversibly inhibit p53 activity, wherein the dose of the
chemotherapeutic agent is greater than a dose of the identical
chemo-therapeutic agent required to treat the cancer in the absence
of the p53 inhibitor.
[0053] These and other aspects of the present invention will become
apparent from the following nonlimiting, detailed description of
the preferred embodiments.
BRIEF DESCRIPTION OF THE FIGURES
[0054] FIG. 1 illustrates screening of a chemical library for a p53
inhibitor;
[0055] FIGS. 2(a) and (b), respectively, show the dependence of
.beta.-galactosidase activity in UV-irradiated Con A cells at 10,
20, and 30 .mu.M and PFT-.alpha. inhibition of p53-responsive
genes;
[0056] FIG. 3 illustrates suppression of p53-dependent apoptosis by
PFT-.alpha. administration;
[0057] FIG. 4 illustrates the specificity of PFT-.alpha. for p53
wild-type cells;
[0058] FIGS. 5(a) and (b) illustrate that PFT-.alpha. delays aging
of rat embryo fibroblasts in vitro;
[0059] FIGS. 6(a)-(e) illustrate the effects of PFT-.alpha. on the
p53 pathway;
[0060] FIGS. 7(a)-(d) contain plots of live animals vs. days after
irradiation, and a plot of weight (%) vs. days after irradiation,
for mice subjected to gamma radiation, and either treated or
untreated with PFT-.alpha.;
[0061] FIG. 8 contains autoradiograms illustrating the effects of
PFT-.alpha. in blocking p53-mediated growth arrest in vivo;
[0062] FIG. 9 shows the small intestine of p53 wild-type mice 24
hours after whole-body gamma radiation;
[0063] FIG. 10 illustrates the selective toxicity of PFT-.alpha. to
p-53-deficient cells treated with taxol and AraC;
[0064] FIGS. 11(a) and (b) show the effect of PFT-.alpha., and time
of application, on the survival of C8 cells after UV radiation;
[0065] FIG. 12(a) and (b) are plots of colony number vs. radiation
dose for C8 and A4-type cells and for human diploid fibroblasts
showing the effect of PFT-.alpha.;
[0066] FIGS. 13(a) and (b) are plots of number of animals vs. days
after irradiation showing that PFT-.alpha. and PFT-.beta., treated
animals are not accompanied by accelerated cancer development;
[0067] FIG. 14 illustrates p53 suppression by PFT-.alpha. and 86B10
in ConA cells treated with doxorubicin;
[0068] FIG. 15 is a plot of tumor volume vs. days for C57BC mice
subjected to treatment with cyclophosphamide, with and without
administration of PFT-.beta.; and
[0069] FIG. 16 compares the toxicity of PFT-.alpha. to
PFT-.beta..
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0070] As previously stated, the effectiveness of chemo- and
radiation therapy has been limited by severe side effects to normal
tissue, including injuries to hematopoietic and lymphoid systems,
intestinal epithelia, and testicular cells. Because p53 is involved
in the induction of such injuries, p53 was investigated as a
potential target for therapeutic suppression to decrease damage to
normal tissue. p53 suppression therapy is especially useful in the
treatment of tumors that lack functional p53, and therefore that
cannot benefit from additional p53 suppression. p53 performs an
important function by eliminating damaged and potentially dangerous
cells by forcing the cell to give its own life for the benefit of
the entire cell society of the organism. Inhibiting p53 activity
during cancer therapy could lead to the survival of genetically
altered cells, which otherwise would be eliminated by p53-dependent
growth arrest or apoptosis. Therefore, p53 suppression has an
inherent danger that damaged cells will not self-destruct and,
therefore, can proliferate. This can increase the risk of a new
cancer induced by suppression of p53 activity.
[0071] It has been found that this inherent danger is offset by
providing a temporary, or reversible, inhibition of p53 activity,
which allows a damaged normal cell to repair itself during the
period of p53 inhibition. When the effects of the p53 inhibitor
have diminished or terminated, p53 then is available to perform its
normal function.
[0072] This mechanism is beneficial in cancer treatment, especially
during the acute phase treatment of a p53-deficient cancer, wherein
normal cells are affected during a cancer treatment, e.g., chemo-
or radiation therapy. In turn, the severe adverse side-effects
attributed to the cancer treatment are reduced or eliminated.
[0073] As also stated previously, a p53 inhibitor useful in therapy
is efficacious at a low concentration, is low in toxicity, does not
cause undesirable side effects at therapeutically effective
concentrations, exhibits reversible, i.e., temporary, p53
inhibition, inhibits p53 for a sufficient time to allow normal
cells to recover from an applied stress, and does not cause a
significant increase in cancer development.
[0074] The term "temporary" or "reversible" inhibition of p53
activity as used herein means inhibition of p53 activity shortly
after administration of a p53 inhibitor, e.g., about 5 minutes to
about one hour after administration, and continuing for about 24 to
about 96 hours after administration of the p53 inhibitor is
completed.
[0075] In identifying useful therapeutic p53 inhibitors, an
important consideration is that activation of p53 leads to
transactivation of p53-responsive genes, and in some cell types
results in apoptosis. Suppression of these effects can be used to
identify therapeutic p53 inhibitors.
[0076] In particular, p53 acts as a nuclear transcription factor
that activates or suppresses a number of p53-responsive genes
through binding with specific DNA sequences. Transcriptional
activation of p53-responsive reporter beta-galactosidase gene
(LacZ) in transgenic mice maps the tissues affected to side effects
of a cancer therapy. Cell lines expressing reporter genes (e.g.,
lacZ, luciferase, GFP, and secreted factors) under the control of
p53-responsive promoters, therefore, can be used to screen
compounds capable of either activating or suppressing p53
transcriptional regulation.
[0077] Specifically, a p53 wild-type Balb 3T3 cell line ConA that
contains LacZ gene under the control of p53-responsive elements
consisting of a p53-binding DNA consensus sequence, p53-binding
site from ribosomal protein promoter in combination with minimal
heat shock gene promoter was used. p53 activation in these cells by
gamma irradiation, UV light, or treatment with various
chemotherapeutic drugs leads to accumulation of beta-galactosidase
that can be detected easily by routine X-gal staining.
[0078] This system has been used previously to identify the
inhibition of p53 activity by sodium salicylate. Sodium salicylate,
however, is not a viable candidate as a therapeutically useful p53
inhibitor because sodium salicylate is therapeutically effective
only at high concentrations starting at 20 mM (millimolar). At this
therapeutically effective concentration, and even at one-half of
the effective concentration, sodium salicylate injections were
lethal to all treated test animals.
[0079] A screening program to detect p53 inhibitors identified the
following classes of compounds as possessing properties that make
the compounds useful in therapeutic applications. In particular,
the following classes of compounds effectively and reversibly
inhibit p53 activation. As discussed in more detail hereafter, the
compounds can be used alone, or, for example, in conjunction with
chemo-therapy or radiation therapy during cancer treatment, to
protect normal cells from p53 programmed death due to stresses
inflicted by a cancer treatment or by a disease or trauma. In
addition, during chemotherapy, both tumor and normal cells are
destroyed. Tumor cells are preferentially killed compared to normal
cells, which is the basis of a successful chemotherapy. By
administering a therapeutic p53 inhibitor, normal cells are
protected, and the dose of the chemotherapeutic agent, therefore,
can be increased to more effectively treat the cancer.
[0080] Examples of therapeutically effective, temporary p53
inhibitors have the general structural formulae (I) through (IV):
3
[0081] wherein X is O, S, or NH,
[0082] m is 0 or 1,
[0083] n is 1 to 4,
[0084] R.sup.1 and R.sup.2, independently, are selected from the
group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,
aralkyl, alkaryl, a heterocycle, heteroaryl, heteroaralkyl,
haloalkyl, haloaryl, alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl,
aral-koxyalkyl, halo, (alkylthio)alkyl, (arylthio)alkyl, and
(aralkylthio)alkyl,
[0085] or R.sup.1 and R.sup.2 are taken together to form an
aliphatic or aromatic, 5 to 8-membered ring, either carbocyclic or
heterocyclic;
[0086] R.sup.3 is selected from the group consisting of hydrogen,
alkyl, haloalkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl,
haloaryl, heteroaralkyl, a hetero-cycle, alkoxy, aryloxy, halo,
NR.sup.4R.sup.5, NHSO.sub.2NR.sup.4R.sup.5, NHSO.sub.2R.sup.4, and
SO.sub.2NR.sup.4R.sup.5- ; and
[0087] R.sup.4 and R.sup.5, independently, are selected from the
group consisting of hydrogen, alkyl, aryl, heteroaryl, and a
heterocycle,
[0088] or R.sup.4 and R.sup.5 are taken together to form an
aliphatic or aromatic, 5- to 8-membered ring, either carbocyclic or
heterocyclic; and
[0089] pharmaceutically acceptable salts and hydrates thereof.
[0090] Compounds of formulae (I) through (IV) contain R.sup.1
through R.sup.5 groups that are unsubstituted or optionally
substituted with one or more, and typically one to three,
substituents. Suitable substituents include, but are not limited
to, alkyl, aryl, OH, NR.sup.4R.sup.5, CN, C(=O)NR.sup.4R.sup.5,
SR.sup.4, SO.sub.2R.sup.4, CO.sub.2R.sup.6 (wherein R.sup.6 is
hydrogen or alkyl), OC(=O)R.sup.6, OR.sup.6, CF.sub.3, halo, and
NO.sub.2.
[0091] As used herein, the term "alkyl," alone or in combination,
is defined to include straight chain or branched chain saturated
hydrocarbon groups from C.sub.1-C.sub.8. The term "lower alkyl" is
defined herein as C.sub.1-C.sub.4. Examples of alkyl groups
include, but are not limited to, methyl, ethyl, n-propyl,
isopropyl, isobutyl, n-butyl, n-hexyl, and the like. The term
"alkyl" also includes "cycloalkyl," which is defined herein to
include cyclic hydrocarbon radicals from C.sub.3-C.sub.7. Examples
of cycloalkyl radicals include, but are not limited to,
cyclopropyl, cyclobutyl, and cyclopentyl. The terms "alkenyl" and
"alkynyl" are defined similarly as "alkyl," but contain at least
one carbon-carbon double bond or triple bond, respectively.
[0092] The term "aryl," alone or in combination, is defined herein
as a monocyclic or polycyclic aromatic group, preferably a
monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl,
that can be unsubstituted or substituted, for example, with one or
more, and in particular one to three, substituents selected from
halo, alkyl, phenyl, hydroxy, hydroxyalkyl, alkoxy, haloalkyl,
nitro, amino, acylamino, alkylthio, alkylsulfinyl, and
alkylsulfonyl. Exemplary aryl groups include phenyl, naphthyl,
tetrahydronaphthyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl,
2-methylphenyl, 4-methylphenyl, biphenyl, 4-iodophenyl,
4-methoxyphenyl, 3-trifluoromethylphenyl, 4-nitrophenyl, and the
like.
[0093] The term "haloaryl" and "haloalkyl" are defined herein as a
previously defined alkyl or aryl group wherein at least one
hydrogen atom has been replaced by a "halo" group as defined
herein.
[0094] The term "heteroaryl" is defined herein as a 5-membered or
6-membered heterocyclic aromatic group, e.g., thienyl, furyl, or
pyridyl, which optionally has a fused benzene ring, and which can
be unsubstituted or substituted, for example, with one or more, and
in particular one to three substituents, like halo, alkyl, hydroxy,
alkoxy, haloalkyl, nitro, amino, acylamino, alkylthio,
alkylsulfinyl, and alkylsulfonyl. Examples of heteroaryl groups
include, but are not limited to, thienyl, furyl, pyridyl,
benzoxazolyl, benzthiazolyl, benzisoxazolyl, oxazolyl, quinolyl,
isoquinolyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl,
pyrazinyl, pyrimidinyl, thiazolyl, thiadiazolyl, benzimidazolyl,
indolyl, benzofuryl, and benzothienyl.
[0095] The term "aralkyl" is defined herein as a previously defined
alkyl group, in which one of the hydrogen atoms is replaced by an
aryl group as defined herein, for example, a phenyl group
optionally having one or more substituents, for example, halo,
alkyl, alkoxy, hydroxy, and the like. An example of an aralkyl
group is benzyl.
[0096] The term "heteroaralkyl" is defined similarly as the term
"aralkyl," however, the hydrogen is replaced by a heteroaryl
group.
[0097] The term "alkaryl" is defined herein as a previously defined
aryl group in which one of the hydrogen atoms is replaced by an
alkyl group as defined herein, either substituted or unsubstituted.
An example of an alkaryl group is 4-methylphenyl.
[0098] The terms "alkoxyalkyl" and "aryloxyalkyl" are defined as an
alkyl group wherein a hydrogen has been replaced by an alkoxy group
or an aryloxy group, respectively. The term "aralkoxyalkyl" is
similarly defined wherein an aralkoxy group is substituted for a
hydrogen of an alkyl group. The terms "(alkylthio)alkyl,"
"(arylthio)alkyl," and "(aralkylthio)allyl" are defined similarly
as the three above groups, except a sulfur atom, rather than an
oxygen atom, is present.
[0099] The term "halogen" or "halo" is defined herein to include
fluorine, chlorine, bromine, and iodine.
[0100] The term "heterocycle" is defined as a C.sub.4 to C.sub.5,
aliphatic ring system, preferably a C.sub.5 to C.sub.6 aliphatic
ring system, containing one to three atoms selected from the group
consisting of oxygen, sulfur, and nitrogen, with the remaining
atoms being carbon. Examples of heterocycles include, but are not
limited to, tetrahydrofuran, tetrahydropyran, morpholine, dioxane,
piperidine, piperazine, pyrrolidine, and morpholine.
[0101] The terms "alkoxy" and "aryloxy" are defined as--OR, wherein
R is alkyl or aryl.
[0102] The term "hydroxy" is defined as--OH.
[0103] The term "hydroxyalkyl" is defined as a hydroxy group
appended to an alkyl group.
[0104] The term "amino" is defined as--NH.sub.2, and the term
"alkylamino" is defined as--NR.sub.2 wherein at least one R is
alkyl and the second R is alkyl or hydrogen.
[0105] The term "acylamino" is defined as RC(=O)N, wherein R is
alkyl or aryl.
[0106] The term "nitro" is defined as--NO.sub.2.
[0107] The term "alkylthio" is defined as--SR, where R is
alkyl.
[0108] The term "alkylsulfinyl" is defined as
R--SO.sub.2.sub..sup.1 , where R is alkyl.
[0109] The term "alkylsulfonyl" is defined as
R--SO.sub.3.sub..sup.1 where R is alkyl.
[0110] In preferred embodiments, X is S or NH; m is 1; n is 1 or 2;
R.sup.1 and R.sup.2, independently, are hydrogen, alkyl, aryl,
aralkyl, alkaryl, or are taken together to form a 5- or 6-membered,
carbocyclic or heterocyclic ring; and R.sup.3 is alkyl, aryl,
alkaryl, aralkyl, haloaryl, or a heterocycle, and salts and
solvates thereof.
[0111] In more preferred embodiments, the compound has a structural
formula (I) or (III); X is S; m is 1; n is 1, R.sup.1 and R.sup.2
are taken together to form a 5- or 6-membered aliphatic carbocyclic
ring; and R.sup.3 is alkyl or phenyl, preferably substituted with
halo (e.g., iodo), alkyl (e.g., methyl), or aryl (e.g.,
phenyl).
[0112] The therapeutic p53 inhibitors include all possible
geometric isomers of compounds of structural formulae (I) through
(IV). The p53 inhibitors also include all possible stereoisomers of
compounds of structural formulae (II) and (IV) including not only
racemic compounds, but also the optically active isomers as well.
When a compound of structural formula (II) or (IV) is desired as a
single enantiomer, it can be obtained either by resolution of the
final product or by stereospecific synthesis from either
isomerically pure starting material or any convenient intermediate.
Resolution of the final product, an intermediate, or a starting
material can be achieved by any suitable method known in the art.
Additionally, in situations where tautomers of the compounds of
structural formulae (I) through (IV) are possible, the present
invention is intended to include all tautomeric forms of the
compounds. For example, a compound of structural formula (I),
wherein m and n each are one, can exist in the following tautomeric
form 4
[0113] Compounds of structural formulae (I) through (IV) which
contain acidic moieties can form pharmaceutically acceptable salts
with suitable cations. Suitable pharmaceutically acceptable cations
include alkali metal (e.g., sodium or potassium) and alkaline earth
metal (e.g., calcium or magnesium) cations. The pharmaceutically
acceptable salts of the compounds of structural formulae (I)
through (IV), which contain a basic center, are acid addition salts
formed with pharmaceutically acceptable acids. Examples include the
hydrochloride, hydrobromide, sulfate or bi-sulfate, phosphate or
hydrogen phosphate, acetate, benzoate, succinate, fumarate,
maleate, lactate, citrate, tartrate, gluconate, methanesulfonate,
benzenesulphonate, and p-toluenesulphonate salts. In light of the
foregoing, any reference to compounds of the present invention
appearing herein is intended to include compounds of structural
formulae (I) through (IV), as well as pharmaceutically acceptable
salts and solvates thereof.
[0114] The compounds of structural formulae (I) through (IV) can be
used to inhibit p53 in any organism that possesses the p53 gene.
Typically, reversible p53 inhibition can be performed in a mammal,
including humans. Therapeutically, a reversible p53 inhibitor, such
as a compound of structural formulae (I) through (IV), can be
administered to a mammal, in a therapeutically effective amount, to
treat any disease, condition, or injury, wherein inhibition of p53
activity provides a benefit.
[0115] As set forth below, administration of a present p53
inhibitor to a mammal has several potential benefits, including,
for example, rescuing damaged cells from death caused by cellular
stress, which occurs in cancer treatments and hyperthermia;
providing a method of treating individuals, like workers in nuclear
power plants and in radiopharmaceuticals, subjected to potentially
harmful radiation dosages; and modulating tissue aging attributed
to senescent cells.
[0116] The temporary p53 inhibitors, like compounds of structural
formulae (I) through (IV), can be therapeutically administered as
the neat chemical, but it is preferable to administer compounds of
structural formulae (I) through (IV) as a pharmaceutical
composition or formulation. Accordingly, the present invention
further provides for pharmaceutical formulations comprising, for
example, a compound of structural formulae (I) through (IV), or
pharmaceutically acceptable salts thereof, together with one or
more pharmaceutically acceptable carriers and, optionally, other
therapeutic and/or prophylactic ingredients. The carriers are
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof.
[0117] The amount of a temporary p53 inhibitor required for use in
therapy varies with the nature of the condition being treated, the
length of time p53 suppression is desired, and the age and the
condition of the patient, and is ultimately determined by the
attendant physician. In general, however, doses employed for adult
human treatment typically are in the range of .001 mg/kg to about
200 mg/kg per day. A preferred dose is about 1 .mu.g/kg to about
100 .mu.g/kg per day. The desired dose can be conveniently
administered in a single dose, or as multiple doses administered at
appropriate intervals, for example as two, three, four or more
subdoses per day. Multiple doses often are desired, or required,
because the suppression of p53 activity is temporary.
[0118] Formulations of the present invention can be administered in
a standard manner, such as orally, parenterally, sublingually,
transdermally, rectally, transmucosally, topically, via inhalation,
or via buccal administration. Parenteral administration includes,
but is not limited to, intravenous, intraarterial, intraperitoneal,
subcutaneous, intramuscular, intrathecal, and intraarticular.
[0119] For veterinary use, a p53 inhibitor, in particular a
compound of formulae (I) through (IV), or a nontoxic salt thereof,
is administered as a suitably acceptable formulation in accordance
with normal veterinary practice. The veterinarian can readily
determine the dosing regimen and route of administration that is
most appropriate for a particular animal.
[0120] A pharmaceutical composition containing a present p53
inhibitor can be in the form of tablets or lozenges formulated in
conventional manner. For example, tablets and capsules for oral
administration can contain conventional excipients such as binding
agents (for example, syrup, accacia, gelatin, sorbitol, tragacanth,
mucilage of starch or polyvinylpyrrolidone), fillers (for example,
lactose, sugar, microcrystalline cellulose, maizestarch, calcium
phosphate, or sorbitol), lubricants (for example, magnesium
stearate, stearic acid, talc, polyethylene glycol, or silica),
disintegrants (for example, potato starch or sodium starch
glycollate), or wetting agents (for example, sodium lauryl
sulfate). The tablets can be coated according to methods well known
in the art.
[0121] Alternatively, the compounds of the present invention can be
incorporated into oral liquid preparations such as aqueous or oily
suspensions, solutions, emulsions, syrups, or elixirs, for example.
Moreover, formulations containing these compounds can be presented
as a dry product for constitution with water or other suitable
vehicle before use. Such liquid preparations can contain
conventional additives, like suspending agents, such as sorbitol
syrup, methyl cellulose, glucose/sugar syrup, gelatin,
hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate
gel, and hydrogenated edible fats; emulsifying agents, such as
lecithin, sorbitan monooleate, or acacia; nonaqueous vehicles
(which can include edibIe oils), such as almond oil, fractionated
coconut oil, oily esters, propylene glycol, and ethyl alcohol; and
preservatives, such as methyl or propyl p-hydroxybenzoate and
sorbic acid.
[0122] Such preparations also can be formulated as suppositories,
e.g., containing conventional suppository bases, such as cocoa
butter or other glycerides. Compositions for inhalation typically
can be provided in the form of a solution, suspension, or emulsion
that can be administered as a dry powder or in the form of an
aerosol using a conventional propellant, such as
dichlorodifluoromethane or trichlorofluoromethane. Typical
transdermal formulations comprise conventional aqueous or
nonaqueous vehicles, such as creams, ointments, lotions, and
pastes, or are in the form of a medicated plaster, patch, or
membrane.
[0123] Additionally, compositions of the present invention can be
formulated for parenteral administration by injection or continuous
infusion. It is envisioned that injection or continuous infusion is
the preferred method of administration. Formulations for injection
can be in the form of suspensions, solutions, or emulsions in oily
or aqueous vehicles, and can contain formulation agents, such as
suspending, stabilizing, and/or dispersing agents. Alternatively,
the active ingredient can be in powder form for reconstitution with
a suitable vehicle (e.g., sterile, pyrogen-free water) before
use.
[0124] A composition in accordance with the present invention also
can be formulated as a depot preparation. Such long acting
formulations can be administered by implantation (for example,
subcutaneously or intramuscularly) or by intramuscular injection.
Accordingly, the compounds of the invention can be formulated with
suitable polymeric or hydrophobic materials (as an emulsion in an
acceptable oil, for example), ion exchange resins, or as sparingly
soluble derivatives (as a sparingly soluble salt, for example).
[0125] A temporary p53 inhibitor, like a compound of formulae (I)
through (IV), also can be used in combination with other
therapeutic agents which can be useful in the treatment of cancer
and other conditions or disease states. The invention thus
provides, in another aspect, a combination of a therapeutic,
temporary p53 inhibitor together with a second therapeutically
active agent.
[0126] A temporary p53 inhibitor, like a compound of formulae (I)
through (IV), can be used in the preparation of a medicament for
coadministration with the second therapeutically active agent in
treatment of conditions where inhibition of p53 activity is
beneficial. In addition, a temporary p53 inhibitor can be used in
the preparation of a medicament for use as adjunctive therapy with
a second therapeutically active compound to treat such conditions.
Appropriate doses of known second therapeutic agents for use in
combination with a temporary p53 inhibitor are readily appreciated
by those skilled in the art.
[0127] For example, a therapeutic, temporary p53 inhibitor can be
used in combination with a cancer therapy, such as radiotherapy or
chemotherapy. In particular, a p53 inhibitor can be used in
conjunction with chemotherapeutic drugs, such as cisplatin,
doxorubicin, Vinca alkaloids, taxol, cyclo-phosphamide,
ifosphamide, chlorambucil, busulfan, mechlorethamine, mitomycin,
dacarbazine, carboplatin, thiotepa, daunorubicin, idarubicin,
mitox-anthrone, bleomycin, esperamicin A.sub.1, dactinomycin,
plicamycin, carmustine, lomustine, tauromustine, streptozocin,
melphalan, dactinomycin, and procarbazine, for example. A
therapeutic p53 inhibitor also can be used in combination with
drugs used to treat stroke, ischemia, or blocked blood supplies; or
in combination with drugs used to treat arthritis or diseases that
cause hyperthermia.
[0128] The combination referred to above can be presented for use
in the form of a single pharmaceutical formulation, and, thus,
pharmaceutical compositions comprising a combination as defined
above together with a pharmaceutically acceptable diluent or
carrier comprise a further aspect of the invention.
[0129] The individual components of such a combination referred to
above, therefore, can be administered either sequentially or
simultaneously from the same or separate pharmaceutical
formulations. As is the case for the present therapeutic p53
inhibitors, a second therapeutic agent can be administered by any
suitable route, for example, by oral, buccal, inhalation,
sublingual, rectal, vaginal, transurethral, nasal, topical,
percutaneous (i.e., transdermal), or parenteral (including
intravenous, intramuscular, subcutaneous, and intracoronary)
administration.
[0130] In some embodiments, a temporary p53 inhibitor, such as a
compound of formulae (I) through (IV), and the second therapeutic
agent are administered by the same route, either from the same or
from different pharmaceutical compositions. However, in other
embodiments, using the same route of administration for the
therapeutic p53 inhibitor and the second therapeutic agent either
is impossible or is not preferred. Persons skilled in the art are
aware of the best modes of administration for each therapeutic
agent, either alone or in a combination.
[0131] Generally, compounds of structural formulae (I) through (IV)
can be prepared according to the following synthetic scheme
disclosed in A. Andreani et al., J. Med. Chem., 38, pp. 1090-1097
(1995), which is incorporated herein by reference. Compounds of
structural formula (I) or (II), wherein n is 1, then can be
converted into a compound of structural formula (III) or (IV). In
the scheme disclosed in the Andreani et al. publication, it is
understood in the art that protecting groups can be employed where
necessary in accordance with general principles of synthetic
chemistry. These protecting groups are removed in the final steps
of the synthesis under basic, acidic, or hydrogenolytic conditions
which are known and readily apparent to those skilled in the art.
By employing appropriate manipulation and protection of any
chemical functionalities, synthesis of compounds of structural
formulae (I) through (IV) not specifically set forth herein can be
accomplished by methods analogous to the schemes set forth below.
Unless otherwise noted, all starting materials were obtained from
commercial suppliers and used without further purification.
[0132] As disclosed in the Andreani et al. publication, the
compounds of general structural formula (I) or (II), wherein X is
S, can be prepared by reacting a 2-aminothiazole with a mole
equivalent amount of a bromoketone compound in acetone at reflux
for about 30 minutes. The reaction mixture then is cooled, and the
product is isolated as the hydrobromide salt. Other compounds of
formula (I) can be prepared identically by reacting a
2-aminoimidazole (X=NH) or a 2-aminooxazole (X=O) with a
bromoketone. The reaction to provide a compound of structural
formula (I), wherein X is S, is illustrated by the following: 5
[0133] The same reaction can be used to provide a compound of
structural formula (II), for example, by reacting
2-aminoimidazolidine with a bromoketone. Alternatively, a compound
of structural formula (I) can be converted to a compound of
structural formula (II).
[0134] A compound of structural formula (I) or (II), wherein m and
n each are 1, can be converted into a compound of structural
formula (III) or (IV), respectively. This conversion is achieved by
heating a composition of structural formula (I) or (II) in a
solvent, such as methanol, ethanol, or isopropyl alcohol, for a
sufficient time, e.g., about 1 to about 10 hours, to cyclize the
compound and yield a compound of structural formula (III) or (IV).
In some cases, a compound of structural formula (I) or (II), in
solution, slowly cyclizes to a compound of structural formula (III)
or (IV) while standing at room temperature. The preparation of a
compound of structural formulae (III) and (IV) from a compound of
structural formulae (I) and (II) is illustrated below: 6
[0135] Particular compounds of structural formulae (I) and (III)
were prepared by the following procedure: 7
[0136] A reaction mixture containing cyclohexanone (1.96 9, 20
mmol), thiourea (1.52 g, 20 mmol), N-bromosuccinimide (NBS) (3.56
g, 20 mmol), and benzoyl peroxide (100 mg) in 40 ml of benzene was
prepared, then heated at reflux overnight. The benzene then was
removed under reduced pressure. The residue was dissolved in water,
then neutralized with sodium carbonate. The resulting precipitate
was filtered, vacuumed to dryness, and recrystallized from hexane
to yield the 2-aminothiazole derivative A (1.81 g, yield 59%).
[0137] A solution of compound A (1.54 g, 10 mmol) and
para-methylphenacyl bromide (2.34 g, 11 mmol) in 50 ml of benzene
was prepared, then stirred at room temperature for 48 hours.
Product (V) (a compound of structural formula (I) where X=S and
n=1) precipitated from the reaction mixture, was filtered, and then
washed with benzene to yield 2.42 g (66% yield) of the compound of
structural formula (V). The compound of structural formula (V) was
a stable, water-soluble compound.
[0138] A solution of compound (V) (1.10 g, 3.0 mmol) in 30 ml
methanol was refluxed for 6 hours. The reaction mixture then was
cooled, mixed with water, and neutralized with sodium carbonate.
The resulting solid product was filtered from the mixture, vacuumed
to dryness, and recrystallized from ethanol to provide 0.45 g
(yield 56%) of compound (VI), i.e., a compound of structural
formula (III).
[0139] These, and other specific, nonlimiting compounds encompassed
by structural formulae (I) and (III) were synthesized and have the
following structures: 8
[0140] The compounds of structural formulae (V)-(XI) are named:
[0141] (V) 2-[2-imino-4,5,6,7-tetrahydro-1,3-
benzothiazol-3(2H)-yl]-1-(4-- methylphenyl)-1-ethanone;
[0142] (VI) 2-(4-methylphenyl)-5,6,7,8-tetra-hydrobenzo
[d]imidazo[2,1-b]thiazole;
[0143] (VII) 2-[2-imino-4,5,6,7-tetrahydro-1,
3-benzothiazol-3(2H)-yl]-1-(- 4-iodophenyl)-1-ethanone;
[0144] (VIII) 2-[2-imino-4,5,6,7-tetrahydro-1,
3-benzothiazol-3(2H)-yl]-1-- (biphenyl)-1-ethanone;
[0145] (IX) 2-phenyl-5,6,7,8-tetrahydrobenzo-
[d]imidazo[2,1-b]thiazole;
[0146] (X) 3-methyl-6-phenylimidazo [2,1-b]-thiazole; and
[0147] (XI) 2, 3-dimethyl-6-phenylimidazo [2, 1-b]thiazole;
respectively. The compound of structural formula (V) also is known
by the trivial names of pifithrin-alpha and PFT-.alpha.. The
compound of structural formula (VI) also is known by the trivial
names pifithrin-beta and PFT-.beta.. The compound of structural
formula (VII) also is known as compound 86B10.
[0148] The compounds of structural formulae (V) and (VI) are
disclosed in Balse et al., Indian J. Chem., Vol. 19B, pp. 293-295
(April, 1980). The compound of structural formula (IX) is disclosed
in Singh et al., Indian J. Chem., Vol. 14B, pp. 997-998 (Dec.,
1976) and in S. Naito et al., J. Heterocyclic Chem., 34, pp.
1763-1767 (1997). The compounds of structural formula (X) and (XI)
are disclosed in P.M. Kochergin et al., J. Gen. Chem. U.S.S.R., 26,
pp. 483-489 (1956).
[0149] The compounds of structural formulae (VII) and (VIII) were
prepared in a scheme identical to compound (V) by using the
appropriate .alpha.-bromo ketone, i.e., bromomethyl
4-(phenyl)ketone and bromomethyl 4-iodophenyl ketone, respectively.
Other compounds of structural formulae (I) and (II) can be prepared
in a similar manner using the appropriate thiazole, imidazole, or
oxazole derivative and bromo ketone.
[0150] Compounds of structural formulae (I)-(IV) also were prepared
by methods disclosed in the above-identified Balse et al. and Singh
et al. publications. Accordingly, the following compounds of
structural formulae (XII)-(XV) also can be util- ized as temporary
p53 inhibitors: 9
[0151] wherein R.sup.7 is hydrogen or alkyl, R.sup.8 is
CO.sub.2R.sup.6 or hydrogen, and R.sup.3 is selected from the group
consisting of phenyl, 4-chlorophenyl, 4-nitrophenyl, 3-nitrophenyl,
4-methylphenyl, 4-phenylphenyl, and 4-bromophenyl. The
3-nitrophenyl derivative is disclosed in WO 98/17267.
[0152] An additional temporary p53 inhibitor of structural formulae
(I)-(IV) has the following structure wherein R.sup.1 and R.sup.2
are taken together to form a 6-membered aromatic ring: 10
[0153] Additional compounds having a structural formula (III),
wherein X is S, are disclosed in JP 11-106340 and JP 7-291976,
incorporated herein by reference.
[0154] The ability of compounds of structural formulae (I) through
(IV) to inhibit p53 activity, both effectively and reversibly, and
their use as therapeutic agents was demonstrated in the following
tests and experiments.
[0155] To demonstrate the ability of a temporary p53 inhibitor,
like a compound of structural formulae (I) through (IV), to
suppress p53 activity, a p53 activator (e.g., doxorubicin or gamma
irradiation) was applied directly to ConA cells followed by X-gal
staining. Then, a p53 inhibitor was applied in a concentration of 1
.mu.M to 20 .mu.M to the test cells in the presence of the p53
activator. Cytotoxicity of the p53 inhibitors also was determined
by standard assays. An estimation of antiapoptotic activity of the
p53 inhibitor compounds was based on an ability to suppress
apoptotic cell death in standard cell systems sensitive to
p53-dependent apoptosis (i.e., mouse embryonic fibroblasts
transformed with Ela+ras, line C8, described by S. W. Lowe et al.,
Cell, 74, pp. 957-968 (1993)) . p53 dependence of the compound
activity was analyzed by testing its effect on p53-deficient cells
(i.e., radiosensitivity or drug sensitivity of p53-/- mouse
embryonic fibroblasts transformed with Ela+ras, line A4, described
by Lowe et al., 1993).
[0156] The results of these tests are illustrated in attached FIGS.
1-16. These figures, in general, are based on tests performed on
compounds of structural formulae (V) and (VI). In the following
Figures, test results illustrated for compound (V), i.e.,
PFT-.alpha., were repeated for compound (VI), i.e., PFT-.beta..
Test results using PFT-.beta., were essentially identical to test
results using PFT-.alpha..
[0157] FIG. 1 illustrates screening a chemical library for
suppression of p53-dependent transcriptional activation. In the
screening test, ConA cells (mouse Balb 3T3 cells expressing
bacterial lacZ gene under the control of p53-responsive promoter,
as described in E. A. Komarova et al., EMBO J., 16, pp. 1391-1400
(1997)), were plated in 96-well plates and treated for 24 hours
with 0.2 .mu.g/ml of doxorubicin (i.e., a p53-activating
chemotherapeutic drug, also known as adriamycin) in combination
with test compounds at concentrations of about 10 to about 20
.mu.M. DMSO (dimethyl sulfoxide) and sodium salicylate were used as
negative and positive controls, respectively (left column). Cells
were fixed and stained by a standard X-gal procedure to monitor
lacZ expression. The well containing pifithrin-alpha is identified
by the arrow in FIG. 1, and illustrates the effectiveness of
PFT-.alpha. in inhibiting p53.
[0158] PFT-.alpha. also blocked activation of p53-responsive lacZ
in ConA cells induced by ultraviolet (UV) light in a dose-dependent
manner, as illustrated in FIG. 2. In particular, FIG. 2(a) shows
that PFT-.alpha., at 10, 20, and 30 .mu.M, affects .beta.-Gal
activity in UV-irradiated (25 J/m.sup.2) ConA cells. The cells were
collected 8 hours after UV treatment, and .beta.-Gal expression in
the extracts was estimated by a standard colorimetric assay. (See,
for example, V. A. Tron et al., Am. J. Pathol., 153, p. 597
(1998).)
[0159] FIG. 2(b) shows that PFT-.alpha. inhibits UV-induced
transactivation of cyclin G, p21/waf1, mdm2, and GAPDH, which are
known p53-responsive genes. FIG. 2(b) contains Northern blots of
RNA from ConA cells as follows: u/t, untreated; PFT, incubated for
8 hours with 10 mM of PFT-.alpha.; UV, 8 hours after UV treatment
(25 J/m.sup.2); UV +PFT, a combination of PFT treatment (10 mm) and
UV treatment.
[0160] However, to be useful therapeutically, a p53 inhibitor must
possess the properties of (a) efficacy at a low concentration, (b)
low toxicity, (c) an absence of adverse side effects, (d)
reversible p53 inhibition, (e) p53 inhibition for a sufficient time
to allow cells to recover from an applied stress, and (f) not
causing a dramatic increase in cancer development.
[0161] FIG. 3 illustrates that pifithrin-alpha suppresses
p53-dependent apoptosis caused by doxorubicin. Equal numbers of
mouse embryo fibroblasts transformed with Ela+ras (line C8, highly
sensitive to p53-dependent apoptosis) were plated in the wells of
6-well plates containing 0, 0.4, and 0.8 .mu.g/ml of doxorubicin,
treated with DMSO and PFT-.alpha. (10 .mu.M) for 48 hours, fixed
with methanol, and stained with crystal violet, followed by elution
of the dye with 1% SDS. Optical density (530 .mu.M) was determined
using a BioTek EL311 microplate reader. The intensity of staining
reflects the number of surviving cells. The results show that at 10
.mu.M PFT-.alpha. inhibited apoptotic death of C8 cells induced by
doxorubicin.
[0162] Identical tests were performed to show that pifithrin-alpha
suppresses p53-dependent apoptosis caused by etoposide, taxol,
cytosine arabinoside, UV light, and gamma radiation (see FIG. 10).
The results are identical to those illustrated in FIG. 3 for
doxorubicin.
[0163] With respect to tests using gamma radiation, it surprisingly
was found that a compound of structural formula (I) or (II), e.g.,
pifithrinalpha, does not protect p53-deficient cells (A4) from
radiation, but to the contrary, at a concentration of 20 .mu.M,
potentiates radiosensitivity of p53-deficient cells. PFT-.alpha.,
therefore, demonstrates the unexpected dual benefit of potentiating
radiation with respect to p53-deficient cancer cells, while
inhibiting p53 activity in p53-containing cells, thereby protecting
such cells from the effects of radiation. FIG. 10 illustrates
selective toxicity of pifithrin-alpha to p53-deficient cells
treated with taxol and AraC (cytosine arabinoside). This data
supports the unexpected dual effect demonstrated by PFT-.alpha.
discussed above in connection with FIG. 3.
[0164] FIG. 4 illustrates that the antiapoptotic activity of
PFT-.alpha. is p53 dependent, i.e., PFT-.alpha. specifically
effects p53 wild-type cells. Sensitivity of C8 cells to UV
irradiation depends on the presence of pifithrin-alpha, while the
sensitivity of C8 having p53 inactivated by GSE56 (a dominant
negative mutant) did not depend on the presence of PFT-.alpha..
PFT-.alpha., therefore, has no effect on survival of p53-deficient
cells after genotoxic stress.
[0165] FIG. 5(a) and (b) illustrate that pifithrin-alpha delays
aging of rat embryo fibroblasts in vitro, i.e., growth stimulation
of presenescent cells by the indicated concentrations of
pifithrin-alpha during 3 days of cell growth. In FIG. 5(a), cell
growth is presented relative to the number of plated cells.
[0166] FIG. 6 shows the effects of PFT-.alpha. on the p53 pathway
and at which stage in the pathway PFT-.alpha. targets p53. FIG.
6(a) demonstrates that PFT-.alpha. inhibits apoptosis in Saos-2
cells transiently expressing p53. Cells were transfected with the
plasmid DNA expressing green fluorescent protein (GFP) with the
5.times. excess of the plasmid carrying either wild-type human p53
(middle and bottom) or with no insert (top). Transfected cells were
maintained with (bottom) or without (top and middle) PFT-.alpha..
The majority of fluorescent cells transfected with p53-expressing
plasmid undergo apoptosis 48 hours after transfection (middle).
Apoptosis was inhibited in the presence of PFT-.alpha.
(bottom).
[0167] FIG. 6(b) shows a comparison of spectra of p53 protein
variants in the lysates of UV-irradiated (25 J/m.sup.2) ConA cells
in the presence of different concentrations of PFT-.alpha. (0, 10,
20, and 30 .mu.M) using two-dimensional protein gel
electrophoresis. FIG. 6(c) shows that PFT-.alpha. partially, and in
a dose-dependent manner, inhibits p53 accumulation in ConA cells
after UV treatment (results of protein immuno-blotting).
PFT-.alpha. was added to the cells before UV treatment and total
cell lysates were prepared 18 hours later.
[0168] FIG. 6(d) illustrates that PFT-.alpha. changes the nuclear
and cytoplasmic distribution of p53. Nuclear and cytoplasmic
fractions were isolated from UV-treated CorA cells 6 hours after UV
irradiation. p53 and p21.sup.waf1 proteins were detected by
immunoblotting. The nuclear and cytoplasmic ratios of p53, but not
p21.sup.waf1, were significantly decreased in the
PFT-.alpha.-treated cells. FIG. 6(e) shows that PFT-.alpha. does
not affect DNA-binding activity of p53. In particular, results of a
gel shift assay using cell lysates from either untreated or
UV-irradiated ConA cells grown in medium containing PFT-.alpha. are
shown. The right-half of the gel shows a supershift of the
p53-binding DNA fragment by monoclonal antibody Pab421. The decline
in the amount of bound DNA is proportional to the overall decrease
in p53 content in the presence of PFT-.alpha..
[0169] The results in FIG. 6(a) suggest that PFT-.alpha. acts
downstream of p53. FIGS. 6(b)-(e) suggest that PFT-.alpha. did not
alter phosphorylation or sequence-specific DNA binding of p53 in
ConA cells after DNA-damaging treatments, as judged by protein
immunoblotting in combination with two-dimensional protein analysis
and gel shift assays. However, PFT-.alpha. slightly lowered the
levels of nuclear, but not cytoplasmic, p53 induced by UV
irradiation. In contrast, PFT-.alpha. did not affect the
nuclear-cytoplasmic ratio of the p53-inducible p21.sup.waf1
protein. These results illustrate that PFT-.alpha. can modulate the
nuclear import or export, or both, of p53, or can decrease the
stability of nuclear p53.
[0170] FIGS. 7(a)-(d) shows the in vivo effect of a single
injection of PFT-.alpha. on the sensitivity of mice to lethal doses
of radiation. In particular, FIG. 7 illustrates that
pifithrin-alpha protects mice from radiation-induced death. In this
test, two different strains of mice (C57BL and Balb(c)) were
treated with lethal and sublethal doses of whole-body gamma
radiation. A comparison was made between (i) untreated unirradiated
mice, (ii) unirradiated mice that received a single intraperitoneal
(i.p.) injection of PFT-.alpha., (iii) untreated gamma-irradiated
mice, and (iv) mice injected intraperitoneally with PFT-.alpha.
immediately before gamma irradiation. PFT-.alpha. treatment
completely rescued mice of both strains from 60% killing doses of
gamma irradiation (8 Gy for C57BL and 6 Gy for Balb/c). Significant
protection also was seen at higher doses of irradiation that were
lethal for control animals (FIG. 7(a)-(c)). PFT-.alpha.-injected
mice lost less weight than irradiated mice that were not pretreated
with the drug (FIG. 7(d)). PFT-.alpha. did not protect p53-null
mice from lethal irradiation, which confirmed that PFT-.alpha. acts
through a p53-dependent mechanism in vivo.
[0171] In the plots of FIG. 7, whole-body gamma irradiated mice (60
total) were divided into four groups. Ten mice from each group were
injected i.p. with pifithrin-alpha (2.2 mg/kg) five minutes prior
to irradiation. Ten mice of each group did not receive an injection
of PFT-.alpha.. FIG. 7 shows the survival curve for the mice in
each of the above three groups. The data in FIG. 7 shows that
temporary p53 inhibitor is an effective radioprotector and that
PFT-.alpha. has a strong rescuing effect in both mouse strains.
PFT-.alpha. injection abrogated the gradual loss of weight by
C57BL6 mice after 8 Gy of gamma irradiation (the observed increase
in the weight of the nonirradiated mice reflects the normal growth
of young 5-week-old animals). The experiments were repeated at
least three times with 10 mice per each experimental subgroup.
[0172] FIG. 8 illustrates that pifithrin-alpha is capable of
blocking p53-mediated growth arrest in vivo in the mouse (single
intraperitoneal injection, 2.2 mg/kg). Four-week-old p53-deficient
mice and p53 wild-type (wt) mice were whole body gamma irradiated
(10 Gy). Pifithrin-alpha was injected in one of the p53 wild-type
animals five minutes before irradiation. .sup.14C-thymidine (10 mCi
per animal) was injected intraperitoneally into each mouse 8 hours
after irradiation. The mice were sacrificed 24 hours after
irradiation and whole body sectioned (25 .mu.m thick) using
cryostatic microtome were prepared and exposed to X-ray film to
monitor the distribution of .sup.14C in the tissue. FIG. 8 presents
autoradiograms of representative sections. Arrows indicate
.sup.14C-thymidine incorporation in the skin and intestine. The
test showed that an injection of PFT-.alpha. inhibits apoptosis in
the skin and small intestine of gamma-irradiated mice.
[0173] FIG. 8 shows that .sup.14C labeling of skin, intestine, and
several other tissues was significantly decreased after gamma
irradiation in p53.sup.+/+ mice but not p53.sup.-/- mice,
reflecting the p53 dependence of the effect. The radiation-induced
decrease in .sup.14C-thymidine incorporation was less pronounced in
PFT-.alpha.-treated mice than in control irradiated animals,
reflecting PFT-.alpha. inhibition of p53 activity. These results
illustrate that PFT-.alpha. attenuates the p53-dependent block of
DNA replication in rapidly proliferating tissues after whole-body
gamma irradiation.
[0174] FIG. 9 contains photographs comparing tissue morphology and
apoptosis (TUNEL staining) in the epithelium of the small intestine
of C57BL6 wild-type mice (PFT-treated (+) and untreated (-)) 24
hours after 10 Gy of whole-body gamma irradiation. Areas of massive
apoptosis are indicated by the arrows. The extensive apoptosis
observed in the crypts and villi of the small intestine was
abrogated in mice treated with PFT-.alpha. before irradiation,
which correlates with the changes in thymidine incorporation
illustrated in FIG. 8.
[0175] FIG. 11 shows the dependence of C8 cell survival after UV
irradiation on the time and duration of PFT-.alpha. application.
FIG. 11 includes a comparison of anti-apoptotic effect of
PFT-.alpha. added at different time intervals to C8 cells treated
with UV radiation (25 J/m.sup.2). Ten .mu.M of PFT-.alpha. were
added to the culture media at different time intervals (FIG.
11(a)). The proportions of surviving cells were estimated using MTT
assay 48 hours after UV treatment and are shown in FIG. 11(b).
[0176] FIGS. 11(a) and (b) show that PFT-.alpha. had little to no
protective effect where administered before (up to 18 hours) and
removed immediately before UV treatment of C8 cells. However, a
short 3-hour incubation with PFT-.alpha. after UV treatment had a
pronounced protective effect, whereas a 24-hour incubation provided
maximal protection. PFT-.alpha. did not rescue UV-irradiated cells
from apoptosis if PFT-.alpha. was administered three hours after UV
irradiation. These results show that PFT-.alpha. can efficiently
inhibit p53-dependent apoptosis and that its effects are reversible
and require the presence of the temporary p53 inhibitor. Because
many cells survived a lethal dose of UV irradiation after only 3
hours of incubation with PFT-.alpha., the UV-induced apoptic death
signal is significantly reduced within several hours and completely
disappears within 24 hours of irradiation.
[0177] The plots of FIG. 12 show that PFT-.alpha. facilitates
long-term survival of p53 wild-type cells, but not p53-deficient
cells after gamma irradiation. Human diploid fibroblasts with
wild-type p53, strain WI38, and p53-deficient fibroblasts from
Li-Fraumeni syndrome patient, line 041, were treated with the
indicated doses of gamma radiation, with or without 20 .mu.M of
PFT-.mu., in the medium (FIG. 12(b)). PFT-.alpha. was removed 48
hours after irradiation and the cells were allowed to grow for an
additional three days. By that time, unirradiated cells reached
complete monolayer. Cell numbers were estimated using crystal
violet staining assay (100% corresponds to confluent cell
cultures). Dashed line indicates the number of cells plated.
p53-wild-type and p53-deficient mouse embryo fibroblasts (MEF)
transformed with Ela+ras, lines C8 and A4, respectively, were
treated with the indicated doses of gamma radiation in the presence
and in the absence of 20 .mu.M of PFT-.alpha. and replated at low
density (10.sup.3 cells per plate) 12 hours after irradiation (FIG.
12(a)). Numbers of growing colonies were calculated in two weeks
and normalized according to the unirradiated control.
[0178] FIG. 13(a) shows the effect of PFT-.alpha. on mice subjected
to whole body radiation. Both wild-type (wt) mice and p53-deficient
mice were irradiated. One group of the wild-type mice were treated
with PFT-.alpha.. FIG. 13(a) shows that the PFT-.alpha. treated
mice survived 300 days after radiation. In contrast, fifteen
untreated wild-type mice died within 100 days. The p53-deficient
mice were unaffected for about 125 days, then all expired over the
next 25-30 days. FIG. 11(b) shows similar protective effects for
PFT-.beta..
[0179] FIG. 14 contains microphotographs of ConA cells stained with
X-gal, and illustrates test results for pifithrin-alpha and 86B10
in a p53 suppression in ConA cells treated with doxorubicin. 86B10
displays a similar, but weaker, p53-inhibition effect than
PFT-.alpha..
[0180] FIG. 15 is a plot of tumor volume vs. days for C57BL mice
treated with cyclophosphamide (CTX), with and without
administration of PFT-.beta.. The mice were treated with CTX at
days 0, 1, and 2, and tumor volume was monitored for sixteen days.
Mice treated with PFT-.beta. exhibited a substantial decrease in
tumor growth.
[0181] As previously stated, the tests and experiments set forth in
FIGS. 1-15 using PFT-.alpha. were repeated using PFT-.beta.. The
results from the tests using PFT-.beta. were essentially identical
to the results from the tests using PFT-.alpha.. However,
PFT-.beta. is a preferred temporary p53 inhibitor because the
toxicity of PFT-.beta. is substantially lower than the toxicity of
PFT-.alpha., as illustrated in FIG. 16.
[0182] The above tests and experiments show that temporary p53
inhibitors, like PFT-.alpha. and PFT-.beta. act downstream of p53
activation. PFT-.alpha. and PFT-.beta. also do not effect either
post-translational modifications of p53 or the DNA binding affinity
of p53. Importantly, the tests show that PFT-.alpha. and PFT-.beta.
and other temporary p53 inhibitors, reduce nuclear accumulation of
p53, which serves as the basis for use of a temporary p53
inhibitor, such as a compound of structural formulae (I) through
(IV), in therapy.
[0183] Suppression of p53 typically results in the survival of
cells that otherwise are eliminated by p53, which can increase the
risk of new cancer development. For example, p53-deficient mice are
extremely sensitive to radiation-induced tumorigenesis. However, no
tumors or any other pathological lesions were found in a group of
30 survivors rescued from lethal gamma irradiation by PFT-.alpha.
seven months after irradiation. Thus, temporary suppression of p53
activity is different from p53 deficiency in terms of cancer
predisposition.
[0184] A temporary p53 inhibitor, like a compound of structural
formulae (I) through (IV), effectively and reversibly inhibits p53
functions. Accordingly, a temporary p53 inhibitor can be applied to
rescue cells having p53 from apoptic death or irreversible growth
arrest caused by genomic stress. Importantly, the cellular effects
of a temporary p53 inhibitor are reversible, and short-lasting,
therefore, p53 suppression requires an essentially constant
presence of the inhibitor. Furthermore, the in vivo effects of a
present inhibitor are dependent on the presence of p53.
[0185] The compounds of structural formulae (I) through (IV), and
especially PFT-.alpha. and PFT-.beta. (a) suppress p53-dependent
radiation-induced growth arrest in rapidly proliferating mouse
tissues, (b) rescue mice from lethal doses of gamma radiation using
a single i.p. injection, (c) reduce the toxicity of
chemotherapeutics, (d) do not reduce the efficacy of chemo- or
radiation therapy of p53 deficient mouse tumors, and (e) do not
result in a high incidence of tumors in irradiated animals, thereby
illustrating the therapeutic use of a temporary p53 inhibitor.
[0186] The above-described tests using PFT-.alpha. and PFT-.beta.
illustrate the therapeutic use of temporary p53 inhibitors to
reduce the side effects of radiation therapy or chemotherapy for
human cancers that have lost functional p53. Because the effects of
PFT-.alpha. and PFT-.beta. are p53 dependent, the compounds do not
affect the sensitivity of such tumors to treatment. In fact, i.p.
injection of PFT-.alpha. did not change the radiation response of
p53-deficient tumor xenografts in p53.sup.+/+ nude mice.
[0187] The temporary p53 inhibitors, like compounds of structural
formulae (I) through (IV), therefore, can be used in the following
applications, for example,
[0188] (a) a therapy using p53 suppression to reduce pathological
consequences of tissue response to variety of stresses associated
with p53 activity (e.g., anticancer radio- and chemotherapy,
ischemias, stroke, hyperthermia, etc.);
[0189] (b) application of a temporary p53 inhibitor, such as a
compound of structural formulae (I) through (IV), e.g., a compound
of structural formula (V) or (VI), as a tool for investigating p53
pathway analysis and modulation;
[0190] (c) administration of a temporary p53 inhibitor, such as a
compound of structural formulae (I) through (IV), as a drug for
rescuing cells from death after a variety of stresses;
[0191] (d) administration of a temporary p53 inhibitor, such as a
compound of structural formulae (I) through (IV), as a drug for
sensitizing p53-deficient cells to anticancer therapy;
[0192] (e) administration of a temporary p53 inhibitor, such as a
compound of structural formulae (I) through (IV), as a potential
antisenescence drug to suppress tissue aging;
[0193] (f) application of a temporary p53 inhibitor, such as a
compound of structural formulae (I) through (IV), to suppress
p53-dependent trans-activation as a tool for p53 pathway analysis
and modulation;
[0194] (g) administration of a temporary p53 inhibitor, such as a
compound of structural formulae (I) through (IV), as a radiation
protector in vivo; and
[0195] (h) administration of a temporary p53 inhibitor, such as a
compound of structural formulae (I) through (IV), in vivo, to
protect cells from a variety of stresses in different pathological
circumstances, including side effects of anticancer therapy, acute
inflammations, injuries (e.g., burns and central nervous system
injuries), cell aging, hyperthermia, seizures, transplant tissues
and organs prior to transplanting, preparation of a host for a bone
marrow transplant, and hypoxias (e.g., ischemia and stroke).
[0196] Obviously, many modifications and variations of the
invention as hereinbefore set forth can be made without departing
from the spirit and scope thereof and, therefore, only such
limitations should be imposed as are indicated by the appended
claims.
* * * * *