U.S. patent application number 09/838821 was filed with the patent office on 2003-07-31 for method for inhibiting c-jun expression using jak-3 inhibitors.
This patent application is currently assigned to Parker Hughes Institute. Invention is credited to Uckun, Fatih M..
Application Number | 20030144178 09/838821 |
Document ID | / |
Family ID | 27616057 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030144178 |
Kind Code |
A1 |
Uckun, Fatih M. |
July 31, 2003 |
Method for inhibiting C-jun expression using JAK-3 inhibitors
Abstract
The invention provides a method for inhibiting c-jun activation
in mammalian or avian cells comprising contacting the cells with a
substance that inhibits the activity of Janus family kinase 3
(JAK-3). The invention also provides a therapeutic method for
preventing or treating a pathological condition in a mammal wherein
c-jun activation is implicated and inhibition of its activation is
desired comprising administering to a mammal in need of such
therapy, an effective amount of a substance that inhibits the
activity of JAK-3. Novel compounds that are JAK-3 inhibitors, as
well as pharmaceutical compositions comprising the compounds are
also provided.
Inventors: |
Uckun, Fatih M.; (White Bear
Lake, MN) |
Correspondence
Address: |
Schwegman, Lundberg, Woessner & Kluth, P.A.
P.O. Box 2938
Minneapolis
MN
55402
US
|
Assignee: |
Parker Hughes Institute
St. Paul
MN
|
Family ID: |
27616057 |
Appl. No.: |
09/838821 |
Filed: |
April 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09838821 |
Apr 19, 2001 |
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09345815 |
Jun 30, 1999 |
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60091150 |
Jun 30, 1998 |
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Current U.S.
Class: |
514/418 ;
514/1.1 |
Current CPC
Class: |
A61K 31/517 20130101;
A61K 31/00 20130101; C07D 239/94 20130101 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 038/17 |
Claims
What is claimed is:
1. A method comprising inhibiting c-jun activation in mammalian or
avian cells by contacting the cells with a substance that inhibits
the activity of Janus family kinase 3 (JAK-3).
2. The method of claim 1 wherein the c-jun activation results from
exposure of the cells to ara-C, a topoisomerase II inhibitor,
ultraviolet radiation, an alkylating agent, or ionizing
radiation.
3. The method of claim 1 wherein the c-jun activation results from
exposure of the cells to ultraviolet radiation or ionizing
radiation.
4. The method of claim 1 wherein the contacting is performed in
vitro.
5. The method of claim 1 wherein the contacting is performed in
vitro.
6. The method of claim 2 wherein the contacting occurs prior to the
exposure.
7. The method of claim 2 wherein the contacting occurs after the
exposure.
8. The method of claim 1 wherein the substance is a protein.
9. The method of claim 1 wherein the substance is a compound of
formula I: 3wherein X is HN, R.sub.11N, S, O, CH.sub.2, or
R.sub.11CH; R.sub.11 is hydrogen, (C.sub.1-C.sub.4)alkyl, or
(C.sub.1-C.sub.4)alkanoyl; R.sub.1-R.sub.8 are each independently
hydrogen, hydroxy, mercapto, amino, nitro, (C.sub.1-C.sub.4)alkyl,
(C.sub.1-C.sub.4)alkoxy, (C.sub.1-C.sub.4)alkylthio, or halo;
wherein two adjacent groups of R.sub.1-R.sub.5 together with the
phenyl ring to which they are attached may optionally form a fused
ring, for example forming a naphthyl or a tetrahydronaphthyl ring;
and further wherein the ring formed by the two adjacent groups of
R.sub.1-R.sub.5 may optionally be substituted by 1, 2, 3, or 4
hydroxy, mercapto, amino, nitro, (C.sub.1-C.sub.4)alkyl,
(C.sub.1-C.sub.4)alkoxy, (C.sub.1-C.sub.4)alkylthio, or halo; and
R.sub.9 and R.sub.10 are each independently hydrogen,
(C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)alkoxy, halo, or
(C.sub.1-C.sub.4)alkanoyl; or R.sub.9 and R.sub.10 together are
methylenedioxy; or a pharmaceutically acceptable salt thereof.
10. The method of claim 1 wherein the substance is
4-(4'-hydroxylphenyl)-a- mino-6,7-dimethoxyquinazoline or
4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-- dimethoxyquinazoline; or
a pharmaceutically acceptable salt thereof.
11. The method of claim 1 wherein the cells are mammalian.
12. The method of claim 1 wherein the cells are human.
13. The method of claim 1 wherein the cells are avian.
14. A therapeutic method for preventing or treating a pathological
condition in a mammal wherein c-jun activation is implicated and
inhibition of its activation is desired comprising administering to
a mammal in need of such therapy, an effective amount of a
substance that inhibits the activity of JAK-3.
Description
PRIORITY OF INVENTION
[0001] This application claims priority under 35 U.S.C. .sctn.19(e)
from U.S. Provisional Application No. 60/091,150, filed Jun. 30,
1998.
BACKGROUND OF THE INVENTION
[0002] The protooncogene c-jun is the cellular counterpart of the
v-jun oncogene of avian sarcoma virus 17. C-jun expression is
activated in response to a diverse set of DNA-damaging agents
including ara-C, UV radiation, topoisomerase II inhibitors,
alkylating agents, and ionizing radiation. As an immediate early
response gene that is rapidly induced by pleiotropic signals, c-jun
may have important regulatory functions for cell cycle progression,
proliferation, and survival. See Ryder, K., Lau, L. F., and
Nathans, D. "A gene activated by growth factors is related to the
oncogene v-jun," Proc Natl Acad Sci USA. 85: 1487-1491, 1988;
Schutte, J., Viallet, J., Nau, M., Segal, S., Fedorko, J., and
Minna, J. "jun-B inhibits and c-fos stimulates the transforming and
trans-activating activities of c-jun, Cell. 59: 987-997, 1989;
Neuberg, M., Adamkiewicz, J., Hunter, J. B., and Mueller, R. "A fos
protein containing the Jun leucine zipper forms a homodimer which
binds to the AP-1 binding site," Nature. 341: 589-590, 1989;
Mitchell, P. J. and Tjian, R. "Transcriptional regulation in
mammalian cells by sequence-specific DNA binding proteins,"
Science. 245: 371-378, 1989; Bohmann, D., Bos, T. J., Admon, T.,
Nishimura, R., Vogt, P. K., and Tijian, R. "Human protooncogene
c-jun encodes a DNA binding protein with structural and functional
properties of transcription factor AP-1," Science. 238: 1386-1392,
1988; Kharbanda, S. M., Sherman, M. L., and Kufe, D. W.
"Transcriptional regulation of c-jun gene expression by
arabinofuranosylcytosine in human myeloid leukemia cells," J Clin
Invest. 86: 1517-1523, 1990; Rosette, C. and Karin, M. "Ultraviolet
light and osmotic stress: activation of the JNK cascade through
multiple growth factor and cytokine receptors," Science. 274:
1194-7, 1996; Rubin, E., Kharbanda, S., Gunji, H., and Kufe, D.
"Activation of the c-jun protooncogene in human myleloid leukemia
cells treated with etoposide," Molecular Pharmacology. 39: 697-701,
1991; Dosch, J. and Kaina, B. "Induction of c-fos, c-jun, junB and
junD mRNA and AP-1 by alkylating mutagens in cells deficient and
proficient for the DNA repair protein O6-methylguanine-DNA
methyltransferase (MGMT) and its relationship to cell death,
mutation induction and chromosomal instability," Oncogene. 13:
1927-35, 1996; Chae, H. P., Jarvis, L. J., and Uckun, F. M. "Role
of tyrosine phosphorylation in radiation-induced activation of
c-jun protooncogene in human lymphohematopoietic precursor cells,"
Cancer Res. 53: 447-51, 1993; and Karin, M., Liu, Z.-G., and Zandi,
E. "AP-1 function and regulation," Current Opinion in Cell Biology.
9: 240-246, 1997.
[0003] C-jun encodes the nuclear DNA-binding protein, JUN, that
contains a leucine-zipper region involved in homo- and
heterodimerization. JUN protein dimerizes with another JUN protein
or the product of c-fos gene and forms the activating protein-1
(AP-1) transcription factor. JUN-JUN homodimers and JUN-FOS
heterodimers preferentially bind to a specific heptameric consensus
sequence found in the promoter region of multiple growth regulatory
genes. Alterations of c-jun protooncogene expression can therefore
modulate the transcription of several growth-regulators affecting
cell proliferation and differentiation. See Ryder, K., Lau, L. F.,
and Nathans, D. "A gene activated by growth factors is related to
the oncogene v-jun," Proc Natl Acad Sci USA. 85: 1487-1491, 1988;
Neuberg, M., Adamkiewicz, J., Hunter, J. B., and Mueller, R. "A fos
protein containing the Jun leucine zipper forms a homodimer which
binds to the AP-1 binding site," Nature. 341: 589-590, 1989; Karin,
M., Liu, Z.-G., and Zandi, E. "AP-1 function and regulation,"
Current Opinion in Cell Biology. 9: 240-246, 1997; Angel, P.,
Allegretto, E. A., Okino, S. T., Hattori, K., Boyle, W. J., Hunter,
T., and Karin, M. "Oncogene jun encodes a sequence-specific
trans-activator similar to AP-1," Nature. 332: 166-170, 1988; and
Musti, A. M., Treier, M., and Bohmann, D. "Reduced
ubiquitin-dependent degradation of c-Jun after phosphorylation by
MAP kinases," Science. 275: 400-402, 1997.
[0004] C-jun plays a pivotal role in Ras-induced transformation and
has also been implicated as a regulator of apoptosis when de novo
protein synthesis is required. C-jun induction is required for
ceramide-induced apoptosis and stress-induced apoptosis after UV
exposure or other forms of DNA damage. This induction is thought to
be triggered by activation of JUN-N-terminal kinases (JNKs) (also
known as stress-activated protein kinases) which leads to enhanced
cjun transcription by phosphorylation of JUN at sites that
increases its ability to activate transcription. Ectopic expression
of a dominant-negative c-jun mutant lacking the N terminus or a
dominant-negative JNK kinase abolishes stress-induced apoptosis.
See Karin, M., Liu, Z.-G., and Zandi, E. "AP-1 function and
regulation," Current Opinion in Cell Biology. 9: 240-246, 1997;
Collotta, F., Polentarutti, N., and Mantovani, A. "Expression and
involvement of c-fos and c-jun protooncogenes in programmed cell
death induced by growth factor deprivation in lymphoid cell lines,"
J. Biol. Chem. 267: 18278-18283, 1992; Ham, J., Babij, C.,
Whitfield, J., Pfarr, C. M., Lallemand, D., Yaniv, M., and Rubin,
L. L. "A c-Jun dominant negative mutant protects sympathetic
neurons against programmed cell death," Neuron. 14: 927-939, 1995;
Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant,
S., Birrer, K M. J., Szabo, E., Zon, L. I., Kyriakis, J. M.,
Haimovitz F A., Fuks, Z., and Kolesnick, R. N. "Requirement for
ceramide-initiated SAPK/JNK signalling in stress-induced
apoptosis," Nature. 380: 75-9, 1996; Hibi, M., Lin, A., Smeal, T.,
Minden, A., and Karin, M. "Identification of an oncoprotein- and
UV-responsive protein kinase that binds and potentiates the c-Jun
activation domain," Genes Dev. 7: 2135-48, 1993; Derijard, B.,
Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and
Davis, R. J. "JNK1: a protein kinase stimulated by UV light and
Ha-Ras that binds and phosphorylates the c-Jun activation domain,"
Cell. 76: 1025-37, 1994; and Chen, Y. R., Wang, X., Templeton, D.,
Davis, R. J., and Tan, T. H. "The role of c-Jun N-terminal kinase
(JNK) in apoptosis induced by ultraviolet C and gamma radiation.
Duration of JNK activation may determine cell death and
proliferation," JBiol Chem. 271: 31929-36, 1996.
[0005] Protein tyrosine kinases (PTK) play important roles in the
initiation and maintenance of biochemical signal transduction
cascades that affect proliferation and survival of B-lineage
lymphoid cells. Oxidative stress has been shown to activate BTK,
SYK, and Src family PTK. It is known that PTK activation precedes
and mandates radiation-induced activation of c-jun protooncogene
expression in human B-lineage lymphoid cells (Chae, H. P., Jarvis,
L. J., and Uckun, F. M. Cancer Res. 53: 447-51, 1993). However, the
identity of the PTK responsible for radiation-induced c-jun
activation is not yet known. See Uckun, F. M., Waddick, K. G.,
Mahajan, S., Jun, X., Takata, M., Bolen, J., and Kurosaki, T. "BTK
as a mediator of radiation-induced apoptosis in DT-40 lymphoma B
cells," Science. 273: 1096-100, 1996; Kurosaki, T. "Molecular
mechanisms in B cell antigen receptor signaling," Curr Opin
Immunol. 9: 309-18, 1997; Uckun F. M, Evans W. E, Forsyth C. J,
Waddick K. G, T-Ahlgren L., Chelstrom L. M, Burkhardt A., Bolen J.,
Myers D. E. "Biotherapy of B-cell precursor leukemia by targeting
genistein to CD19-associated tyrosine kinases." Science
267:886-891, 1995; Myers D. E., Jun X., Waddick K. G., Forsyth C.,
Chelstrom L. M., Gunther R. L., Turner N. E, Bolen J., Uckun F. M.
"Membrane-associated CD19-LYN complex is an endogenous
p53-independent and bcl-2-independent regulator of apoptosis in
human B-lineage lymphoma cells. "Proc Nat'l Acad Sci USA 92:
9575-9579, 1995; Tuel Ahlgren, L., Jun, X., Waddick, K. G., Jin,
J., Bolen, J., and Uckun, F. M. "Role of tyrosine phosphorylation
in radiation-induced cell cycle-arrest of leukemic B-cell
precursors at the G2-M transition checkpoint," Leuk Lymphoma. 20:
417-26, 1996; Qin, S., Minami, Y., Hibi, M., Kurosaki, T., and
Yamamura, H. "Syk-dependent and--independent signaling cascades in
B cells elicited by osmotic and oxidative stress," J Biol Chem.
272: 2098-103, 1997; Saouaf, S. J., Mahajan, S., Rowley, R. B.,
Kut, S., Fargnoli, J., Burkhardt, A. L., Tsukada, S., Witte, O. N.,
and Bolen, J. B. "Temporal differences in the activation of three
classes of non-transmembrane protein tyrosine kinases following B
cell antigen receptor surface engagement," Proc Natl Acad Sci USA.
91: 9524-28, 1994; Law, D. A., Chan, V. F. W., Datta, S. K., and
DeFranco, A. L. "B-cell antigen receptor motifs have redundant
signalling capabilities and bind the tyrosine kinases PTK72,Lyn and
Fyn," Curr Biol. 3: 645-57, 1993; Hibbs, M. L., Tarlinton, D. M.,
Armes, J., Grail, D., Hodgson, G., Maglitto, R., Stacker, S. A.,
and Dunn, A. R. "Multiple defects in the immune system of
Lyn-deficient mice, culminating in autoimmune disease," Cell. 83:
301-311, 1995; Aoki, Y., Isselbacker, K. J., and Pilai, S. "Bruton
tyrosine kinase is tyrosine phosphorylated and activated in pre-B
lymphocytes and receptor-ligated B cells," Proc Natl Acad Sci USA.
91: 10606-10609, 1994; Jugloff, L. S. and Jongstra Bilen, J.
"Cross-linking of the IgM receptor induces rapid translocation of
IgM-associated Ig alpha, Lyn, and Syk tyrosine kinases to the
membrane skeleton, J Immunol. 159: 1096-106, 1997; Thomis, D. S.,
Gurniak, C. B., Tivol, E., Sharpe, A. H., and Berg, L. J." Defects
in B lymphocyte maturation and T lymphocyte activation in mice
lacking Jak 3," Science. 270: 794-797, 1995; Nosaka, T., Van
Deursen, J. M., Tripp, R.A., Thierfelder, W. E., Witthuhn, B. A.,
McMickle, A. P., Doherty, P. c., Grosveld, G. C., and Ihle, J. N.
"Defective lymphoid development in mice lacking Jak 3," Science.
270: 800-802, 1995.
[0006] U.S. patent application Ser. No. 09/087,479 (entitled
Quinazolines For Treating Brain Tumor; filed May 28, 1998)
discloses hydroxyquinazoline derivatives that exhibit potent
cytotoxicity against human glioblastoma cells (i.e. brain tumor
cells). Because JAK-3 is not known to be present in these
glioblastoma cells, the cytotoxic activity of the
hydroxyquinazoline derivatives is not believed to result from
inhibition of JAK-3 activity. Additionally, the cytotoxic activity
of the hydroxyquinazoline derivatives is not known to result from
the inhibition of c-jun activation.
[0007] There is currently a need for therapeutic agents and methods
that are useful for preventing or reducing cell damage that results
from exposure to radiation and chemical agents that cause
DNA-damage. There is also a need for chemical agents as well as in
vitro and in vivo methods that can be used to further investigate
the biological pathways associated with DNA-damage that results
from exposure to radiation or chemical agents.
SUMMARY OF THE INVENTION
[0008] The invention provides a method comprising inhibiting c-jun
expression in cells (e.g. mammalian or avian) by contacting the
cells (in vitro or in vivo) with a substance that inhibits the
activity of Janus family kinase 3 (JAK-3).
[0009] The invention also provides a therapeutic method for
preventing or treating a pathological condition in a mammal (e.g. a
human) wherein c-jun activation is implicated and inhibition of its
expression is desired comprising administering to a mammal in need
of such therapy, an effective amount of a substance that inhibits
the activity of JAK-3.
[0010] The invention also provides novel compounds of formula I as
well as processes and intermediates useful for their
preparation.
[0011] The invention also provides substances that are effective to
inhibit JAK-3 for use in medical therapy (preferably for use in
treating conditions that result from exposure to radiation or to
chemical agents that cause DNA damage), as well as the use of
substances that inhibit JAK-3 for the manufacture of a medicament
for the treatment of a condition that is associated with exposure
to radiation, or to chemical agents that cause DNA damage.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1. Radiation-induced c-jun activation in wild-type DT40
lymphoma B-cells. [A]. Dose response for induction of c-jun mRNA.
DT-40 chicken cells were irradiated at the indicated doses
(0,10,15,20 Gy). Total RNA was extracted after a 2 hours or 4 hours
post-irradiation time period. RNA (20 mg) was loaded on a Northern
gel and transferred by capillary blotting to a nylon membrane. The
Northern blot was hybridized with a .sup.32P labeled chicken c-jun
probe (top panel) or a chicken GAPDH probe (bottom panel). The
inset shows the values for the c-jun/GAPDH transcript expression
ratios as determined with a Bio Rad Storage Phosphor Imager and
corresponding SI values [B]. Effect of the PTK inhibitor genistein
on induction of c-jun mRNA. Cells were treated with 30 mg/ml of
genistein for 24 hours at 37.degree. C. prior to exposure to 20 Gy
ionizing radiation. c-jun expression levels were determined as in
[A].
[0013] FIG. 2. Radiation-induced activation of c-jun in BTK-DT-40
cells. Two representative experiments (shown in [A] and [B])
showing induction of c-jun mRNA expression by ionizing radiation in
wild type (WT) and BTK.sup.- DT-40 cells. Poly (A).sup.+ RNA was
isolated from non-irradiated cells as well as irradiated cells (20
Gy, with a 2 hours post-radiation recovery period). Northern blots
of 2 mg of poly (A)+were hybridized with c-jun probe (top panel),
(-actin probe (middle panel in [A] only), and GAPDH probe (bottom
panel). The inset below each panel shows the relative expression of
c-jun normalized for RNA load (c-jun/GAPDH ratio) and SI (fold
induction over non-irradiated controls).
[0014] FIG. 3. Induction of c-jun mRNA expression by ionizing
radiation in wild type and mutant DT40 cell lines. DT-40, BTK.sup.-
DT-40, SYK.sup.- DT-40 (shown in [A]), as well as LYN.sup.- DT-40
and LYN SYK DT 40 cells (shown in [B]) were irradiated with 20 Gy
and poly (A).sup.+ RNA (in [A]) or total RNA (in [B]) was harvested
after a 2 hour recovery period. RNA from non-irradiated cells was
used as a control. Northern blots containing 2 mg of poly (A).sup.+
(in [A]) or 20 mg of total RNA (in [B]) from each cell line were
hybridized with both .sup.32 P labeled c-jun probe(top panel) and
GAPDH probe (bottom panel). The insets below the panels show the
relative expression of c-jun normalized for RNA loading
(c-jun/GAPDH ratios) as well as the SI (fold induction over
non-irradiated controls).
[0015] FIG. 4. JAK-3 Inhibitors. [A]. Structures of JAK-3
inhibitors. [B] Specificity of JAK-3 inhibitors. Sf21 cells
infected with baculovirus expression vectors for JAK-1 JAK-2 or
JAK-3 were subjected to immunoprecipitation with anti-JAK
antibodies. JAK-1 (shown in B.1), JAK-2 (shown in B.2) and JAK-3
(shown in B.3 and B.4 which illustrate results from 2 independent
experiments) immune complexes were treated with 1% DMSO (vehicle
control =CON), Compound 1, or Compound 2 for 1 hour prior to hot
kinase assays, as described (20,22). Both compounds inhibited JAK-3
when used at 10 .mu.g/ml whereas they did not inhibit JAK-1 or
JAK-2 even at 75 .mu.g/ml [C]. EMSAs of 32Dc22-IL-2R.beta. cells.
Compound 1(100 (g/ml) and Compound 2 (100 (g/ml) inhibited IL-2
triggered JAK-3-dependent STAT activation but not IL-3-triggered
JAK-1/JAK-2-dependent STAT activation in 32Dc11-IL-2R.beta.
cells.
[0016] FIG. 5. Effects of a JAK-3 inhibitor on c-jun induction in
irradiated DT40 cells. Cells were treated with the quinazoline
derivative
4-(3'-Bromo-4'-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline (100
mg/ml) for 24 hours at 37.degree. C. prior to exposure to 20 Gy
ionizing radiation. c-jun expression levels were determined as
outlined in FIGS. 1-3.
DETAILED DESCRIPTION
[0017] As used herein, the term "inhibit" means to reduce by a
measurable amount, or prevent entirely; and the phrase "inhibit
c-jun activation" includes the inhibition of RNA production and the
inhibition of the production of the protein encoded by the RNA.
[0018] Applicants examined the potential involvement of BTK, SYK
and LYN in radiation-induced c-jun activation, using DT40 chicken
lymphoma B-cell clones rendered deficient for these specific PTK by
targeted gene disruption. It was found that BTK plays no role in
radiation-induced c-jun activation. Similarly, neither LYN nor SYK
are required for activation of c-jun after radiation exposure.
However, their participation may influence the magnitude of the
c-jun response. It was unexpectedly discovered, however, that an
inhibitor of Janus family kinase 3 (JAK-3) abrogated
radiation-induced c-jun activation.
[0019] C-jun expression can be activated by exposure to chemical
agents that damage DNA such as ara-C, a topoisomerase II
inhibitors, or alkylating agents. C-jun activation can also result
from exposure to ultraviolet radiation or ionizing radiation.
According to the invention, inhibitors of JAK-3 can be used to
inhibit c-jun expression resulting from exposure to radiation or
exposure to chemical agents.
[0020] The methods of the invention can be carried out in vitro.
Such in vitro methods are also useful for studying the biological
processes associated with cell response to DNA damaging agents. The
methods of the invention can also be carried out in vivo. Such
methods can also be used to study the biological processes
associated with cell response to DNA damaging agents, as well as
for treating pathological conditions in mammals (e.g. humans) that
result from exposure to DNA-damaging agents.
[0021] Pathological conditions that result from exposure to
DNA-damaging agents include conditions that result from oxidative
stress, such as tissue or organ (e.g. heart, liver, or kidney)
damage, inflammation, and hair loss, as well as the negative
effects that are produced by oxygen free radicals during
chemotherapy. Oxidative stress may result from exposure to external
agents, or may result from internal processes. Thus, JAK-3
inhibitors are also useful for treating conditions resulting from
the action of internally generated oxygen free radicals, such as
aging and amyelotrophic lateral sclerosis (ALS).
[0022] According to the invention, the JAK-3 inhibitors may be
administered prophylactically, i.e. prior to exposure to the
DNA-damaging agent, or the JAK-3 inhibitors may be administered
after exposure to the DNA damaging agent.
[0023] The JAK-3 inhibitors useful in the methods of the invention
include all compounds capable of inhibiting the activity of JAK-3,
it being well known in the art how to measure a compounds ability
to inhibit JAK-3, for example, using standard tests similar to the
test described hereinbelow in Example 2 under the heading "Effects
of a JAK-3 inhibitor on radiation-induced c-jun activation in DT40
cells."
[0024] JAK-3 inhibitors that are useful in the methods of the
invention include compounds of formula I: 1
[0025] wherein
[0026] X is HN, R.sub.11N, S, O, CH.sub.2, or R.sub.11CH;
[0027] R.sub.11 is hydrogen, (C.sub.1-C.sub.4)alkyl, or
(C.sub.1-C.sub.4)alkanoyl;
[0028] R.sub.1-R.sub.8 are each independently hydrogen, hydroxy,
mercapto, amino, nitro, (C.sub.1-C.sub.4)alkyl,
(C.sub.1-C.sub.4)alkoxy, (C.sub.1-C.sub.4)alkylthio, or halo;
wherein two adjacent groups of R.sub.1R.sub.5 together with the
phenyl ring to which they are attached may optionally form a fused
ring, for example forming a naphthyl or a tetrahydronaphthyl ring;
and further wherein the ring formed by the two adjacent groups of
R.sub.1-R.sub.5 may optionally be substituted by 1, 2, 3, or 4
hydroxy, mercapto, amino, nitro, (C.sub.1-C.sub.4)alkyl,
(C.sub.1-C.sub.4)alkoxy, (C.sub.1-C.sub.4)alkylthio, or halo;
and
[0029] R.sub.9 and R.sub.10 are each independently hydrogen,
(C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)alkoxy, halo, or
(C.sub.1-C.sub.4)alkanoyl; or R.sub.9 and R.sub.10 together are
methylenedioxy; or a pharmaceutically acceptable salt thereof.
[0030] The following definitions are used, unless otherwise
described: halo is fluoro, chloro, bromo, or iodo. Alkyl, alkanoyl,
etc. denote both straight and branched groups; but reference to an
individual radical such as "propyl" embraces only the straight
chain radical, a branched chain isomer such as "isopropyl" being
specifically referred to. (C.sub.1-C4)Alkyl includes methyl, ethyl,
propyl, isopropyl, butyl, iso-butyl, and sec-butyl;
(C.sub.1-C.sub.4)alkoxy includes methoxy, ethoxy, propoxy,
isopropoxy, butoxy, iso-butoxy, and sec-butoxy; and
(C.sub.1-C.sub.4)alkanoyl includes acetyl, propanoyl and
butanoyl.
[0031] A specific group of compounds are compounds of formula I
wherein R.sub.1-R.sub.5 are each independently hydrogen, mercapto,
amino, nitro, (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)alkoxy,
(C.sub.1-C.sub.4)alkylth- io, or halogen.
[0032] Another specific group of compounds are compounds of formula
I wherein R.sub.9 and R.sub.10 are each independently hydrogen,
(C.sub.1-C.sub.4)alkyl, halo, or (C.sub.1-C.sub.4)alkanoyl; or
R.sub.9 and R.sub.10 together are methylenedioxy; or a
pharmaceutically acceptable salt thereof.
[0033] JAK-3 inhibitors that are useful in the methods of the
invention also include compounds of formula I as described in U.S.
patent application Ser. No. 09/087,479 (entitled Quinazolines For
Treating Brain Tumor; filed May 28, 1998).
[0034] Preferred JAK-3 inhibitors include
4-(4'-hydroxylphenyl)-amino-6,7-- dimethoxyquinazoline and
4-(3'-bromo-4'-hydroxylphenyl)-arnino-6,7-dimetho- xyquinazoline,
or a pharmaceutically acceptable salt thereof.
[0035] Substances that inhibit JAK-3 ("the Substance(s)") can be
formulated as pharmaceutical compositions and administered to a
mammalian host, such as a human patient in a variety of forms
adapted to the chosen route of administration, i.e., orally or
parenterally, by intravenous, intramuscular, topical or
subcutaneous routes.
[0036] Thus, the Substances may be systemically administered, e.g.,
orally, in combination with a pharmaceutically acceptable vehicle
such as an inert diluent or an assimilable edible carrier. They may
be enclosed in hard or soft shell gelatin capsules, may be
compressed into tablets, or may be incorporated directly with the
food of the patient's diet. For oral therapeutic administration,
the Substance may be combined with one or more excipients and used
in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 0.1% of the
Substance. The percentage of the compositions and preparations may,
of course, be varied and may conveniently be between about 2 to
about 60% of the weight of a given unit dosage form. The amount of
Substance in such therapeutically useful compositions is such that
an effective dosage level will be obtained.
[0037] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
Substance may be incorporated into sustained-release preparations
and devices.
[0038] The Substances may also be administered intravenously or
intraperitoneally by infusion or injection. Solutions of the
Substance can be prepared in water, optionally mixed with a
nontoxic surfactant. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, triacetin, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0039] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the Substance which are adapted for the
extemporaneous preparation of sterile injectable or infusible
solutions or dispersions, optionally encapsulated in liposomes. In
all cases, the ultimate dosage form must be sterile, fluid and
stable under the conditions of manufacture and storage. The liquid
carrier or vehicle can be a solvent or liquid dispersion medium
comprising, for example, water, ethanol, a polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycols, and the
like), vegetable oils, nontoxic glyceryl esters, and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the formation of liposomes, by the maintenance of the
required particle size in the case of dispersions or by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars, buffers or sodium chloride.
Prolonged absorption of the injectable compositions can be brought
about by the use in the compositions of agents delaying absorption,
for example, aluminum monostearate and gelatin.
[0040] Sterile injectable solutions are prepared by incorporating
the Substance in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying techniques, which yield a powder of the active ingredient
plus any additional desired ingredient present in the previously
sterile-filtered solutions.
[0041] For topical administration, the Substances may be applied in
pure form, i.e., when they are liquids. However, it will generally
be desirable to administer them to the skin as compositions or
formulations, in combination with a dermatologically acceptable
carrier, which may be a solid or a liquid.
[0042] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the Substances can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers.
[0043] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0044] Examples of useful dermatological compositions which can be
used to deliver the Substances to the skin are known to the art;
for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria
(U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157)
and Wortzman (U.S. Pat. No. 4,820,508).
[0045] Useful dosages of the compounds of formula I can be
determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known
to the art; for example, see U.S. Pat. No. 4,938,949.
[0046] Generally, the concentration of the Substance in a liquid
composition, such as a lotion, will be from about 0.1-25 wt-%,
preferably from about 0.5-10 wt-%. The concentration in a
semi-solid or solid composition such as a gel or a powder will be
about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
[0047] The amount of the Substance required for use in treatment
will vary not only with the particular salt selected but also with
the route of administration, the nature of the condition being
treated and the age and condition of the patient and will be
ultimately at the discretion of the attendant physician or
clinician.
[0048] In general, however, a suitable dose will be in the range of
from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75
mg/kg of body weight per day, such as 3 to about 50 mg per kilogram
body weight of the recipient per day, preferably in the range of 6
to 90 mg/kg/day, most preferably in the range of 15 to 60
mg/kg/day.
[0049] The Substance is conveniently administered in unit dosage
form; for example, containing 5 to 1000 mg, conveniently 10 to 750
mg, most conveniently, 50 to 500 mg of active ingredient per unit
dosage form.
[0050] Ideally, the Substance should be administered to achieve
peak plasma concentrations of from about 0.5 to about 75 .mu.M,
preferably, about 1 to 50 .mu.M, most preferably, about 2 to about
30 .mu.M. This may be achieved, for example, by the intravenous
injection of a 0.05 to 5% solution of the Substance, optionally in
saline, or orally administered as a bolus containing about 1-100 mg
of the Substance. Desirable blood levels may be maintained by
continuous infusion to provide about 0.01-5.0 mg/kg/hr or by
intermittent infusions containing about 0.4-15 mg/kg of the
Substance.
[0051] The Substance may conveniently be presented in a single dose
or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
[0052] The invention will now be illustrated by the following
non-limiting Examples.
EXAMPLES
Example 1
[0053] Chemical synthesis and Characterization of JAK-3
Inhibitors
[0054] Melting points are uncorrected. .sup.1H NMR spectra were
recorded using a Varian Mercury 300 spectrometer in DMSO-d.sub.6 or
CDCl.sub.3. Chemical shifts are reported in parts per million (ppm)
with tetramethylsilane (TMS) as an internal standard at zero ppm.
Coupling constant (J) are given in hertz and the abbreviations s,
d, t, q, and m refer to singlet, doublet, triplet, quartet and
multiplet, respectively. Infrared spectra were recorded on a
Nicolet PROTEGE 460-IR spectrometer. Mass spectroscopy data were
recorded on a FINNIGAN MAT 95, VG 7070E-HF G.C. system with an HP
5973 Mass Selection Detector. UV spectra were recorded on BECKMAN
DU 7400 and using MeOH as the solvent. TLC was performed on a
precoated silica gel plate (Silica Gel KGF; Whitman Inc). Silica
gel (200-400 mesh, Whitman Inc.) was used for all column
chromatography separations. All chemicals were reagent grade and
were purchased from Aldrich Chemical Company (Milwaukee, Wis) or
Sigma Chemical Company (St. Louis, Mo.).
[0055] The common synthetic precursor
4-chloro-6,7-dimethoxyquinazoline (7), used for preparing compounds
(1) and (2), was prepared using liturature procedures as
illustrated in Scheme 1. 2
[0056] 4,5-Dimethoxy-2-nitrobenzoic acid (3) was treated with
thionyl chloride and then reacted with ammonia to give
4,5-dimethoxy-2-nitrobenza- mide (4) as described by F. Nomoto et
al. Chem. Pharm. Bull. 1990, 38, 1591-1595. The nitro group in
compound (4) was reduced with sodium borohydride in the presence of
copper sulfate (see C. L. Thomas Catalytic Processes and Proven
Catalysts Academic Press, New York (1970)) to give
4,5-dimethoxy-2-aminobenzamide (5) which was cyclized by refluxing
with formic acid to give 6,7-dimethoxyquinazoline-4(3H)-one (6).
Compound (6) was refluxed with phosphorus oxytrichloride to provide
the common synthetic precursor (7).
[0057] Compounds 1 and 2 (FIG. 4) were prepared from the common
synthetic precursor (7) and the requsite aniline as follows.
[0058] 4-(4'-Hydroxylphenyl)-amino-6, 7-dimethoxyquinazoline (1). A
mixture of 448 mg (2 mmol) of 4-chloro-6,7-dimethoxy-quinazoline
(7) and 2.5 mmol of 4-hydroxyaniline in 20 ml of alcohol (EtOH or
MeOH) was refluxed for 8 hours. After cooling triethylamine was
added to basify the solution, and the solvent was concentrated to
give material that was recrystallized from DMF to give compound
(1); 84.29%; m.p. 245.0-248.0.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 11.21(s, 1H, -NH), 9.70(s, 1H, -OH),
8.74(s, 1H, 2-H), 8.22(s, 1H, 5-H), 7.40(d, 2H, J=8.9 Hz, 2',
6'-H), 7.29(s, 1H, 8-H), 6.85(d, 2H, J=8.9 Hz, 3', 5'-H), 3.98(s,
3H, --OCH.sub.3), 3.97(s, 3H, --OCH.sub.3). UV(MeOH)
.lambda..sub.max(e): 203.0, 222.0, 251.0, 320.0 nm.
IR(KBr)u.sub.max: 3428, 2836, 1635, 1516, 1443, 1234 cm.sup.-1.
GC/MS m/z 298 (M.sup.++1, 100.00), 297(M.sup.+, 26.56),
296(M.sup.+-1, 12.46).
[0059]
4-(3'-Bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxy-quinazoline (2).
A mixture of 448 mg (2 mmol) of 4-chloro-6,7-dimethoxy-quinazoline
(7) and 2.5 mmol of 3-bromo-4-hydroxyaniline in 20 ml of alcohol
(EtOH or MeOH) was refluxed for 8 hours. After cooling,
triethylamine was added to basify the solution, and the solvent was
concentrated to give material that was recrystallized from DMF to
give compound (2); 89.90%; m.p. 233.0-233.5.degree. C.; .sup.1H
NMR(DMSO-d.sub.6): .delta. 10.08(s, 1H, --NH), 9.38(s, 1H, -OH),
8.40(s, 1H, 2-H), 7.89(d, 1H, J.sub.2', 5'=2.7 Hz, 2'-H), 7.75(s,
1H, 5-H), 7.55(dd, 1H, J.sub.5', 6'=9.0 Hz, J.sub.2', 6'=2.7 Hz,
6'-H), 7.14(s, 1H, 8-H), 6.97(d, 1H, J.sub.5', 6'=9.0 Hz, 5'-H),
3.92(s, 3H, --OCH.sub.ER), 3.90(s, 3H, --OCH.sub.3).
UV(MeOH).lambda..sub.max(e): 203.0, 222.0, 250.0, 335.0 nm.
IR(KBr)u.sub.max: 3431(br), 2841, 1624, 1498, 1423, 1244 cm.sup.-1.
GC/MS m/z 378(M.sup.++2, 90.68), 377(M.sup.+1, 37.49), 376(M.sup.+,
100.00), 360(M.sup.+3.63), 298(18.86), 282 (6.65).
Example 2
Biological Screening
[0060] Materials and Methods
[0061] Cell Lines. The establishment and characterization of
BTK-deficient, SYK-deficient, and LYN-deficient clones and
reconstituted SYK-deficient cell lines of DT-40 chicken lymphoma
B-cells were previously reported. The culture medium was RPMI 1640
(Life Technologies; Gaithersburg, Md.), supplemented with 1%
chicken serum (Sigma; St. Louis, Mo.), 5% fetal bovine serum
(Hyclone, Logan, Utah) and 1% penicillin-streptomycin (Life
Technologies). See Uckun, F. M., Waddick, K. G., Mahajan, S., Jun,
X., Takata, M., Bolen, J., and Kurosaki, T. Science. 273: 1096-100,
1996; Kurosaki, T. Curr Opin Immunol. 9: 309-18, 1997; Kurosaki,
T., Johnson, S. A., Pao, L., Sada, K., Yamamura, H., and Cambier,
J. C. J. Exp. Med. 182: 1815-1823, 1995; and Dibirdik I.,
Kristupaitis D., Kurosaki T., Tuel-Ahlgren L., Chu A., Pond D.,
Tuong D., Luben R., Uckun F. M. J. Biol. Chem. 273(7),
pp:4035-4039, 1998.
[0062] Use of PTK Inhibitors. Cells (2.times.10.sup.6/ml) were
treated for 24 hours at 37.degree. C. with either (1) the PTK
inhibitory isoflavone genistein (Calbiochem, La Jolla, Calif.) at
111 mM (30 mg/ml) concentration or (2) the Janus family kinase, 3
(JAK-3)-specific PTK inhibitor
4-(3'-bromo-4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline,
C.sub.16,H.sub.14Br(N.sub.3O.sub.3), kindly provided by Dr.
Xing-Ping Liu, Alexander and Parker Pharmaceutical Inc., Roseville,
Minn.) at 270 MM (100 mg/ml) prior to radiation in order to assess
the effects of these agents on radiation-induced c-jun
activation.
[0063] Irradiation of cells. Cells (2.times.10.sup.6cells/ml) in
plastic tissue culture flasks were irradiated with 10-20 Gy at a
dose rate of 4 Gy/min during log phase growth and under aerobic
conditions using a .sup.137Cs irradiator (J.L. Shephard, Glendale,
Calif., as previously described by Tuel Ahlgren, L., Jun, X.,
Waddick, K. G., Jin, J., Bolen, J., and Uckun, F. M. "Role of
tyrosine phosphorylation in radiation-induced cell cycle-arrest of
leukemic B-cell precursors at the G2-M transition checkpoint," Leuk
Lymphoma. 20: 417-26, 1996; and Uckun, F. M., Jaszcz, W., Chandan
Langlie, M., Waddick, K. G., Gajl Peczalska, K. and Song, C.W.
"Intrinsic radiation resistance of primary clonogenic blasts from
children with newly diagnosed B-cell precursor acute lymphoblastic
leukemia," J Clin Inves. 91:1044-1051, 1993. In some experiments,
cells were preincubated with PTK inhibitors for 24 hours prior to
irradiation.
[0064] c-jun probe. A 506 basepair (bp) c-jun probe was obtained by
polymerase chain reaction (PCR) amplification of chicken genomic
DNA. Primer sequences were determined based upon the sequence of
chicken c-jun (GenBank accession code CHKJUN). Two primers:
5'-ACTCTGCACC CAACTACAACGC-3' (SEQ. ID NO: 1) and 5'-CTTCTACCGT
CAGCTTTACGCG-3' (SEQ ID NO: 2) were used for amplification.
Amplification was performed with a mix of Taq polymerase and a
proof reading polymerase (eLONGase:Taq polymerase plus Pyrococcus
species GB-D polymerase, Gibco BRL, Grand Island, N.Y.) on an
thermocycler, Ericomp Delta II cycler, using a hot start. PCR
products were subsequently cloned into the cloning vector, PCR 2.1
(Invitrogen, San Diego, Calif.). An insert of the proper size (506
basepair) was identified as chicken c-jun by sequence analysis
using PRISM dye terminator cycle sequencing (AmpliTaq.RTM. DNA
Polymerase, FS) and analyzed on an automated sequencer, ALF express
sequencer (Pharmacia Biotech, Piscataway, N.J.). A 538 base pair
chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe was
generated by reverse transcription and subsequent PCR amplification
(RT-PCR) from chicken RNA with the following primers:
5'-AGAGGTGCTGCCCAGAACATCATC-3' (SEQ ID NO: 3) and
5'-GTGGGGAGACAGAAGGGAACAGA-3' (SEQ ID NO: 4). A 413 bp chicken
B-actin probe was generated by RT-PCR amplification from chicken
RNA with the following primers: 5'-GCCCTCTTCCAGCATCTTTCTT-3' (SEQ
ID NO: 5) and 5'-TTTATGCGCATTTATGGGTT-3' (SEQ ID NO: 6). The
amplified cDNAs were cloned into PCR 2.1.
[0065] RNA isolation and Northern blot hybridization analysis.
Total RNA was extracted from approximately 2.5.times.10.sup.7 cells
with Trizol Reagent, a monophasic solution of phenol and guanidine
isothiocyanate as described by Chomcznski, P. and Sacchi, N.
"Single-step method of RNA isolation by
guanidinium-thiocyanate-phenol-chloroform extraction," Anal.
Biochem. 162: 156-159, 1987. Poly (A).sup.+ RNA was isolated
directly from 1-3.times.10.sup.8 cells with an Invitrogen Fast Trak
2.0 mRNA isolation kit. In brief, cells were lysed in a sodium
dodecyl sulfate (SDS) lysis buffer containing a proprietary mixture
of proteases. The lysate was directly incubated with oligo-dT for
absorption and subsequent elution of poly (A).sup.+ RNA.
[0066] Two micrograms of poly (A).sup.+ or 20 micrograms of total
RNA were denatured in formaldehyde/formamide loading dye at
65.degree. prior to loading onto a 1% agarose-formaldehyde
denaturing gel. Transcript sizes were determined relative to RNA
markers of 0.5-9 kb. The gels were stained with Radiant Red in
H.sub.2O to check loading and integrity of RNA prior to transfer.
The RNA was subsequently transferred to positively charged nylon
membrane with 20.times. standard sodium citrate(SSC) transfer
buffer (LXSSC=0.15 M sodium chloride-0.015 M sodium citrate) by
downward capillary transfer. The c-jun fragment was radiolabeled by
random priming with [(-.sup.32P]-dCTP (3000 Ci/mM) [Amersham,
Arlington Heights, Ill.] (40). Northern blots were hybridized
overnight at 42.degree. C. in prehybridization/hybridization
solution (50% formamide with proprietary blocking and background
reduction reagents; Ambion, Austin, Tex.) for 16-24 hours and
unbound probe was removed by washing to a final stringency of 0.1%
SDS, 0.1.times.SSC (65.degree. C.). The blots were analyzed both by
autoradiography and using the BioRad Storage Phosphor Imager System
(BioRad, Hercules, Calif.) for quantitative scanning. The blots
were subsequently stripped in boiling 0.1% SDS, and then
rehybridized with a chicken GAPDH and/or chicken (62 -actin probe
to normalize for loading differences.
[0067] Results and Discussion
[0068] Exposure of DT40 chicken lymphoma B-cells to ionizing
radiation activates the c-jun protooncogene. Exposure of human
lymphoma B-cells to 10-20 Gy-rays results in enhanced c-jun
expression with a maximum response at 1-2 hours (Chae, H. P.,
Jarvis, L. J., and Uckun, F. M. Cancer Res. 53: 447-51, 1993). It
has also been reported that ionizing radiation triggers in DT-40
chicken lymphoma B-cells biochemical and biological signals similar
to those in human lymphoma B-cells (Uckun, F. M., Waddick, K. G.,
Mahajan, S., Jun, X., Takata, M., Bolen, J., and Kurosaki, T.
Science. 273: 1096-100, 1996). In order to determine if DT-40
chicken lymphoma B-cells show a similar c-jun response to ionizing
radiation, DT-40 cells were irradiated with 5,10,15 or 20 Gy and
examined total RNA harvested from cells 2 or 4 hours after
radiation exposure for expression levels of 1.8 kb chicken c-jun
transcripts by quantitative Northern blot analysis. As shown in
FIG. 1A, radiation exposure increased the level of c-jun
transcripts in a dose-and time-dependent manner without
significantly affecting the GAPDH transcript levels with a maximum
stimulation index (SI) [as determined by comparison of the
c-jun/GAPDH ratios in non-irradiated versus irradiated cells] of
3.1, 4 hours after 20 Gy. In seven additional independent
experiments, the stimulation index for 20 Gy ionizing radiation at
2 hours after radiation exposure ranged from 2.4 to 3.8 (mean
(SE=2.9.+-.0.4).
[0069] The role of PTK in radiation-induced activation of c-jun
expression in chicken lymphoma B cells was examined next, since PTK
inhibitors were shown to prevent radiation-induced c-jun activation
in human lyrnphoma B-cells. As shown in FIG. 1B, ionizing radiation
did not significantly enhance c-jun expression levels in DT-40
cells treated with the PTK-inhibitory isoflavone, genistein
(stimulation index=1.1) indicating that activation of a PTK is
required for radiation-induced c-jun expression in chicken lymphoma
B cells as well. These findings established DT40 chicken lymphoma
B-cells as a suitable model to further elucidate the molecular
mechanism of radiation-induced c-jun activation.
[0070] Cytoplasmic protein tyrosine kinases BTK, LYN, and SYK are
not required for radiation induced c-jun activation. BTK is
abundantly expressed in lymphoma B-cells and its activation has
been shown to be required for radiation-induced apoptosis of DT-40
cells (Uckun, F. M., Waddick, K. G., Mahajan, S., Jun, X., Takata,
M., Bolen, J., and Kurosaki, T. Science. 273: 1096-100, 1996).
DT-40 cells rendered BTK-deficient by targeted disruption of the
BTK genes do not undergo apoptosis after radiation exposure.
Therefore, we set out to determine if BTK could be the PTK
responsible for radiation-induced c-jun activation as well, by
comparing the levels of c-jun induction in BTK-deficient (BTK)
versus wild-type DT-40 cells. Contrary to our expectations, 20 Gy
ionizing radiation did not fail to induce c-jun expression in
BTK-deficient DT-40 cells in any of the three independent
experiments performed. The stimulation indices ranged from 1.6 to
3.9 (mean.+-.SE=2.4.+-.0.5) (FIG. 2). Thus, ionizing
radiation-induced increases in c-jun transcript levels do not
depend upon the presence of BTK.
[0071] Since SYK is also abundantly expressed in DT-40 cells and is
rapidly activated after ionizing radiation, we next examined if SYK
might be the PTK responsible for radiation-induced increases in
c-jun transcript levels. As shown in FIG. 3A, 20 Gy ionizing
radiation enhanced c-jun expression in SYK DT-40 cells rendered
SYK-deficient by targeted gene disruption even though the
stimulation indices observed in five independent experiments were
lower than from those in wild-type cells (1.9.+-.0.2, vs
2.9.+-.0.4,p<0.01). Thus, SYK is not required for
radiation-induced c-jun activation in DT-40 cells but it may
participate in generation of an optimal signal.
[0072] DT40 cells express high levels of LYN but do not express
other members of the Src PTK family, including BLK, HCK, SRC, FYN,
or YES at detectable levels (see Uckun, F. M., Waddick, K. G.,
Mahajan, S., Jun, X., Takata, M., Bolen, J., and Kurosaki, T.
Science. 273: 1096-100, 1996; Kurosaki, T., Johnson, S. A., Pao,
L., Sada, K., Yamamura, H., and Cambier, J. C. "Role of the Syk
autophosphorylation site and SH2 domains in B cell antigen receptor
signaling," J. Exp. Med. 182: 1815-1823, 1995; and Takata, M.,
Homma, Y., and Kurosaki, T. "Requirement of phospholipase
C-.gamma.2 activation in surface immunoglobulin M-induced B cell
apoptosis.," J Exp Med. 182: 907-914, 1995. Since it has previously
been demonstrated that SRC family PTK are essential for
UV-stimulated increases in c-jun expression, we postulated that the
predominant SRC-family member, LYN, might mediate radiation-induced
c-jun expression in DT-40 cells. To test this hypothesis, we
examined the ability of ionizing radiation to activate c-jun
expression in DT-40 cells rendered LYN-deficient by targeted gene
disruption. LYN-deficient (LYN) cells showed enhanced c-jun
expression after irradiation, however the stimulation indices were
lower than those in wild-type DT-40 (FIG. 3B). Since LYN and SYK
have been shown to cooperate in the generation of other signals in
B-cells (see Kurosaki, T. "Molecular mechanisms in B cell antigen
receptor signaling," Curr Opin Immunol. 9: 309-18, 1997), the
ability of ionizing radiation to induce c-jun expression in
LYN.sup.-SYK.sup.- DT-40 cells, generated by targeted disruption of
the syk gene in LYN.sup.- deficient DT-40 cells was examined. As
shown in FIG. 3B, LYN SYK DT-40 cells showed elevated c-jun
transcript levels after irradiation, indicating that the c-jun
response does not depend on either of these PTK, either alone or in
cooperation. Similar to SYK, LYN is not required for
radiation-induced c-jun activation in DT-40 cells but it may
participate in generation of an optimal response.
[0073] Interestingly, in four independent experiments, we observed
higher baseline expression levels of c-jun in SYK.sup.- DT-40 cells
than in wild-type DT-40 cells (Range: 1.4-2.3-fold,
mean.+-.SE=1.6+0.2-fold), suggesting that Syk may be involved in
regulation of baseline c-jun levels. To further explore this
possibility, we compared c-jun levels in SYK.sup.- cells to those
of SYK.sup.- cells reconstituted with wild-type or kinase domain
mutant (K.sup.-) syk gene. We observed that reconstitution with
wild-type syk reduced the higher baseline expression levels of
c-jun in SYK.sup.- cells, whereas reconstitution with a K.sup.- syk
failed to reduce c-jun levels (data not shown). These results
implicate SYK as a negative regulator of c-jun expression. This
novel function of SYK seems to depend on its kinase domain.
[0074] Effects of a JAK-3 inhibitor on radiation-induced c-jun
activation in DT40-cells. B-cell signal transduction events direct
fundamental decisions regarding cell survival during periods of
oxidative stress. A better understanding of the dynamic interplay
between B-cell signaling pathways is needed to determine how vital
decisions are dictated during intracellular oxidation changes. STAT
proteins (signal transducers and activators of transcription) are a
family of DNA binding proteins that were identified during a search
for interferon (IFN) a- or g-stimulated gene transcription targets.
There are presently seven STAT family members. The JAK family of
cytoplasmic protein kinases were originally demonstrated to also
function in IFN signaling, and are now known to participate in a
broad range of receptor-activated signal cascades. Different
ligands and cell activators employ specific JAK and STAT family
members. The basic model for STAT activation suggests that in
unstimulated cells, latent forms of STATs are predominantly
localized within the cytoplasm. Ligand binding induces STAT
proteins to associate with intracellular phosphotyrosine residues
of transmembrane receptors. Once STATs are bound to receptors,
receptor-associated JAK kinases phosphorylate the STAT proteins.
STAT proteins then dimerize through specific reciprocal
SH2-phosphotyrosine interactions and may form complexes with other
DNA-binding proteins. STAT complexes translocate to the nucleus and
interact with DNA response elements to enhance transcription of
target genes. Signaling events regulating apoptotic responses have
been shown to utilize STAT proteins. Notably, a recent study
demonstrated JAK activation by tyrosine phosphorylation in cells
that are exposed to reactive oxygen intermediates, which in-turn
lead to tyrosine phosphorylation and activation of STAT-1, STAT-3
and STAT-6.
[0075] After establishing that LYN, BTK, and SYK kinases are not
required for radiation-induced c-jun activation, we set out to
determine if c-jun activation is functionally linked to the
JAK-STAT pathway. To this end, we examined the effects of a JAK-3
inhibitory novel quinazoline derivative on c-jun expression levels
in irradiated DT-40 cells. To identify a potent JAK-3 specific
inhibitor, the effects of two novel quinazoline derivatives on the
enzymatic activity of JAK-1; JAK-2, and JAK-3 were examined using
Sf21 cells that were infected with baculovirus expression vectors
for these kinases, using standard methods (FIG. 4). Infected cells
were harvested, JAKs were immunoprecipitated with appropriate
antibodies (anti-JAK-1: (HR-785), cat# sc-277, rabbit polyclonal
IgG affinity purified, 0.1 mg/ml, Santa Cruz Biotechnology;
anti-JAK-2: (C-20)-G, cat # sc-294-G, goat polyclonal IgG affinity
purified, 0.2 mg/ml, Santa Cruz Biotechnology; anti-JAK-3: (C-21),
cat # sc-513, rabbit polyclonal IgG affinity purified, 0.2 mg/ml,
Santa Cruz Biotechnology), and kinase assays were performed
following a 1 hour exposure of the immunoprecipitated Jaks to the
quinazoline compounds, as described by Uckun, F. M., Waddick, K.
G., Mahajan, S., Jun, X., Takata, M., Bolen, J., and Kurosaki, T.
Science. 273: 1096-100, 1996; Uckun F. M, Evans W. E, Forsyth C. J,
Waddick K. G, T-Ahlgren L., Chelstrom L. M, Burkhardt A., Bolen J.,
Myers D. E. Science 267:886-891, 1995; and Myers D. E., Jun X.,
Waddick K. G., Forsyth C., Chelstrom L. M., Gunther R. L., Turner
N. E, Bolen J., Uckun F. M. Proc Nat'l Acad Sci USA 92: 9575-9579,
1995; and Tuel Ahlgren, L., Jun, X., Waddick, K. G., Jin, J.,
Bolen, J., and Uckun, F. M. Leuk Lymphoma. 20: 417-26, 1996.
[0076] As shown in FIG. 4B, both compounds inhibited JAK-3 (FIGS.
B.3 and B.4) but not JAK-1 (FIG. B.1) or JAK-2 (FIG. B.2) (FIG.
4D). Electrophoretic Mobility Shift Assays (EMSAs) were performed
to examine the effects of both compounds on cytokine-induced STAT
activation. Specifically, 32Dc11/IL2R.beta. cells (gift from James
Ihle, St. Jude Children's Research Hospital) were exposed at
8.times.10.sup.6/ml in RPMI supplemented with FBS to the JAK-3
inhibitors at a final concentration of 10 .mu.g/ml in 1% DMSO) for
1 hour and subsequently stimulated with IL2 or IL3 as indicated.
Cells were collected after 15 minutes and resuspended in lysis
buffer (100 mM Tris-HCI pH 8.0, 0.5% NP-40, 10% glycerol, 100 mM
EDTA, 0.1 mM NaVO3, 50 mM NaF, 150 mM Nacl, 1 mM DTT, 3 (g/ml
Aprotinin, 2 g/ml Pepstatin A, 1 (g/ml Leupeptin and 0.2 mM PMSF).
Lysates were precleared by centrifugation for 30 minutes. Cell
extracts (approximately 10 g) were incubated with 2 .mu.g of
poly(dI-dC) for 30 minutes, followed by a 30 minute incubation with
1 ng of poly nucleotide kinase-".sup.32P labeled double stranded
DNA oligonucleotide representing the IRF-1 STAT DNA binding
sequence (Santa Cruz Biotechnology, Santa Cruz, Calif.). Samples
were resolved by nondenaturing PAGE and visualized by
autoradiography. As shown in FIG. 4C, both compounds inhibited the
JAK-3-dependent STAT activation after stimulation with IL-2, but
they did not affect the JAK-1/JAK-2-dependent STAT activation after
stimulation with IL-3. Compound 2 was selected for further
experiments designed to examine the effects of JAK-3 inhibition on
radiation-induced c-jun activation.
[0077] As shown in FIG. 5, ionizing radiation failed to induce
c-jun expression in DT-40 cells treated with the JAK-3 inhibitor.
This demonstrates that JAK-3 inhibitors are capable of inhibiting
radiation induced c-jun expression.
[0078] In untreated cells, c-jun expression is induced by exposure
to DNA-damaging chemical agents and by exposure to radiation. Thus,
c-jun expression is an early marker of cellular response to such
DNA-damaging agents. It has been shown that compounds that inhibit
JAK-3 are capable of inhibiting the expression of c-jun.
Accordingly, JAK-3 inhibitors may be useful to prevent or treat
diseases or conditions that result from exposure to DNA-damaging
agents.
[0079] JAK-3 maps to human chromosome 19p12-13.1. A cluster of
genes encoding protooncogenes and transcription factors is also
located near this region. JAK-3 expression has been demonstrated in
mature B-cells as well as B-cell precursors. JAK-3 has also been
detected in leukemic B-cell precursors and lymphoma B-cells. The
physiological roles for JAK-3 have been borne out through targeted
gene disruption studies in mice, the genetic analysis of patients
with severe combined immunodeficiency, and biochemical studies of
JAK-3 in cell lines. A wide range of stimuli result in JAK-3
activation in B-cells, including interleukin 7 and interleukin 4.
The B-cell marker CD40 constitutively associates with JAK-3 and
ligation of CD40 results in JAK-3 activation which has been shown
to be mandatory for CD40-mediated gene expression. Constitutive
activity of JAK-3 has been observed in v-abl transformed pre-B
cells and coimmunoprecipitations show that v-abl physically
associates with JAK-3 implicating JAK-3 in v-abl induced cellular
transformation. See Ihle, J. N. "Janus kinases in cytokine
signalling," Philos Trans R Soc Lond B Biol Sci 351:159-66, 1996;
Leonard, W. J. "STATs and cytokine specificity," Nat Med 2:968-9,
1996; Levy, D. E. "The house that Jak/Stat built," Cytokine Growth
Factor Rev 8:81-90, 1997; Riedy, M. C. et al. "Genomic sequence,
organization, and chromosomal localization of human JAK-3,"
Genomics 37, 57-61, 1996; Safford, M. G., Levenstein, M., Tsifrina,
E., Amin, S., Hawkins, A. L., Griffin, C.A., Civin, C.I. and Small,
D. "JAK-3: expression and mapping to chromosome 19p12-13.1"
[published erratum appears in Exp Hematol 1997 July; 25(7):650].
Exp Hematol 25, 374-86, 1997; Kumar, A., Toscani, A., Rane, S. and
Reddy, E. P. "Structural organization and chromosomal mapping of
JAK-3 locus," Oncogene 13, 2009-14, 1996; Hoffman, S. M., Lai, K.
S., Tomfohrde, J., Bowcock, A., Gordon, L. A. and Mohrenweiser, H.
W. "JAK-3 maps to human chromosome 19p12 within a cluster of
proto-oncogenes and transcription factors," Genomics 43, 109-111,
1997; Tortolani, P. J. et al. "Regulation of JAK-3 expression and
activation in human B cells and B cell malignancies," J Immunol
155, 5220-6, 1995; Sharfe, N., Dadi, H. K., J J, O. S. and Roifnan,
C. M. "JAK-3 activation in human lymphocyte precursor cells," Clin
Exp Immunol 108, 552-6, 1997; Gurniak, C. B. and Berg, L. J.
"Murine JAK-3 is preferentially expressed in hematopoietic tissues
and lymphocyte precursor cells," Blood 87, 3151-60, 1996; Rolling,
C., Treton, D., Beckmann, P., Galanaud, P. and Richard, Y. "JAK-3
associates with the human interleukin 4 receptor and is tyrosine
phosphorylated following receptor triggering," Oncogene 10,
1757-61, 1995; Rolling, C., Treton, D., Pellegrini, S., Galanaud,
P. and Richard, Y. "IL4 and IL13 receptors share the gamma c chain
and activate STAT6, STAT3 and STAT5 proteins in normal human B
cells," FEBSLett 393, 53-6, 1996; Hanissian, S. H. and Geha, R. S.
"JAK-3 is associated with CD40 and is critical for CD40 induction
of gene expression in B cells," Immunity 6, 379-87, 1997; Danial,
N. N., Pernis, A. and Rothman, P. B. "Jak-STAT signaling induced by
the v-abl oncogene," Science 269, 1875-7, 1995.
[0080] Summary
[0081] Exposure of B-lineage lymphoid cells to ionizing radiation
induces an elevation of c-jun protooncogene mRNA levels. This
signal is abrogated by protein tyrosine kinase (PTK) inhibitors,
indicating that activation of an as yet unidentified PTK is
mandatory for radiation-induced c-jun expression. Experimental
evidence shows that the cytoplasmic tyrosine kinases BTK, SYK and
LYN are not required for this signal. Lymphoma B-cells rendered
deficient for LYN, SYK or both by targeted gene disruption showed
increased c-jun expression levels after radiation exposure, but the
magnitude of the stimulation was lower than in wild-type cells.
Thus, these PTK may participate in the generation of an optimal
signal. Notably, inhibitors of Janus family kinase 3 (JAK-3)
abrogated radiation-induced c-jun activation. This suggests that
JAKs are important regulators of radiation-induced c-jun
activation, and that JAK-3 inhibitors are useful for preventing or
treating diseases or conditions that result from chemical-induced
or radiation-induced c-jun activation.
[0082] All publications, patents, and patent documents are
incorporated by reference herein, as though individually
incorporated by reference. The invention has been described with
reference to various specific and preferred embodiments and
techniques. However, it should be understood that many variations
and modifications may be made while remaining within the spirit and
scope of the invention.
* * * * *