U.S. patent application number 16/452884 was filed with the patent office on 2020-04-09 for methods to treat allergic conditions.
The applicant listed for this patent is National Jewish Health. Invention is credited to Erwin W. Gelfand, Meiqin Wang.
Application Number | 20200108119 16/452884 |
Document ID | / |
Family ID | 45464077 |
Filed Date | 2020-04-09 |
View All Diagrams
United States Patent
Application |
20200108119 |
Kind Code |
A1 |
Gelfand; Erwin W. ; et
al. |
April 9, 2020 |
METHODS TO TREAT ALLERGIC CONDITIONS
Abstract
Disclosed are methods to treat allergic conditions, including
pulmonary and non-pulmonary conditions, in a subject by
administering a composition that inhibits Pim kinase. Also
disclosed are methods to treat allergic conditions in a subject by
administering a composition that induces expression of Runx3.
Inventors: |
Gelfand; Erwin W.;
(Englewood, CO) ; Wang; Meiqin; (Glendale,
CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Jewish Health |
Denver |
CO |
US |
|
|
Family ID: |
45464077 |
Appl. No.: |
16/452884 |
Filed: |
June 26, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15664550 |
Jul 31, 2017 |
10369194 |
|
|
16452884 |
|
|
|
|
14319252 |
Jun 30, 2014 |
|
|
|
15664550 |
|
|
|
|
13293911 |
Nov 10, 2011 |
8802099 |
|
|
14319252 |
|
|
|
|
61412194 |
Nov 10, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
29/00 20180101; A61P 11/06 20180101; A61P 37/08 20180101; A61P 1/00
20180101; A61P 11/00 20180101; A61K 31/4045 20130101; A61P 17/00
20180101; A61P 11/02 20180101; A61K 31/553 20130101; A61K 38/1719
20130101; A61K 31/7052 20130101; A61K 31/00 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 31/00 20060101 A61K031/00; A61K 31/4045 20060101
A61K031/4045; A61K 31/7052 20060101 A61K031/7052; A61K 31/553
20060101 A61K031/553 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was supported in part with funding provided
by NIH Grant Nos. HL-36577, HL-61005 and AI-77609 awarded by the
National Institutes of Health. The government of the United States
has certain rights to this invention.
Claims
1. A method to treat an allergic condition in a subject who has or
is at risk of having an allergic condition, comprising
administering to the subject a composition that inhibits Pim1
kinase.
2. The method of claim 1, wherein the allergic condition is
selected from the group consisting of allergic rhinitis, asthma,
airway hyperresponsiveness, airway inflammation, a food allergy,
eosinophilic esophagitis, chronic urticaria, atopic dermatitis,
occupational allergy, allergic conjunctivitis, hay fever, airborne
allergic sensitivities, stinging insect allergy, hypersensitivity
pneumonitis, eosinophilic lung diseases, inflammatory bowel
disease, ulcerative colitis, Crohn's disease and drug
allergies.
3. The method of claim 1, wherein the allergic condition is
asthma.
4. The method of claim 1, wherein the allergic condition is
rhinitis.
5. The method of claim 1, wherein the allergic condition is a food
allergy.
6. The method of claim 5, wherein the food allergy is a peanut
allergy.
7. The method of claim 1, wherein the subject has been sensitized
to an allergen and has been exposed to, or is at risk of being
exposed to, the allergen.
8. The method of claim 7, wherein the allergen is selected from the
group consisting of a food, a plant, a gas, a pathogen, a metal, a
glue and a drug.
9. The method of claim 1, wherein the composition comprises a
compound selected from the group consisting of a small molecule
inhibitor, an antibody, a chemical entity, a nucleotide, a peptide,
and a protein.
10. The method of claim 1, wherein the composition comprises a
small molecule inhibitor.
11. The method of claim 10, wherein the small molecule inhibitor is
a Pim1 kinase inhibitor.
12. Then method of claim 11, wherein the Pim1 kinase inhibitor is
selected from the group consisting of AR460770, AR440, SimI4A,
staurosporine, and bisindolylmaleimide.
13. The method of claim 11, wherein administration of the Pim1
kinase inhibitor induces expression of Runx3.
14. The method of claim 11, wherein administration of the Pim1
kinase inhibitor reduces CD4.sup.+ and CD8.sup.+ cell
proliferation.
15. The method of claim 11, wherein administration of the Pim1
kinase inhibitor suppresses Th2 differentiation.
16. The method of claim 11, wherein administration of the Pim1
kinase inhibitor suppresses Th17 differentiation.
17. The method of claim 1 wherein the composition is administered
by a delivery method selected from the group consisting of aerosol
delivery, parenteral delivery and oral delivery.
18-31. (canceled)
32. A method to treat an allergic condition in a subject who has or
is at risk of having an allergic condition, comprising
administering to the subject a composition that inhibits expression
of Pim1 kinase, wherein the allergic condition does not comprise a
pulmonary condition.
33. The method of claim 32, wherein the allergic condition is
selected from the group consisting of a food allergy, eosinophilic
esophagitis, chronic urticaria, atopic dermatitis, occupational
allergy, allergic conjunctivitis, airborne allergic sensitivities,
stinging insect allergy, inflammatory bowel disease, ulcerative
colitis, Crohn's disease and drug allergies.
34-57. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 15/664,550, filed Jul. 31, 2017, which is a
continuation application of U.S. application Ser. No. 14/319,252,
filed Jun. 30, 2014, now abandoned, which is a divisional
application of U.S. application Ser. No. 13/293,911, filed Nov. 10,
2011, now issued U.S. Pat. No. 8,802,099, which claims priority to
U.S. Provisional Application No. 61/412,194 having a filing date of
Nov. 10, 2010, the entire contents of each of which are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention is directed to a novel method for
treating conditions related to allergic disease, such as asthma,
airway hyperresponsivness (AHR) and intestinal allergy by
inhibition of Pim kinase as well as by inducing expression of Runx3
(Runt-related transcription factor-3).
BACKGROUND OF THE INVENTION
[0004] Allergic conditions now affect almost 1 in 3 individuals
during their lifetime and pose a major socio-economic burden on
society. These conditions, including asthma, allergic rhinitis,
eosinophilic esophagitis, atopic dermatitis, and intestinal allergy
most often have a genetic basis. Genetic susceptibility alone
cannot account for these conditions but gene-environment
interactions are responsible for the induction/inception of an
allergic disease and for the maintenance and progression of the
disease. Understanding the mechanisms underlying these conditions
is central to developing appropriate treatment strategies and even
preventative interventions. Defining the pathophysiology of these
atopic diseases/conditions has, on one hand been very fruitful,
with identification of critical cell-cell interactions, mediators
such as cytokines and chemokines, and unique signaling pathways,
but direct targeting of many of these circuits has not sustained in
the clinic. To a large extent this may be the result of targeting
individual downstream processes and not their upstream control or
convergence points. Attempts to block key mediators singly such as
anti-histamines, cytokines (IL-4, IL-5, IL-13) or cells
(eosinophils) have had limited success. The primary therapy for
treatment of these diseases/conditions remains corticosteroids, a
therapy without downstream specificity but multiple actions
upstream in the pathways. Corticosteroids are not immunomodulatory
but are anti-inflammatory. Moreover, when stopped, all disease
manifestations return. New therapeutic approaches are needed,
especially those that have the potential to modify existing disease
and prevent progression.
[0005] The survival kinases are defined as cytoplasmic
serine/threonine kinases that phosphorylate substrates which
contribute to the control of cell proliferation, survival,
differentiation, apoptosis, and tumorigenesis (Amaravadi, R., et
al. 2005. J. Clin Invest 115:2618-2624). Akt is a well-studied
survival kinase, in which gene amplifications have been
demonstrated in several cancers (Cheng, J. Q., et al. 1992. Proc.
Natl. Acad. Sci. USA 89:9267-9271; Bellacosa, A. D., et al. 1995.
Int J. Cancer 64:280-285; Stahl., J. M., et al. 2004. Cancer Res.
64:7002-7010) and inhibitors assessed for treatment of malignancy
(Masure, S. B., et al. 1999. Eur. J. Biochem. 265:353-360; Yang,
L., et al. 2004. Cancer Res. 64:4394-4399; Kondapaka, S. B., et al.
2003. Mol. Cancer Ther. 2:1093-1103). The provirus integration site
for Moloney murine leukemia virus (PIM), Pim kinase is another
potent survival kinase that has been implicated in cell survival
through suppression of myc-induced apoptosis (van Lohuizen, M., et
al. 1989. Cell 56:673-682). There are three subtypes of Pim kinases
(serine/threonine kinases) that control cell survival,
proliferation, differentiation, and apoptosis (Bachmann, M., et al.
2005. Int. J. Biochem. Cell Biol. 37:726-730; Wang, Z., et al.
2001. J. Vet. Sci. 2:167-179; Amaravadi R., et al. 2005. J. Clin
Invest. 115:2618-2624). Pim1 kinase and Pim2 kinase are primarily
restricted to hematopoietic cells and Pim3 kinase primarily is
expressed in brain, kidney, and mammary tissue (Mikkers, H., et al.
2004. Mol Cell Biol 24:6104-6115). Unlike other serine/threonine
kinases, these kinases are regulated via JAK/STAT activation driven
transcription of the Pim gene rather than by membrane recruitment
and phosphorylation (Fox, C. J., et al. 2005. J. Exp. Med.
201:259-266). Overexpression of Pim kinase has been demonstrated in
various human lymphoma, leukemic and prostatic cancers and the role
of Pim-induced oncogenic transformation has been extensively
studied in hematopoietic tumors (Amson, R., et al. 1989. Proc.
Natl. Acad. Sci. USA 86:8857-8861; Valdman, A., et al. 2004.
Prostrate 60:367-371; Cibull, T. L., et al. 2006. J. Clin. Pathol.
59:285-288; Nieborowska-Skorska, M., et al. 2002. Blood
99:4531-4539). Despite intensive studies on the role of Pim kinase
in the development of tumor cells, the role of Pim kinase in immune
cells has been less well studied. In human, Pim kinases are
expressed in eosinophils, and play a major role in IL-5-induced
eosinophil survival (Temple, R., et al. 2001. Am. J. Respir. Cell
Mol. Biol. 25:425-433; Andina, N., et al. 2009. J. Allergy Clin.
Immunol. 123:603-611). In addition, Pim1 expression was increased
in eosinophils from BAL fluid compared to blood from asthmatic
patients after allergen provocation (Stout, B. A., et al. 2004. J.
Immunol 173:6409-6417). In a recent study, Pim kinase was also
shown to promote cell survival in T cells (Fox, C. J., et al. 2005.
J. Exp Med. 201:259-266).
[0006] Pim1 kinase is involved in cell proliferation and
differentiation (Wang Z, et al. J Vet Sci. 2001; 2(3):167-179) and
has been implicated in cytokine-dependent signaling in
hematopoietic cells and T cells (Aho T L, et al. BMC Cell Biol.
2006; 7:21-29; Rainio E M, et al. J Immunol. 2002; 168(4): 1524-7).
It has been showed that Pim1 expression is enhanced during T cell
activation in a protein kinase C dependent manner (Wingett D, et
al. J Immunol. 1996; 156(2):549-57). Pim1 increased T cell
proliferation by enhancing activity of nuclear factor activated
T-cells (NFAT) thereby increasing IL-2 production in T cells
(Rainio E M, et al. J Immunol. 2002; 168(4): 1524-7).
[0007] Although it is well known that survival kinases regulate
common substrates like Bad or 4EBP1 to induce cell survival and
proliferation (Yan, B., et al. 2003. J. Biol. Chem.
278:45358-45367), the downstream activities of each kinases are
different. To date, the precise downstream target of Pim kinase is
not known. However, c-Myc, suppressor of cytokine signaling-1
(SOCS-1), PAP-1, PTP-U2S, and heterochromatin protein 1 (HP-1) all
are potential downstream targets of Pim kinase (van Lohuizen, M.,
et al. 1989. Cell 56:673-682; Chen, X. P., et al. 2002. Proc. Natl.
Acad. Sci. USA 99:2175-2180; Maita, H., et al. 2000. Eur. J.
Biochem. 267:5168-5178; Koike, N., et al. 2000. FEBS Lett
467:17-21; Wang, Z., et al. 2001. Arch Biochem Biophys 390:9-18).
Recently, nuclear factor of activated T-cells (NFATc1) was reported
to be a potential downstream substrate of Pim kinase (Rainio, E. M.
et al. 2002. J. Immunol 168:1524-1527). As the regulation of NFAT
activity has been shown to be important for normal selection of
thymocytes, NFAT may play a role in the functional development of T
cells (Patra, A. K. 2006. J. Immunol. 177:4567-4576) as well as in
the suppression of CD4.sup.+ and CD8.sup.+ T cell proliferation and
T cell cytokine production as a downstream substrate of Pim
kinase.
[0008] CD4.sup.+ T cells play a central role in controlling
allergic inflammation (Buss, W. W., et al. 1995. Am J Respir Crit
Care Med. 152:388-393). CD4.sup.+ T cells, especially Th2 cells
producing IL-4, IL-5, and IL-13, have been identified in BAL fluid
and airway tissues in asthmatics (Robinson, D. S., et al. 1992. N.
Engl J. Med. 326:298-304). The transfer of Th2 cells followed by
airway allergen challenge in mice was sufficient to induce airway
eosinophilia and AHR (Cohn, L., et al. 1997. J. Exp. Med.
186:1731-1747; Hogan, S. P., et al. 1998. J. Immunol.
161:1501-1509). Conversely, CD8.sup.+ T cells, which are also key
components of adaptive immunity, have drawn limited attention in
the pathogenesis of asthma. However, recent studies demonstrated
the increased numbers of CD8.sup.+ T cells in the lung tissues of
asthmatics (Azzawi, M., et al. 1990. Am. Rev. Respir. Dis.
142:1407-1413) and recent reports suggested that not only CD4.sup.+
T cells but also CD8.sup.+ T cells were essential to the
development of AHR and allergic inflammation (Hamelmann, E., et al.
1996. J. Exp Med. 183:1719-1729; Isogai, S., et al. 2004. J.
Allergy. Clin. Immunol. 114:1345-1352; Miyahara, N., et al. 2004.
J. Immunol. 172:2549-2558; Miyahara, N., et al. 2004. Nat. Med.
10:865-869). Subsets of CD8.sup.+ T cells, which produce IL-4,
IL-5, and IL-13 but not IFN-.gamma., labeled as Tc2 cells, are
known to increase AHR and airway inflammation (Croft, M., et al.
1994. J. Exp Med. 180:1715-1728; Seder, R. A., et al. 1992. J.
Immunol. 148:1652-1656; Coyle, A. J., et al. 1995. J. Exp. Med.
181:1229-1233). Thus, both CD4.sup.+ T cells and CD8.sup.+ T cells
play key roles in the pathogenesis of asthma.
[0009] Asthma is a multifactorial inflammatory disorder
characterized by persistent airway inflammation and airway
hyperresponsiveness (AHR) as a result of the cellular and molecular
responses induced by allergen exposure, infectious pathogens, or
chemical agents (Buss, W. W., et al. 2001. N. Engl. J. Med.
344:350-362; Umetsu, D. T., et al. 2002. Nat. Immunol. 3:715-720).
Several clinical and experimental investigations have shown that T
cells, especially Th2-type cells, play a pivotal role in the
development of AHR and eosinophilic inflammation through the
secretion of a variety of Th2 cytokines, including IL-4, IL-5, and
IL-13 (Wills-Karp, M., et al. 1998. Science 282:2258-2261;
Robinson, D. S., et al. 1992. N. Engl. J. Med. 326:298-304). These
cytokines bind to the extracellular Janus kinase (JAK) receptors
and subsequently induce the phosphorylation and activation of
signal transducers and activators of transcription (STAT), which
translocates into the nucleus, where it binds to DNA and affects
basic cell functions, cellular growth, differentiation and death
Aaronson, D. S., et al. 2002. Science 296:1653-1655.
[0010] Knowledge of the pathogenesis of atopic diseases/conditions
was originally interpreted within the framework of a binary T
helper 1 (Th1)/Th2 paradigm. This has now been broadened to
incorporate other T cell subsets. Importantly, the differentiation
and commitment of these populations of T cells is shaped by
transcriptional circuits that center on key transcriptional
regulators, the proteins that bind DNA to activate or repress gene
expression. Runt-related transcription factors (Runx), are a novel
family of transcription factors which are key regulators of
lineage-specific gene expression, and are responsible for the
development of allergic responses (Fainaru, O., et al. 2004. EMBO
J. 23:969-979; Fainaru, O., et al. 2005. Proc. Natl. Acad. Sci. USA
102:10598-10603). There are three mammalian Runx genes: Runx1,
Runx2, and Runx3. Runx1 is required for hematopoiesis (Okuda, T.,
et al. 1996. Cell 84:321-330) and Runx2 is critical regulator of
osteogenesis (Ducy, P., et al. 1997. Cell 89:747-745). The Runx3
gene resides on human chromosome 1p36.1 (Levanon, D., et al. 1994.
Genomics 23:425-432), which maps to a region containing
susceptibility genes for asthma (Haagerup, A., et al. 2002. Allergy
57:680-686) and on mouse chromosome 4 (Calabi, F., et al. 1995.
Genomics 26:607-610), which contains a susceptibility gene for
atopic dermatitis (Christensen U., et al. 2009. 126:549-557). Runx3
is thought to play a critical role in regulating T-cell
development, the differentiation of Th1/Th2 cells and Th1/Th2
cytokine production and the development of an allergic
disease/condition. It has been reported that Pim1 kinase regulates
Runx expression in vitro (Aho T L, et al. BMC Cell Biol. 2006;
7:21-29) and loss of Runx3 results in spontaneous development IBD,
as well as allergic asthma (Brenner O, et al. Proc Natl Acad Sci
USA. 2004; 101(45):16016-21; Fainaru O, et al. EMBO J. 2004; 23(4):
969-79). The Runx transcription factors are also key regulators of
lineage-specific gene expression (Komine O, et al. J Exp Med. 2003;
198 (1): 51-61).
[0011] Peanut allergy is one of the most common food allergies
characterized by acute allergic diarrhea and intestinal
inflammation. During an allergic reaction, several cell types,
including Th2 cells (T-helper cells), mast cells, and eosinophils,
are recruited to the intestine and activated to release cytokines
and chemokines, contributing to increased intestinal inflammation
(Kweon M N, et al. J Clin Invest 2000; 106:199-206; Wang M, et al.
J. Allergy Clin Immunol 2010, 126 (2): 306-316). CD4T cells (i.e. T
cells that express CD4), especially Th2 cells, which are known to
produce interleukin-4 (IL-4) and interleukin-13 (IL-13), are
considered critical in the development of allergic diarrhea and
intestinal inflammation (Knight A K, et al. Am J Physiol
Gastrointest Liver Physiol. 2007; 293(6): G1234-43; Kweon M N, et
al. J Clin Invest. 2000; 106(2): 199-206). In patients with food
allergies, increased numbers of activated T cells have been
correlated with elevated levels of Th2 cytokines as well as the
degree of gastrointestinal (GI) inflammation and dysfunction
(Eigenmann P A. Pediatr Allergy Immunol 2002; 13:162-71; Eigenmann
P A, et al. Adv Exp Med Biol 1996; 409:217). It has been shown that
after treatment with oral peanut immunotherapy, levels of
peanut-specific Th2-cytokine (IL-4 and IL-5) production by
peripheral blood mononuclear cells (PBMCs) was significantly
decreased in children with peanut anaphylaxis (Blumchen K, et al. J
Allergy Clin Immunol. 2010; 126(1):83-91).
[0012] More evidence in humans and mice has shown that Th17 cells,
a novel subset of IL-17-producing CD4.sup.+ T cells, play an
important role in the pathogenesis of immune-mediated diseases,
including asthma and inflammatory bowel disease (IBD) (Tesmer L A,
et al. Immunol Rev. 2008, 223:87-113 Kolls J K and Linden A.
Immunity. 2004, 21:467-476). Th17 cells exist and are found
constitutively in the small intestine of naive mice housed under
conventional conditions (Ivanov I I, et al. Cell. 2006; 126(6):
1121-33). Increased levels of IL-17A (a member of the IL-17 family)
have been found in the small intestine of peanut allergy mouse
models as well as in the small intestine or in the peripheral blood
of food allergy patients (Wang M, et al. J. Allergy Clin Immunol
2010, 126 (2): 306-316). The level of IL-17A is associated with the
severity of diarrhea and intestinal inflammation. These data
suggested that CD4.sup.+ T cells that produce Th2 and Th17
cytokines play an important role in food allergy. However, the
signal pathway involved in Th2-, Th17-cells responding to allergic
food reactions has not been well defined.
SUMMARY OF INVENTION
[0013] The present invention provides for a method to treat an
allergic condition in a subject having or at risk of having an
allergic condition, comprising administering a composition that
inhibits Pim1 kinase. In one aspect, administration of the Pim1
kinase inhibitor induces expression of Runx3. In another aspect,
administration of the Pim1 kinase inhibitor reduces CD4+ and CD8+
proliferation. In yet another aspect, administration of the Pim1
kinase inhibitor suppresses Th2 differentiation. In still another
aspect, administration of the Pim1 kinase inhibitor suppresses Th17
differentiation.
[0014] In another embodiment, the present invention is directed
toward a method to treat an allergic condition in a subject having
or at risk of having an allergic condition, comprising
administering a composition that induces expression of Runx3. In
one aspect the composition interacts with a regulator of Runx3
expression. In another aspect, the regulator of Runx3 expression is
selected from a Pim kinase, CxCL12, core binding factor-beta,
transducin-like enhancer protein 1, IL-7, Stat 5, ETS-1, interferon
regulatory factor 4 (IRF-4) or other regulators of Runx3. In still
another aspect, the composition inhibits the activity of a compound
selected from IRF-4 or Pim kinase. In yet another aspect the
composition comprises an IRF-4 inhibitor. In a preferred
embodiment, the composition comprises a Pim kinase inhibitor
selected from a Pim1 kinase inhibitor, a Pim2 kinase inhibitor or a
Pim3 kinase inhibitor.
[0015] In still another embodiment, the present invention is
directed toward a method to treat an allergic condition in a
subject who has or is at risk of having an allergic condition,
comprising administering to the subject a composition that inhibits
Pim1 kinase, wherein the allergic condition does not comprise a
pulmonary condition. In another aspect the allergic condition is
selected from a food allergy, eosinophilic esophagitis, chronic
urticaria, atopic dermatitis, occupational allergy, allergic
conjunctivitis, airborne allergic sensitivities, stinging insect
allergy, inflammatory bowel disease, ulcerative colitis, Crohn's
disease or drug allergies. In one aspect, the food allergy is a
peanut allergy. In one aspect, the Pim1 kinase inhibitor induces
expression of Runx3. In another aspect, the administration of the
Pim1 kinase inhibitor reduces CD4+ and CD8+ proliferation. In yet
another aspect, administration of the Pim1 kinase inhibitor
suppresses Th2 differentiation. In still another aspect,
administration of the Pim1 kinase inhibitor suppresses Th17
differentiation.
[0016] In yet another embodiment, the present invention is directed
toward a method to treat an allergic condition in a subject who has
or is at risk of having an allergic condition, comprising
administering to the subject a composition that induces expression
of Runx3, wherein the composition does not comprise a Pim kinase
inhibitor. In one aspect the composition interacts with a regulator
of Runx3 expression. In another aspect, the regulator of Runx3
expression is selected from CxCL12, core binding factor-beta,
transducin-like enhancer protein 1, IL-7, Stat 5, ETS-1, and IRF-4.
In still another aspect, the composition inhibits the activity of
IRF-4. In yet another aspect the composition comprises an IRF-4
inhibitor.
[0017] In another aspect of the present invention, the composition
activates the activity of a compound selected from CxCL12, core
binding factor-beta, transducin-like enhancer protein 1,
interleukin-7 (IL-7), Stat 5, or ETS-1. In another aspect, this
activator is selected from G proteins, phosphatidylinositol-3
kinase (PI3K), JAK kinases, Rho GTPases, or focal
adhesion-associated proteins.
[0018] In other embodiments of the present invention, the allergic
condition is selected from allergic rhinitis, asthma, airway
hyperresponsiveness, a food allergy, eosinophilic esophagitis,
chronic urticaria, atopic dermatitis, occupational allergy,
allergic conjunctivitis, hay fever, airborne allergic
sensitivities, stinging insect allergy, hypersensitivity
pneumonitis, eosinophilic lung diseases, inflammatory bowel
disease, ulcerative colitis, Crohn's disease or drug allergies. In
a preferred embodiment, the allergic disease is asthma. In still
another aspect, the allergic disease is rhinitis. In another
preferred embodiment, the allergic disease is a food allergy. In
still another preferred embodiment, the allergic disease is a
peanut allergy.
[0019] In another embodiment of the present invention, the methods
further provide that the subject has been sensitized to an allergen
and has been exposed to, or is at risk of being exposed to, the
allergen. In one aspect, the allergen is selected from a food, a
plant, a gas, a pathogen, a metal, a glue or a drug.
[0020] In another embodiment of the present invention, the
composition comprises a compound selected from a small molecule
inhibitor, an antibody, a chemical entity, a nucleotide, a peptide
or a protein. In a preferred embodiment, the composition comprises
a small molecule inhibitor. In one aspect, the small molecule
inhibitor is a Pim kinase inhibitor selected from a Pim1 kinase
inhibitor, a Pim2 kinase inhibitor or a Pim3 kinase inhibitor. In a
preferred embodiment, the Pim kinase inhibitor is a Pim1 kinase
inhibitor. In still another aspect the Pim1 kinase inhibitor is
selected from AR460770 (also referred to as AARY-770 and
AR00460770), AR440, SimI4A, staurosporine, bisindolymaleimide or
other Pim1 kinase inhibitors.
[0021] In still another aspect of the present invention, the
composition is administered by a delivery method selected from
aerosol delivery, parenteral delivery or oral delivery.
[0022] All patents and publications referenced herein are
incorporated by reference in their entireties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A-1E shows data that Pim1 kinase is expressed in the
mouse small intestine. FIG. 1A is a protocol for induction of
PE-induced intestinal allergy. FIG. 1B is a western blot analysis
of Pim1 kinase expression in the jejunum from sensitized and
challenged mice and control mice. FIG. 1C shows the relative mRNA
expression levels of Pim family members determined by quantitative
RT-PCR. FIG. 1D shows a representative immunohistochemical staining
for Pim1 and Pim3 kinases in intestinal tissue from PE/PE and
control PBS/PE mice. FIG. 1E shows the quantitation of mucosal Pim1
and Pim3 kinase-expressing cells in the jejunum. Results were
obtained from 3 independent experiments, and each experiment
included 4 mice per group (n=12). *P<0.01 between PE/PE and
PBS/PE groups. PBS/PE, sham sensitized but PE challenged; PE/PE, PE
sensitized and challenged.
[0024] FIGS. 2A-2C shows the expression of Runx3 in the mouse small
intestine. FIG. 1A shows the relative expression of Runx3, Runx1,
and Cbf.beta. mRNA levels in the jejunum of sensitized and
challenged mice determined by quantitative RT-PCR. FIG. 2B is a
western blot analysis of Runx3 protein levels in the jejunum of
sensitized and challenged mice and control mice. FIG. 2C shows the
quantitation of mucosal Runx3-expressing cell numbers in the
jejunum of PBS/PE and PE/PE mice. Results are representative of 3
independent experiments, and each experiment included 4 mice per
group. *P<0.05, **P<0.01, #P<0.001 between PE/PE and
PBS/PE groups. PBS/PE, nonsensitized but challenged with PE group;
PE/PE, sensitized and challenged with PE group.
[0025] FIGS. 3A-3G shows inhibition of Pim1 kinase reduces
intestinal responses. FIG. 3A shows the kinetics of development of
diarrhea assessed 30 minutes after the last challenge in mice
treated with or without AR460770. FIG. 3B shows the symptom scores
as assessed 30 minutes after oral challenge. FIG. 3C shows the
plasma levels of histamine as assessed within 30 minutes after the
last oral challenge. FIG. 3D shows the quantitation of mucosal mast
cell numbers in the jejunum using chloroacetate esterase staining.
FIG. 3E shows the quantitation of mucosal eosinophil numbers in the
jejunum using immunohistochemistry and anti-MBP antibody. FIG. 3F
shows the quantitation of mucosal goblet cells numbers in the
jejunal epithelium by PAS staining 24 hrs after the last challenge.
For mast cells and eosinophils, results are expressed as the number
of chloroacetate esterase- or anti-major basic protein-stained
cells per square millimeter of lamina propria, respectively. For
goblet cells, the number of PAS.sup.+ cells was divided by the
total number of epithelial cells in the villi. FIG. 3G shows the
quantitative analysis of numbers of CD4 and CD8 T cells in jejunal
tissue. Results were obtained from 3 independent experiments, and
each experiment included 4 mice per group. #P<0.01 between
PBS/PE/vehicle and PE/PE/vehicle groups. *P<0.01, **P<0.001
comparing vehicle-treated with AR460770-treated sensitized and
challenged mice.
[0026] FIGS. 4A-4C shows the effect of inhibition of Pim1 kinase on
cytokines and transcription factor expression. FIG. 4A shows the
quantitative RT-PCR analysis of Th1, Th2, and Th17 cytokine mRNA
expression in the jejunum of PBS/PE/vehicle mice and PE/PE mice
treated with AR460770 or vehicle. FIG. 4B shows the key
transcription factors for Th1, Th2, and Th17 cytokine expression
levels in the jejunum of PBS/PE/vehicle mice and PE/PE mice treated
with AR460770 or vehicle. Relative mRNA expression levels of T-bet,
ROG, GATA43, NFATc1, and ROR.gamma.t in the jejunum determined by
quantitative RT-PCR. FIG. 4C shows the percentage of
IFN-.gamma..sup.+CD3.sup.+CD4.sup.+, IL-4.sup.+CD3.sup.+CD4.sup.+,
IL-13.sup.+CD3.sup.+CD4.sup.+ and IL-17A.sup.+CD3.sup.+CD4.sup.+
cells in the MLN of PBS/PE/vehicle mice and PE/PE mice treated with
the inhibitor or vehicle. The data shown are representative of 3
independent experiments. #P<0.05; ##P<0.01 between
PBS/PE/vehicle and PE/PE/vehicle groups (n=12). *P<0.05;
**P<0.01 between PE/PE mice treated with AR460770 (100 mg/kg)
and vehicle.
[0027] FIGS. 5A-5D show Pim1 kinase regulates Runx3 transcription
factor expression in intestine. FIG. 5A shows Runx3 protein is
upregulated by Pim1 kinase inhibitor treatment in the jejunum of
PE/PE mice. Representative Western blots show Runx3 in jejunal
extracts from PBS/PE/vehicle mice and PE/PE mice treated with or
without AR460770 analyzed for the expression of Runx3, .beta.-actin
was used as a loading control. A representative of 3 independent
experiments is shown. FIG. 5B shows the quantitative RT-PCR
analysis of the induction of Runx mRNA expression in the jejunum of
PBS/PE/vehicle mice and PE/PE mice treated with or without
AR460770. FIG. 5C shows the quantitation of mucosal Runx3-positive
cell numbers in the jejunum. Results are expressed as the number of
Runx3-stained cells per square millimeter of lamina propria.
Results are from 3 independent experiments with 4 mice per group.
#P<0.01 between PBS/PE/vehicle and PE/PE/vehicle groups.
*P<0.01 between PE/PE mice treated with AR460770 and control
vehicle. FIG. 5D: Runx1, Runx3 and Cbf.beta. (core binding factor
beta; a transcriptional co-activator that is known to enhance
DNA-binding of Runx proteins) mRNA expression was detected in the
intestine of mice. Non-sensitized peanut challenged mice are
indicated by "PBS/PE". Sensitized-peanut challenged mice are
indicated by "PE/PE" and peanut challenged mice treated with the
Pim1 kinase inhibitor is indicated as "PE/PE+AR770": 1 mg/kg, 10
mg/kg, 30 mg/kg or 100 mg/kg.
[0028] FIGS. 6A-6E show Pim1 kinase inhibitor modulates Runx3
expression and suppresses the differentiation of naive CD4 T cells
into the Th2 and Th17 lineage in vitro. FIG. 6A shows the cell
proliferation reported as number of cells. FIG. 6B shows the cell
proliferation as measured by .sup.3H-thymidine incorporation (cpm)
and expressed as a % of the vehicle-treated group. FIG. 6C shows
the levels of cytokine production in the supernatants of cultured
CD4 T cells. CD4 T cells were cultured under Th1, Th2, and Th17
polarizing conditions in the presence or absence of the inhibitor
for 6 days after which the cells were stimulated with anti-CD3 and
anti-CD28 for 24 hrs and supernatants were collected and assayed
for cytokines by ELISA. FIG. 6 D shows Pim1 kinase regulates Runx3
and cell-specific transcription factor mRNA expression in naive CD4
T cells differentiated in vitro into Th2 or Th17 cells as shown by
quantitative RT-PCR. FIG. 6E shows a western blot analysis of Runx3
protein levels in the polarized Th1, Th2, and Th17 cells. The cells
were cultured as previously described in FIG. 6C and on day 6 cells
were lysed and processed for Western blot analysis with anti-Runx3.
.beta.-actin was used as a loading control. A representative
Western blot from one of 3 similar experiments is shown. For other
panels, results are from 3 independent experiments, 4 mice/group
(n=12). *P<0.05; **P<0.01 comparing vehicle-treated and
AR460770 treated-cells. NS: nonsignificant comparing
vehicle-treated and AR460770 treated-cells.
[0029] FIGS. 7A-7B show the expression levels of Pim1 kinase in
lungs following OVA sensitization and challenge. Pim1 kinase levels
were determined by Western blot in lungs of mice which were
sensitized and challenged with OVA or received sham sensitization
and OVA challenge. Expression levels were examined at three time
points: 6 hrs after the second OVA challenge, 6 hrs after the third
OVA challenge, and 24 hrs after third OVA challenge. Experiments
were repeated at least 3 times. GAPDH was used as a loading control
(FIG. 7A) and the average optical densitometry was expressed by
standardizing to total ERK (FIG. 7B).
[0030] FIGS. 8A-8E show the effect of Pim1 kinase inhibition on
airway responses following primary allergen challenge. The effects
of a Pim1 kinase inhibitor were determined in the primary allergen
challenge model. (FIG. 8A) Changes in pulmonary resistance (RL) in
response to increasing doses of methacholine (MCh), (FIG. 8B) Cell
composition in BAL fluid. Macro; macrophages, Lympho; lymphocytes,
Eos; eosinophils, Neu; neutrophils. (FIG. 8C) BAL fluid cytokine
levels. (FIG. 8D) Lung tissue histology following staining with
hematoxylin and eosin (H&E) and (FIG. 8E) periodic acid-Schiff
(PAS). Quantitative analysis of inflammatory and PAS.sup.+ cells in
lung tissue was performed as described in Materials and Methods.
Mice were sham sensitized followed by OVA challenge (PBS/OVA) or
sensitized and challenged with OVA (OVA/OVA). Pim1 inhibitor,
AR00460770, was administered at doses of 1, 10, 30, or 100 mg/kg.
Control groups received vehicle. (n=8). *p<0.05; compared to
OVA/OVA vehicle. #p<0.05; compared to PBS/OVA vehicle.
**p<0.05; compared to OVA/OVA AR00460770 1 mg/kg. ##p<0.05:
compared to PBS/OVA AR00460770 30 mg/kg.
[0031] FIGS. 9A-9E show the effect of Pim1 kinase inhibition on
airway responses in the secondary allergen challenge model. The
effects of Pim1 kinase inhibition were determined in the secondary
allergen challenge model. (FIG. 9A) Changes in pulmonary resistance
(RL) in response to increased dose of methacholine (MCh), (FIG. 9B)
Cell composition in BAL fluid, (FIG. 9C) BAL fluid cytokine levels,
(FIG. 9D) lung tissue histology following staining with hematoxylin
and eosin (H&E), and (FIG. 9E) periodic acid-Schiff (PAS).
Quantitative analysis of inflammatory and goblet cells was as
described in Materials and Methods. Mice were sham sensitized
followed by OVA challenge (PBS/OVA) or sensitized and challenged
with OVA (OVA/OVA). Pim1 inhibitor was administered at doses of 1,
10, 30, or 100 mg/kg. Control groups received vehicle. (n=8).
*p<0.05; compared to OVA/OVA vehicle or OVA/OVA AR00460770 1
mg/kg. #p<0.05 compared to OVA/OVA AR00460770 10 mg/kg.
**p<0.05 compared to OVA/OVA vehicle or OVA/OVA AR00460770 1
mg/kg.
[0032] FIG. 10 shows the effects of Pim kinase inhibition on
numbers of CD4.sup.+ and CD8.sup.+ T cells. In OVA sensitized and
challenged mice, the numbers of CD4.sup.+ T cells (CD4) and
CD8.sup.+ T cells (CD8) in the lungs of mice treated with a Pim1
kinase inhibitor (OVA/OVA AR00460770) or vehicle (OVA/OVA vehicle)
were determined. MNCs isolated from lungs were stained with
anti-CD3, anti-CD4, and anti-CD8 for flow cytometry analysis as
described in Materials and Methods. The data shown were
representative of 3 independent experiments. *p<0.05 compared to
vehicle.
[0033] FIGS. 11A-11C show the effect of Pim1 kinase inhibition on
cell proliferative responses and cytokine production from CD4.sup.+
and CD8.sup.+ T cells. Purified spleen CD4.sup.+ and CD8.sup.+ T
cells were preincubated with the Pim1 kinase inhibitor followed by
anti-CD3 and anti-CD28 stimulation. FIG. 11A shows the cell
proliferation assays carried out 24 hrs after anti-CD3/anti-CD28
stimulation and calculated from the uptake of tritium-labeled
thymidine. (n=8). FIG. 11B shows the quantitation of cytokine
levels in supernates from anti-CD3/anti-CD28 stimulated CD4.sup.+
and in FIG. 11C CD8.sup.+ T cells. CPM; count per minutes.
*p<0.05; compared to vehicle-treated cells.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention generally relates to methods of
treating an allergic condition in a subject who has or who is at
risk of having the allergic condition. In a preferred embodiment,
the method comprises administering a composition that inhibits Pim1
kinase. In still another preferred embodiment, the method comprises
administering a composition that induces Runx3 expression.
[0035] The inventors have identified a critical role of Pim1 kinase
in the development of allergic conditions. In particular,
inhibition of Pim1 kinase effectively reduces the development of a
full spectrum of allergen-induced responses, including lung
inflammatory responses and intestinal inflammatory response at
least in part through limiting the expansion and activities of
effector CD4.sup.+ and CD8.sup.+ T cells. As such, inhibition of
Pim1 kinase expression and/or activity represents a novel
therapeutic target in the treatment of various allergic
conditions.
[0036] The inventors have also identified a critical role of Runx3
in the development of allergic conditions. In particular, loss of
Runx3 results in the development of atopy. In addition, the
inventors have determined that Runx3 mRNA and protein levels are
decreased following allergen sensitization and challenge. In turn,
Runx3 mRNA and protein levels are increased when tolerance to an
inciting allergen is induced and is associated with decreased mast
cell, eosinophil, and goblet cell accumulation in the tissues as
well as decreases in Th2 cytokine production, thus providing that
Runx3 plays an important role in regulating the development of
allergy.
[0037] More particularly, the inventors have shown that Pim1 kinase
is essential to the development of peanut-induced intestinal
allergy. Inhibition of Pim1 kinase prevented peanut-induced
diarrhea and intestinal inflammation in vivo as well as impairing
Th2 or Th17 cell differentiation in vitro by enhancing Runx3
expression and repressing nuclear factor of activated T-cells
cytoplasmic 1 (NFATc1) expression (the transcriptional activity of
NFATc1 is enhanced by Pim1 kinase (Wang, M., et al. 2010. J.
Allergy Clin. Immunol. 126:306-316).
[0038] In addition the inventors have determined that following
administration of a Pim1 kinase inhibitor to sensitized and
challenged mice, all of the clinical manifestations of the
disease/condition and the accumulation of mast cells, and
eosinophils in the tissues were prevented. In particular, the
decreases in Runx3 expression were also prevented. Therefore, the
data identify the transcription factor Runx3 as an upstream
convergence point involved in the regulation of atopy development
and is responsible for the protection against disease through
induction of tolerance. Targeting the regulation of Runx3
expression represents a novel approach for the prevention of
allergic conditions and inhibition of Pim1 kinase expression and/or
activity is a means for achieving repression of allergic responses
through induction (i.e. upregulation) or sustaining expression of
Runx3.
[0039] As described in the examples below, together with results
which demonstrate the reduction of both Th2 and Th1 cytokines in
BAL fluid and the reduction of both CD4.sup.+ and CD8.sup.+ T cells
following Pim inhibitor treatment, concerns that the suppressive
effects of Pim kinase inhibition on allergen-induced airway
responses were due to its toxic effects on immune cells were
considered. However, as the cell numbers and viability were not
altered in in vitro cultures of CD4.sup.+ and CD8.sup.+ T cells
with up to 10 .mu.M of the inhibitor, the effects on airway
responses were not likely induced by drug-mediated cell toxicity.
Furthermore, Pim inhibitor treatment in sham-sensitized but
OVA-challenged mice did not alter airway responsiveness to MCh,
further indicating that the Pim inhibitor did not exhibit toxic
effects on lung resident cells, including airway smooth muscle.
[0040] In addition, the inventors have determined that Pim1 kinase
inhibition alters the activities of the CD4.sup.+ and CD8.sup.+ T
effector cells in airways, and determined that the numbers of
CD4.sup.+ and CD8.sup.+ T cells in the lungs are dramatically
decreased in Pim inhibitor-treated, sensitized and challenged mice.
Further, in in vitro experiments Pim inhibitor treatment
demonstrates suppressive effects on the cell proliferation of
CD4.sup.+ and CD8.sup.+ T cells in response to stimulation with
anti-CD3 and anti-CD28. These results show that the of inhibition
of Pim1 kinase limits responses through interference with the
expansion of critical effector cells, CD4.sup.+ and CD8.sup.+ T
cells in the airways, and possibly eosinophils.
[0041] According to the present invention, allergic conditions,
include but are not limited to pulmonary conditions such as
allergic rhinitis, asthma, airway hyperresponsiveness, and hay
fever as well as other allergic conditions including but not
limited to a food allergy, eosinophilic esophagitis, chronic
urticaria, atopic dermatitis, occupational allergy, allergic
conjunctivitis, airborne allergic sensitivities, stinging insect
allergy, hypersensitivity pneumonitis, eosinophilic lung diseases,
inflammatory bowel disease, ulcerative colitis, Crohn's disease and
drug allergies. More specifically, symptoms of the allergies,
including but not limited to diarrhea and intestinal inflammation
as well as asthma and airway hyperresponsiveness, is apparently or
obviously, directly or indirectly triggered by an allergen to which
a subject has previously been sensitized. Sensitization to an
allergen refers to being previously exposed one or more times to an
allergen such that an immune response is developed against the
allergen. Responses associated with an allergic reaction (e.g.,
histamine release, edema, vasodilatation, bronchial constriction,
airway inflammation, airway hyperresponsiveness, asthma, allergic
rhinitis (hay fever), nasal congestion, sneezing, running nose,
skin rash, diarrhea including acute allergic diarrhea and
intestinal inflammation), typically do not occur when a naive
subject is exposed to the allergen for the first time, but once a
cellular and humoral immune response is produced against the
allergen, the subject is "sensitized" to the allergen. Allergic
reactions then occur when the sensitized individual is re-exposed
to the same allergen (e.g., an allergen challenge). Once a subject
is sensitized to an allergen, the allergic reactions can become
worse with each subsequent exposure to the allergen, because each
re-exposure not only produces allergic symptoms, but further
increases the level of antibody produced against the allergen and
the level of T cell response against the allergen.
[0042] According to the present invention, inflammation is
characterized by the release of inflammatory mediators (e.g.,
cytokines or chemokines) which recruit cells involved in
inflammation to a tissue. A condition or disease associated with
allergic inflammation is a condition or disease in which the
elicitation of one type of immune response (e.g., a Th2-type immune
response) against a sensitizing agent, such as an allergen, can
result in the release of inflammatory mediators that recruit cells
involved in inflammation in a subject, the presence of which can
lead to tissue damage and sometimes death. A Th2-type immune
response is characterized in part by the release of cytokines which
include IL-4, IL-5, IL-13 and IL-17. The present invention is
particularly useful for treating allergen-induced food allergies
(such as peanut allegories) and airway hyperresponsiveness and
airway inflammation, including, allergen-induced asthma and
rhinitis.
[0043] Accordingly, various embodiments of the present invention
include treating a patient that has been sensitized to an allergen
and has been or is at risk of becoming exposed to the allergen.
Such allergens can be related to a food, a plant, a gas, a
pathogen, a metal, a glue or a drug. Examples of food allergens
include but are not limited to groundnuts such as peanuts; nuts
from trees including Brazilian nuts, hazelnuts, almonds, walnuts;
fruit, milk, eggs, fish, shellfish, wheat, or gluten. Examples of
plant allergens include but are not limited to pollen, trees,
grass, weeds, ragweed, poison Oak or poison ivy. Examples of gas
allergens include but are not limited to environmental tobacco
smoke, and carbon monoxide. Examples of pathogen allergens include
but are not limited to mold, viruses or bacteria. Examples of metal
allergens include but are not limited to lead, nickel, chromate, or
cobalt. Examples of drug allergens include but are not limited to
penicillin, sulfur, or aspirin. Additional allergens include but
are not limited to latex, dust mites, pet dander (skin flakes),
droppings from cockroaches, rodents and other pests or insects.
[0044] According to the present invention, "airway
hyperresponsiveness" or "AHR" refers to an abnormality of the
airways that allows them to narrow too easily and/or too much in
response to a stimulus capable of inducing airflow limitation. AHR
can be a functional alteration of the respiratory system resulting
from inflammation in the airways or airway remodeling (e.g., such
as by collagen deposition). Airflow limitation refers to narrowing
of airways that can be irreversible or reversible. Airflow
limitation or airway hyperresponsiveness can be caused by collagen
deposition, bronchospasm, airway smooth muscle hypertrophy, airway
smooth muscle contraction, mucous secretion, cellular deposits,
epithelial destruction, alteration to epithelial permeability,
alterations to smooth muscle function or sensitivity, abnormalities
of the lung parenchyma and infiltrative diseases in and around the
airways. Many of these causative factors can be associated with
inflammation. AHR can be triggered in a patient with a condition
associated with the above causative factors by exposure to a
provoking agent or stimulus. Such stimuli include, but are not
limited to, an allergen.
[0045] According to the present invention, treatment of a subject
having an allergic condition can commence as soon as it is
recognized (i.e., immediately) by the subject or by a clinician
that the subject has been exposed or is about to be exposed to an
allergen. Treating the subject can comprise administering a
composition including but not limited to a small molecule
inhibitor, an antibody, a chemical entity, a nucleotide, a peptide
or a protein that inhibits Pim kinase. Inhibiting Pim kinase
includes both direct inhibition of the kinase as well as inhibition
of the expression of the kinase. Inhibition of a Pim kinase can be
by any mechanism, including, without limitations, decreasing
activity of the Pim kinase, increasing inhibition of Pim kinase,
degradation of Pim kinase, a reduction or elimination of expression
of Pim kinase. For example, the action of Pim kinase can be
decreased by blocking or reducing the production of Pim kinase,
"knocking out" the gene encoding Pim kinase, reducing Pim kinase
activity, or inhibiting the activity of Pim kinase. Additionally,
binding to Pim kinase to prevent its wild-type enzymatic activity,
including competitive and noncompetitive inhibition, inhibiting
transcription, and regulating expression can also inhibit Pim
kinase. Small molecule inhibitors include but are not limited to a
Pim1 kinase inhibitor, a Pim2 kinase inhibitor, and a Pim3 kinase
inhibitor. Various Pim1 kinase inhibitors include but are not
limited to AR460770 (also referred to as AR00460770, ARRY770 and
AR770; ARRAY Biopharma), AR440 (ARRAY Biopharma), SimI4A,
staurosporine, bisindolylmaleimide or triazolopyridine Pim kinase
inhibitor compounds (as described for example in U.S. Patent
Publication Nos. US2011/014485 and US2011/0144100). In one
embodiment an antibody prevents or inhibits expression and/or
activity of a Pim kinase. In one aspect, the antibody prevents or
inhibits expression and/or activity of Pim1 kinase.
[0046] In accordance with the present invention, acceptable
protocols to administer the composition including the route of
administration and the effective amount of the composition to be
administered to a subject can be determined by those skilled in the
art. The composition of the present invention can be administered
in vivo or ex vivo. Suitable in vivo routes of administration can
include, but are not limited to, aerosol, oral, nasal, inhaled,
topical, intratracheal, transdermal, rectal, or parenteral routes.
Preferred parenteral routes can include, but are not limited to,
subcutaneous, intradermal, intravenous, intramuscular, or
intraperitoneal routes.
[0047] According to the present invention, Pim1 kinase inhibitors
are able to induce expression of Runx3. These inhibitors also
reduce expression of CD4.sup.+ and CD8.sup.+ T cell proliferation
and have the ability to suppress Th2 differentiation and/or Th17
differentiation. The administration of a Pim1 kinase inhibitor
prevents the development of AHR, airway inflammation and BAL
cytokine production in subjects (for example mice) sensitized and
challenged to allergen and attenuates the consequences of secondary
challenges in previously sensitized and challenged subjects. These
suppressive effects are manifested on both CD4+ and CD8+ T
cells.
[0048] In one embodiment, the method of treating an allergic
condition can comprise administering a composition comprising a
compound that interacts with a regulator of Runx3 expression (mRNA
or protein expression). Examples of a regulator include but are not
limited to a Pim kinase. Other examples include transcriptional
factors and regulators such as CxCL12 (chemokine (C-X-C motif)
ligand 12), core binding factor-beta (CBFbeta, also known as
polyomavirus enhancer binding protein 2 beta and is known to form a
heterodimer with Runx1), transducin-like enhancer protein 1 (TLE1,
a transcriptional co-repressor that is known to bind Runx1 and
Runx3), interleukin-7 (IL-7), signal transducer and activator of
transcription 5A (Stat 5), ETS-1, interferon regulatory factor 4
(IRF-4; known to be important in the regulation of interferons in
response to infection by viruses and is also lymphocyte specific
and negatively regulates Toll-like receptor (TLR) signaling that is
central to the activation of innate and adaptive immune responses)
or demethylating agents. In a preferred embodiment, the composition
inhibits the activity of a compound such as Pim kinase and IRF4.
Pim kinases include but are not limited to Pim1 kinase, Pim2
kinase, Pim3 kinase or a combination. In a preferred embodiment,
the Pim kinase is Pim1 kinase. In another aspect, the compound is
an antibody including but not limited to anti-CxCL12, anti-CBFbeta,
anti-TLE1, anti-IL-7, anti-Stat 5, anti-ETS-1 or anti-IRF-4.
[0049] In another embodiment, the regulator that is capable of
inducing Runx3 expression of the present invention may be a Pim
kinase inhibitor. The Pim kinase inhibitor includes but is not
limited to Pim1 kinase inhibitor, Pim2 kinase inhibitor or Pim3
kinase inhibitor. In a preferred embodiment, the Pim kinase
inhibitor is a Pim1 kinase inhibitor. Examples of Pim kinase
inhibitors include but are not limited to AR460770 (also referred
to as AR00460770, ARRY770 and AR770; ARRAY Biopharma), AR440 (ARRAY
Biopharma), SimI4A, staurosporine, bisindolylmaleimide or
triazolopyridine Pim kinase inhibitor compounds.
[0050] In still another embodiment of the present invention, the
composition comprises an regulator that is an activator that
increases expression of Runx3. The activators include but are not
limited to G proteins, phosphatidylinositol-3 kinase (PI3K), JAK
kinases, Rho GTPases or focal adhesion-associated proteins.
[0051] According to the methods of the present invention, an
effective amount of a composition to administer to a subject
comprises an amount that is capable of inhibiting expression and/or
activity of Pim1 kinase and/or inducing Runx3 expression (mRNA
and/or protein) without being toxic to the subject. An amount that
is toxic to a subject comprises any amount that causes damage to
the structure or function of a subject (i.e., poisonous).
[0052] In addition, according to the present invention, the
composition can comprise a pharmaceutically acceptable excipient.
According to the present invention, the composition, may be
administered with a pharmaceutically acceptable carrier, which
includes pharmaceutically acceptable excipients and/or delivery
vehicles, for delivering the agent to a subject (e.g., a liposome
delivery vehicle). As used herein, a pharmaceutically acceptable
carrier refers to any substance suitable for delivering a
therapeutic composition useful in the method of the present
invention to a suitable in vivo or ex vivo site. Preferred
pharmaceutically acceptable carriers are capable of maintaining the
composition of the present invention in a form that, upon arrival
of the composition to a target cell, the composition is capable of
entering the cell and inhibiting Pim1 kinase and/or inducing Runx3
expression (mRNA and/or protein) in the cell. Suitable excipients
of the present invention include excipients or formularies that
transport or help transport, but do not specifically target a
nucleic acid molecule to a cell (also referred to herein as
non-targeting carriers). Examples of pharmaceutically acceptable
excipients include, but are not limited to water, phosphate
buffered saline, Ringer's solution, dextrose solution,
serum-containing solutions, Hank's solution, other aqueous
physiologically balanced solutions, oils, esters and glycols.
Aqueous carriers can contain suitable auxiliary substances required
to approximate the physiological conditions of the recipient, for
example, by enhancing chemical stability and isotonicity.
[0053] Suitable auxiliary substances include, for example, sodium
acetate, sodium chloride, sodium lactate, potassium chloride,
calcium chloride, and other substances used to produce phosphate
buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances
can also include preservatives, such as thimerosal, m- or o-cresol,
formalin and benzol alcohol. Compositions of the present invention
can be sterilized by conventional methods and/or lyophilized.
[0054] According to the methods of the present invention, the
subject can be any animal subject, and particularly, in any
vertebrate mammal, including, but not limited to, primates,
rodents, livestock or domestic pets. Preferred mammals for the
methods of the present invention include humans.
[0055] Another embodiment of the present invention, the present
invention is directed toward a method to treat an allergic
condition in a subject who has or is at risk of having an allergic
disease, comprising administering to the subject a composition that
inhibits Pim1 kinase, wherein the allergic condition is not a
pulmonary condition. In one aspect, the allergic condition can be a
food allergy, eosinophilic esophagitis, chronic urticaria, atopic
dermatitis, occupational allergy, allergic conjunctivitis, airborne
allergic sensitivities, stinging insect allergy, inflammatory bowel
disease, ulcerative colitis, Crohn's disease or drug allergies. In
another aspect, the food allergy is a peanut allergy. In yet
another aspect, the subject has been sensitized to an allergen and
has been exposed to, or is at risk of being exposed to, the
allergen. In one aspect, the allergen is selected from a food, a
plant, a gas, a pathogen, a metal, a glue or a drug. In another
aspect, the composition comprises a compound selected from a small
molecule inhibitor, an antibody, a chemical entity, a nucleotide, a
peptide or a protein. In one aspect, the small molecule inhibitor
can be a Pim kinase inhibitor selected from a Pim1 kinase
inhibitor, a Pim2 kinase inhibitor and a Pim3 kinase inhibitor. In
a preferred aspect, the Pim kinase inhibitor is a Pim1 kinase
inhibitor. In still another aspect the Pim1 kinase inhibitor is
selected from AA460770, AR440, SimI4A, staurosporine,
bisindolymaleimide or triazolopyridine Pim kinase inhibitor
compounds. In one aspect, the Pim1 kinase inhibitor induces
expression of Runx3. In another aspect, the Pim1 kinase inhibitor
reduces CD4+ and CD8+ proliferation. In yet another aspect, the
Pim1 kinase inhibitor suppresses Th2 differentiation. In still
another aspect, the Pim1 kinase inhibitor suppresses Th17
differentiation.
[0056] In yet another embodiment, the present invention is directed
toward a method to treat an allergic condition in a subject who has
or is at risk of having an allergic disease, comprising
administering to the subject a composition that induces expression
of Runx3, wherein the composition does not comprise a Pim kinase
inhibitor. In one aspect the composition interacts with a regulator
of Runx3 expression. In another aspect, the regulator of Runx3
expression is selected from CxCL12, core binding factor-beta,
transducin-like enhancer protein 1, IL-7, Stat 5, ETS-1, and IRF-4.
In still another aspect, the composition inhibits the activity of
IRF-4. In yet another aspect the composition comprises an IRF-4
inhibitor. In another aspect, the allergic condition can be
allergic rhinitis, asthma, airway hyperresponsiveness, a food
allergy, eosinophilic esophagitis, chronic urticaria, atopic
dermatitis, occupational allergy, allergic conjunctivitis, hay
fever, airborne allergic sensitivities, stinging insect allergy,
hypersensitivity pneumonitis, eosinophilic lung diseases,
inflammatory bowel disease, ulcerative colitis, Crohn's disease and
drug allergies. In another aspect, the subject has been sensitized
to an allergen and has been exposed to, or is at risk of being
exposed to, the allergen. In one aspect, the allergen is selected
from a food, a plant, a gas, a pathogen, a metal, a glue or a
drug.
[0057] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following exemplary claims.
[0058] The following examples are provided for illustrative
purposes, and are not intended to limit the scope of the invention
as claimed herein. Any variations which occur to the skilled
artisan are intended to fall within the scope of the present
invention. All references cited in the present application are
incorporated by reference herein to the extent that there is no
inconsistency with the present disclosure.
EXAMPLES
Materials and Methods for Examples 1-7 Described Below:
[0059] Mice: Five- to 6-week-old female wild-type (WT) BALB/cByJ
mice were purchased from the Jackson Laboratory (Bar Harbor,
Me.).
[0060] Preparation of peanut protein: Crude peanut extract (PE) was
prepared from defatted raw flours (Golden Peanut Company,
Alpharetta, Ga.) as previously described (Wang, M., et al. 2010. J.
Allergy Clin. Immunol. 126:306-316).
[0061] Sensitization and intragastric challenge: The experimental
protocol for sensitization and challenge to peanut as described
(Wang, M., et al. 2010. J. Allergy Clin. Immunol. 126:306-316)
(FIG. 1A).
[0062] Assessment of hypersensitivity reactions: Anaphylactic
symptoms were evaluated 30 minutes after the oral challenge, as
reported (Li, X. M., et al. 2000. J. Allergy Clin. Immunol.
106:150-158). Scoring of symptoms was performed in a blinded manner
by an independent observer.
[0063] Histology: The jejunum was fixed in 10% formalin, embedded
in paraffin, and cut into 5-.mu.m sections for immunohistochemical
analysis.
[0064] Cytokines levels in tissue and cell culture: The preparation
of intestine homogenates and analyses were performed as described
(Wang, M., et al. 2010. J. Allergy Clin. Immunol. 126:306-316).
[0065] Measurement of peanut-specific antibody: Serum
peanut-specific IgE, IgG1, and IgG2a levels were measured by ELISA,
as described (Li, X. M., et al. 2000. J. Allergy Clin. Immunol.
106:150-158).
[0066] Histamine levels in plasma: Levels of histamine in plasma
were measured using an enzyme immunoassay histamine kit (Beckman
Coulter, Fullerton, Calif.), as described by the manufacturer. The
concentration of histamine was calculated from a standard curve
provided by the manufacturer.
[0067] T-cell differentiation and treatment with the Pim1 kinase
inhibitor in vitro: Differentiation of Th1, Th2, or Th17 cells were
performed as described (Komine, O., et al. 2003. J. Exp. Med.
198:51-61; Ashino, S., et al. 2010. Intl Immunol. 22:503-513).
[0068] Western blot analysis: Proteins were prepared from jejunal
tissue and cultured cells were lysed as described (Wang, M., et al.
2010. J. Allergy Clin. Immunol. 126:306-316; Ohnishi, H., et al.
2008. J. Allergy Clin Immunol. 121:864-871).
[0069] Quantitative real-time PCR: RNA was extracted from jejunal
tissue homogenates or from CD4 T cells cultured in vitro using
Trizol (Invitrogen) according to the manufacturer's protocol. cDNA
was generated using the iScript cDNA synthesis kit (Bio-Rad
Laboratories, Hercules, Calif.). Quantitative real-time PCR was
performed on the ABI Prism 7300 sequence detection system (Applied
Biosystems, Foster City, Calif.). Primers and probes for murine
IL4, IL6, IL13, IL17A, IFNg, Pim1, Pim2, Pim3, Runx1, Runx3,
CBF.beta., T-bet, ROG (repressor of Gata3), GATA3, NFATc1,
ROR.gamma.t, and GAPDH were purchased as Tagman Gene Expression
Assays from Applied Biosystems. Fold change was calculated using
the Delta Delta cycle threshold (.DELTA..DELTA.C.sub.T) method.
[0070] Intracellular cytokine staining and flow cytometry: Cells
from MLN or differentiated CD4 T cells were labeled with anti-CD3
and anti-CD4 antibodies (eBiosciences). For intracellular staining,
MLN cells or differentiated CD4 T cells were stimulated with 5
ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) for 6 hrs in the
presence of 10 mg/ml brefeldin A (Sigma-Aldrich). Following
staining for cell surface markers, cells were fixed with 4%
paraformaldehyde in PBS, permeabilized with 0.1% saponin, and
stained for intracytoplasmic IL-4, IL-13, IL-17A, and IFN-.gamma.
using antibodies from BD Biosciences. Stained cells were analyzed
on FACSCalibur (BD Biosciences) using CellQuest software (BD
Biosciences).
[0071] Cell proliferation: Th1-, Th2-, or Th17-polarized CD4 T
cells were incubated with anti-CD3 and anti-CD28 (eBioscience) at
37.degree. C. for 24 hrs. To monitor the degree of cell
proliferation, .sup.3H-thymidine (PerkinElmer, Boston, Mass.) was
added to the cultures for another 6 hrs prior to harvesting the
cells and incorporation was measured in a liquid scintillation
counter (Packard Bioscience Company, Meriden, Conn.).
[0072] Cell viability and apoptosis: Cell viability was determined
using trypan blue exclusion assay. Cell apoptosis was detected by
flow cytometry using surface staining with 7AAD and annexin V (BD
Biosciences).
[0073] Statistical analysis: ANOVA was used to determine the levels
of difference between all groups. Comparisons for all pairs
utilized the Tukey Kramer highest significance difference test. P
values for significance were set at 0.05. All results were
expressed as the mean.+-.SEM.
Example 1
[0074] This example shows that Pim1 kinase is upregulated in the
small intestine of peanut sensitized and challenged mice.
[0075] Pim1 kinase protein expression was increased in the jejunum
of PE sensitized and challenged mice (FIG. 1B). Pim1 kinase mRNA
levels were 2-fold higher in the jejunum of PE sensitized and
challenged mice (FIG. 1C); Pim2 and Pim3 mRNA levels were not
altered following sensitization and challenge. Pim1 was expressed
predominantly in the lamina propria of jejunal tissues of
sensitized and challenged mice and the numbers of positive cells
were increased by approximately 5-fold in PE sensitized and
challenged mice (FIGS. 1D, 1E). The numbers of Pim3-positive cells
were lower with little alteration following PE sensitization and
challenge.
Example 2
[0076] This example shows that Runx3 is downregulated in the small
intestine of peanut sensitized and challenged mice.
[0077] Runx3 associates with Pim1 and catalytically active Pim1
kinase regulates the transcriptional activity of Runx3 (Aho, T. L.,
et al. 2006. BMC Cell Biol. 7:21-29). Runx3 and Runx/core binding
factor .beta. (Cbf.beta.) mRNA levels were decreased in the small
intestine of PE sensitized and challenged mice. The levels of Runx3
and Cbf.beta. mRNA but not Runx1 were approximately 2-fold lower in
the jejunum of PE sensitized and challenged mice compared to
control mice (FIG. 2A). In parallel, Runx3 protein expression was
also decreased in the jejunum of PE sensitized and challenged mice
(FIG. 2B). Immunohistochemical analysis of jejunal tissues revealed
that Runx3 protein was mainly expressed in the lamina propria and
levels of expression were decreased by 3-fold in PE sensitized and
challenged mice (FIG. 2C).
Example 3
[0078] This example demonstrates inhibition of Pim1 kinase can
attenuate PE-induced intestinal responses in vivo.
[0079] Based on the data showing increased levels of Pim1 mRNA and
protein in the jejunum after peanut sensitization and challenge in
wild-type (WT) mice, it was thought that Pim1 plays an essential
role in the development of intestinal allergy. Whether inhibition
of Pim1 kinase alters the severity of PE-induced intestinal allergy
using the small molecule inhibitor, AR460770 was determined.
Sensitized mice were given the inhibitor twice daily by mouth
during the 7 days of PE challenge. AR460770 administration resulted
in a dose-dependent inhibitory effect on peanut-induced intestinal
allergy induction; 30-100 mg/kg AR460770 prevented development of
diarrhea and symptoms in PE sensitized and challenged mice (FIGS.
3A, 3B).
[0080] As mast cells were shown to be involved in the response to
PE sensitization and challenge (Wang, M., et al. 2010. J. Allergy
Clin. Immunol. 126:306-316), mast cell degranulation by
quantitating plasma levels of histamine within 30 minutes of the
last challenge were monitored. Levels of histamine in AR460770 (30
and 100 mg/kg)-treated mice were significantly decreased following
sensitization and challenge (FIG. 3C).
[0081] When administered after sensitization and during challenge,
the inhibitor had no effect on peanut-specific antibody production
as shown by unaltered levels of serum peanut-specific IgE, IgG1,
and IgG2a.
[0082] AR460770 inhibited mast cell and eosinophil accumulation and
goblet cell metaplasia in the small intestinal tissues in a
dose-dependent manner. PE sensitized and challenged mice treated
with AR460770 at a dose of 30-100 mg/kg demonstrated markedly
reduced numbers of mast cells, eosinophils, and PAS' goblet cells
in the mucosa of the small intestine. The small intestine lamina
propria in the sham sensitized group contained few CD4 and CD8 T
cells. These numbers were significantly increased in the untreated
PE sensitized and challenged group and reduced to baseline levels
in the treated (100 mg/kg) group (FIG. 3G).
[0083] Collectively, these results show that Pim1 kinase activation
plays an essential role in enhancing allergic diarrhea, intestinal
inflammation, and goblet cell metaplasia.
Example 4
[0084] This example shows that Pim1 kinase can regulate IL-13 and
IL-17 production.
[0085] In addition to Th2 cells, Th17 cells have been implicated in
allergic disease (Tesmer, L. A., et al. 2008. Immunol. Rev
223:87-113; Kolls J. K., et al. 2004. Immunity 21:467-476; Lajoie,
S., et al. 2010. Nat Immunol. 11:928-935). The effect of Pim1
kinase inhibition in vivo on Th2 and Th17 cytokine levels were
examined. After 7 days of PE challenges, intestinal tissue from
sensitized mice demonstrated significant increases in IL4, IL6,
IL13, and IL17A but not IFNG mRNA expression (FIG. 4A). After
treatment with AR460770, mRNA expression levels for these cytokines
returned to control levels. The levels of the key transcription
factors regulating differentiation were also assessed. The
expression levels of GATA3, NFATc1, and ROR.gamma.t mRNA were also
significantly increased in PE sensitized and challenged mice while
IFNG, T-bet, and ROG mRNA levels were not altered (FIG. 4B).
Following treatment with the inhibitor, these increased levels also
returned to control levels.
[0086] To assess the impact of Pim1 kinase inhibition on T
lymphocyte cytokine production, MLN CD4 T cells were isolated from
mice treated with the inhibitor or vehicle and stimulated them with
anti-CD3/anti-CD28. PE sensitization and challenge resulted in
significant increases in the numbers of IL-4-, IL-13- and
IL-17A-producing CD4 T cells (FIG. 4C). Mice treated with the
inhibitor exhibited a 2-4-fold decrease in the numbers of these CD4
cytokine-producing cells. The percentages of
CD4.sup.+IFN-.gamma..sup.+ cells were not altered by sensitization
and challenge or treatment with the inhibitor.
Example 5
[0087] This example demonstrates that Pim1 can regulate Runx3
transcription factor expression.
[0088] In view of reports that Runx3 plays a critical role in the T
cell response to antigen (Collins, A., et al. 2009. Nat. Rev.
Immunol. 9:106-115) and has been linked to Pim1 kinase activation
(Aho, T. L., et al. 2006. BMC Cell Biol. 7:21-29), inhibition of
Pim1 kinase affecting Runx3 expression was determined. Protein was
extracted from jejunal tissues and western blot analysis showed a
decrease in levels of Runx3 in PE sensitized and challenged mice
(FIG. 5A). In mice treated with AR460770, the levels of Runx3 were
restored to baseline levels. In parallel, levels of Runx3 and
Cbf.beta. mRNA were also decreased in the PE sensitized and
challenged mouse tissue and these levels were restored to control
values following treatment with the inhibitor (FIGS. 5B and 5D).
Runx3 protein expression was decreased in the jejunum of PE
sensitized and challenged mice but was similarly restored after
treatment with the inhibitor (FIG. 5C). Taken together, these data
indicated that PE sensitization and challenge resulted in Pim1
activation and Runx3 inhibition and the latter could be reversed by
inhibition of Pim1.
[0089] In addition, Pim1 mRNA and protein levels were increased in
the jejunum following peanut sensitization and challenge whereas
the levels of Runx3 mRNA and protein were significantly decreased.
Administration of the Pim1 kinase inhibitor (AR770) reduced the
incidence of diarrhea in a dose-dependent manner. In parallel, mast
cell and eosinophil accumulation and goblet cell metaplasia in the
small intestinal tissues were markedly decreased. Mesenteric lymph
node and small intestine Th2 and Th17 cytokine production were also
significantly decreased. In contrast, mice treated with the Pim1
kinase inhibitor (AR770) had increased levels of Runx3 mRNA and
protein in the small intestine. In vitro, the Pim1 kinase inhibitor
repressed Th2 and Th17 but not Th1 cell differentiation and
proliferation in a dose-dependent manner and enhanced Runx3
expression in Th2 cell differentiation.
Example 6
[0090] This example demonstrates the effects of Pim1 kinase
inhibition on Th1, Th2 and Th17 cell differentiation and Runx3
expression in vitro.
[0091] Considering the effects of AR460770 on Runx3 and Th2/Th17
cytokine production in vivo, the effect of Pim1 kinase inhibition
on Runx3 expression and T cell differentiation and function in
vitro was determined. Isolated naive CD4.sup.+CD45RB.sup.+ T cells
were cultured under Th1, Th2, and Th17 polarizing conditions in the
presence or absence of the inhibitor for 6 days and then stimulated
with the combination of anti-CD3/anti-CD28. The Pim1 kinase
inhibitor suppressed Th2 and Th17 cell expansion in a
dose-dependent manner; 0.1-1 .mu.M AR460770 inhibited cell number
increases (FIG. 6A) and Th2 and Th17 cell proliferation assessed by
.sup.3H-thymidine incorporation (FIG. 6B); Th1 cell expansion was
not significantly affected. In parallel, the levels of Th2 and Th17
cytokines in the polarized T cell cultures (IL-4, IL-13, and
IL-17A, respectively) were decreased in the presence of 1 .mu.M
AR460770 (FIG. 6C); levels of IFN-.gamma. were not affected by the
inhibitor. These effects were not due to altered cell
viability.
[0092] In the polarized T cell cultures the effects of AR460770 on
the expression of Runx3 and lineage-specific transcription factors
was determined by quantitative RT-PCR. In polarized Th2 cultures
treated with the inhibitor, Runx3 mRNA expression was upregulated
compared to vehicle treatment (FIG. 6D). Runx3 protein levels were
also increased in polarized Th2 cells (FIG. 6E). Similar results
were seen in Th17 polarized cells. In parallel, levels of IL13 and
GATA3, and IL17A and RO.gamma.t mRNA expression were also decreased
in Th2 and Th17 cells, respectively (FIG. 6D). No effects were
detected in Th1 cells. These data demonstrated that inhibition of
Pim1 kinase impacts Th2 and Th17 but not Th1 differentiation and
promoted expression of Runx3. Thus, Pim1 kinase functions as a
positive regulator for Th2 and Th17 differentiation and expansion
and as a negative regulator of Runx3 expression.
Example 7
[0093] This example demonstrates a Pim1 kinase inhibitor and
treatment in vivo.
[0094] To determine the role of Pim1 kinase in the development of
allergen-induced AHR and airway inflammation, a small molecule Pim1
kinase inhibitor, AR460770 (ARRAY Biopharma, Boulder, Colo.) was
used. The inhibitor was tested at 10 mM against 230 kinases in
enzymatic assays (Millipore Kinase Profiler, Millipore, Billerica,
Mass.) and shown to be selective for the three PIM isoforms (Table
1). Cellular IC.sub.50 for Pim kinase inhibition were determined by
assessing Ser112 phosphorylation of transiently infected Bad in HEK
293 cells engineered to express Pim1, Pim2 or Pim3 (Fainaru, O., et
al. 2005. Proc. Natl. Acad. Sci. USA 102:10598-10603) (Table 2).
Sensitized mice were administered the inhibitor twice daily by
mouth (1-100 mg/kg) or vehicle (50 mM citric buffer, pH 4.0) during
the 7 days of PE challenge. To determine the concentration of
AR460770 achieved at the doses used, a satellite group of mice were
dosed in a similar fashion and on day 30 plasma was collected 2 hrs
after the last dose (peak level). Compound levels were determined
to be 17, 124, 180, and 1100 nM at doses of 1, 10, 30, and 100
mg/kg, respectively (data not shown). Based on the cellular
activity of AR460770 (Table 2), Pim1 inhibition should be attained
at all but the 1-10 mg/kg dose, inhibition of Pim3 would be
achieved only at the 100 mg/kg dose and there should be no
inhibition of Pim2 at any dose tested.
TABLE-US-00001 TABLE 1 Characterization of PIM inhibitor AR00460770
Enzyme Kinase IC.sub.50 (nm) Pim-1 Proto-oncogene
serine/threonine-protein kinase 0.300 Pim-2 71 Pim-3 4 PASK
proline-alanine-rich STE20-related kinase 62 TNK2 tyrosine kinase,
non-receptor2 3000 CAMK2.gamma. Ca.sup.2+/calmodulin-dependent
protein kinase II 6000 gamma Flt3 fms-like tyrosine kinase
receptor-3 >10000 PDGFR Platelet-derived growth factor receptors
>10000 MARK1 MAP/microtubule affinity-regulating kinase 1
>10000 CAMK2.beta. Ca.sup.2+/calmodulin-dependent protein kinase
II >10000 beta AMPK 5' AMP-activated protein kinase >10000
RSK ribosomal s6 kinase >10000
[0095] AR00460770 was tested at 10 .mu.M against 230 kinases in
enzymatic assays (Millipore KinaseProfiler). It was determined to
be selective for the 3 PIM isoforms.
TABLE-US-00002 [0095] TABLE 2 Cellular IC50s for PIM Inhibition
PIM1 PIM2 PIM3 AR00460770 93 9200 340
Cellular IC.sub.50s for PIM inhibition were determined for
AR00460770 by assessing Ser112 phosphorylation of transiently
transfected BAD in HEK cell lines engineered to express PIM 1, PIM2
or PIM3. Cellular IC.sub.50s in nM are shown.
Materials and Methods for Examples 8-12 Below:
[0096] Animals: Female BALB/c mice, 8-12 weeks of age and free of
pathogens were purchased from The Harlan Laboratory (Indianapolis,
Ind.). The animals were maintained on an OVA-free diet. Experiments
were conducted under a protocol approved by the Institutional
Animal Care and Use Committee of National Jewish Health.
Sensitization and challenge with allergen: The experimental
protocol for sensitization and primary and secondary challenge to
allergen utilized described procedures (Takeda, K., et al. 2005.
Eur. Respir. J. 26:577-585). Briefly, in the primary allergen
challenge protocol, mice were sensitized by intraperitoneal (ip)
injection of 20 .mu.g of OVA (Fisher Scientific, Pittsburgh, Pa.)
emulsified in 2.0 mg alum (AlumImuject: Pierce, Rockford, Ill.) on
days 1 and 14 followed by aerosolized OVA challenge (1%/o in saline
for 20 minutes) on days 28, 29, and 30. The control mice were
sensitized with PBS followed by OVA challenge in the same way. In
the secondary allergen challenge protocol, mice were sensitized
with 10 .mu.g of OVA with alum on days 1 and 7 followed by 0.2% OVA
challenge on days 14 to 16 (primary allergen challenge). Fourteen
days after the last primary allergen challenge, mice were
challenged again with 1% OVA for 20 minutes (secondary allergen
challenge). A group of mice were sensitized with PBS followed by
primary and secondary challenge with OVA. In all groups, assays
were carried out 48 hrs after the last allergen challenge.
[0097] Pim kinase inhibitor treatment: To determine the role of
Pim1 kinase in the development of allergen-induced AHR and airway
inflammation, the Pim1 kinase inhibitor, AR00460770 (ARRAY
Biopharma, Boulder, Colo.). To characterize AR00460770 in vitro,
the cellular half maximal inhibitory concentration (IC.sub.50) and
kinase selectivity assays were determined. Cellular IC.sub.50 of
AR00460770 was analyzed by Ser112 phosphorylation of transiently
transfected BAD in HEK-293 cell lines engineered to express human
Pim1 and Pim2 (Millipore, Billerica, Mass.) and rat Pim3 (Array
BioPharma, Boulder, Colo.) in conjunction with a DNA vector
construct directing the expression of the Pim kinase substrate
GST-BAD (pEBG-mBAD). Cells were treated with serial dilutions of
AR00460770 for 1.5 hrs and then labeled with an antibody specific
for phospho-BAD (Ser112) and an antibody against GST (Cell
Signaling Technology, Danvers, Mass.) as a normalization control.
Immunoreactivity was detected using IR fluorophore-conjugated
secondary antibodies and quantified on the imager (Aerius, LI-COR,
Lincoln, Nebr.). The kinase selectivity of AR00460770 was evaluated
using the KinaseProfiler service (Millipore) (Lopez-Ramos, M., et
al. 2010. FASEB J. 24:3171-3185; Yan Bin, et al. 2003. J. Biol.
Chem. 278:45358-45367; Fox, C. J., et al. 2003. Genes Dev.
17:1841-1854). The properties and specificity of the inhibitor are
described in Tables 1 and 2.
[0098] Western blot analysis: Lung tissues were homogenized,
lysates cleared of debris and resuspended in an equal volume of
2.times. Lamelli buffer. Lysates were loaded onto a 4-10% gradient
reducing gel, subjected to electrophoresis, and transferred to
nitrocellulose membranes. The membranes were blotted with goat
anti-Pim1 (Santa Cruz, Santa Cruz, Calif.) and rabbit anti-GAPDH
(R&D Systems, Minneapolis, Minn.), anti-goat IgG (Invitrogen,
Carlsbad, Calif.) and anti-rabbit IgG (Rockland, Gilbertson, Pa.).
Images were captured and quantitatively analyzed using the Odyssey
infrared imager (Li-cor, Lincoln, Nebr.).
Assessment of airway function: Airway responsiveness was assessed
as previously described by measuring changes in airway resistance
in response to increasing doses of inhaled methacholine (MCh,
Sigma-Aldrich, St. Louis, Mo.) in anesthetized and ventilated mice
(Takeda, K., et al. 1997. J. Exp. Med. 186:449-454). The values of
peak airway responses to inhaled MCh were recorded.
[0099] Bronchoalveolar lavage (BAL) and lung histology: Lungs were
lavaged with 1 ml of Hanks balanced salt solution through the
trachea immediately after assessment of AHR. Numbers of total
leukocyte were counted with a hemocytometer and cell
differentiation was performed on the cytospin slides prepared with
Wright-Giemsa stain. The numbers of inflammatory and
mucus-containing cells were quantitated as described (Tomkinson,
A., et al. 2001. Am J. Respir. Crit. Care Med. 163:721-730).
[0100] Measurement of cytokines: Cytokine levels in the BAL fluid
and cell culture supernatants were measured by ELISA as described
(Tomkinson, A., et al. 2001. Am J. Respir. Crit. Care Med.
163:721-730).
[0101] Isolation of lung mononuclear cells (MNCs) and flow
cytometry: Lung MNCs were isolated as described previously using
collagenase digestion and cell composition identified as described
(Oshiba, A., et al. 1996. J. Clin Invest. 97:1398-1408).
[0102] CD4.sup.+ and CD8.sup.+ T cell purification and cell
proliferation assay: Purification of CD4.sup.+ and CD8.sup.+ T
cells was conducted as described (Miyahara, N., et al. 2004. J.
Immunol. 172:2549-2558). Purity of CD4.sup.+ and CD8.sup.+ T cell
populations exceeded 95% as assessed by flow cytometry.
[0103] In cell proliferation assays, an anti-mouse CD3e mAb (5
.mu.g/mL, R&D Systems, Minneapolis, Minn.) was immobilized on
96-well flat-bottom plates overnight at 4.degree. C. Purified
CD4.sup.+ and CD8.sup.+ T cells incubated with inhibitor or PBS as
vehicle, (2.times.10.sup.5 cells/well) and anti-CD28 mAb (5 pig/mL,
R&D Systems) were added to the anti-CD3-precoated plates and
incubated at 37.degree. C. for 24 hrs. After 24 hrs, 1 .mu.Ci
tritium-labeled thymidine per well (PerkinElmer, Boston, Mass.) was
added to 96-well plates for 6 hrs and harvested with distilled
water followed by counting in a microplate scintillation and
luminescence counter (Packard, Meriden, Conn.). Cell viability of
CD4.sup.+ and CD8.sup.+ T cells was assessed 24 hrs after
incubation with 10 .mu.M of inhibitor by a vital stain with trypan
blue and determined using an automated cell counter (Countess,
Invitrogen, Carlsbad, Calif.).
[0104] Statistical analysis: Results were expressed as the
mean.+-.SEM. The t test was used to determine differences between
the two groups. For comparisons between multiple groups, the
Tukey-Kramer test was used. Nonparametric analysis using the
Mann-Whitney U test or Kruskal-Wallis test was also used to confirm
that the statistical differences remained significant even if the
underlying distribution was uncertain. Differences were regarded as
statistically significant when the p-value was lower than 0.05.
Example 8
[0105] This example demonstrates that lung Pim1 kinase levels can
be increased after sensitization and challenge with allergen.
[0106] To determine the importance of Pim1 kinase following
allergen challenge, protein expression levels of Pim1 kinase in
lung tissue after OVA challenge of sensitized mice was determined.
Pim1 expression levels in OVA sensitized mice were markedly
increased following OVA challenge compared with levels seen in
non-sensitized, challenged only mice. This upregulation was
detected in OVA sensitized mice 6 hrs after the second OVA
challenge, and remained high up to 24 hrs after the third OVA
challenge (FIGS. 7A and 7B).
Example 9
[0107] This example shows Pim1 kinase inhibitor treatment can
prevent development of AHR and airway inflammation following
primary allergen challenge.
[0108] To determine the effect of Pim1 kinase inhibitor treatment
on allergen-induced airway inflammation and AHR, mice were treated
with the inhibitor or vehicle during the OVA challenge phase in the
primary allergen challenge model. As shown in FIGS. 8A and 8B,
vehicle-treated mice developed higher airway responses to MCh and
eosinophil numbers in BAL fluid following sensitization and
challenge with OVA compared to sham-sensitized, OVA challenged
mice. Mice treated with the Pim1 inhibitor at doses of 10, 30, or
100 mg/kg developed significantly lower airway responsiveness to
inhaled MCh and lower BAL eosinophil numbers compared to the
vehicle-treated group. Sham-sensitized but OVA challenged mice were
treated with 100 mg/kg of the inhibitor to assess potential effects
on smooth muscle contraction. Treatment with the inhibitor in this
way did not alter the development of increasing RL to increasing
concentrations of inhaled MCh.
[0109] As shown in FIG. 8C, inhibitor treatment of sensitized and
challenged mice reduced the levels of IL-4, IL-5, IL-13, and
IFN-.gamma. in BAL fluid in a dose-dependent manner with
significant changes seen primarily at the highest administered dose
of the inhibitor (100 mg/kg).
[0110] Histopathological analysis of lung tissue sections revealed
that the numbers of inflammatory cells, including eosinophils in
the peribronchial and perivascular areas, were increased in mice
after OVA sensitization and challenge compared to sham-sensitized
and challenged mice (FIG. 8D). Similarly, the numbers of PAS.sup.+
mucus-containing goblet cells were increased in the sensitized and
challenged mice (FIG. 8E). Administration of the inhibitor
significantly decreased the numbers of inflammatory cells and
PAS.sup.+ mucus-containing goblet cells in the lung tissues in a
dose-dependent manner (FIG. 8E).
Example 10
[0111] This example shows inhibition of Pim1 kinase can attenuate
development of AHR and airway inflammation in the secondary
allergen challenge model.
[0112] The airway responses in the primary allergen challenge model
reflect the first immune responses in the lungs, where adaptive
immunity is initiated in response to airborne allergen exposure.
For the most part, asthmatics have already developed allergic
airway inflammation and airway dysfunction prior to initiation of
treatment, immune responses to allergen and tissue remodeling of
the airways are generally already established. The secondary
allergen challenge model is an approach to examine the response to
allergen provocation where allergen-induced airway inflammation has
been previously established. To determine the effects of Pim1
kinase inhibition in the secondary allergen challenge model, AHR,
cell composition, and cytokine levels in BAL fluid was measured 48
hrs after a single provocative allergen challenge. As in the
primary allergen challenge model, vehicle-treated mice developed
significantly higher airway responsiveness to MCh and eosinophils
in BAL fluid following OVA sensitization and secondary allergen
challenge. Similar to the results observed in the primary allergen
challenge model, treatment with the Pim1 kinase inhibitor (10, 30,
and 100 mg/kg) significantly decreased levels of airway
responsiveness and the number of eosinophils in BAL fluid in a
dose-dependent manner compared to the vehicle-treated groups (FIGS.
9A and 9B). Assays of BAL cytokine levels demonstrated that 11-4,
IL-5, IL-13, and IFN-g were decreased in Pim1 kinase inhibitor (100
mg/kg)-treated mice that had been sensitized and challenged with
OVA (FIG. 9C). Histopathological analysis revealed that Pim1 kinase
inhibition decreased numbers of inflammatory cell in the lungs and
goblet cell metaplasia along the airways (FIG. 9D).
Example 11
[0113] This example demonstrates that a decrease of CD4.sup.+ and
CD8.sup.+ T cells in the lungs of sensitized and challenged mice
follows treatment with the Pim1 kinase inhibitor.
[0114] As both CD4.sup.+ and CD8.sup.+ T cells are potent effector
cells in the development of allergic inflammation, their numbers
were examined after inhibitor treatment in sensitized and
challenged mice. Lungs from OVA sensitized and challenged mice
which received either inhibitor or vehicle were excised and lung
MNCs were purified. Numbers of CD4.sup.+ and CD8.sup.+ T cells were
determined by flow cytometry. As shown in FIG. 10, the overall
number of CD4.sup.+ T cells was significantly lower in the
inhibitor-treated mice (1.48.+-.0.26.times.10.sup.6 cells/lung vs.
3.09.+-.0.35.times.10.sup.6 cells/lung in vehicle-treated mice).
CD8.sup.+ T cells were also decreased following Pim1 kinase
inhibition from 0.57.+-.0.21.times.10.sup.6 cells/lung to
0.29.+-.0.06.times.10.sup.6 cells/lung. These results demonstrate
that Pim1 kinase inhibition in vivo reduces the numbers of
CD4.sup.+ and CD8.sup.+ T cells that accumulate in the lungs of
sensitized and challenged mice.
Example 12
[0115] This example shows a reduction of CD4.sup.+ and CD8.sup.+ T
cell proliferation and cytokine production in vitro following Pim1
kinase inhibitor treatment can occur.
[0116] To examine the proliferative capacity of T cells following
inhibition of Pim1 kinase, CD4 and CD8 T cells were isolated and
purified from spleen and incubated with a combination of anti-CD3
and anti-CD28 for 24 hrs. Cell viabilities of CD4.sup.+ or
CD8.sup.+ T cells were determined in the presence of 10 mM of the
inhibitor. After 24 hrs, inhibitor treatment did not show
significant effects on cell viabilities compared to vehicle control
(from 90.0 to 90.3% in CD4+ T cells and from 80.2-82.8% in CD8+ T
cells, respectively). In a dose-dependent manner, the Pim1 kinase
inhibitor reduced CD4.sup.+ and CD8.sup.+ T cell proliferation
triggered by the combination of anti-CD3/anti-CD28. In stimulated
cell cultures, increased levels of IL-4, IL-5, IL-13, and IFN-g
were detected. Treatment with the inhibitor decreased the levels of
all of these cytokines in a dose-dependent fashion (FIGS. 11A, 11B
and 11C).
[0117] The foregoing description of the present invention has been
presented for purposes of illustration. The description is not
intended to limit the invention to the form disclosed herein.
Consequently, variations and modifications commensurate with the
above teachings, and the skill or knowledge of the relevant art,
are within the scope of the present invention. The embodiments
described hereinabove are further intended to explain the best mode
known for practicing the invention and to enable others skilled in
the art to utilize the invention in such, or other, embodiments and
with various modifications required by the particular applications
or uses of the present invention. It is intended that the appended
claims be construed to include alternative embodiments to the
extent permitted by the prior art. Each publication and reference
cited herein is incorporated herein by reference in its
entirety.
REFERENCES
[0118] 1. Gavett, S. H., X. Chen, F. Finkelman, and M. Wills-Karp.
1994. Depletion of murine CD4+T lymphocytes prevents
antigen-induced airway hyperreactivity and pulmonary eosinophilia.
Am J Respir Cell Mol Biol 10:587-593. [0119] 2. Umetsu, D. T., J.
J. McIntire, O. Akbari, C. Macaubas, and R. H. DeKruyff. 2002.
Asthma: an epidemic of dysregulated immunity. Nat Immunol
3:715-720. [0120] 3. Hogan, S. P., K. I. Matthaei, J. M. Young, A.
Koskinen, I. G. Young, and P. S. Foster. 1998. A novel T
cell-regulated mechanism modulating allergen-induced airways
hyperreactivity in BALB/c mice independently of IL-4 and IL-5. J
Immunol 161:1501-1509. [0121] 4. Wills-Karp, M., J. Luyimbazi, X.
Xu, B. Schofield, T. Y. Neben, C. L. Karp, and D. D. Donaldson.
1998. Interleukin-13: central mediator of allergic asthma. Science
282:2258-2261. [0122] 5. Robinson, D. S., Q. Hamid, S. Ying, A.
Tsicopoulos, J. Barkans, A. M. Bentley, C. Corrigan, S. R. Durham,
and A. B. Kay. 1992. Predominant TH2-like bronchoalveolar
T-lymphocyte population in atopic asthma. N Engl J Med 326:298-304.
[0123] 6. Aaronson, D. S., and C. M. Horvath. 2002. A road map for
those who don't know JAK-STAT. Science 296:1653-1655. [0124] 7.
Bachmann, M., and T. Moroy. 2005. The serine/threonine kinase
Pim-1. Int J Biochem Cell Biol 37:726-730. [0125] 8. Wang, Z., N.
Bhattacharya, M. Weaver, K. Petersen, M. Meyer, L. Gapter, and N.
S. Magnuson. 2001. Pim-1: a serine/threonine kinase with a role in
cell survival, proliferation, differentiation and tumorigenesis. J
Vet Sci 2:167-179. [0126] 9. Amaravadi, R., and C. B. Thompson.
2005. The survival kinases Akt and Pim as potential pharmacological
targets. J Clin Invest 115:2618-2624. [0127] 10. Fox, C. J., P. S.
Hammerman, and C. B. Thompson. 2005. The Pim kinases control
rapamycin-resistant T cell survival and activation. J Exp Med
201:259-266. [0128] 11. Amson, R., F. Sigaux, S. Przedborski, G.
Flandrin, D. Givol, and A. Telerman. 1989. The human protooncogene
product p33pim is expressed during fetal hematopoiesis and in
diverse leukemias. Proc Natl Acad Sci USA 86:8857-8861. [0129] 12.
Valdman, A., X. Fang, S. T. Pang, P. Ekman, and L. Egevad. 2004.
Pim-1 expression in prostatic intraepithelial neoplasia and human
prostate cancer. Prostate 60:367-371. [0130] 13. Cibull, T. L., T.
D. Jones, L. Li, J. N. Eble, L. Ann Baldridge, S. R. Malott, Y.
Luo, and L. Cheng. 2006. Overexpression of Pim-1 during progression
of prostatic adenocarcinoma. J Clin Pathol 59:285-288. [0131] 14.
Nieborowska-Skorska, M., G. Hoser, P. Kossev, M. A. Wasik, and T.
Skorski. 2002. Complementary functions of the antiapoptotic protein
A1 and serine/threonine kinase pim-1 in the BCR/ABL-mediated
leukemogenesis. Blood 99:4531-4539. [0132] 15. Temple, R., E.
Allen, J. Fordham, S. Phipps. H. C. Schneider, K. Lindauer, I.
Hayes, J. Lockey, K. Pollock, and R. Jupp. 2001. Microarray
analysis of eosinophils reveals a number of candidate survival and
apoptosis genes. Am J Respir Cell Mol Biol 25:425-433. [0133] 16.
Andina, N., S. Didichenko, J. Schmidt-Mende, C. A. Dahinden, and H.
U. Simon. 2009. Proviral integration site for Moloney murine
leukemia virus 1, but not phosphatidylinositol-3 kinase, is
essential in the antiapoptotic signaling cascade initiated by IL-5
in eosinophils. J Allergy Clin Immunol 123:603-611. [0134] 17.
Stout, B. A., M. E. Bates, L. Y. Liu, N. N. Farrington, and P. J.
Bertics. 2004. IL-5 and granulocyte-macrophage colony-stimulating
factor activate STAT3 and STATS and promote Pim-1 and cyclin D3
protein expression in human eosinophils. J Immunol 173:6409-6417.
[0135] 18. Busse, W. W., R. L. Coffman, E. W. Gelfand, A. B. Kay,
and L. J. Rosenwasser. 1995. Mechanisms of persistent airway
inflammation in asthma. A role for T cells and T-cell products. Am
J Respir Crit Care Med 152:388-393. [0136] 19. Cohn, L., R. J.
Homer, A. Marinov, J. Rankin, and K. Bottomly. 1997. Induction of
airway mucus production By T helper 2 (Th2) cells: a critical role
for interleukin 4 in cell recruitment but not mucus production. J
Exp Med 186:1737-1747. [0137] 20. Hogan, S. P., A. Koskinen, K. I.
Matthaei, I. G. Young, and P. S. Foster. 1998.
Interleukin-5-producing CD4+ T cells play a pivotal role in
aeroallergen-induced eosinophilia, bronchial hyperreactivity, and
lung damage in mice. Am J Respir Crit Care Med 157:210-218. [0138]
21. Azzawi, M., B. Bradley, P. K. Jeffery, A. J. Frew, A. J.
Wardlaw, G. Knowles, B. Assoufi, J. V. Collins, S. Durham, and A.
B. Kay. 1990. Identification of activated T lymphocytes and
eosinophils in bronchial biopsies in stable atopic asthma. Am Rev
Respir Dis 142:1407-1413. [0139] 22. Hamelmann, E., A. Oshiba, J.
Paluh, K. Bradley, J. Loader, T. A. Potter, G. L. Larsen, and E. W.
Gelfand. 1996. Requirement for CD8+ T cells in the development of
airway hyperresponsiveness in a marine model of airway
sensitization. J Exp Med 183:1719-1729. [0140] 23. Isogai, S., R.
Taha, M. Tamaoka, Y. Yoshizawa, Q. Hamid, and J. G. Martin. 2004.
CD8+ alphabeta T cells can mediate late airway responses and airway
eosinophilia in rats. J Allergy Clin Immunol 114:1345-1352. [0141]
24. Miyahara, N., K. Takeda, T. Kodama, A. Joetham, C. Taube, J. W.
Park, S. Miyahara, A. Balhorn, A. Dakhama, and E. W. Gelfand. 2004.
Contribution of antigen-primed CD8+ T cells to the development of
airway hyperresponsiveness and inflammation is associated with
IL-13. J Immunol 172:2549-2558. [0142] 25. Miyahara, N., B. J.
Swanson, K. Takeda, C. Taube, S. Miyahara, T. Kodama, A. Dakhama,
V. L. Ott, and E. W. Gelfand. 2004. Effector CD8+ T cells mediate
inflammation and airway hyper-responsiveness. Nat Med 10:865-869.
[0143] 26. Croft, M., L. Carter, S. L. Swain, and R. W. Dutton.
1994. Generation of polarized antigen-specific CD8 effector
populations: reciprocal action of interleukin (IL)-4 and IL-12 in
promoting type 2 versus type 1 cytokine profiles. J Exp Med
180:1715-1728. [0144] 27. Seder, R. A., J. L. Boulay, F. Finkelman,
S. Barbier, S. Z. Ben-Sasson, G. Le Gros, and W. E. Paul. 1992.
CD8+ T cells can be primed in vitro to produce IL-4. J Immunol
148:1652-1656. [0145] 28. Coyle, A. J., F. Erard, C. Bertrand, S.
Walti, H. Pircher, and G. Le Gros. 1995. Virus-specific CD8+ cells
can switch to interleukin 5 production and induce airway
eosinophilia. J Exp Med 181:1229-1233. [0146] 29. Takeda, K., N.
Miyahara, T. Kodama, C. Taube, A. Balhorn, A. Dakhama, K. Kitamura,
A. Hirano, M. Tanimoto, and E. W. Gelfand. 2005.
S-carboxymethylcysteine normalises airway responsiveness in
sensitised and challenged mice. Eur Respir. 126:577-585. [0147] 30.
Takeda, K., E. Hamelmann, A. Joetham, L. D. Shultz, G. L. Larsen,
C. G. Irvin, and E. W. Gelfand. 1997. Development of eosinophilic
airway inflammation and airway hyperresponsiveness in mast
cell-deficient mice. J Exp Med 186:449-454. [0148] 31. Tomkinson,
A., G. Cieslewicz, C. Duez, K. A. Larson, J. J. Lee, and E. W.
Gelfand. 2001. Temporal association between airway
hyperresponsiveness and airway eosinophilia in ovalbumin-sensitized
mice. Am J Respir Crit Care Med 163:721-730. [0149] 32. Oshiba, A.,
E. Hamelmann, K. Takeda, K. L. Bradley, J. E. Loader, G. L. Larsen,
and E. W. Gelfand. 1996. Passive transfer of immediate
hypersensitivity and airway hyperresponsiveness by
allergen-specific immunoglobulin (Ig) E and IgG1 in mice. J Clin
Invest 97:1398-1408. [0150] 33. Cheng, J. Q., A. K. Godwin, A.
Bellacosa, T. Taguchi, T. F. Franke. T. C. Hamilton, P. N.
Tsichlis, and J. R. Testa. 1992. AKT2, a putative oncogene encoding
a member of a subfamily of protein-serine/threonine kinases, is
amplified in human ovarian carcinomas. Proc Natl Acad Sci USA
89:9267-9271. [0151] 34. Bellacosa, A., D. de Feo, A. K. Godwin, D.
W. Bell, J. Q. Cheng, D. A. Altomare, M. Wan, L. Dubeau, G.
Scambia, V. Masciullo, G. Ferrandina, P. Benedetti Panici, S.
Mancuso, G. Neri, and J. R. Testa. 1995. Molecular alterations of
the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer
64:280-285. [0152] 35. Stahl, J. M., A. Sharma, M. Cheung, M.
Zimmerman, J. Q. Cheng, M. W. Bosenberg, M. Kester, L.
Sandirasegarane, and G. P. Robertson. 2004. Deregulated Akt3
activity promotes development of malignant melanoma. Cancer Res
64:7002-7010. [0153] 36. Masure, S., B. Haefner, J. J. Wesselink,
E. Hoefnagel, E. Mortier, P. Verhasselt, A. Tuytelaars, R. Gordon,
and A. Richardson. 1999. Molecular cloning, expression and
characterization of the human serine/threonine kinase Akt-3. Eur J
Biochem 265:353-360. [0154] 37. Yang, L., H. C. Dan, M. Sun, Q.
Liu, X. M. Sun, R. I. Feldman, A. D. Hamilton, M. Polokoff. S. V.
Nicosia, M. Herlyn, S. M. Sebti, and J. Q. Cheng. 2004. Akt/protein
kinase B signaling inhibitor-2, a selective small molecule
inhibitor of Akt signaling with antitumor activity in cancer cells
overexpressing Akt. Cancer Res 64:4394-4399. [0155] 38. Kondapaka,
S. B., S. S. Singh, G. P. Dasmahapatra, E. A. Sausville, and K. K.
Roy. 2003. Perifosine, a novel alkylphospholipid, inhibits protein
kinase B activation. Mol Cancer Ther 2:1093-1103. [0156] 39. van
Lohuizen, M., S. Verbeek, P. Krimpenfort, J. Domen, C. Saris, T.
Radaszkiewicz, and A. Berns. 1989. Predisposition to
lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc
and N-myc in murine leukemia virus-induced tumors. Cell 56:673-682.
[0157] 40. Mikkers, H., M. Nawijn, J. Allen, C. Brouwers, E.
Verhoeven, J. Jonkers, and A. Berns. 2004. Mice deficient for all
PIM kinases display reduced body size and impaired responses to
hematopoietic growth factors. Mol Cell Biol 24:6104-6115. [0158]
41. Katakami, N., H. Kaneto, H. Hao, Y. Umayahara, Y. Fujitani, K.
Sakamoto, S. Gorogawa, T. Yasuda, D. Kawamori, Y. Kajimoto, M.
Matsuhisa, C. Yutani, M. Hori, and Y. Yamasaki. 2004. Role of pim-1
in smooth muscle cell proliferation. J Biol Chem 279:54742-54749.
[0159] 42. Chen, L. S., S. Redkar, D. Bearss, W. G. Wierda, and V.
Gandhi. 2009. Pim kinase inhibitor, SGI-1776, induces apoptosis in
chronic lymphocytic leukemia cells. Blood 114:4150-4157. [0160] 43.
Hu, X. F., J. Li, S. Vandervalk, Z. Wang, N. S. Magnuson, and P. X.
Xing. 2009. PIM-1-specific mAb suppresses human and mouse tumor
growth by decreasing PIM-1 levels, reducing Akt phosphorylation,
and activating apoptosis. J Clin Invest 119:362-375. [0161] 44.
Borgonovo, B., G. Casorati, E. Frittoli, D. Gaffi, E. Crimi, and S.
E. Burastero. 1997. Recruitment of circulating allergen-specific T
lymphocytes to the lung on allergen challenge in asthma. J Allergy
Clin Immunol 100:669-678. [0162] 45. Yan, B., M. Zemskova, S.
Holder, V. Chin, A. Kraft, P. J. Koskinen, and M. Lilly. 2003. The
PIM-2 kinase phosphorylates BAD on serine 112 and reverses
BAD-induced cell death. J Biol Chem 278:45358-45367. [0163] 46.
Chen, X. P., J. A. Losman, S. Cowan, E. Donahue, S. Fay, B. Q.
Vuong, M. C. Nawijn, D. Capece, V. L. Cohan, and P. Rothman. 2002.
Pim serine/threonine kinases regulate the stability of Socs-1
protein. Proc Natl Acad Sci USA 99:2175-2180. [0164] 47. Maita, H.,
Y. Harada, D. Nagakubo, H. Kitaura, M. Ikeda, K. Tamai, K.
Takahashi, H. Ariga, and S. M. Iguchi-Ariga. 2000. PAP-1, a novel
target protein of phosphorylation by pim-1 kinase. Eur J Biochem
267:5168-5178. [0165] 48. Koike, N., H. Maita, T. Taira, H. Ariga,
and S. M. Iguchi-Ariga. 2000. Identification of heterochromatin
protein 1 (HP1) as a phosphorylation target by Pim-1 kinase and the
effect of phosphorylation on the transcriptional repression
function of HP1(1). FEBS Lett 467:17-21. [0166] 49. Wang, Z., N.
Bhattacharya, M. K. Meyer, H. Seimiya, T. Tsuruo, J. A. Tonani, and
N. S. Magnuson. 2001. Pim-1 negatively regulates the activity of
PTP-U2S phosphatase and influences terminal differentiation and
apoptosis of monoblastoid leukemia cells. Arch Biochem Biophys
390:9-18. [0167] 50. Rainio, E. M., J. Sandholm, and P. J.
Koskinen. 2002. Cutting edge: Transcriptional activity of NFATc1 is
enhanced by the Pim-1 kinase. J. Immunol 168:1524-1527. [0168] 51.
Patra, A. K., T. Drewes, S. Engelmann, S. Chuvpilo, H. Kishi, T.
Hunig, E. Serfling, and U. H. Bommhardt. 2006. PKB rescues
calcineurin/NFAT-induced arrest of Rag expression and pre-T cell
differentiation. J Immunol 177:4567-4576. [0169] 52. Busse, W. W.,
and R. F. Lemanske, Jr. 2001. Asthma. N Engl J Med 344:350-362.
[0170] 53. Rosenberg, H. F., S. Phipps, and P. S. Foster. 2007.
Eosinophil trafficking in allergy and asthma. J Allergy Clin
Immunol 119:1303-1310; quiz 1311-1302. [0171] 54. Larche, M., D. S.
Robinson, and A. B. Kay. 2003. The role of T lymphocytes in the
pathogenesis of asthma. J Allergy Clin Immunol 111:450-463; quiz
464. [0172] 55. Mullarkey, M. F., B. A. Blumenstein, W. P. Andrade,
G. A. Bailey, I. Olason, and C. E. Wetzel. 1988. Methotrexate in
the treatment of corticosteroid-dependent asthma. A double-blind
crossover study. N Engl J Med 318:603-607. [0173] 56. Alexander, A.
G., N. C. Barnes, and A. B. Kay. 1992. Trial of cyclosporin in
corticosteroid-dependent chronic severe asthma. Lancet 339:324-328.
[0174] 57. Kweon M N, Yamamoto M, Kajiki M, Takahashi I, Kiyono H.
Systemically derived large intestinal CD4 (+) Th2 cells play a
central role in STAT6-mediated allergic diarrhea. J Clin Invest
2000; 106:199-206 [0175] 58. Wang M, Takeda K, Shiraishi Y, Okamoto
M, Dakhama A, Joetham A, Gelfand E W. Peanut-Induced Intestinal
Allergy is Mediated Through a Mast Cell-IgE-Fc.epsilon.RI-IL-13
Pathway. J. Allergy Clin Immunol 2010, 126 (2): 306-316 [0176] 59.
Knight A K, Blazquez A B, Zhang S, Mayer L, Sampson H A, Berin M C.
CD4 T cells activated in the mesenteric lymph node mediate
gastrointestinal food allergy in mice. Am J Physiol Gastrointest
Liver Physiol. 2007; 293(6): G1234-43 [0177] 60. Kweon M N,
Yamamoto M, Kajiki M, Takahashi I, Kiyono H. Systemically derived
large intestinal CD4(+) Th2 cells play a central role in
STAT6-mediated allergic diarrhea. J Clin Invest. 2000; 106(2):
199-206 [0178] 61. Eigenmann P A. T lymphocytes in food allergy:
overview of an intricate network of circulating and organ-resident
cells. Pediatr Allergy Immunol 2002; 13:162-71. [0179] 62.
Eigenmann P A, Huang S K, Ho D G, Sampson H A. Human T cell clones
and cell lines specific to ovomucoid recognize different domains
and consistently express IL-5. Adv Exp Med Biol 1996; 409:217
[0180] 63. Blumchen K, Ulbricht H, Staden U, Dobberstein K,
Beschorner J, de Oliveira L C, Shreffler W G, Sampson H A,
Niggemann B, Wahn U, Beyer K. Oral peanut immunotherapy in children
with peanut anaphylaxis. J Allergy Clin Immunol. 2010; 126(1):83-91
[0181] 64. Kolls J K, Linden A. Interleukin-17 family members and
inflammation. Immunity. 2004, 21:467-476 [0182] 65. Ivanov I L
McKenzie B S, Zhou L, Tadokoro C E, Lepelley A, Lafaille J J, Cua D
J, Littman D R. The orphan nuclear receptor RORgammat directs the
differentiation program of proinflammatory IL-17+T helper cells.
Cell. 2006; 126(6): 1121-33. [0183] 66. Wang Z, Bhattacharya N,
Weaver M, Petersen K, Meyer M, Gapter L, Magnuson N S. Pim-1: a
serine/threonine kinase with a role in cell survival,
proliferation, differentiation and tumorigenesis. J Vet Sci. 2001;
2(3): 167-179
[0184] 67. Aho T L, Sandholm J, Peltola K J, Ito Y, Koskinen P J.
Pim-1 kinase phosphorylates RUNX family transcription factors and
enhances their activity. BMC Cell Biol. 2006; 7:21-29 [0185] 68.
Rainio E M, Sandholm J, Koskinen P J. Transcriptional activity of
NFATc1 is enhanced by the Pim-1 kinase. J Immunol. 2002; 168(4):
1524-7 [0186] 69. Wingett D, Long A, Kelleher D, Magnuson N S.
pim-1 proto-oncogene expression in anti-CD3-mediated T cell
activation is associated with protein kinase C activation and is
independent of Raf-1. J Immunol. 1996; 156(2):549-57. [0187] 70.
Tesmer L A, Lundy S K, Sarkar S, Fox D A. Th17 cells in human
disease. Immunol Rev. 2008, 223:87-113 [0188] 71. Brenner O,
Levanon D, Negreanu V, Golubkov O, Fainaru O, Woolf E, Groner Y.
Loss of Runx3 function in leukocytes is associated with
spontaneously developed colitis and gastric mucosal hyperplasia.
Proc Natl Acad Sci USA. 2004; 101(45): 16016-21 [0189] 72. Fainaru
O, Woolf E, Lotem J, Yarmus M, Brenner O, Goldenberg D, Negreanu V,
Bernstein Y, Levanon D, Jung S, Groner Y. Runx3 regulates mouse
TGF-beta-mediated dendritic cell function and its absence results
in airway inflammation. EMBO J. 2004; 23(4): 969-79 [0190] 73.
Komine O, Hayashi K, Natsume W, Watanabe T, Seki Y, Seki N, Yagi R,
Sukzuki W, Tamauchi H, Hozumi K, Habu S, Kubo M, Satake M. The
Runx1 transcription factor inhibits the differentiation of naive
CD4+ T cells into the Th2 lineage by repressing GATA3 expression. J
Exp Med. 2003; 198 (1): 51-61 [0191] 74. Nawijn M C, Alendar A,
Berns A. For better or for worse: the role of Pim oncogenes in
tumorigenesis. Nat Rev Cancer. 2011; 11:23-34. [0192] 75. Kicic A,
Hallstrand T S, Sutanto E N, Stevens P T, Kobor M S, Taplin C, Pare
P D, Beyer R P, Stick S M, Knight D A. Decreased fibronectin
production significantly contributes to dysregulated repair of
asthmatic epithelium. Am J Respir Crit Care Med. 2010; 181:889-98.
[0193] 76. Lack G, Renz H, Saloga J, Bradley K L, Loader J E, Leung
D Y M, Larsen G L, Gelfand E W. Nebulized but not parenteral IFN-L
decreases IgE production and normalizes airway function in a murine
model of allergen sensitization. J Immunol 1994; 152:25446-25454.
[0194] 77. Flaishon L, Topilaki I, Shoseyov D, Hershkoviz R,
Fireman E, Levo Y, Marmor S, Shachar I. Anti-inflammatory
properties of low levels of IFN-.quadrature.. J Immunol 2002;
168:3707-3711. [0195] 78. Yoshida M, Leigh R, Matsumoto K, Wattie
J, Ellis R, O'Byrne P M, Inman M D. Effect of
interferon-.quadrature. on allergic airway responses in
interferon-gamma-deficient mice. Amer J Resp Crit Care Med 2002,
166:451-456. [0196] 79. Hessel E M, Van Oosterhout A J, Van Ark I,
Van Esch B, Hofman G, Van Loveren H, Savelkoul H F, Nijkamp F P.
Development of airway hyperresponsiveness is dependent on
interferon-gamma and independent of eosinophil infiltration. Amer J
Resp Cell Molec Biol 1997; 16:325-334. [0197] 80. ten Hacken N H T,
Oosterhoff Y, Kauffman H F, Guevarra L, Satoh T, Tollerud D J,
Postma D S. Elevated serum interferon-.gamma. in atopic asthma
correlates with increased airways responsiveness and circadian peak
expiratory flow variation. Eur Resp J 1998; 11:312-316. [0198] 81.
Heaton T. Rowe J, Turner S. Aalberse R C, de Klerk N,
Suriyaarachchi D, Serralha M, Holt B J, Hollams E, Yerkovich S,
Holt K, Sly P D, Goldblatt J, Le Souef P, Holt P G. An
immunoepidemiological approach to asthma: Identification of
in-vitro T cell response patterns associated with different
wheezing phenotypes in children. Lancet 2005; 365:142-149. [0199]
82. Lopez A F, Sanderson C J, Gamble J R, Campbell H D, Young I G,
Vadas M A. Recombinant human interleukin 5 is a selective activator
of human eosinophil function. J Exp Med 1988; 167:219-224. [0200]
83. Laoukili J, Perret E, Willems T, Minty A, Parthoens E, Houcine
O, Coste A, Jorissen M, Marano F, Caput D, Tournier F. IL-13 alters
mucociliary differentiation and ciliary beating of human
respiratory epithelial cells. J Clin Invest 2001; 108:1817-1824.
[0201] 84. Cohn L, Tepper J S, Bottomly K. IL-4-independent
induction of airway hyperresponsiveness by Th2, but not Th1, cells.
J Immunol 1998; 161:3813-3816. [0202] 85. Perrier C, Thierry A C,
Mercenier A, Corthesy B. Allergen-specific antibody and cytokine
responses, mast cell reactivity and intestinal permeability upon
oral challenge of sensitized and tolerized mice. Clin Exp Allergy
2010; 40:153-162. [0203] 86. Taniuchi I, Osato M, Egawa T, Sunshine
M J, Bae S C, Komori T, Ito Y, Littman D R. Differential
requirements for Runx proteins in CD4 repression and epigenetic
silencing during T lymphocyte development. Cell 2002; 111:621-633.
[0204] 87. Cruz-Guilloty F, Pipkin M E, Djuretic I M, Levanon D,
Lotem J, Lichtenheld M G, Groner Y, Rao A. Runx3 and T-box proteins
cooperate to establish the transcriptional program of effector
CTLs. J Exp Med 2009; 206:51-59. [0205] 88. Djuretic I M, Levanon
D, Negreanu V, Groner Y, Rao A, Ansel K M. Transcription factors
T-bet and Runx3 cooperate to activate IFN.quadrature. and silence
IL4 in T helper type 1 cells. Nat Immunol 2007; 8:145-153. [0206]
89. Kohu K, Ohmori H, Wong W F, Onda D, Wakoh T, Kon S, Yamashita
M, Nakayama T, Kubo M, Satake M. The Runx3 transcription factor
augments Th1 and down-modulates Th2 phenotypes by interacting with
and attenuating GATA3. J Immunol 2009; 183:7817-7824. [0207] 90.
Lee S H, Jeong H M, Choi J M, Cho Y C, Kim T S, Lee K Y, Kang B Y.
Runx3 inhibits 1-4 production in T cells via physical interaction
with NFAT. Biochem Biophys Res Commun 2009; 381:214-217. [0208] 91.
Aujla M. Targeted therapies: Pim kinase inhibition and
chemoresistance. Nat Rev Clin Oncol 2010; 7:3. [0209] 92. Brault L,
Gasser C, Bracher F, Huber K, Knapp S, Schwaller L. PIM
serine/threonine kinases in the pathogenesis and therapy of
hematologic malignancies and solid cancers. Haematologica 2010;
95:1004-1015. [0210] 93. Aho T L, Lund R J, Ylikoski E K,
Matikainen S, Lahesmaa R, Koskinen P J. Expression of human pim
family genes is selectively up-regulated by cytokines promoting T
helper type 1, but not T helper type 2, cell differentiation.
Immunology 2005; 116:82-88. [0211] 94. Dautry F, Weil D, Yu J,
Dautry-Varsat A. Regulation of pim and myb mRNA accumulation by
interleukin 2 and interleukin 3 in murine hematopoietic cell lines.
J Biol Chem 1988; 263:17615-17620. [0212] 95. Lilly M, Le T,
Holland P, Hendrickson S L. Sustained expression of the pim-1
kinase is specifically induced in myeloid cells by cytokines whose
receptors are structurally related. Oncogene 1992; 7:727-732.
[0213] 96. Matikainen S, Sareneva T, Ronni T, Lehtonen A, Koskinen
P J, Julkunen I. Interferon-.alpha. activates multiple STAT
proteins and up-regulates proliferation-associated IL-2R.alpha.,
c-myc, and pim-1 genes in human T cells. Blood 1999; 93:1980-1991.
[0214] 97. Jackson L J, Wright D, Gross S, Robinson J, Marmsater F
P, Allen S, Munson M, Carter L L. Inhibition of T cell function in
vitro and in vivo using small molecule antagonists of PIM kinases.
J Immunol 2009; 182:35.33. [0215] 98. Walter D M, McIntire J J,
Berry G, McKenzie A N, Donaldson D D, DeKruyff R H, Umetsu D T.
Critical role for IL-13 in the development of allergen induced
airway hyperreactivity. J Immunol 2001; 167:4668-4675. [0216] 99.
Miyahara S, Miyahara N, Matsubara S, Takeda K, Koya T, Gelfand E W.
IL-13 is essential to the late-phase response in allergic rhinitis.
J Allergy Clin Immunol 2006; 118:1110-1116. [0217] 100. Donaldson D
D, Whitters M J. Fitz L J, Neben T Y, Finnerty H, Henderson S L,
O'Hara Jr R M, Beier D R, Turner K J, Wood C R, Collins M. The
murine IL-13 receptor alpha 2: molecular cloning, characterization,
and comparison with murine IL-13 receptor alpha 1. J Immunol 1998;
161:2317-2324. [0218] 101. Taube C, Miyahara N, Ott V, Swanson B,
Takeda K, Loader J, Shultz L D, Tager A M, Luster A D, Dakhama A,
Gelfand E W. The leukotriene B4 receptor (BLT1) is required for
effector CD8+ T cell-mediated, mast cell-dependent airway
hyperresponsiveness. J Immunol 2006; 176:3157-3164. [0219] 102.
Wang Y H, Voo K S, Liu B, Chen C Y, Uygungil B, Spoede W, Bernstein
J A, Huston D P, Liu Y J. A novel subset of CD4 (+) T(H)2
memory/effector cells that produce inflammatory IL-17 cytokine and
promote the exacerbation of chronic allergic asthma. J Exp Med
2010; 207:2479-2491. [0220] 103. Szabo S J, Kim S T, Costa G L,
Zhang X, Fathman C G, Glimcher L H. A novel transcription factor,
bet, directs Th1 lineage commitment. Cell 2000; 100:655-669. [0221]
104. Zhang D H, Cohn L, Ray P, Bottomly K, Ray A. Transcription
factor GATA-3 is differentially expressed in murine Th1 and Th2
cells and controls Th2-specific expression of the interleukin-5
gene. J Biol Chem 1997; 272:21597-21603. [0222] 105. Zheng W,
Flavell R A. The transcription factor GATA-3 is necessary and
sufficient for Th2 cytokine gene expression in CD4 T cells. Cell
1997; 89:587-596. [0223] 106. Zhou L, Lopes J E, Chong M M, Ivanov
I I, Min R, Victora G D, Shen Y, Du J, Rubtsov Y P, Rudensky A Y,
Ziegler S F, Littman D R. TGF-beta-induced Foxp3 inhibits T(H)17
cell differentiation by antagonizing RORgammat function. Nature
2008; 453:236-240. [0224] 107. Okamoto K, Iwai Y, Oh-Hora M,
Yamamoto M, Morio T, Aoki K, Ohya K, Jetten A M, Akira S. Muta T,
Takayanagi H. IkappaBzeta regulates T(H)17 development by
cooperating with ROR nuclear receptors. Nature 2010; 464:1381-1385.
[0225] 108. Ichiyama K, Yoshida H, Wakabayashi Y, Chinen T, Saeki
K. Nakaya M, Takaesu G, Hori S, Yoshimura A, Kobayashi T. Foxp3
inhibits RORgammat-mediated IL-17A mRNA transcription through
direct interaction with RORgammat. J Biol Chem 2008;
283:17003-17008. [0226] 1109. Zhou L, Ivanov I I, Spolski R, Min R,
Shenderov K, Egawa T, Levy D E, Leonard W J, Littman D R. IL-6
programs T(H)-17 cell differentiation by promoting sequential
engagement of the IL-21 and IL-23 pathways. Nat Immunol 2007;
8:967-974. [0227] 110. Li X M, Serebrisky D, Lee S Y, Huang C K,
Bardina L, Schofield B H, Stanley J S, Burks A W, Bannon G A,
Sampson H A. A murine model of peanut anaphylaxis: T- and B-cell
responses to a major peanut allergen mimic human responses. J
Allergy Clin Immunol 2000; 106:150-158.
* * * * *