U.S. patent application number 17/555786 was filed with the patent office on 2022-09-08 for aurora kinase and janus kinase inhibitors for prevention of graft versus host disease.
The applicant listed for this patent is H. LEE MOFFITT CANCER CENTER AND RESEARCH INSTITUTE, INC.. Invention is credited to Claudio Anasetti, Brian Betts, Harshani Lawrence, Nicholas Lawrence, Joseph Pidala, Said M. Sebti.
Application Number | 20220281828 17/555786 |
Document ID | / |
Family ID | 1000006351573 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220281828 |
Kind Code |
A1 |
Betts; Brian ; et
al. |
September 8, 2022 |
AURORA KINASE AND JANUS KINASE INHIBITORS FOR PREVENTION OF GRAFT
VERSUS HOST DISEASE
Abstract
Disclosed herein are compounds and methods for reducing the risk
of developing, preventing, or treating graft versus host disease
(GVHD) in a subject. The compounds can concurrently block Aurora
kinase A and JAK2 signal transduction which synergistically
suppresses alloreactive human T-cells in vitro, prevents xenogeneic
graft-versus-host disease without impairing anti-tumor responses,
and promotes the development and suppressive potency of CD39+
inducible T.sub.reg. In certain aspects, disclosed are compounds of
Formula I-V. ##STR00001## ##STR00002##
Inventors: |
Betts; Brian; (Tampa,
FL) ; Sebti; Said M.; (Tampa, FL) ; Lawrence;
Harshani; (Tampa, FL) ; Lawrence; Nicholas;
(Tampa, FL) ; Anasetti; Claudio; (Tampa, FL)
; Pidala; Joseph; (Tampa, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
H. LEE MOFFITT CANCER CENTER AND RESEARCH INSTITUTE, INC. |
Tampa |
FL |
US |
|
|
Family ID: |
1000006351573 |
Appl. No.: |
17/555786 |
Filed: |
December 20, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16083681 |
Sep 10, 2018 |
11203576 |
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PCT/US2017/022074 |
Mar 13, 2017 |
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17555786 |
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62307030 |
Mar 11, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/505 20130101; A61P 35/00 20180101; C07D 401/12 20130101;
C07D 403/12 20130101; A61K 31/506 20130101; C07D 239/48
20130101 |
International
Class: |
C07D 239/48 20060101
C07D239/48; C07D 401/12 20060101 C07D401/12; C07D 403/12 20060101
C07D403/12; A61P 35/00 20060101 A61P035/00; A61K 31/506 20060101
A61K031/506; A61K 31/505 20060101 A61K031/505; A61K 45/06 20060101
A61K045/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government Support under Grant
No. K08 HL11654701A1 and Grant No. P30-CA076292 awarded by the
National Institutes of Health. The Government has certain rights in
the invention.
Claims
1-18. (canceled)
19. A compound comprising the formula IIIC: ##STR00040## wherein
R.sup.1 is selected from the group consisting of H, Cl, F, Br, I,
C.sub.1-C.sub.6 alkyl, CN, NO.sub.2, and NH.sub.2; R.sup.2 is
selected from the group consisting of H, F, and Cl; and each
R.sup.4 is selected, independently, from the group consisting of H,
COOH, CONH.sub.2, CONR.sup.5, SO.sub.2NH.sub.2, CONSO.sub.2R.sup.5,
tetrazole, 4-morpholine, and COR.sup.5; each R.sup.5 is selected,
independently, from the group consisting of C.sub.1-C.sub.6 alkyl,
cycloalkyl, heteroaryl, and heteroalkyl; and m is 1-5, or a
pharmaceutically acceptable salt thereof.
20. The compound of claim 19, wherein m is 1 and R.sup.4 is
selected from the group consisting of COOH, 2-CONH.sub.2,
4-CONH.sub.2, SO.sub.2NH.sub.2, tetrazole, and 4-morpholine.
21. The compound of claim 19, wherein R.sup.1 is Cl, F, Br, or I
and R.sup.2 is H.
22. The compound of claim 19, wherein the compound is:
##STR00041##
23-24. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Application 62/307,030, filed Mar. 11, 2016, which is
incorporated by reference herein.
FIELD
[0003] The subject matter disclosed herein relates generally to
graft versus host disease (GVHD). More specifically, the subject
matter disclosed herein relates to inhibitors of Aurora kinase and
JAK2 and their use in preventing or treating GVHD.
BACKGROUND
[0004] Graft-versus-host disease (GVHD) is a leading cause of
non-relapse mortality after allogeneic hematopoietic cell
transplantation (alloHCT). Broadly acting calcineurin inhibitors
(CNI) are often used to prevent GVHD, but exert undesirable
antagonistic effects on T-cell receptor signaling and regulatory
T-cell (T.sub.reg) differentiation and function (M. Vaeth et al.,
Selective NFAT targeting in T cells ameliorates GvHD while
maintaining antitumor activity. Proc. Natl. Acad. Sci. USA 112,
1125 (2015); K. Singh et al., Superiority of rapamycin over
tacrolimus in preserving nonhuman primate T.sub.reg half-life and
phenotype after adoptive transfer. Am. J. Transplant.: 14, 2691
(2014); R. Zeiser et al., Inhibition of CD4+CD25+ regulatory T-cell
function by calcineurindependent interleukin-2 production. Blood
108, 390 (2006)). The lack of immune selectivity by CNIs places the
alloHCT recipient at risk for opportunistic infections and relapse
of their underlying hematologic malignancy. An alternative approach
at GVHD prevention is to concurrently target CD28 costimulation and
IL-6 receptor activation of T-cells by inhibiting key signal
transduction molecules in these pathways.
[0005] CD28 costimulation contributes to T-cell alloreactivity and
GVHD. GVHD in rodents is ameliorated by transplantation of CD28
negative compared to wild type T-cells (P. Tan et al., Induction of
alloantigen-specific hyporesponsiveness in human T lymphocytes by
blocking interaction of CD28 with its natural ligand B7/BB1. J.
Experi. Med. 177, 165 (1993); X. Z. Yu et al., CD28-specific
antibody prevents graft versus-host disease in mice. J. Immunol.
164, 4564 (2000)). Blockade of ligand interactions between
CD80/CD86 and CD28 with neutralizing antibody also reduces murine
GVHD. CD28 signal transduction activates mTOR and Aurora kinase in
T-cells (J. Song et al., The kinases aurora B and mTOR regulate the
G1-S cell cycle progression of T lymphocytes. Nature Immunol. 8, 64
(2007)). mTOR is a known pharmacologic target in GVHD prophylaxis
(C. Cutler et al., Tacrolimus/sirolimus vs tacrolimus/methotrexate
as GVHD prophylaxis after matched, related donor allogeneic HCT.
Blood 124, 1372 (2014); J. Pidala et al., Prolonged sirolimus
administration after allogeneic hematopoietic cell transplantation
is associated with decreased risk for moderate-severe chronic graft
vs. host disease. Haematologica (2015); J. Pidala et al., A
randomized phase II study to evaluate tacrolimus in combination
with sirolimus or methotrexate after allogeneic hematopoietic cell
transplantation. Haematologica 97, 1882 (2012)), and its blockade
is selectively toxic to T.sub.conv compared to T.sub.reg (R. Zeiser
et al., Differential impact of mammalian target of rapamycin
inhibition on CD4+CD25+Foxp3+ regulatory T cells compared with
conventional CD4+ T cells. Blood 111, 453 (2008)). However, GVHD
prevention with sirolimus, an mTOR inhibitor, is inadequate if not
combined with a CNI (L. Johnston et al., Sirolimus and
mycophenolate mofetil as GVHD prophylaxis in myeloablative,
matched-related donor hematopoietic cell transplantation. Bone
Marrow Transplant. 47, 581 (2012)). Aurora kinase isoforms
ubiquitously regulate mitotic progression and cellular polarity in
human cells (M. Carmena, W. C. Earnshaw, The cellular geography of
aurora kinases. Nature Rev. Mol. Cell Biol. 4, 842 (2003); S. M.
Lens et al., Shared and separate functions of polo-like kinases and
aurora kinases in cancer. Nature Rev. Cancer 10, 825 (2010)), but
also mediate T-cell costimulation. Aurora kinase is able to
activate substrates required for T-cell proliferation that are
shared with mTOR, and is only partially curtailed by sirolimus.
Complete inhibition of Aurora activity requires direct blockade of
the molecule or targeting upstream phosphatidylinositol-3-OH kinase
(PI(3)K). Increased Aurora kinase A expression was recently
correlated with acute GVHD in human recipients of alloHCT, as well
as experimental models studying murine and nonhuman primate (NHP)
hosts. Accordingly, treatment with sirolimus did not control Aurora
activity in the transplanted NHPs. Moreover, pharmacologic blockade
of this novel pathway with alisertib, an Aurora kinase A inhibitor,
significantly delayed the onset of GVHD in mice. These data
revealed that Aurora kinase A alone does not fully control
alloreactivity (S. N. Furlan et al., Transcriptome analysis of GVHD
reveals aurora kinase A as a targetable pathway for disease
prevention. Sci. Translational Med. 7, 315ra191 (2015)).
[0006] IL-6 receptor signaling polarizes T.sub.H1 and T.sub.H17
cells that are effectors in GVHD, and impairs T.sub.regs that
modulate GVHD (B. C. Betts et al., Janus kinase-2 inhibition
induces durable tolerance to alloantigen by human dendritic
cell-stimulated T cells yet preserves immunity to recall antigen.
Blood 118, 5330 (2011); B. C. Betts, A. Veerapathran, J. Pidala, X.
Z. Yu, C. Anasetti, STAT5 polarization promotes iT.sub.regs and
suppresses human T-cell alloresponses while preserving CTL
capacity. J. Leukocyte Biol. 95, 205 (2014); J. Choi et al.,
Pharmacologic blockade of JAK1/JAK2 reduces GvHD and preserves the
graft-versus-leukemia effect. PloS one 9, e109799 (2014); A.
Laurence et al., STAT3 transcription factor promotes instability of
nT.sub.reg cells and limits generation of iT.sub.reg cells during
acute murine graft-versus-host disease. Immunity 37, 209 (2012); R.
Zeiser et al., Ruxolitinib in corticosteroid-refractory
graft-versus-host disease after allogeneic stem cell
transplantation: a multi-center survey. Leukemia, (2015)). IL-6
activates JAK2 and leads to downstream phosphorylation of STAT3 (B.
C. Betts et al., Anti-IL6-receptor-alpha (tocilizumab) does not
inhibit human monocyte-derived dendritic cell maturation or
alloreactive T-cell responses. Blood 118, 5340 (2011)). It was
observed that CD4+ T-cell JAK2 activity by IL-6 is increased among
alloHCT recipients who later develop GVHD (B. C. Betts et al., CD4+
T cell STAT3 phosphorylation precedes acute GVHD, and subsequent
T.sub.H17 tissue invasion correlates with GVHD severity and
therapeutic response. J. Leukocyte Biol., (2015)). Anti-IL-6
receptor antibody combined with a CNI ameliorates human GVHD, but
it does not influence T.sub.H1, T.sub.H17, or T.sub.reg
differentiation (G. A. Kennedy et al., Addition of interleukin-6
inhibition with tocilizumab to standard graft-versus-host disease
prophylaxis after allogeneic stem-cell transplantation: a phase 1/2
trial. The Lancet. Oncology 15, 1451 (2014)). JAK2 inhibition
conversely polarizes natural T.sub.reg responses, and inhibits
T.sub.H1 and T.sub.H17 development in vitro. However, selective
blockade of JAK2 alone does not provide lasting protection in
murine GVHD. This observation is distinct from JAK1/JAK2
inhibition, where co-blockade of JAK1 acts broadly to reduce GVHD
(Blood 123, 3832 (2014)) as well as beneficial anti-viral CTL (S.
Spoerl et al., Activity of therapeutic JAK 1/2 blockade in
graft-versus-host disease. Blood 122, 3843 (2013); A. Heine, P.
Brossart, D. Wolf, Ruxolitinib is a potent immunosuppressive
compound: is it time for anti-infective prophylaxis? Blood 122,
1192 (2013)). These data show that JAK2 activation selective
inhibition is insufficient to completely prevent GVHD despite
favorable immune effects.
[0007] Thus there is a need for new compositions and methods for
treating GVHD. The compositions and methods disclosed herein
address these and other needs.
SUMMARY
[0008] In accordance with the purposes of the disclosed materials
and methods, as embodied and broadly described herein, the
disclosed subject matter, in one aspect, relates to compounds,
compositions and methods of making and using compounds and
compositions. In specific aspects, the disclosed subject matter
relates to reducing the risk of, preventing, or treating graft
versus host disease (GVHD) in a subject. More specifically, the
subject matter disclosed herein relates to inhibitors of Aurora
kinase A and JAK2. In more specific examples, the disclosed subject
matter relates to concurrent inhibition of Aurora kinase A and JAK2
and their use in reducing the risk of, preventing, or treating
GVHD. GVHD can be attributed to a solid organ transplant, tissue
graft, or a cellular transplant.
[0009] The methods described herein can include administering to a
subject at risk of developing or having GVHD, a composition
comprising a compound of the following formula
##STR00003##
wherein [0010] R.sup.1 is selected from the group consisting of H,
Cl, F, Br, I, CN, NO.sub.2, NH.sub.2, CF.sub.3, CO.sub.2H,
CO.sub.2NH.sub.2, CO.sub.2NHR.sup.5, CO.sub.2R.sup.5, C(O)R.sup.5,
C(O)NH.sub.2, C(O)NHR.sup.5, and C.sub.1-C.sub.6 alkyl optionally
substituted with alkoxy, alkenyl, alkynyl, aryl, heteroaryl,
aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy,
ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or
thiol; [0011] R.sup.2 is selected from the group consisting of H,
OH, CN, NO.sub.2, NH.sub.2, optionally substituted C.sub.1-C.sub.6
alkyl, optionally substituted cycloalkyl, optionally substituted
aryl, and optionally substituted heteroaryl, wherein optionally
substituted substituents are optionally substituted with alkoxy,
alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic
acid, ester, ether, halide, hydroxy, ketone, nitro, silyl,
sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol; [0012] or
R.sup.1 and R.sup.2 together form a fused cycloalkyl,
cycloheteroalkyl, aryl, or heteraryl group; [0013] each R.sup.3 is
selected, independently, from the group consisting of
SO.sub.2NH.sub.2, SO.sub.2NHR.sup.5, NHSO.sub.2R.sup.5,
NHCO.sub.2R.sup.5, NHC(O)R.sup.5, NHCONHR.sup.5, F, Cl, Br, I,
NO.sub.2, optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 alkoxy, optionally substituted
cycloheteroaryl, and optionally substituted fused cycloheteroalkyl,
wherein optionally substituted substituents are optionally
substituted with sulfonyl; [0014] each R.sup.4 is selected,
independently, from the group consisting of F, Cl, Br, I, NO.sub.2,
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 alkoxy, COOH, C(O)NH.sub.2,
C(O)R.sup.5, C(O)NHR.sup.5, CH.sub.2C(O)R.sup.5, SO.sub.2NH.sub.2,
SO.sub.2NHR.sup.5, CONHSO.sub.2R.sup.5, optionally substituted
phenyl, optionally substituted OPhenyl, tetrazole, piperadinyl,
piperazinyl, and morpholinyl, wherein optionally substituted
substituents are optionally substituted with alkoxy, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, oxo, silyl, sulfo-oxo,
sulfonyl, sulfone, sulfoxide, or thiol; [0015] each R.sup.5 is
selected, independently, from optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocycloalkyl, and optionally
substituted heteroalkyl, wherein optionally substituted
substituents are optionally substituted with C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, cycloalkyl, cycloheteroalkyl, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, oxo, silyl, sulfo-oxo,
sulfonyl, sulfone, sulfoxide, or thiol; [0016] R.sup.7 is selected
from the group consisting of H, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, halide, hydroxyl, cyano, nitro, and amino;
[0017] n is 0-5; and [0018] m is 1-5, or a pharmaceutically
acceptable salt thereof.
[0019] In certain aspects, the composition can include a compound
of the following formula:
##STR00004##
wherein [0020] R.sup.1 is selected from the group consisting of H,
Cl, F, Br, I, C.sub.1-C.sub.6 alkyl, CN, NO.sub.2, and NH.sub.2;
[0021] R.sup.2 is selected from the group consisting of H, F, and
Cl; [0022] each R.sup.3 is selected, independently, from the group
consisting of Cl, Br, F, COOH, CF.sub.3, CN, phenyl, OCH.sub.3,
COR.sup.5, CONH.sub.2, CONR.sup.5, and COONH.sub.2; and [0023] each
R.sup.4 is selected, independently, from the group consisting of H,
COOH, CONH.sub.2, CONR.sup.5, SO.sub.2NH.sub.2, CONSO.sub.2R.sup.5,
tetrazole, 4-morpholine, and COR.sup.5; [0024] each R.sup.5 is
selected, independently, from the group consisting of optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
cycloalkyl, optionally substituted heteroaryl, and optionally
substituted heteroalkyl, wherein optionally substituted
substituents are optionally substituted with C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, cycloalkyl, cycloheteroalkyl, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, oxo, silyl, sulfo-oxo,
sulfonyl, sulfone, sulfoxide, or thiol; [0025] n is 0-5; and [0026]
m is 1-5, or a pharmaceutically acceptable salt thereof.
[0027] In certain aspects, the composition can include a compound
having one of the following formulas:
##STR00005##
wherein [0028] R.sup.1 is selected from the group consisting of H,
Cl, F, Br, I, CH.sub.3 and NH.sub.2; [0029] R.sup.2 is selected
from the group consisting of H, F, and Cl; [0030] R.sup.3 is
selected from the group consisting of 2-Cl, 2-Br, 2-F, 2-COOH,
2-CF.sub.3, 2-CN, 2-phenyl, 2-OCH.sub.3, 2-COONH.sub.2, 4-COOH, and
4-OCH.sub.3; and [0031] R.sup.4 is selected from the group
consisting of H, COOH, 2-CONH.sub.2, 4-CONH.sub.2,
SO.sub.2NH.sub.2, tetrazole, and 4-morpholine.
[0032] In certain examples of Formulas I, II, and IIIA, R.sup.3 is
2-Cl, and n is 1. In some examples, R.sup.4 is 4-COOH, and m is 1.
In other examples, R.sup.4 is 4-CONH.sub.2, and m is 1. In some
examples, R.sup.3 is 2-Cl, n is 1, m is 1, and R.sup.4 is COOH,
COR.sup.5, CONH.sub.2, CONR.sup.5, or CONSO.sub.2R.sup.5, wherein
R.sup.5 is C.sub.1-C.sub.6 alkyl, cycloalkyl, heteroaryl, or
heteroalkyl. In other examples, R.sup.3 is H, m is 1, and R.sup.4
is COOH, COR.sup.5, CONH.sub.2, CONR.sup.5, or CONSO.sub.2R.sup.5,
wherein R.sup.5 is C.sub.1-C.sub.6 alkyl, cycloalkyl, heteroaryl,
or heteroalkyl. In a preferred example, R.sup.1 is Cl, R.sup.2 is
H, R.sup.3 is H, m is 1, and R.sup.4 is 4-CONH.sub.2. In other
examples n is 0.
[0033] The compositions described herein can be administered at a
dose of about 0.1 mg/kg to about 100 mg/kg.
[0034] Additional advantages will be set forth in part in the
description that follows, and in part will be obvious from the
description, or may be learned by practice of the aspects described
below. The advantages described below will be realized and attained
by means of the elements and combinations particularly pointed out
in the appended claims. It is to be understood that both the
foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive.
BRIEF DESCRIPTION OF THE FIGURES
[0035] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
described below.
[0036] FIGS. 1A-1E show synergistic immune suppression with
combined inhibition of Aurora kinase A and JAK2. In FIG. 1A,
T-cells were stimulated with DCs (DC:T-cell ratio 1:30) exposed to
either TG101348 (JAK2 inhibitor), alisertib (Aurora kinase A
inhibitor), or both at a fixed ratio of 1:5 respectively at varying
concentrations once on day 0. Proliferation was determined by
fluorescence assay on day 5, with % proliferation based on DMSO
control. Graph depicts combination synergy (IC.sub.50=TG101348 350
nM and alisertib 1.75 .mu.M), showing 1 representative independent
experiment of 2 performed in triplicate. Combination index (CI)
calculated per Chou and Talalay method. In FIG. 1B, AlloMLR
(DC:T-cell ratio 1:30) was treated with bisanilinopyrimidine (I)
(dual JAK2/Aurora kinase A inhibitor) or DMSO once on day 0.
Proliferation was determined by colorimetric assay on day 5, with %
proliferation based on DMSO control. IC.sub.50=100 nM. Graph shows
average triplicate means.+-.SEM from 3 independent experiments
(ANOVA). In FIGS. 1C-1D, bar graphs depict mean gated CD3.sup.+
Tcell STAT3 (target of JAK2) or H3 Ser10 (target of Aurora)
phosphorylation.+-.SD following 5-day allogeneic DC stimulation
treated with bisanilinopyrimidine (I) or DMSO from 3 independent
experiments (unpaired t-test). Representative contour plots are
shown with phosphorylation based on isotype control. In FIG. 1E, a
bar graph shows mean T-cell viability.+-.SD by LIVE/DEAD Yellow
exclusion from 4 independent experiments (unpaired t-test).
*P<0.05.
[0037] FIGS. 2A-2I show concurrent blockade of Aurora kinase A and
JAK2 selectively suppresses alloreactive T.sub.conv, while sparing
responder T.sub.regs. T-cells were stimulated with DCs (DC:Tcell
ratio 1:30) and treated with bisanilinopyrimidine (I) or DMSO once
on day 0. In FIGS. 2A-2D, bar graphs show percent means of
CD4.sup.+ alloreactive T.sub.conv (CD25.sup.+, CD127.sup.+),
CD4.sup.+ T.sub.reg (CD25.sup.+, CD127.sup.-), and T.sub.reg:allo
T.sub.conv ratio.+-.SD from 5 independent experiments on day 5 of
culture (unpaired t-test). Representative contour plots show
CD4.sup.+ alloreactive T.sub.conv (CD25+, CD127+) and CD4+T.sub.reg
(CD25+, CD127-) populations exposed to bisanilinopyrimidine (I) or
DMSO after 5 days of DCallostimulation. In FIG. 2E, box plots
depict mean T.sub.reg versus T.sub.conv proliferation by Cell Trace
Violet dilution.+-.SD among identically treated alloMLRs exposed to
bisanilinopyrimidine (I) or DMSO from 3 independent experiments
(unpaired t-test). FIG. 2F is a representative histogram that shows
proliferation in each T-cell compartment with respect to
bisanilinopyrimidine (I) or DMSO treatment. In FIG. 2G, a bar graph
shows mean gated CD4.sup.+T-cell STAT5 phosphorylation.+-.SD
following brief IL-2 stimulation treated with bisanilinopyrimidine
(I) or DMSO from 3 independent experiments (ANOVA). In FIG. 2H, a
histogram depicts intracellular Foxp3 expression among CD4.sup.+,
CD25.sup.+, CD127.sup.- T.sub.regs following 5-day
DCallostimulation exposed to bisanilinopyrimidine (I) or DMSO. Data
representative of 5 experiments. In FIG. 2I, AlloMLRs (DC:T-cell
ratio 1:30) were treated with alisertib (Aurora kinase A
inhibitor), TG101348 (JAK2 inhibitor), a combination of both, or
DMSO once on day 0. Representative contour plots show CD4.sup.+
alloreactive T.sub.conv (CD25.sup.+, CD127.sup.+) and CD4.sup.+
T.sub.reg (CD25.sup.+, CD127.sup.-) populations exposed to either
inhibitor, the combination, or DMSO after 5 days of
DCallostimulation. Data are from one representative experiment of
2. NS=not significant, *P<0.05, **P=0.001-0.01.
[0038] FIGS. 3A-3E show dual blockade of Aurora kinase A and JAK2
selectively increases the ratio of iT.sub.reg to alloreactive
T.sub.conv. In FIG. 3A, a representative contour plots show
T.sub.reg (CD25.sup.+, CD127.sup.-) depletion of naive CD4.sup.+
T-cell responders at outset of alloMLR (DC:T-cell ration 1:30),
followed by induction of iT.sub.reg versus allo T.sub.conv
(CD25.sup.+, CD127.sup.+) after 5 days of culture exposed to
AJI-214 or DMSO. In FIGS. 3B-3C, bar graphs show mean frequency of
CD4.sup.+ iT.sub.reg and allo T.sub.conv in 5-day allogeneic
co-cultures treated with bisanilinopyrimidine (I) or DMSO.+-.SD
from 4 independent experiments (unpaired t-test). In FIG. 3D, a bar
graph depicts triplicate mean % demethylation of Foxp3.+-.SEM among
iT.sub.regs in alloMLRs treated with bisanilinopyrimidine (I) or
DMSO from 4 independent experiments (unpaired t-test). In FIG. 3E,
bar graphs show replicate means of absolute numbers of iT.sub.reg,
allo T.sub.conv, and the ratio of iT.sub.reg:allo T.sub.conv.+-.SD
from 5-day MLRs treated with Alisertib 1.75 .mu.M, TG101348 350 nM,
a combination of both, bisanilinopyrimidine (I) 750 nM, or DMSO
control (ANOVA). Data are from one representative experiment of 2
performed in triplicate.
[0039] FIGS. 4A-4B show combined inhibition of Aurora kinase A and
JAK2 enhances antigen-specific iT.sub.reg suppressive potency. In
FIG. 4A, the suppressive capacity of sorted, DC-allostimulated
iT.sub.regs previously exposed to bisanilinopyrimidine (I) or DMSO
was tested at different ratios of iT.sub.reg to self T-cell
responders stimulated by original allogeneic DCs (DC:responder
T-cell ratio 1:30) in fresh alloMLRs. No additional
bisanilinopyrimidine (I) or DMSO was added. The bar graph shows
triplicate means of % proliferation.+-.SEM based on 3H-thymidine
incorporation on day 6 from 3 independent experiments (ANOVA). In
FIG. 4B, the potency of iT.sub.regs generated in the presence of
alisertib (Aurora kinase A inhibitor), TG101348 (JAK2 inhibitor), a
combination of both, or DMSO was tested in standard suppression
assays. No additional small molecule inhibitors or DMSO was added.
The bar graph shows triplicate means of % proliferation.+-.SD based
on 3H-thymidine incorporation on day 6 (unpaired t-test). Data are
from one representative experiment of 2 performed in
triplicate.
[0040] FIGS. 5A-5J show targeting Aurora kinase A and JAK2
increases CD39 expression and ATP scavenging among iT.sub.reg. In
FIG. 5A, contour plots show the CD4.sup.+ iT.sub.reg and
non-T.sub.reg gating strategy after 5-day alloMLR treated with
bisanilinopyrimidine (I) (750 nM) or DMSO. In FIGS. 5B-5D, CD39
expression (% and geometric MFI) was increased by among iT.sub.reg
generated in the presence of bisanilinopyrimidine (I) (750 nM). The
bar graphs show mean data.+-.SD from 3 independent experiments
(unpaired t-test). In FIG. 5E, bar graphs show replicate means of
ATP consumption.+-.SD after stimulating 75,000 iT.sub.reg with 50
.mu.M of ATP for 45 minutes. ATP measured by luminescence assay.
Data are from one representative experiment of 2 performed in
triplicate (unpaired t-test). In FIG. 5F, AlloMLRs of naive,
T.sub.reg-depleted CD4.sup.+ T-cells, and allogeneic DCs (DC:T-cell
ratio 1:30) were treated with either bisanilinopyrimidine (I),
ARL67156 (CD39 inhibitor), both, or DMSO. Proliferation was
determined by colorimetric assay on day 5, with % proliferation
based on DMSO control. Graph shows average triplicate means.+-.SEM
from 5 independent experiments (unpaired t-test). In FIGS. 5G-5H,
bar graphs depict mean fold MFI of LAG3 and CTLA4.+-.SD on
iT.sub.regs harvested from alloMLRs treated with
bisanilinopyrimidine (I) or DMSO from 3 independent experiments
(unpaired t-test). In FIGS. 5I-5J, bar graphs show triplicate mean
concentrations of IL-10 and TGF-beta.+-.SEM among
PMA/ionomycin-stimulated iT.sub.regs previously exposed to
bisanilinopyrimidine (I) or DMSO during co-culture from 4
independent experiments (unpaired t-test). *P<0.05,
**P=0.001-0.01, ****P<0.0001.
[0041] FIGS. 6A-6B show Aurora kinase A inhibition does not impair
T.sub.H17 differentiation. Isolated naive CD4.sup.+ T-cells were
stimulated with allogeneic DCs for 5 days with alisertib, AJI-214,
TG101348, or DMSO. Media was supplemented with IL-6, TGF-beta, and
anti-IFN-.gamma.mAb to promote RORgammaT expression. The bar graphs
depict replicate means of relative RORgammaT expression.+-.SEM
among drug- and control-treated CD4.sup.+ T-cells (unpaired
t-test). Data are from one representative experiment of 2 performed
in triplicate for each figure. *P<0.05, **P=0.001-0.01.
[0042] FIGS. 7A-7H show blockade of Aurora kinase A and JAK2
reduces xenogeneic GVHD and preserves anti-tumor CTL function. In
FIG. 7A, AlloMLR (DC:T-cell ratio 1:30) was treated with
bisanilinopyrimidine (II) (dual Aurora kinase A/JAK2 inhibitor) or
DMSO once on day 0. Proliferation was determined by colorimetric
assay on day 5 (ANOVA). IC.sub.50=200 nM. Graph shows triplicate
means.+-.SD from 1 representative experiment of 2. In FIG. 7B, a
representative histogram shows bisanilinopyrimidine (II) inhibits
CD3.sup.+ T-cell STAT3 phosphorylation in vitro. Data are from one
representative experiment of 2. NSG mice received human PBMCs
(30.times.10.sup.6 cells), with bisanilinopyrimidine (II) (50 mg/kg
daily) or vehicle administered from day 0 to day +14. In FIG. 7C, a
representative histogram shows H3 ser10 phosphorylation in human
CD3.sup.+ T-cells harvested at day +14 among each group. In FIGS.
7D-7F, graphs show mean % weight change.+-.SEM, GVHD scores.+-.SEM
(unpaired t-test), and survival of pooled data from 2 independent
experiments (long-rank test). n=8 mice per each group. In FIG. 7G,
a graph depicts mean specific lysis.+-.SD by human CTL generated in
vivo using NSG mice transplanted with human PBMCs and irradiated
U937 cells (10.sup.7) on days 0 and +7. Results shown are from 1 of
2 independent experiments, using a total of 7 mice per arm. In FIG.
7H, a graph shows mean specific lysis.+-.SD of CD8.sup.+ CTL
generated in vitro while exposed to bisanilinopyrimidine (I) or
DMSO for 10 days. Results shown are from 1 of 2 independent
experiments. U937 lysis was measured by released fluorescence after
4 hours. **P=0.001-0.01, ***P=0.0001-0.001.
[0043] FIGS. 8A-8K show targeting Aurora kinase A and JAK2
increases the proportion of T.sub.reg to allo T.sub.conv in vivo,
and reduces T-cell homing to recipient livers. Xenotransplanted NSG
mice were treated with bisanilinopyrimidine (II) (50 mg/kg daily)
or vehicle daily starting at day 0, then euthanized on day +14.
Recipient spleens and livers were harvested, and tissue-resident
T-cells were evaluated. Dot plots show absolute number of total
spleen cells (FIG. 8A), human CD3.sup.+ T-cells (FIG. 8B), %
CD4.sup.+, CD25.sup.+, CD127.sup.-, Foxp3.sup.+ T.sub.reg (FIG.
8C), % CD4+, CD25.sup.+, CD127.sup.+ allo T.sub.conv (FIG. 8D), and
the ratio of T.sub.reg to allo T.sub.conv (FIG. 8E) (unpaired
t-test). In FIG. 8F, representative contour plots show the %
CD4.sup.+ T.sub.reg and % CD4.sup.+ alloreactive T.sub.conv
residing in spleens of bisanilinopyrimidine (II)- or
vehicle-treated mice at day +14. FIG. 8G, the representative
histograms show corresponding expression of Foxp3 within the
CD4+T.sub.regs. In FIGS. 8H-8I, sections of recipient livers show
bisanilinopyrimidine (II) significantly reduces the amount of
tissue-resident human T-cells, compared to vehicle control
(red=CD3). Dot plots show the number of human T-cells per high
power field (FIG. 8J) and % CD4.sup.+ cell expressing Foxp3 (FIG.
8K) in the livers at day +14 (unpaired t-test). Pooled data from 2
independent experiments. n=7-8 mice per each group. NS=not
significant, *P<0.05, ***P=0.0001-0.001.
[0044] FIGS. 9A-9H show synergistic immune suppression with
combined inhibition of Aurora kinase A and JAK2. In FIG. 9A, Human
T cells were stimulated with DCs (DC/T cell ratio of 1:30) exposed
to TG101348 (JAK2 inhibitor), alisertib (Aurora kinase A
inhibitor), or both TG101348 and alisertib at a fixed ratio of 1:5,
respectively, at varying concentrations once on day 0.
Proliferation was determined by fluorescence assay on day 5, with %
proliferation based on dimethyl sulfoxide (DMSO) control. Graph
depicts combination synergy (IC.sub.50 values for TG101348 and
alisertib were 350 nM and 1.75 mM, respectively), showing one
representative independent experiment of two performed with
triplicate technical replicates. The CI was calculated using the
Chou-Talalay method. AlloMLR (DC/T cell ratio of 1:30) treated with
AJI-214 (FIG. 9B), AJI-100 (FIG. 9C) (dual JAK2/Aurora kinase A
inhibitors), or DMSO once on day 0 is shown. IC.sub.50 values for
AJI-214 and AJI-100 were 100 and 200 nM, respectively. Graph shows
average triplicate means.+-.SEM from two to three independent
experiments [analysis of variance (ANOVA)]. In FIG. 9D, a bar graph
depicts T cell proliferation when exposed to alisertib (1.75 mM),
TG101348 (350 nM), a combination of alisertib and TG101348 (combo),
AJI-214 (750 nM), or AJI-100 (750 nM) in alloMLRs. Means.+-.SEM
from four independent experiments (ANOVA) are shown using
triplicate technical replicates. In FIGS. 9E-9H, bar graphs depict
the mean gated CD3.sup.+ T cell H3Ser10 (target of Aurora) or STAT3
(target of JAK2) phosphorylation.+-.SD from three independent
experiments after stimulation with allogeneic DCs (5 days) or IL-6
(15 min), respectively (ANOVA). Representative contour plots show
H3Ser10 and STAT3 phosphorylation, respectively.
[0045] FIGS. 10A-10H show immunosuppressive effect of Aurora kinase
A/JAK2 blockade on responder T.sub.conv and T.sub.H subsets. In
FIG. 10A-10B, T cells were stimulated with allogeneic DCs (DC/T
cell ratio of 1:30) and treated with kinase inhibitors or DMSO once
on day 0. Bar graphs show replicate mean absolute numbers of
activated CD4.sup.+, CD25.sup.+, CD127.sup.+ or activated
CD8.sup.+, CD25.sup.+ T.sub.conv.+-.SEM at day +5 from six
independent experiments (ANOVA and paired t test). In FIG. 10C, a
bar graph shows the mean number of IL-17 spots per well.+-.SD from
triplicate technical replicates among DC-allostimulated CD4.sup.+ T
cells. One of three representative experiments is shown. In FIG.
10D, a bar graph shows mean % CD4.sup.+, IFN-.gamma..sup.+ T
cells.+-.SEM at day +5 of alloMLR from five independent experiments
with technical replicates performed in triplicate. In FIGS.
10E-10F, representative contour plots show T.sub.reg (CD25.sup.+
and CD127.sup.-) depletion of CD4.sup.+ T cell responders at outset
of alloMLR (DC/T cell ratio of 1:30), followed by induction of
iT.sub.reg (CD127.sup.-, CD25.sup.+, and Foxp3.sup.+) after 5 days
of culture exposed to kinase inhibitors or DMSO. In FIG. 10G, a bar
graph shows mean absolute numbers of iT.sub.regs.+-.SEM from seven
independent experiments performed with two to three technical
replicates (ANOVA and paired t test). In FIG. 10H, a representative
histograms depict pSTAT5 expression among IL-2-stimulated CD3.sup.+
T cells while exposed to kinase inhibitors or DMSO. Geometric mean
fluorescence intensity (MFI) of pSTAT5 is shown along the right
margin. One of three representative experiments is shown.
*P<0.05, **P=0.001 to 0.01. Alisertib (1.75 mM), TG101348 (350
nM), AJI-214 (750 nM), and AJI-100 (750 nM).
[0046] FIGS. 11A-11C show combined inhibition of Aurora kinase A
and JAK2 enhances antigen-specific iT.sub.reg-suppressive potency.
In FIG. 11A, a Bar graph depicts mean % demethylation of
Foxp3.+-.SEM among iT.sub.regs in alloMLRs treated with AJI-214
(750 nM) or DMSO from four independent experiments using triplicate
technical replicates. In FIG. 11B, the suppressive capacity of
sorted, DC-allostimulated iT.sub.regs previously exposed to AJI-214
or DMSO was tested at different ratios of iT.sub.reg to T cell
responders stimulated by fresh allogeneic DCs (DC/responder T cell
ratio of 1:30) in alloMLRs. No additional AJI-214 or DMSO was
added. Bar graph shows means of % proliferation.+-.SEM based on
[.sup.3H]thymidine incorporation on day 6 from three independent
experiments with triplicate technical replicates (ANOVA). In FIG.
11C, the potency of iT.sub.regs generated in the presence of
alisertib (1.75 mM), TG101348 (350 nM), a combination of alisertib
and TG101348, or DMSO was tested in standard suppression assays. No
additional small-molecule inhibitors or DMSO was added. Bar graph
shows means of % proliferation.+-.SD based on [.sup.3H]thymidine
incorporation on day 6 (paired t test). Data are from one
representative experiment of two performed using triplicate
technical replicates. *P<0.05.
[0047] FIGS. 12A-12J show targeting Aurora kinase A and JAK2
increases CD39 expression and ATP scavenging among iT.sub.reg. In
FIG. 12A, contour plots show the CD4.sup.+ iT.sub.reg and
non-T.sub.reg gating strategy after 5-day alloMLR treated with
AJI-214 (750 nM) or DMSO. In FIGS. 12B-12D, CD39 density [geometric
MFI (gMFI)] is increased by among iT.sub.reg generated in the
presence of AJI-214 (750 nM). Bar graphs show mean data.+-.SD from
three independent experiments (paired t test). In FIG. 12E, bar
graphs show replicate means of ATP consumption.+-.SD after
stimulating 75,000 iT.sub.regs with 50 mM ATP for 45 min. ATP was
measured by luminescence assay. Data are from one representative
experiment of two performed using triplicate technical replicates
(paired t test). In FIG. 12F, AlloMLRs of naive, T.sub.reg-depleted
CD4.sup.+ T cells and allogeneic DCs (DC/T cell ratio of 1:30) were
treated with AJI-214, ARL67156 (CD39 inhibitor), both AJI-214 and
ARL67156, or DMSO. Proliferation was determined by colorimetric
assay on day 5, with % proliferation based on DMSO control. Graph
shows means.+-.SEM from five independent experiments using three
technical replicates (paired t test). In FIGS. 12G-12H, bar graphs
depict mean fold MFI of LAG3 and CTLA4.+-.SD on iT.sub.regs
harvested from alloMLRs treated with AJI-214 or DMSO from three
independent experiments. In FIG. 12I-12J, bar graphs show mean
concentrations of IL-10 and TGF-0.+-.SEM among PMA (phorbol
12-myristate 13-acetate)/ionomycin-stimulated iT.sub.regs
previously exposed to AJI-214 or DMSO during coculture from four
independent experiments using three technical replicates.
*P<0.05, **P=0.001 to 0.01, ****P<0.0001.
[0048] FIGS. 13A-13G show Blockade of Aurora kinase A and JAK2
reduces xenogeneic GVHD and preserves the in vivo generation of
potent antitumor CTL. NSG mice received human PBMCs
(30.times.10.sup.6 cells) by intraperitoneal injection, with
alisertib (30 mg/kg daily), TG101348 (45 mg/kg twice a day), a
combination of alisertib and TG101348, or vehicle administered by
oral gavage from day 0 to day +14. In FIG. 13A, percent survival is
shown among the four groups (log-rank test). In FIG. 13B, a graph
shows mean GVHD clinical scores.+-.SEM for each group of mice
(P=0.02 at day +30, vehicle versus combo, Mann-Whitney). Pooled
data are from two independent experiments. n=7 to 8 mice per group.
NSG mice were transplanted with human PBMCs as described, with
AJI-100 (50 mg/kg daily) or vehicle administered ip from day 0 to
day +14. In FIGS. 13C-13D, percent survival (log-rank test) and
mean GVHD clinical scores.+-.SEM (Mann-Whitney) are demonstrated.
Pooled data are from two independent experiments. n=8 mice per
group. In FIG. 13E, representative contour plots show expression of
H3Ser.sup.10 and STAT3 phosphorylation among human CD3.sup.+ T
cells harvested from recipient spleens at day +14. In FIG. 13F, a
bar graph shows the mean % pH3Ser.sup.10+ and % pSTAT3.sup.+ T
cells.+-.SEM among AJI-100-treated and vehicle-treated mice at day
+14 (n=6 mice per group, two independent experiments,
Mann-Whitney). In FIG. 13G, a graph depicts mean specific
lysis.+-.SD by human CD8.sup.+ CTL generated in vivo using NSG mice
transplanted with human PBMCs and vaccinated with irradiated U937
cells (1.times.10.sup.7) on days 0 and +7. Results shown are from
one of two independent experiments, using a total of seven mice per
group. U937 lysis was measured by released fluorescence after 4
hours (vehicle versus AJI-100, not significant, Mann-Whitney).
*P<0.05, **P=0.001 to 0.01, ***P=0.0001 to 0.001,
****P<0.0001.
[0049] FIGS. 14A-14M show targeting Aurora kinase A and JAK2
increases the proportion of T.sub.reg to activated T.sub.conv and
reduces T.sub.H17 and T.sub.H1 cells in xenotransplanted recipient
mice. In FIGS. 14A-14D, xenotransplanted NSG mice were treated with
AJI-100 (50 mg/kg) or vehicle daily starting at day 0 and then
euthanized on day +14. Recipient spleens, livers, and lungs were
harvested, and tissue-resident T cells were evaluated. Bar graphs
show replicate mean absolute number of human CD4.sup.+ T cells
(FIG. 14A), CD4.sup.+ T.sub.regs (FIG. 14B), CD4.sup.+ activated
T.sub.conv (CD25.sup.+ and CD127.sup.+) (FIG. 14C), and the ratio
of T.sub.reg to activated T.sub.conv (FIG. 14D).+-.SEM
(Mann-Whitney). In FIG. 14E, representative contour plots show the
% CD4.sup.+ T.sub.reg and % CD4.sup.+ activated allo-T.sub.conv
residing in spleens of AJI-100-treated or vehicle-treated mice at
day +14. The representative histograms show corresponding
expression of Foxp3 within the CD4.sup.+ T.sub.regs. In FIG. 14F, a
bar graph shows the replicate mean number of IL-17 spots per
well.+-.SEM among human lymphocytes harvested from recipient
spleens at day +14 (Mann-Whitney). In FIG. 14G-14H, Bar graph and
representative contour plots depict the amount of CD4+,
IFN-.gamma..sup.+ T cells.+-.SEM from AJI-100 or vehicle-treated
mice at day +14 (Mann-Whitney). In FIG. 14I, sections of recipient
livers (top) and lung (bottom) show that AJI-100 significantly
reduces GVHD damage in recipient target organs, compared to vehicle
control. In FIG. 14J-14K bar graphs depict the mean GVHD pathology
scores.+-.SEM for host liver and lung at day +14. In FIG. 14L-14M,
bar graphs shows that the mean number of human CD3.sup.+ T
cells.+-.SEM [per high-power field (HPF)] infiltrating liver or
lung at day +14 is significantly reduced by AJI-100 compared to
vehicle (Mann-Whitney). Pooled data are from at least two
independent experiments. n=6 to 14 mice per group. *P<0.05,
**P=0.001 to 0.01, ****P<0.0001.
[0050] FIGS. 15A-15C are micrographs of representative H&E
stained human, xenogenic shin grafts at day +21. Mice were treated
with AJI-100. Mice receiving skin alone and no PBMC's (allogenic
peripheral blood mononuclear cells) were considered as
non-rejection control (n=1 experiment, 12 mice).
DETAILED DESCRIPTION
[0051] The materials, compounds, compositions, and methods
described herein may be understood more readily by reference to the
following detailed description of specific aspects of the disclosed
subject matter, the Figures, and the Examples included therein.
[0052] Before the present materials, compounds, compositions, and
methods are disclosed and described, it is to be understood that
the aspects described below are not limited to specific synthetic
methods or specific reagents, as such may, of course, vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular aspects only and is not intended
to be limiting.
[0053] Also, throughout this specification, various publications
are referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which the disclosed matter pertains. The references disclosed are
also individually and specifically incorporated by reference herein
for the material contained in them that is discussed in the
sentence in which the reference is relied upon.
General Definitions
[0054] In this specification and in the claims that follow,
reference will be made to a number of terms, which shall be defined
to have the following meanings:
[0055] Throughout the specification and claims the word "comprise"
and other forms of the word, such as "comprising" and "comprises,"
means including but not limited to, and is not intended to exclude,
for example, other additives, components, integers, or steps.
[0056] As used in the description and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a composition" includes mixtures of two or more such
compositions, reference to "an inhibitor" includes mixtures of two
or more such inhibitors, reference to "the kinase" includes
mixtures of two or more such kinase, and the like.
[0057] "Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not.
[0058] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the disclosure are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contain certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Furthermore, when numerical ranges of varying scope are set forth
herein, it is contemplated that any combination of these values
inclusive of the recited values may be used. Further, ranges can be
expressed herein as from "about" one particular value, and/or to
"about" another particular value. When such a range is expressed,
another aspect includes from the one particular value and/or to the
other particular value. Similarly, when values are expressed as
approximations, by use of the antecedent "about," it will be
understood that the particular value forms another aspect. It will
be further understood that the endpoints of each of the ranges are
significant both in relation to the other endpoint, and
independently of the other endpoint. Unless stated otherwise, the
term "about" means within 5% (e.g., within 2% or 1%) of the
particular value modified by the term "about."
[0059] By "reduce" or other forms of the word, such as "reducing"
or "reduction," is meant lowering of an event or characteristic
(e.g., the risk of having GVHD). It is understood that this is
typically in relation to some standard or expected value, in other
words it is relative, but that it is not always necessary for the
standard or relative value to be referred to.
[0060] By "prevent" or other forms of the word, such as
"preventing" or "prevention," is meant to stop a particular event
or characteristic, to stabilize or delay the development or
progression of a particular event or characteristic, or to minimize
the chances that a particular event or characteristic will occur.
Prevent does not require comparison to a control as it is typically
more absolute than, for example, reduce. As used herein, something
could be reduced but not prevented, but something that is reduced
could also be prevented. Likewise, something could be prevented but
not reduced, but something that is prevented could also be reduced.
It is understood that where reduce or prevent are used, unless
specifically indicated otherwise, the use of the other word is also
expressly disclosed.
[0061] As used herein, "treatment" refers to obtaining beneficial
or desired clinical results. Beneficial or desired clinical results
include, but are not limited to, any one or more of: alleviation of
one or more symptoms (such as GVHD), diminishment of extent of
GVHD, stabilized (i.e., not worsening) state of GVHD, preventing or
delaying occurrence or recurrence of GVHD, delay or slowing of GVHD
progression, and amelioration of the GVHD state.
[0062] The term "patient" preferably refers to a human in need of
treatment for any purpose, and more preferably a human in need of
such a treatment to treat GVHD. However, the term "patient" can
also refer to non-human animals, preferably mammals such as dogs,
cats, horses, cows, pigs, sheep and non-human primates, among
others, that are in need of treatment.
Chemical Definitions
[0063] As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results, directly
or indirectly, from combination of the specified ingredients in the
specified amounts.
[0064] References in the specification and concluding claims to
parts by weight of a particular element or component in a
composition denotes the weight relationship between the element or
component and any other elements or components in the composition
or article for which a part by weight is expressed. Thus, in a
mixture containing 2 parts by weight of component X and 5 parts by
weight component Y, X and Y are present at a weight ratio of 2:5,
and are present in such ratio regardless of whether additional
components are contained in the mixture.
[0065] A weight percent (wt. %) of a component, unless specifically
stated to the contrary, is based on the total weight of the
formulation or composition in which the component is included.
[0066] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic, and
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
below. The permissible substituents can be one or more and the same
or different for appropriate organic compounds. For purposes of
this disclosure, the heteroatoms, such as nitrogen, can have
hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valencies of
the heteroatoms. This disclosure is not intended to be limited in
any manner by the permissible substituents of organic compounds.
Also, the terms "substitution" or "substituted with" include the
implicit proviso that such substitution is in accordance with
permitted valence of the substituted atom and the substituent, and
that the substitution results in a stable compound, e.g., a
compound that does not spontaneously undergo transformation such as
by rearrangement, cyclization, elimination, etc.
[0067] The term "aliphatic" as used herein refers to a non-aromatic
hydrocarbon group and includes branched and unbranched, alkyl,
alkenyl, or alkynyl groups.
[0068] The term "alkyl" as used herein is a branched or unbranched
saturated hydrocarbon group of 1 to 24 carbon atoms, such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,
pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl,
hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can
also be substituted or unsubstituted. The alkyl group can be
substituted with one or more groups including, but not limited to,
alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl,
heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,
hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone,
sulfoxide, or thiol, as described below.
[0069] The symbols A.sup.n is used herein as merely a generic
substituent in the definitions below.
[0070] The term "alkoxy" as used herein is an alkyl group bound
through a single, terminal ether linkage; that is, an "alkoxy"
group can be defined as --OA.sup.1 where A.sup.1 is alkyl as
defined above.
[0071] The term "alkenyl" as used herein is a hydrocarbon group of
from 2 to 24 carbon atoms with a structural formula containing at
least one carbon-carbon double bond. Asymmetric structures such as
(A.sup.1A.sup.2)C.dbd.C(A.sup.3A.sup.4) are intended to include
both the E and Z isomers. This may be presumed in structural
formulae herein wherein an asymmetric alkene is present, or it may
be explicitly indicated by the bond symbol C.dbd.C. The alkenyl
group can be substituted with one or more groups including, but not
limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl,
aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,
halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol, as described below.
[0072] The term "alkynyl" as used herein is a hydrocarbon group of
2 to 24 carbon atoms with a structural formula containing at least
one carbon-carbon triple bond. The alkynyl group can be substituted
with one or more groups including, but not limited to, alkyl,
halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl,
aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy,
ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or
thiol, as described below.
[0073] The term "aryl" as used herein is a group that contains any
carbon-based aromatic group including, but not limited to, benzene,
naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The
term "heteroaryl" is defined as a group that contains an aromatic
group that has at least one heteroatom incorporated within the ring
of the aromatic group. Examples of heteroatoms include, but are not
limited to, nitrogen, oxygen, sulfur, and phosphorus. The term
"non-heteroaryl," which is included in the term "aryl," defines a
group that contains an aromatic group that does not contain a
heteroatom. The aryl and heteroaryl group can be substituted or
unsubstituted. The aryl and heteroaryl group can be substituted
with one or more groups including, but not limited to, alkyl,
halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl,
aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy,
ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or
thiol as described herein. The term "biaryl" is a specific type of
aryl group and is included in the definition of aryl. Biaryl refers
to two aryl groups that are bound together via a fused ring
structure, as in naphthalene, or are attached via one or more
carbon-carbon bonds, as in biphenyl.
[0074] The term "cycloalkyl" as used herein is a non-aromatic
carbon-based ring composed of at least three carbon atoms. Examples
of cycloalkyl groups include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, etc. The term
"heterocycloalkyl" is a cycloalkyl group as defined above where at
least one of the carbon atoms of the ring is substituted with a
heteroatom such as, but not limited to, nitrogen, oxygen, sulfur,
or phosphorus. The cycloalkyl group and heterocycloalkyl group can
be substituted or unsubstituted. The cycloalkyl group and
heterocycloalkyl group can be substituted with one or more groups
including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl,
aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,
halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol as described herein.
[0075] The term "cycloalkenyl" as used herein is a non-aromatic
carbon-based ring composed of at least three carbon atoms and
containing at least one double bound, i.e., C.dbd.C. Examples of
cycloalkenyl groups include, but are not limited to, cyclopropenyl,
cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl,
cyclohexadienyl, and the like. The term "heterocycloalkenyl" is a
type of cycloalkenyl group as defined above where at least one of
the carbon atoms of the ring is substituted with a heteroatom such
as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.
The cycloalkenyl group and heterocycloalkenyl group can be
substituted or unsubstituted. The cycloalkenyl group and
heterocycloalkenyl group can be substituted with one or more groups
including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl,
aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,
halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol as described herein.
[0076] The term "cyclic group" is used herein to refer to either
aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl,
cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic
groups have one or more ring systems that can be substituted or
unsubstituted. A cyclic group can contain one or more aryl groups,
one or more non-aryl groups, or one or more aryl groups and one or
more non-aryl groups.
[0077] The term "aldehyde" as used herein is represented by the
formula --C(O)H. Throughout this specification "C(O)" is a short
hand notation for C.dbd.O.
[0078] The terms "amine" or "amino" as used herein are represented
by the formula NA.sup.1A.sup.2A.sup.3, where A.sup.1, A.sup.2, and
A.sup.3 can be, independently, hydrogen, an alkyl, halogenated
alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group
described above.
[0079] The term "carboxylic acid" as used herein is represented by
the formula --C(O)OH. A "carboxylate" as used herein is represented
by the formula --C(O)O--.
[0080] The term "ester" as used herein is represented by the
formula --OC(O)A.sup.1 or --C(O)OA.sup.1, where A.sup.1 can be an
alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl,
cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl
group described above.
[0081] The term "ether" as used herein is represented by the
formula A.sup.1OA.sup.2, where A.sup.1 and A.sup.2 can be,
independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl,
heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or
heterocycloalkenyl group described above.
[0082] The term "ketone" as used herein is represented by the
formula A.sup.1C(O)A.sup.2, where A.sup.1 and A.sup.2 can be,
independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl,
heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or
heterocycloalkenyl group described above.
[0083] The term "halide" as used herein refers to the halogens
fluorine, chlorine, bromine, and iodine.
[0084] The term "hydroxyl" as used herein is represented by the
formula --OH.
[0085] The term "nitro" as used herein is represented by the
formula --NO.sub.2.
[0086] The term "cyano" as used herein is represented by the
formula --CN The term "azido" as used herein is represented by the
formula --N.sub.3.
[0087] The term "oxo" as used herein is represented by .dbd.O.
[0088] The term "sulfonyl" is used herein to refer to the sulfo-oxo
group represented by the formula --S(O).sub.2A.sup.1, where A.sup.1
can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl,
aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or
heterocycloalkenyl group described above. The term "sulfoxide" is
used herein to refer to the sulfo-oxo group represented by the
formula --OS(O).sub.2A.sup.1, where A.sup.1 can be hydrogen, an
alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl,
cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl
group described above.
[0089] The term "sulfonylamino" or "sulfonamide" as used herein is
represented by the formula --S(O).sub.2NH.sub.2.
[0090] The term "thiol" as used herein is represented by the
formula --SH.
[0091] It is to be understood that the compounds provided herein
may contain chiral centers.
[0092] Such chiral centers may be of either the (R-) or (S-)
configuration. The compounds provided herein may either be
enantiomerically pure, or be diastereomeric or enantiomeric
mixtures. It is to be understood that the chiral centers of the
compounds provided herein may undergo epimerization in vivo. As
such, one of skill in the art will recognize that administration of
a compound in its (R-) form is equivalent, for compounds that
undergo epimerization in vivo, to administration of the compound in
its (S-) form.
[0093] As used herein, substantially pure means sufficiently
homogeneous to appear free of readily detectable impurities as
determined by standard methods of analysis, such as thin layer
chromatography (TLC), nuclear magnetic resonance (NMR), gel
electrophoresis, high performance liquid chromatography (HPLC) and
mass spectrometry (MS), gas-chromatography mass spectrometry
(GC-MS), and similar, used by those of skill in the art to assess
such purity, or sufficiently pure such that further purification
would not detectably alter the physical and chemical properties,
such as enzymatic and biological activities, of the substance. Both
traditional and modern methods for purification of the compounds to
produce substantially chemically pure compounds are known to those
of skill in the art. A substantially chemically pure compound may,
however, be a mixture of stereoisomers.
[0094] Unless stated to the contrary, a formula with chemical bonds
shown only as solid lines and not as wedges or dashed lines
contemplates each possible isomer, e.g., each enantiomer,
diastereomer, and meso compound, and a mixture of isomers, such as
a racemic or scalemic mixture.
[0095] A "pharmaceutically acceptable" component is one that is
suitable for use with humans and/or animals without undue adverse
side effects (such as toxicity, irritation, and allergic response)
commensurate with a reasonable benefit/risk ratio.
[0096] "Pharmaceutically acceptable salt" refers to a salt that is
pharmaceutically acceptable and has the desired pharmacological
properties. Such salts include those that may be formed where
acidic protons present in the compounds are capable of reacting
with inorganic or organic bases. Suitable inorganic salts include
those formed with the alkali metals, e.g., sodium, potassium,
magnesium, calcium, and aluminum. Suitable organic salts include
those formed with organic bases such as the amine bases, e.g.,
ethanolamine, diethanolamine, triethanolamine, tromethamine,
N-methylglucamine, and the like. Such salts also include acid
addition salts formed with inorganic acids (e.g., hydrochloric and
hydrobromic acids) and organic acids (e.g., acetic acid, citric
acid, maleic acid, and the alkane- and arene-sulfonic acids such as
methanesulfonic acid and benzenesulfonic acid). When two acidic
groups are present, a pharmaceutically acceptable salt may be a
mono-acid-mono-salt or a di-salt; similarly, where there are more
than two acidic groups present, some or all of such groups can be
converted into salts.
[0097] "Pharmaceutically acceptable excipient" refers to an
excipient that is conventionally useful in preparing a
pharmaceutical composition that is generally safe, non-toxic, and
desirable, and includes excipients that are acceptable for
veterinary use as well as for human pharmaceutical use. Such
excipients can be solid, liquid, semisolid, or, in the case of an
aerosol composition, gaseous.
[0098] A "pharmaceutically acceptable carrier" is a carrier, such
as a solvent, suspending agent or vehicle, for delivering the
disclosed compounds to the patient. The carrier can be liquid or
solid and is selected with the planned manner of administration in
mind. Liposomes are also a pharmaceutical carrier. As used herein,
"carrier" includes any and all solvents, dispersion media,
vehicles, coatings, diluents, antibacterial and antifungal agents,
isotonic and absorption delaying agents, buffers, carrier
solutions, suspensions, colloids, and the like. The use of such
media and agents for pharmaceutical active substances is well known
in the art. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions is contemplated.
[0099] The term "therapeutically effective amount" as used herein
means that amount of active compound or pharmaceutical agent that
elicits the biological or medicinal response in a tissue, system,
animal or human that is being sought by a researcher, veterinarian,
medical doctor or other clinician. In some embodiments, an
effective amount is an amount sufficient to delay development. In
some embodiments, an effective amount is an amount sufficient to
prevent or delay occurrence and/or recurrence. An effective amount
can be administered in one or more doses.
[0100] Effective amounts of a compound or composition described
herein for treating a mammalian subject can include about 0.1 to
about 1000 mg/Kg of body weight of the subject/day, such as from
about 1 to about 100 mg/Kg/day, especially from about 10 to about
100 mg/Kg/day. The doses can be acute or chronic. A broad range of
disclosed composition dosages are believed to be both safe and
effective.
[0101] Reference will now be made in detail to specific aspects of
the disclosed materials, compounds, compositions, articles, and
methods, examples of which are illustrated in the accompanying
Examples and Figures.
Compounds
[0102] Disclosed are compounds that are Aurora kinase inhibitors,
e.g., Aurora A, B, and/or C kinase inhibitors and Janus kinase 2
(JAK2) inhibitors. The compounds can concurrently block Aurora
kinase A and JAK2 signal. These disclosed compounds can be used in
various compositions to reduce the risk of developing, prevent, or
treat GVHD in a subject. These disclosed compounds can be used in
various compositions to synergistically suppress alloreactive human
T-cells in vitro, prevents xenogeneic graft-versus-host disease
(GVHD) without impairing anti-tumor responses, and promotes the
development and suppressive potency of CD39.sup.+ inducible
T.sub.reg.
[0103] In certain embodiments, the disclosed compounds have the
chemical structure shown in Formula I.
##STR00006##
wherein [0104] R.sup.1 is selected from the group consisting of H,
Cl, F, Br, I, CN, NO.sub.2, NH.sub.2, CF.sub.3, CO.sub.2H,
CO.sub.2NH.sub.2, CO.sub.2NHR.sup.5, CO.sub.2R.sup.5, C(O)R.sup.5,
C(O)NH.sub.2, C(O)NHR.sup.5, and C.sub.1-C.sub.6 alkyl optionally
substituted with alkoxy, alkenyl, alkynyl, aryl, heteroaryl,
aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy,
ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or
thiol; [0105] R.sup.2 is selected from the group consisting of H,
OH, CN, NO.sub.2, NH.sub.2, optionally substituted C.sub.1-C.sub.6
alkyl, optionally substituted cycloalkyl, optionally substituted
aryl, and optionally substituted heteroaryl, wherein optionally
substituted substituents are optionally substituted with alkoxy,
alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic
acid, ester, ether, halide, hydroxy, ketone, nitro, silyl,
sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol; [0106] or
R.sup.1 and R.sup.2 together form a fused cycloalkyl,
cycloheteroalkyl, aryl, or heteraryl group; [0107] each R.sup.3 is
selected, independently, from the group consisting of
SO.sub.2NH.sub.2, SO.sub.2NHR.sup.5, NHSO.sub.2R.sup.5,
NHCO.sub.2R.sup.5, NHC(O)R.sup.5, NHCONHR.sup.5, F, Cl, Br, I,
NO.sub.2, optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 alkoxy, optionally substituted
cycloheteroaryl, and optionally substituted fused cycloheteroalkyl,
wherein optionally substituted substituents are optionally
substituted with sulfonyl; [0108] each R.sup.4 is selected,
independently, from the group consisting of F, Cl, Br, I, NO.sub.2,
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 alkoxy, COOH, C(O)NH.sub.2,
C(O)R.sup.5, C(O)NHR.sup.5, CH.sub.2C(O)R.sup.5, SO.sub.2NH.sub.2,
SO.sub.2NHR.sup.5, CONHSO.sub.2R.sup.5, optionally substituted
phenyl, optionally substituted OPhenyl, tetrazole, piperadinyl,
piperazinyl, and morpholinyl, wherein optionally substituted
substituents are optionally substituted with alkoxy, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, oxo, silyl, sulfo-oxo,
sulfonyl, sulfone, sulfoxide, or thiol; [0109] each R.sup.5 is
selected, independently, from the group consisting of optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
cycloalkyl, optionally substituted heteroaryl, and optionally
substituted heteroalkyl, wherein optionally substituted
substituents are optionally substituted with C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, cycloalkyl, cycloheteroalkyl, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, oxo, silyl, sulfo-oxo,
sulfonyl, sulfone, sulfoxide, or thiol; [0110] R.sup.7 is selected
from the group consisting of H, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, halide, hydroxyl, cyano, nitro, and amino;
[0111] n is 0-5; and [0112] m is 1-5, or a pharmaceutically
acceptable salt thereof.
[0113] Thus, in the disclosed compounds there can be from none to 5
different substituents R.sup.3 and from 1 to 5 different
substituents R.sup.4. Pharmaceutically acceptable salts of these
compounds are also disclosed. In some examples, R.sup.1 is F,
R.sup.2 is hydrogen, and R.sup.3 is 2-Cl. In one specific example,
R.sup.1 is Cl or F, R.sup.2 is hydrogen, R.sup.3 is hydrogen or
2-Cl, and R.sup.4 is 4-CONH.sub.2.
[0114] In certain embodiments, the disclosed compounds have the
chemical structure shown in Formula II.
##STR00007##
wherein R.sup.1-R.sup.4 and m and n are as defined above for
Formula I.
[0115] In some examples of Formula II, R.sup.1 is selected from the
group consisting of H, Cl, F, Br, I, C.sub.1-C.sub.6 alkyl, CN,
NO.sub.2, and NH.sub.2. Also in Formula II, R.sup.2 is selected
from the group consisting of H, F, and Cl.
[0116] Additionally in Formula II, each R.sup.3 is selected,
independently, from the group consisting of Cl, Br, F, COOH,
CF.sub.3, CN, phenyl, OCH.sub.3, COR.sup.5, CONH.sub.2, CONR.sup.5,
and COONH.sub.2. When n is 0, there is no R.sup.3.
[0117] Further in Formula II, each R.sup.4 is selected,
independently, from the group consisting of H, COOH, CONH.sub.2,
CONR.sup.5, SO.sub.2NH.sub.2, CONSO.sub.2R.sup.5, tetrazole,
4-morpholine, and COR.sup.5. Each R.sup.5 is selected,
independently, from the group consisting of optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted cycloalkyl,
optionally substituted heteroaryl, and optionally substituted
heteroalkyl. In some examples R.sup.5 is an unsubstituted
substituent.
[0118] Still further in Formula II, n is 0-5 (e.g., 0, 1, 2, 3, 4,
or 5) and m is 1-5 (e.g., 1, 2, 3, 4, or 5). Thus, in the disclosed
compounds there can be from none to 5 different substituents
R.sup.3 and from 1 to 5 different substituents R.sup.4. In certain
examples of Formula II, R.sup.1 is F, R.sup.2 is H, m is 3 and
R.sup.4 is 3,5-di-F, 4-OH. Pharmaceutically acceptable salts of
these compounds are also disclosed.
[0119] In certain preferred aspects, the compound has Formula II,
wherein R.sup.3 is 2-Cl, and n is 1. In other examples, the
compound has Formula II, n is 0 and there is no R.sup.3. In other
examples, the compound has Formula II, n is 2 and one R.sup.3 is an
ortho-Cl and the other R.sup.3 is a para hydroxyl, methoxyl, or
cyano group. In other examples, the compound has Formula II,
wherein R.sup.4 is 4-CONH.sub.2, and m is 1. In other examples, the
compound has Formula II, wherein R.sup.4 is 4-CONHR.sup.5, and m is
1 In still other examples, the compound has Formula II, wherein
R.sup.3 is 2-Cl, n is 1, m is 1, and R.sup.4 is COOH, COR.sup.5,
CONH.sub.2, CONR.sup.5, or CONSO.sub.2R.sup.5, wherein R.sup.5 is
C.sub.1-C.sub.6 alkyl, cycloalkyl, heteroaryl, or heteroalkyl. In
other examples, the compound has Formula II, wherein R.sup.3 is
absent, n is 0, m is 1, and R.sup.4 is COOH, COR.sup.5, CONH.sub.2,
CONR.sup.5, or CONSO.sub.2R.sup.5, wherein R.sup.5 is
C.sub.1-C.sub.6 alkyl, cycloalkyl, heteroaryl, or heteroalkyl.
[0120] Still further, the disclosed compounds can have the
following Formula IIIA or IIIB:
##STR00008##
wherein [0121] R.sup.1 is selected from the group consisting of H,
Cl, F, Br, I, CH.sub.3 and NH.sub.2; [0122] R.sup.2 is selected
from the group consisting of H, F, and Cl; [0123] R.sup.3 is
selected from the group consisting of 2-Cl, 2-Br, 2-F, 2-COOH,
2-CF.sub.3, 2-CN, 2-phenyl, 2-OCH.sub.3, 2-COONH.sub.2, 4-COOH, and
4-OCH.sub.3; and [0124] R.sup.4 is selected from the group
consisting of H, COOH, 2-CONH.sub.2, 4-CONH.sub.2,
SO.sub.2NH.sub.2, tetrazole, and 4-morpholine.
[0125] In Formula IIIA or IIIB, R.sup.1 is selected from the group
consisting of H, Cl, F, Br, I, CH.sub.3 and NH.sub.2; R.sup.2 is
selected from the group consisting of H, F, and Cl.
[0126] Also in Formula IIIA, R.sup.3 is selected from the group
consisting of 2-Cl, 2-Br, 2-F, 2-COOH, 2-CF.sub.3, 2-CN, 2-phenyl,
2-OCH.sub.3, 2-COONH.sub.2, 4-COOH, and 4-OCH.sub.3.
[0127] Additionally in Formula IIIA or IIIB, R.sup.4 is selected
from the group consisting of H, COOH, 2-CONH.sub.2, 4-CONH.sub.2,
SO.sub.2NH.sub.2, tetrazole, and 4-morpholine.
[0128] Still further, the disclosed compounds can have the
following Formula IV:
##STR00009##
[0129] In Formula IV, R.sup.1, R.sup.2, R.sup.4, and m are as
defined herein.
[0130] In other examples, disclosed herein are compounds of Formula
IA.
##STR00010##
wherein [0131] X is CH or N; [0132] R.sup.1 is selected from the
group consisting of H, Cl, F, Br, I, CN, NO.sub.2, NH.sub.2,
CF.sub.3, CO.sub.2H, CO.sub.2NH.sub.2, CO.sub.2NHR.sup.5,
CO.sub.2R.sup.5, C(O)R.sup.5, C(O)NH.sub.2, C(O)NHR.sup.5, and
C.sub.1-C.sub.6 alkyl optionally substituted with alkoxy, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol; [0133] R.sup.2 is H, OH, CN,
NO.sub.2, NH.sub.2, optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted cycloalkyl, optionally substituted aryl, and
optionally substituted heteroaryl; wherein optionally substituted
substituents are optionally substituted with alkoxy, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol; [0134] or R.sup.1 and R.sup.2
together form a fused cycloalkyl, cycloheteroalkyl, aryl or
heteraryl group; [0135] each R.sup.3 is selected, independently,
from the group consisting of SO.sub.2NH.sub.2, SO.sub.2NHR.sup.5,
NHSO.sub.2R.sup.5, NHCO.sub.2R.sup.5, NHC(O)R.sup.5, NHCONHR.sup.5,
F, Cl, Br, I, NO.sub.2, optionally substituted C.sub.1-C.sub.6
alkyl, optionally substituted C.sub.1-C.sub.6 alkoxy, optionally
substituted cycloheteroaryl, and optionally substituted fused
cycloheteroalkyl, wherein optionally substituted substituents are
optionally substituted with sulfonyl; [0136] each R.sup.5 is
selected, independently, from optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocycloalkyl, and optionally
substituted heteroalkyl, wherein optionally substituted
substituents are optionally substituted with C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, alkenyl, alkynyl, aryl, heteroaryl,
aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy,
ketone, nitro, oxo, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,
or thiol; [0137] R.sup.6 is selected from the group consisting of
H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl-OH,
CO.sub.2R.sup.5, CO.sub.2H, and CO.sub.2NHR.sup.5; [0138] R.sup.7
is selected from the group consisting of H, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, halide, hydroxyl, cyano, nitro, and amino;
[0139] R.sup.8 is OH or =0; [0140] n is 0-5; and [0141] p is 1 or 2
or a pharmaceutically acceptable salt thereof.
[0142] In other examples, disclosed herein are compounds of Formula
IB.
##STR00011##
wherein [0143] X is N or CH; [0144] R.sup.1 is selected from the
group consisting of H, Cl, F, Br, I, CN, NO.sub.2, NH.sub.2,
CF.sub.3, CO.sub.2H, CO.sub.2NH.sub.2, CO.sub.2NHR.sup.5,
CO.sub.2R.sup.5, C(O)R.sup.5, C(O)NH.sub.2, C(O)NHR.sup.5, and
C.sub.1-C.sub.6 alkyl optionally substituted with alkoxy, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol; [0145] R.sup.2 is selected from the
group consisting of H, OH, CN, NO.sub.2, NH.sub.2, optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
cycloalkyl, optionally substituted aryl, and optionally substituted
heteroaryl, wherein optionally substituted substituents are
optionally substituted with C.sub.1-C.sub.6 alkyl, cycloalkyl,
aryl, or heteroaryl substituted with alkoxy, alkenyl, alkynyl,
aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,
halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol; [0146] or R.sup.1 and R.sup.2
together form a fused cycloalkyl, cycloheteroalkyl, aryl or
heteraryl group; [0147] each R.sup.3 is selected, independently,
from the group consisting of SO.sub.2NH.sub.2, SO.sub.2NHR.sup.5,
NHSO.sub.2R.sup.5, NHCO.sub.2R.sup.5, NHC(O)R.sup.5, NHCONHR.sup.5,
F, Cl, Br, I, NO.sub.2, optionally substituted C.sub.1-C.sub.6
alkyl, optionally substituted C.sub.1-C.sub.6 alkoxy, optionally
substituted cycloheteroaryl, and optionally substituted fused
cycloheteroalkyl, wherein optionally substituted substituents are
optionally substituted with sulfonyl; [0148] each R.sup.5 is
selected, independently, from the group consisting of optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
cycloalkyl, optionally substituted heteroaryl, and optionally
substituted heteroalkyl, wherein optionally substituted
substituents are optionally substituted with C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, cycloalkyl, cycloheteroalkyl, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, oxo, silyl, sulfo-oxo,
sulfonyl, sulfone, sulfoxide, or thiol; [0149] R.sup.6 is selected
from the group consisting of H, C.sub.1-C.sub.6 alkyl,
CO.sub.2R.sup.5, CO.sub.2H, and CO.sub.2NHR.sup.5; [0150] R.sup.7
is selected from the group consisting of H, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, halide, hydroxyl, cyano, nitro, and amino;
[0151] R.sup.8 is OH or =0; [0152] n is 0-5; and [0153] p is 1 or 2
or a pharmaceutically acceptable salt thereof.
[0154] In other examples, disclosed herein are compounds of Formula
V.
##STR00012##
wherein [0155] X is N or CH; [0156] L is selected from the group
consisting of O, S, C.sub.1-4alkyl, C(O)NH, NHC(O), CH.sub.2C(O),
C(O)CH.sub.2, CH.sub.2CH.sub.2C(O), CH.sub.2C(O)CH.sub.2,
CH.sub.2C(O)NH, and NH(CO)CH.sub.2; R.sup.1 is selected from the
group consisting of H, Cl, F, Br, I, CN, NO.sub.2, NH.sub.2,
CF.sub.3, CO.sub.2H, CO.sub.2NH.sub.2, CO.sub.2NHR.sup.5,
CO.sub.2R.sup.5, C(O)R.sup.5, C(O)NH.sub.2, C(O)NHR.sup.5, and
C.sub.1-C.sub.6 alkyl optionally substituted with alkoxy, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol; [0157] R.sup.2 is selected from the
group consisting of H, OH, CN, NO.sub.2, NH.sub.2, optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
cycloalkyl, optionally substituted aryl, and optionally substituted
heteroaryl, wherein optionally substituted substituents are
optionally substituted with alkoxy, alkenyl, alkynyl, aryl,
heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,
hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone,
sulfoxide, or thiol; [0158] or R.sup.1 and R.sup.2 together form a
fused cycloalkyl, cycloheteroalkyl, aryl, or heteraryl group;
[0159] each R.sup.3 is selected, independently, from the group
consisting of SO.sub.2NH.sub.2, SO.sub.2NHR.sup.5,
NHSO.sub.2R.sup.5, NHCO.sub.2R.sup.5, NHC(O)R.sup.5, NHCONHR.sup.5,
F, Cl, Br, I, NO.sub.2, optionally substituted C.sub.1-C.sub.6
alkyl, optionally substituted C.sub.1-C.sub.6 alkoxy, optionally
substituted cycloheteroaryl, and optionally substituted fused
cycloheteroalkyl, wherein optionally substituted substituents are
optionally substituted with sulfonyl; [0160] each R.sup.5 is
selected, independently, from the group consisting of optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
cycloalkyl, optionally substituted heteroaryl, and optionally
substituted heteroalkyl, wherein optionally substituted
substituents are optionally substituted with C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, cycloalkyl, cycloheteroalkyl, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, oxo, silyl, sulfo-oxo,
sulfonyl, sulfone, sulfoxide, or thiol; [0161] R.sup.6 is selected
from the group consisting of H, C.sub.1-C.sub.6 alkyl,
CO.sub.2R.sup.5, CO.sub.2H, and CO.sub.2NHR.sup.5; [0162] R.sup.7
is selected from the group consisting of H, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, halide, hydroxyl, cyano, nitro, and amino;
[0163] R.sup.8 is OH or =0; [0164] n is 0-5; and [0165] p is 1 or 2
or a pharmaceutically acceptable salt thereof.
[0166] In certain specific examples of Formula I-V, R.sup.1 and
R.sup.2 together form a fused cycloalkyl, cycloheteroalkyl, aryl or
heteraryl group. In other examples, R.sup.1 and R.sup.2 together
form a fused furan. In other examples, R.sup.1 and R.sup.2 together
form a fused cyclopentyl or fused cyclohexyl. In other examples,
R.sup.1 and R.sup.2 together form a fused phenyl.
[0167] In certain specific examples of Formula I-V, R.sup.1 is
C.sub.1-8 alkyl or heteroalkyl. In other examples, R.sup.1 is
methyl, ethyl, or trifluoromethyl. In other examples, R.sup.1 is
chloro, bromo, or fluoro. In other examples, R.sup.1 is
CO.sub.2C.sub.1-8alkyl, CO.sub.2H, CO.sub.2NH.sub.2, or
CO.sub.2NHC.sub.1-8 alkyl.
[0168] In certain specific examples of Formula I-V, R.sup.2 is
C.sub.1_.sub.8 alkyl or heteroalkyl. In other examples, R.sup.2 is
hydrogen.
[0169] In the disclosed compounds there can be from 1 to 5
different substituents R.sup.3, i.e., n can be 1 to 5, though
preferable n can be 1 to 3. In some examples, there is no R.sup.3
substituent, i.e., n is 0. In specific examples, R.sup.3 is
SO.sub.2NH.sub.2, SO.sub.2NHR.sup.5, or NHSO.sub.2R.sup.5, wherein
R.sup.5 is C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 cycloalkyl
optionally substituted with C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxyl, hydroxyl, or halide. In other examples, R.sup.3 is
NHC(O)R.sup.5, wherein R.sup.5 is C.sub.1-C.sub.6 alkyl or
C.sub.1-C.sub.6 cycloalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxyl, cycloalky,
cycloheteroalkyl, hydroxyl, or halide. In other examples, R.sup.3
is C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 cycloalkyl. In other
examples, R.sup.3 is C.sub.1-C.sub.6 alkoxyl. In other examples,
R.sup.3 is halide. In other examples, n is 2 and each R.sup.3 is
selected from the group consisting of C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, halide, SO.sub.2NH.sub.2,
SO.sub.2NHR.sup.5, and NHSO.sub.2R.sup.5, wherein R.sup.5 is
C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 cycloalkyl optionally
substituted with C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxyl,
hydroxyl, or halide. In other examples, n is 2 and each R.sup.3 is
selected from the group consisting of C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxyl, and halide. In other examples, n is 3 and
each R.sup.3 is selected from the group consisting of
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxyl, and halide. In
other examples, n is 2 and each R.sup.3 together form a fused
hetercycloalkyl.
[0170] In certain specific examples of the above formula, R.sup.1
is F and R.sup.2 is H.
[0171] In certain specific examples, R.sup.4 is C(O)NHR.sup.5.
[0172] In certain specific examples, R.sup.6 is C.sub.1-8 alkyl. In
other examples, R.sup.6 is methyl. In other examples, R.sup.6 is
hydrogen.
[0173] In certain specific examples, R.sup.7 is chloro, bromo, or
fluoro. In other examples, R.sup.7 is hydrogen.
[0174] In some examples, X is N.
[0175] In specific examples, L is CH.sub.2(O) or C(O)NH.
[0176] In specific examples, n and m are both 1. In other examples,
n is 0.
[0177] In specific examples R.sup.8 is oxo and p is 1. In other
examples R.sup.8 is oxo and p is 2.
[0178] Pharmaceutically acceptable salts of these compounds are
also disclosed.
[0179] Specific examples of compounds having Formula I, IA, IB, II,
III, IV, and V are disclosed herein are in Tables 1-4.
TABLE-US-00001 TABLE 1 Bisanilinipyrimidine analogs. ##STR00013##
Cmpd Entry ID # R.sup.1 R.sup.2 R.sup.3 R.sup.4 1 1 H H ortho-COOH
para-COOH 2 3a H H ortho-COOH ortho-CONH.sub.2 3 3b H H ortho-COOH
H 4 3c H H ortho-COOH para-morpholine 5 3d H H ortho-COOH
ortho-COOH 6 3e H H ortho-COOMe para-COOMe 7 3f H H H H 8 3g H H
ortho-COONH.sub.2 para-CONH.sub.2 9 3h H H H para-COOH 10 3i H H
para-COOH para-COOH 11 3j CH.sub.3 H ortho-COOH para-COOH 12 3k H
CH.sub.3 ortho-COOH para-COOH 13 3l H H ortho-Cl para-COOH 14 3m F
H ortho-COOH All H 15 3n F H ortho-COOH para-COOH 16 3o F H
ortho-Cl para-COOH 17 3p F H ortho-Cl H 18 3q H H ortho-COOH
meta-COOH 19 3r F H ortho-COOH meta-COOH 20 4c Cl Cl ortho-COOH
para-COOH 21 4d Cl H ortho-COOH para-COOH 22 4a NH.sub.2 H
ortho-COOH para-COOH 23 4b H NH.sub.2 ortho-COOH para-COOH
TABLE-US-00002 TABLE 2 Bisanilinipyrimidine analogs. ##STR00014##
Cmpd Entry ID # R.sup.1 R.sup.2 R.sup.3 R.sup.4 R.sup.5 24 6a H H
ortho-F para-COOH H 25 6b H H ortho-CF.sub.3 All H H 26 6c H H
2-Cl-4-F para-COOH H 27 6d H H ortho-OCF.sub.3 para-COOH H 28 6e H
H ortho-OMe para-COOH H 29 6f H H ortho-OMe All H H 30 6g H H
ortho-CN All H H 31 6h H H ortho-CF.sub.3 para-COOH H 31 6i H H
ortho-Br para-COOH H 33 6j H H ortho-Cl para- H CH.sub.2--COOH 34
6k H H Ortho-Cl para-COOH, H meta-OH 35 6l H H Ortho-F All H H 36
6m H H Ortho-I para-COOH H 37 6n H H Ortho-CN para-COOH H 38 6o H H
Ortho-Cl meta-COOH H 39 6p H H ortho-Cl para-CONH.sub.2 H 40 6q H H
ortho-phenyl para-COOH H 41 6r H H ortho-Cl para-COOH CH.sub.3 42
6s H H ortho-Cl para-COOH CH.sub.3--CH.sub.2 43 6t F H ortho-Cl
meta-COOH H
TABLE-US-00003 TABLE 3 Bisanilinipyrimidine analogs. Entry Compound
ID 44 9a (RE1-043) ##STR00015## 45 9b (Re1-032) ##STR00016## 46 9c
(RE1-031) ##STR00017## 47 9d (RE1-025) ##STR00018## 48 9e (RE1-039)
##STR00019## 49 9f (RE1-019) ##STR00020## 50 9g (HM5-018-2)
##STR00021## 51 9h (YL5-146-4) ##STR00022## 52 6p (YL5-145)
##STR00023## 53 9i (YL5-146-3) ##STR00024## 54 9j (HM6-007-1)
##STR00025## 55 9k (HM6-020-2) ##STR00026## 56 9l (HM4-153-2)
##STR00027## 57 9m (HM6-021-4) ##STR00028## 58 9n (HM6-029-1)
##STR00029## 59 13a (SO2-162) ##STR00030## 60 13b (SO3-033)
##STR00031## 61 13c (SO3-036) ##STR00032## 62 13d (SO3-035)
##STR00033##
TABLE-US-00004 TABLE 4 Bisanilinipyrimidine analogs. ##STR00034##
Cmpd CompoundID # R.sup.1 R.sup.2 R.sup.3 R.sup.4 RK2-014 14 H H
meta-CF.sub.3 para-COOH RK2-017-01 15 H H meta-CF.sub.3
para-CONH.sub.2 RK2-037 16 H H meta-CF.sub.3 meta- isobutyramide
RK2-025 17 H H meta-CF.sub.3 meta-CF.sub.3 RK2-015-03 18 H H
meta-CF.sub.3 ortho-COOH RK2-017-02 19 H H meta-CF.sub.3
meta-CONH.sub.2 RK2-053 20 H H meta-CF.sub.3 meta-acetamide
RK2-015-02 21 H H meta-CF.sub.3 meta-COOH RK2-056 22 H H
meta-CF.sub.3 meta-butyramide RK2-046-02 23 H H meta-CF.sub.3 meta-
propionamide RK2-013 24 H H meta-CF.sub.3 meta- cyclopropane
carboxamide RK2-015-01 25 H H meta-CF.sub.3 All H RK2-046-01 26 H H
meta-CF.sub.3 meta-.sup.tbutyl- carboxyamide RK2-044 27 H H
meta-CF.sub.3 meta- cyclopentyl- carboxamide RK2-052 28 H H
meta-CF.sub.3 meta-isobutyl- carboxamide RK2-043 29 H H
meta-CF.sub.3 meta-(4- chlorobenzyl) carboxamide RK2-049 30 H H
meta-CF.sub.3 meta-benzyl- carboxamide YL5-048 31 H Me.sub.2N H All
H YL5-050 32 H Me.sub.2N ortho- All H COOH YL5-068 33 NH.sub.2 H
ortho- para-COOC.sub.2H.sub.5 COOH YL5-146-5 34 H H ortho-Cl
para-OCH.sub.3 YL5-080 35 Me H ortho- para-COOH COOH
[0180] In further example, the compound can be one of the following
compounds.
##STR00035##
[0181] In still further examples, the compound can have Formula
IIIC:
##STR00036##
wherein [0182] R.sup.1 is selected from the group consisting of H,
Cl, F, Br, I, C.sub.1-C.sub.6 alkyl, CN, NO.sub.2, and NH.sub.2;
[0183] R.sup.2 is selected from the group consisting of H, F, and
Cl; and [0184] each R.sup.4 is selected, independently, from the
group consisting of H, COOH, CONH.sub.2, CONR.sup.5,
SO.sub.2NH.sub.2, CONSO.sub.2R.sup.5, tetrazole, 4-morpholine, and
COR.sup.5; [0185] each R.sup.5 is selected, independently, from the
group consisting of C.sub.1-C.sub.6 alkyl, cycloalkyl, heteroaryl,
and heteroalkyl; and [0186] m is 1-5,
[0187] or a pharmaceutically acceptable salt thereof.
[0188] In specific examples of Formula IIIC, m is 1 and R.sup.4 is
selected from the group consisting of COOH, 2-CONH.sub.2,
4-CONH.sub.2, SO.sub.2NH.sub.2, tetrazole, and 4-morpholine. In
other examples, R.sup.1 is Cl, F, Br, or I. In further examples,
R.sup.2 is H. In still further examples, the compound is:
##STR00037##
[0189] Further specific examples, include
##STR00038##
wherein R.sup.3 and n are as defined in Formula I, and R.sup.10 is
hydrogen, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkyl-OH.
[0190] Scheme I describes the general synthetic route used for
preparation of dianilinipyrimidine (1) from readily available
building blocks. The 2,4-dichloropyrimidine was initially reacted
with the requisite commercially available anilines with the method
predominantly using isopropanol as the solvent, with reflux heating
to obtain the required analog.
Method
[0191] Further provided herein are methods of reducing the risk of
developing, preventing, or treating graft versus host disease
(GVHD) in a subject. The method can include administering to the
subject an effective amount of a compound or composition as
disclosed herein. The methods can further include administering a
second compound or composition, such as, for example, an
immunosuppressant.
[0192] Also disclosed are methods for treating GVHD in a patient.
In one embodiment, an effective amount of one or more compounds or
compositions disclosed herein is administered to a patient at risk
of developing or have GVHD and who is in need of treatment thereof.
The patient can be a human or other mammal, such as a primate
(monkey, chimpanzee, ape, etc.), dog, cat, cow, pig, or horse, or
other animals at risk of developing or have GVHD. GVHD may be due
to a transplatation procedure involving the implantation of
immunogenic tissue including but are not limited to, solid organ
transplants (such as heart, kidney, and liver), tissue grafts (such
as skin, intestine, pancreas, cornea, gonad, bone, and cartilage),
and cellular transplants (such as cells from pancreas, brain and
nervous tissue, muscle, skin, bone, cartilage, and liver). In such
procedures, organ rejection is an obstacle to complete recovery.
The individual's immune system recognizes antigens (HLA or minor H
antigens) on the implanted tissue as foreign and mounts an immune
response against it, which injures and destroys the implanted
tissue.
[0193] Further provided herein are methods of treating or
preventing cancer in a subject, comprising administering to the
subject an effective amount of a compound or composition as
disclosed herein. The methods can further comprise administering a
second compound or composition, such as, for example, anticancer
agents or anti-inflammatory agents. Additionally, the method can
further comprise administering an effective amount of ionizing
radiation to the subject.
[0194] Methods of killing a tumor cell are also provided herein.
The methods comprise contacting a tumor cell with an effective
amount of a compound or composition as disclosed herein. The
methods can further include administering a second compound or
composition (e.g., an anticancer agent or an anti-inflammatory
agent) or administering an effective amount of ionizing radiation
to the subject.
[0195] Also provided herein are methods of radiotherapy of tumors,
comprising contacting the tumor with an effective amount of a
compound or composition as disclosed herein and irradiating the
tumor with an effective amount of ionizing radiation.
[0196] Also disclosed are methods for treating oncological
disorders in a patient. In one embodiment, an effective amount of
one or more compounds or compositions disclosed herein is
administered to a patient having an oncological disorder and who is
in need of treatment thereof. The disclosed methods can optionally
include identifying a patient who is or can be in need of treatment
of an oncological disorder. The patient can be a human or other
mammal, such as a primate (monkey, chimpanzee, ape, etc.), dog,
cat, cow, pig, or horse, or other animals having an oncological
disorder. Oncological disorders include, but are not limited to,
cancer and/or tumors of the anus, bile duct, bladder, bone, bone
marrow, bowel (including colon and rectum), breast, eye, gall
bladder, kidney, mouth, larynx, esophagus, stomach, testis, cervix,
head, neck, ovary, lung, mesothelioma, neuroendocrine, penis, skin,
spinal cord, thyroid, vagina, vulva, uterus, liver, muscle,
pancreas, prostate, blood cells (including lymphocytes and other
immune system cells), and brain. Specific cancers contemplated for
treatment include carcinomas, Karposi's sarcoma, melanoma,
mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer,
leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic,
chronic myeloid, and other), and lymphoma (Hodgkin's and
non-Hodgkin's), and multiple myeloma.
Administration
[0197] The disclosed compounds can be administered in combination
with pharmaceutical formulations. Appropriate doses will be readily
appreciated by those skilled in the art.
[0198] The term "administration" and variants thereof (e.g.,
"administering" a compound) in reference to a compound of the
invention means introducing the compound or a prodrug of the
compound into the system of the animal in need of treatment. When a
compound of the invention or prodrug thereof is provided in
combination with one or more other active agents (e.g., a cytotoxic
agent, etc.), "administration" and its variants are each understood
to include concurrent and sequential introduction of the compound
or prodrug thereof and other agents.
[0199] In vivo application of the disclosed compounds, and
compositions containing them, can be accomplished by any suitable
method and technique presently or prospectively known to those
skilled in the art. For example, the disclosed compounds can be
formulated in a physiologically- or pharmaceutically-acceptable
form and administered by any suitable route known in the art
including, for example, oral, nasal, rectal, topical, and
parenteral routes of administration. As used herein, the term
parenteral includes subcutaneous, intradermal, intravenous,
intramuscular, intraperitoneal, and intrasternal administration,
such as by injection. Administration of the disclosed compounds or
compositions can be a single administration, or at continuous or
distinct intervals as can be readily determined by a person skilled
in the art.
[0200] The compounds disclosed herein, and compositions comprising
them, can also be administered utilizing liposome technology, slow
release capsules, implantable pumps, and biodegradable containers.
These delivery methods can, advantageously, provide a uniform
dosage over an extended period of time. The compounds can also be
administered in their salt derivative forms or crystalline
forms.
[0201] The compounds disclosed herein can be formulated according
to known methods for preparing pharmaceutically acceptable
compositions. Formulations are described in detail in a number of
sources which are well known and readily available to those skilled
in the art. For example, Remington's Pharmaceutical Science by E.
W. Martin (1995) describes formulations that can be used in
connection with the disclosed methods. In general, the compounds
disclosed herein can be formulated such that an effective amount of
the compound is combined with a suitable carrier in order to
facilitate effective administration of the compound. The
compositions used can also be in a variety of forms. These include,
for example, solid, semi-solid, and liquid dosage forms, such as
tablets, pills, powders, liquid solutions or suspension,
suppositories, injectable and infusible solutions, and sprays. The
preferred form depends on the intended mode of administration and
therapeutic application. The compositions also preferably include
conventional pharmaceutically-acceptable carriers and diluents
which are known to those skilled in the art. Examples of carriers
or diluents for use with the compounds include ethanol, dimethyl
sulfoxide, glycerol, alumina, starch, saline, and equivalent
carriers and diluents. To provide for the administration of such
dosages for the desired therapeutic treatment, compositions
disclosed herein can advantageously comprise between about 0.1% and
99%, and especially, 1 and 15% by weight of the total of one or
more of the subject compounds based on the weight of the total
composition including carrier or diluent.
[0202] Formulations suitable for administration include, for
example, aqueous sterile injection solutions, which can contain
antioxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient; and
aqueous and nonaqueous sterile suspensions, which can include
suspending agents and thickening agents. The formulations can be
presented in unit-dose or multi-dose containers, for example sealed
ampoules and vials, and can be stored in a freeze dried
(lyophilized) condition requiring only the condition of the sterile
liquid carrier, for example, water for injections, prior to use.
Extemporaneous injection solutions and suspensions can be prepared
from sterile powder, granules, tablets, etc. It should be
understood that in addition to the ingredients particularly
mentioned above, the compositions disclosed herein can include
other agents conventional in the art having regard to the type of
formulation in question.
[0203] Compounds disclosed herein, and compositions comprising
them, can be delivered to a cell either through direct contact with
the cell or via a carrier means. Carrier means for delivering
compounds and compositions to cells are known in the art and
include, for example, encapsulating the composition in a liposome
moiety. Another means for delivery of compounds and compositions
disclosed herein to a cell comprises attaching the compounds to a
protein or nucleic acid that is targeted for delivery to the target
cell. U.S. Pat. No. 6,960,648 and U.S. Application Publication Nos.
20030032594 and 20020120100 disclose amino acid sequences that can
be coupled to another composition and that allows the composition
to be translocated across biological membranes. U.S. Application
Publication No. 20020035243 also describes compositions for
transporting biological moieties across cell membranes for
intracellular delivery. Compounds can also be incorporated into
polymers, examples of which include poly (D-L lactide-co-glycolide)
polymer for intracranial tumors; poly [bis(p-carboxyphenoxy)
propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL);
chondroitin; chitin; and chitosan.
[0204] For the treatment of GVHD, the compounds disclosed herein
can be administered to a patient at risk of developing GVHD or in
need of treatment in combination with other known treatments for
GVHD. These other substances or treatments can be given at the same
as or at different times from the compounds disclosed herein. For
example, the compounds disclosed herein can be used in combination
with an immunosuppressive agent such as IMUREK.TM. (azathioprine
sodium), brequinar sodium, SPANIDIN.TM. (gusperimus
trihydrochloride, also known as deoxyspergualin), mizoribine (also
known as bredinin), CELLCEPT.TM. (mycophenolate mofetil),
NEORAL.TM. (Cyclosporin A; also marketed as a different formulation
under the trademark SANDIMMUNE.TM.), PROGRAF.TM. (tacrolimus, also
known as FK-506), RAPIMMUNE.TM. (sirolimus, also known as
rapamycin), leflunomide (also known as HWA-486), ZENAPAX.TM.,
glucocortcoids, such as prednisolone and its derivatives,
corticosteroids, antibodies such as orthoclone (OKT3),
cyclophosphamide, methotrexate, 6-mercaptopurine, vincristine,
antithymyocyte globulins, such as thymoglobulins; an Aurora A
inhibitor; or a Janus kinase 2 inhibitor. A conventional
immunosuppressant drug, such as those above, may thus be
administered in an amount substantially less (e.g. 20% to 50% of
the standard dose) than when the compound is administered alone.
The compounds described herein can be administered at regular
intervals over a time period of at least 2 weeks.
[0205] Therapeutic application of compounds and/or compositions
containing them can be accomplished by any suitable therapeutic
method and technique presently or prospectively known to those
skilled in the art. Further, compounds and compositions disclosed
herein have use as starting materials or intermediates for the
preparation of other useful compounds and compositions.
[0206] Compounds and compositions disclosed herein can be locally
administered at one or more anatomical sites, such as sites of a
transplant, optionally in combination with a pharmaceutically
acceptable carrier such as an inert diluent. Compounds and
compositions disclosed herein can be systemically administered,
such as intravenously or orally, optionally in combination with a
pharmaceutically acceptable carrier such as an inert diluent, or an
assimilable edible carrier for oral delivery. They can be enclosed
in hard or soft shell gelatin capsules, can be compressed into
tablets, or can be incorporated directly with the food of the
patient's diet. For oral therapeutic administration, the active
compound can be combined with one or more excipients and used in
the form of ingestible tablets, buccal tablets, troches, capsules,
elixirs, suspensions, syrups, wafers, aerosol sprays, and the
like.
[0207] The tablets, troches, pills, capsules, and the like can also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring can be added. When the unit dosage form is a capsule, it
can contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials can be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules can be coated with gelatin, wax,
shellac, or sugar and the like. A syrup or elixir can contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
active compound can be incorporated into sustained-release
preparations and devices.
[0208] Compounds and compositions disclosed herein, including
pharmaceutically acceptable salts, hydrates, or analogs thereof,
can be administered intravenously, intramuscularly, or
intraperitoneally by infusion or injection. Solutions of the active
agent or its salts can be prepared in water, optionally mixed with
a nontoxic surfactant. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, triacetin, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations can contain a preservative to prevent the growth
of microorganisms.
[0209] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient, which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. The ultimate dosage form should be sterile, fluid and
stable under the conditions of manufacture and storage. The liquid
carrier or vehicle can be a solvent or liquid dispersion medium
comprising, for example, water, ethanol, a polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycols, and the
like), vegetable oils, nontoxic glyceryl esters, and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the formation of liposomes, by the maintenance of the
required particle size in the case of dispersions or by the use of
surfactants. Optionally, the prevention of the action of
microorganisms can be brought about by various other antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
buffers or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the inclusion of agents that
delay absorption, for example, aluminum monostearate and
gelatin.
[0210] Sterile injectable solutions are prepared by incorporating a
compound and/or agent disclosed herein in the required amount in
the appropriate solvent with various other ingredients enumerated
above, as required, followed by filter sterilization. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and the freeze drying techniques, which yield a powder of the
active ingredient plus any additional desired ingredient present in
the previously sterile-filtered solutions.
[0211] For topical administration, compounds and agents disclosed
herein can be applied in as a liquid or solid. However, it will
generally be desirable to administer them topically to the skin as
compositions, in combination with a dermatologically acceptable
carrier, which can be a solid or a liquid. Compounds and agents and
compositions disclosed herein can be applied topically to a
subject's skin to reduce the size (and can include complete
removal) of malignant or benign growths, or to treat an infection
site. Compounds and agents disclosed herein can be applied directly
to the growth or infection site. Preferably, the compounds and
agents are applied to the growth or infection site in a formulation
such as an ointment, cream, lotion, solution, tincture, or the
like. Drug delivery systems for delivery of pharmacological
substances to dermal lesions can also be used, such as that
described in U.S. Pat. No. 5,167,649.
[0212] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers, for example.
[0213] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user. Examples of
useful dermatological compositions which can be used to deliver a
compound to the skin are disclosed in U.S. Pat. Nos. 4,608,392;
4,992,478; 4,559,157; and 4,820,508.
[0214] Useful dosages of the compounds and agents and
pharmaceutical compositions disclosed herein can be determined by
comparing their in vitro activity, and in vivo activity in animal
models. Methods for the extrapolation of effective dosages in mice,
and other animals, to humans are known to the art; for example, see
U.S. Pat. No. 4,938,949.
[0215] Also disclosed are pharmaceutical compositions that comprise
a compound disclosed herein in combination with a pharmaceutically
acceptable carrier. Pharmaceutical compositions adapted for oral,
topical or parenteral administration, comprising an amount of a
compound constitute a preferred aspect. The dose administered to a
patient, particularly a human, should be sufficient to achieve a
therapeutic response in the patient over a reasonable time frame,
without lethal toxicity, and preferably causing no more than an
acceptable level of side effects or morbidity. One skilled in the
art will recognize that dosage will depend upon a variety of
factors including the condition (health) of the subject, the body
weight of the subject, kind of concurrent treatment, if any,
frequency of treatment, therapeutic ratio, as well as the severity
and stage of the pathological condition.
Kits
[0216] Kits for practicing the methods of the invention are further
provided. By "kit" is intended any manufacture (e.g., a package or
a container) comprising at least one reagent, e.g., anyone of the
compounds described in Table 1. The kit may be promoted,
distributed, or sold as a unit for performing the methods of the
present invention. Additionally, the kits may contain a package
insert describing the kit and methods for its use. Any or all of
the kit reagents may be provided within containers that protect
them from the external environment, such as in sealed containers or
pouches.
[0217] To provide for the administration of such dosages for the
desired therapeutic treatment, in some embodiments, pharmaceutical
compositions disclosed herein can comprise between about 0.1% and
45%, and especially, 1 and 15%, by weight of the total of one or
more of the compounds based on the weight of the total composition
including carrier or diluents. Illustratively, dosage levels of the
administered active ingredients can be: intravenous, 0.01 to about
20 mg/kg; intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous,
0.01 to about 100 mg/kg; intramuscular, 0.01 to about 100 mg/kg;
orally 0.01 to about 200 mg/kg, and preferably about 1 to 100
mg/kg; intranasal instillation, 0.01 to about 20 mg/kg; and
aerosol, 0.01 to about 20 mg/kg of animal (body) weight.
[0218] Also disclosed are kits that comprise a composition
comprising a compound disclosed herein in one or more containers.
The disclosed kits can optionally include pharmaceutically
acceptable carriers and/or diluents. In one embodiment, a kit
includes one or more other components, adjuncts, or adjuvants as
described herein. In another embodiment, a kit includes one or more
immunosuppressant agents, such as those agents described herein. In
one embodiment, a kit includes instructions or packaging materials
that describe how to administer a compound or composition of the
kit. Containers of the kit can be of any suitable material, e.g.,
glass, plastic, metal, etc., and of any suitable size, shape, or
configuration. In one embodiment, a compound and/or agent disclosed
herein is provided in the kit as a solid, such as a tablet, pill,
or powder form. In another embodiment, a compound and/or agent
disclosed herein is provided in the kit as a liquid or solution. In
one embodiment, the kit comprises an ampoule or syringe containing
a compound and/or agent disclosed herein in liquid or solution
form.
Examples
[0219] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
Material and Methods:
[0220] Monoclonal antibodies and flow cytometry:
Fluorochrome-conjugated mouse anti-human monoclonal antibodies
included anti-CD3, CD4, CD25, CD39, CD107a, CD127, CTLA4, Foxp3,
LAG3, phosphorylated STAT3 Y705, phosphorylated STAT5 Y694, and
phosphorylated H3 serine 10 (BD Biosciences, San Jose, Calif.;
eBioscience San Jose, Calif.; Cell Signaling Technology, Boston,
Mass.). LIVE/DEAD Fixable Yellow Dead Cell Stain (Life
Technologies, Grand Island, N.Y. USA) was used to determine
viability. Live events were acquired on a FACSCalibur or LSRII flow
cytometer (FlowJo software, ver. 7.6.4; TreeStar, Ashland,
Oreg.).
[0221] Allogeneic mixed leukocyte reactions: Bulk donor T-cells
were allostimulated with allogeneic DCs (DC:T-cell ratio of 1:30)
as previously described (OneBlood, Tampa, Fla.)(15, 16, 20). For
synergy assays, TG101348 (JAK2 inhibitor, Chemietek, Indianapolis,
Ind.), Alisertib (Aurora kinase A inhibitor, Selleckchem, Houston,
Tex.), or both TG101348 and Alisertib (ratio 1:5, respectively)
were added once on day 0. bisanilinopyrimidine (I) (dual JAK2 and
Aurora kinase A inhibitor(27, 29), Moffitt Chemical Biology Core,
Tampa, Fla.) or DMSO was added once on day 0 at concentrations
ranging from 0.078-2.5 .mu.M. As indicated, alloMLRs consisting of
T.sub.reg depleted, naive CD4.sup.+ T-cells (Miltenyi Biotec Inc,
San Diego, Calif.) were treated with a combination of ARL67156
(CD39 inhibitor, 125 .mu.M, Sigma), AJI, or DMSO on day 0 to study
the role of CD39 and ATP in this system. T-cell proliferation was
measured on day 5 by a colorimetric assay (CellTiter 96 Aqueous One
Solution Cell Proliferation Assay [MTS]; or CellTiter Blue,
Promega, Madison, Wis.). Absorbance/optical density (OD) was
analyzed at 490 nm or 590 nm, respectively. Proliferation (%)=(OD
treated alloMLR-OD T-cells alone)/(OD DMSO alloMLR-OD T-cells
alone).times.100.
[0222] Protein phosphorylation in T-cells: T-cells were cultured
with allogeneic DCs (DC:T-cell ratio of 1:30) for 5 days in
RPMI/10% pooled human serum, with alisertib (1.75 .mu.M), TG101348
(350 nM), a combination of both inhibitors, AJI-214 (750 nM),
AJI-100 (750 nM), or DMSO control added once on day 0. After 5
days, T cells were then harvested and directly fixed (Cytofix, BD
Biosciences) for 10 min at 37.degree. C. After washing with
phosphate-buffered saline, the T cells were permeabilized with
icecold methanol (90%, v/v) for at least 20 min at -20.degree. C.
The cells were stained for expression of CD3 and pH3Ser.sup.10.
[0223] STAT3 and STAT5 phosphorylation: As indicated, T cells were
serum-starved in RPMI treated with DMSO diluent control, alisertib,
TG101348, a combination of both inhibitors, AJI-214, or AJI-100 for
4 hours. IL-6-induced pSTAT3 or IL-2-induced pSTAT5 (Y694) was
measured by flow cytometry.
[0224] NSG mice: After transplantation and treatment with either
AJI-100 or vehicle control, human T cells were isolated from
recipient mouse spleens at day +14, stained for pH3Ser.sup.10 or
pSTAT3 (+IL-6 stimulation), and analyzed as described.
[0225] Effect of dual pathway inhibition on effector CD4 T cell
differentiation: Purified human T-cells were allostimulated with
DCs at a DC:T-cell ratio of 1:30 in RPMI/10% pooled human serum.
DMSO, alisertib (1.75 mM), TG101348 (350 nM), both alisertib and
TG101348, AJI-214 (750 nM), or AJI-100 (750 nM) was added once on
day 0. The T cells were harvested and surface-stained on day 5 for
CD3, CD4, CD25, and CD127. Activated CD4+T.sub.conv were
characterized by expression of CD25 and CD127, and activated
CD8.sup.+ T.sub.conv (CD3.sup.+ and CD4.sup.-) were identified by
CD25 expression. The absolute number of CD4.sup.+ and CD8.sup.+
T.sub.conv was calculated by flow cytometry using CountBright beads
(Life Technologies). T.sub.H1 cells were characterized by
expression of CD3, CD4, and intracellular IFN-.gamma. (after an
additional 4 to 5 hours of stimulation with PMA/ionomycin). For
T.sub.H1 experiments, purified CD4.sup.+ T cells were used as
opposed to bulk T cells.
[0226] iT.sub.reg differentiation and potency: iT.sub.regs were
generated as previously described in the presence of alisertib
(1.75 mM), TG101348 (350 nM), both alisertib and TG101348, AJI-214
(750 nM), AJI-100 (750 nM), or DMSO. On day 5, iT.sub.regs were
isolated and washed to minimize drug carry-over as reported. The T
cells were harvested and surface-stained on day 5 for CD3, CD4,
CD25, and CD127, followed by fixation and permeabilization
(eBioscience) and Foxp3 staining. The absolute number of iT.sub.reg
was calculated by flow cytometry using CountBright beads (Life
Technologies). The purified iT.sub.regs were titrated against
alloMLRs consisting of responder CD4+CD25- T cells
(5.times.10.sup.4) from the iT.sub.regs donor and fresh allogeneic
DCs (1.6.times.10.sup.3) to determine suppressive potency. T cell
proliferation was determined by pulsing cells with
[.sup.3H]thymidine (1 mCi per well). Surface expression of CD39 and
LAG3 was evaluated on the iT.sub.regs. iT.sub.regs production of
CTLA4 was assessed by intracellular staining after a 5-hour
treatment of PMA/ionomycin, with GolgiStop added during the last 4
hours of incubation. iT.sub.regs synthesis of IL-10 and TGF-.beta.
(Quansys Biosciences) was quantified from supernatants using
multiplex cytokine assays after PMA/ionomycin stimulation.
[0227] ATP Hydrolysis assay: iT.sub.regs generated in the presence
of AJI-241 (750 nM) or DMSO were plated in V-bottom 96 well plates
in serum free media at a concentration of 75,000 cells per 100
.mu.L. ARL67156 (125 .mu.M) was added or not as indicated. A fixed
dose of ATP (50 .mu.M) was added to the cells and incubated at
37.degree. C. for 45 minutes. ATP consumption was measured by a
luminescence assay per the manufacturer's instructions (Promega,
CellTiter-Glo Luminescent Cell Viability Assay) and read by a
spectrofluorimeter. Percent consumption was calculated as
(luminescence of test supernatant/luminescence of 50 .mu.M ATP
cell-free control supernatant).times.100.
[0228] Foxp3 TSDR demethylation analysis: Foxp3 TSDR demethylation
was analyzed among magnetic bead-purified (Miltenyi),
allostimulated bisanilinopyrimidine (I)- and DMSO-treated
iT.sub.regs. The primer selection, procedure for amplifying
methylation and demethylation specific TSDR products, genomic DNA
isolation, bisulfite conversion, and qPCR were performed as
previously reported.
[0229] RORgammaT expression by RT-PCR: Naive CD4.sup.+ T cells were
purified and allostimulated (DC/T cell ratio of 1:30) as reported
(B. C. Betts et al., CD4.sup.+ T cell STAT3 phosphorylation
precedes acute GVHD, and subsequent T.sub.H17 tissue invasion
correlates with GVHD severity and therapeutic response. J.
Leukocyte Biol. (2015)). Alisertib (1.75 mM), TG101348 (350 nM),
both alisertib and TG101348, AJI-214 (750 nM), AJI-100 (750 nM), or
DMSO control was added once on day 0. Medium was supplemented with
IL-6 (1.times.10.sup.5 IU/mL), TGF-.beta. (4 ng/mL; R&D
Systems), and anti-IFN-.gamma. monoclonal antibody (10 mg/ml;
eBioscience) to polarize T.sub.H17. After 5 days, the T cells were
harvested, washed, and then plated at 35,000 cells per well in an
IL-17 ELISPOT plate (R&DSystems). The CD4.sup.+ T cells were
stimulated with PMA/ionomycin, and the ELISPOT assay was performed
according to the manufacturer's instructions.
[0230] Xenogeneic GVHD model: NOD scid gamma (NSG) mice (male or
female, 6-24 weeks old) were purchased from Jackson Laboratory (Bar
Harbor, Me., USA) and raised per an IACUC-approved protocol in
adherence to the NIH Guide for the Care and Use of Laboratory
Animals. Mice received either (i) alisertib (30 mg/kg daily),
TG101348 (45 mg/kg twice a day), a combination of alisertib and
TG101348, or vehicle (methylcellulose) by oral gavage or (ii)
AJI-100 (50 mg/kg daily) or vehicle (50% polyethylene glycol, 15%
2-hydroxypropyl-b-cyclodextrin, and 10% DMSO in sterile saline) ip
from day 0 to day +14.
[0231] Mice were monitored for GVHD clinical scores (K. R. Cooke et
al., An experimental model of idiopathic pneumonia syndrome after
bone marrow transplantation: I. The roles of minor H antigens and
endotoxin. Blood 88, 3230 (1996)), weight, and premoribund status.
As indicated, mice were euthanized on day +14 to study recipient
spleen T.sub.conv, T.sub.regs, T.sub.H1, T.sub.H17, B cells, and
T-cell signal transduction. Human CD4.sup.+ T.sub.reg (CD25.sup.+,
CD127.sup.-, Foxp3.sup.+), T.sub.conv (CD25.sup.+, CD127.sup.+),
T.sub.H1, T.sub.H17, and CD19.sup.+ B cells residing in recipient
spleens were quantified by flow cytometry. T-cell pH3 ser10 and
PSTAT3 were evaluated by flow cytometry. IL-17 ELISPOTs were
performed using isolated human T cells from recipient mouse spleen
as described above. Tissue samples were prepared, stained (Ventana
Medical Systems, Tucson, Ariz.), and imaged (Vista, Calif., USA) to
identify human T-cells as previously described. All vertebrate
animal work was IACUC-approved.
[0232] CTL generation and tumor lysis assays: NSG mice were
transplanted with human PBMCs as described above, and treated with
either AJI-100 (50 mg/kg daily) or vehicle control. Additionally,
recipient mice received an inoculum of irradiated U937 cells (ATCC,
10.sup.7/mouse) on days 0 and +7. Mice were euthanized between days
+10 to +12, spleens were harvested, and human CD8.sup.+ T-cells
were isolated by magnetic bead separation. Fresh U937 target cells
were labeled with Calcein-AM for 30 minutes, washed, and then
cultured with the purified CD8.sup.+ T-cells at varying effector to
target ratios for 4 hours at 37.degree. C. No additional drugs were
added during this final culture. The amount of supernatant
fluorescence released by the target cells was measured using a
spectrofluorimeter (485 nm excitation and 535 nm emission) (S.
Neri, E. Mariani, A. Meneghetti, L. Cattini, A. Facchini,
Calcein-acetyoxymethyl cytotoxicity assay: standardization of a
method allowing additional analyses on recovered effector cells and
supernatants. Clinical Diagnostic Lab. Immunol. 8, 1131 (2001)).
Percent lysis was calculated as follows: [(test
fluorescence-spontaneous fluorescence)/(maximum
fluorescence-spontaneous fluorescence)].times.100. For in vitro CTL
generation, human PBMCs were cultured with irradiated U937 cells at
a ratio of 1:1 in the presence of bisanilinopyrimidine (I) 750 nM
or DMSO for 10 days. Cultures were replenished with media and
inhibitors on days +3 and +7, and fresh irradiated U937 cells added
on day +7. On day +10, the cells were harvested and CD8 T-cells
were isolated by magnetic bead separation (Miltenyi).
[0233] The tumor lysis assay was performed as described using
fresh, Calcein-AM labeled U937 targets.
[0234] Statistical Analysis: For comparisons of paired data sets,
the paired t test was used. ANOVA was used for group comparisons.
Survival comparisons were made using the log-rank test. The
Mann-Whitney test was used for all others. The statistical analysis
was conducted using Prism software version 5.04 (GraphPad).
Statistical significance was defined by P<0.05 (two-tailed).
[0235] For drug combination experiments, the results were analyzed
for synergistic, additive, or antagonistic effects using the
combination index (CI) method developed by Chou and Talalay.
[0236] The dose-effect curve for each drug alone was determined
based on experimental observations using the median-effect
principle and then compared to the effect achieved with a
combination of the two drugs to derive a CI value. For this
analysis,
[0237] XLfit software (IDBS) was used to create log-log
dose-fractional effect plots for each drug and combination and to
regress a straight line through the points and was used to
calculate the values of D.sub.m and m for use in the median-effect
equation as follows: f.sub.a/f.sub.u=(D/D.sub.m).sub.m, where D is
the dose of the drug, D.sub.m is the dose required for a 50% effect
(analogous to IC.sub.50), f.sub.a and f.sub.u are the affected and
unaffected fractions, respectively (f.sub.a=1-f.sub.u), and m is
the exponent signifying the sigmoidicity of the dose-effect curve.
The CI calculation chosen for the analysis of the drug combinations
was the isobologram equation for mutually nonexclusive drugs with
different modes of action as follows:
CI=(D).sub.1/(D.sub.x).sub.1+(D).sub.2/(D.sub.x).sub.2+(D).sub.1(D).sub.2-
/(D.sub.x)1(D.sub.x)2, where (D.sub.x).sub.1 and (D.sub.x).sub.2 in
the denominators are the concentrations for drug 1 and drug 2 alone
that gave x % inhibition, whereas (D).sub.1 and (D).sub.2 in the
numerators are the concentrations of drug 1 and drug 2 in
combination that also inhibited x % (that is, isoeffective).
[0238] Synthesis of bisanilinopyrimidine (I) and
bisanilinopyrimidine (II): The bisanilinopyrimidine
bisanilinopyrimidine (I) was prepared using a method previously
reported (Lawrence, et al., Development of o-chlorophenyl
substituted pyrimidines as exceptionally potent aurora kinase
inhibitors. J. Med. Chem. 2012, 55, 7392-416.). The
bisanilinopyrimidine bisanilinopyrimidine (II) was prepared using a
two step route (Scheme I) to prepare other 2,4-dianilinopyrimidines
(1). Reaction of 2,4-dichloro-5-fluoropyrimidine with aniline
provided the intermediate 1. Intermediate 1 was reacted further
with 4-aminobenzamide to give the required bisanilinopyrimidine
(II), with HPLC purity >99%.
##STR00039##
[0239] 2-Chloro-5-fluoro-N-phenylpyrimidin-4-amine (intermediate
1): To a solution of 5-fluoro-2,4-dichloropyrimidine (2.00 g, 11.98
mmol) and diethylisopropylamine (2.50 mL, 14.37 mmol) in
isopropanol (12 mL) was added aniline (1.09 mL, 11.98 mmol). The
mixture was stirred at reflux for 1 h. The reaction mixture was
concentrated under reduced pressure. The residue was dissolved in
dichloromethane (20 mL) and washed with water (10 mL) and brine (10
mL). The organic layer was dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The resulting solid was
triturated using EtOAc/hexanes to give the title compound as a
white solid (1.35 g, 50%). Mp: 135-136.degree. C. .sup.1H NMR (400
MHz, DMSO-d6): .delta. 9.99 (s, 1H, disappeared on D.sub.2O shake),
8.30 (d, J=3.5 Hz, 1H), 7.66 (d, J=8.0 Hz, 2H), 7.37 (t, J=8.0 Hz,
2H), 7.13 (t, J=8.0 Hz, 1H). .sup.19F NMR (376 MHz, DMSO-d6):
.delta. -153.7 (s). HPLC-MS (ESI.sup.+): m/z 226.1 [40%,
(M.sub.37Cl+H).sup.+], 224.1 [100%, (M.sub.35Cl+H).sup.+].
[0240]
5-Fluoro-N4-phenyl-N2-[4-(4-carboxamide)phenyl]pyrimidine-2,4-diami-
ne (bisanilinopyrimidine (II)): A mixture of intermediate 1 (1.00
g, 4.47 mmol), 4-aminobenzamide (0.609 g, 4.47 mmol), and methanol
(4.5 mL) was heated at 100.degree. C. for 14 h. The reaction
mixture was cooled to room temperature and the precipitate filtered
and washed with MeOH (2.times.10 mL). The resulting solid was
sonicated in saturated sodium bicarbonate solution (10 mL) for 2
min, then filtered, washed with water (3.times.20 mL), MeOH
(2.times.10 mL), and dried to give bisanilinopyrimidine (II) as a
white solid (1.07 g, 74%). Mp: 248-249.degree. C. HPLC: 99.9%
[tR=10.9 min, 50% MeOH, 50% water (with 0.1% TFA), 20 min]. .sup.1H
NMR (400 MHz, DMSO-d6): .delta. 9.51 (s, 1H, disappeared on
D.sub.2O shake), 9.44 (s, 1H, disappeared on D.sub.2O shake), 8.14
(d, J=3.7 Hz, 1H), 7.77 (brs, 1H, disappeared on D.sub.2O shake),
7.76 (d, J=7.8 Hz, 2H), 7.72 (s, 4H), 7.36 (t, J=7.8 Hz, 2H), 7.12
(brs, 1H, disappeared on D.sub.2O shake), 7.10 (t, J=7.8 Hz, 1H).
.sup.19F NMR (376 MHz, DMSO-d6): .delta. -163.2 (s). HPLC-MS
(ESI+): m/z 324.2 [100%, (M+H).sup.+]. LC-MS (ESI.sup.+): 992.3
[20%, (3M+Na).sup.+], 669.2 [50%, (2M+Na).sup.+], 346.1 [30%,
(M+Na).sup.+], 324.1 [100%, (M+H).sup.+]. HRMS (ESI+): m/z calcd
for C.sub.17H.sub.14FN.sub.5O (M+H).sup.+ 324.1255, found
324.1262.
Study 1:
[0241] Synergistic immune suppression with combined inhibition of
Aurora kinase A and JAK2 Using allogeneic mixed leukocyte reactions
(alloMLRs), the JAK2 inhibitor, TG101348, reduced alloreactive
T-cell proliferation at concentrations of 350 nM and greater as
previously reported(15) (FIG. 1A). The Aurora kinase A inhibitor,
alisertib (M. G. Manfredi et al., Characterization of Alisertib
(MLN8237), an investigational small molecule inhibitor of aurora A
kinase using novel in vivo pharmacodynamic assays. Clinical Cancer
research 17, 7614 (2011); H. Yang et al., Dual Aurora A and JAK2
kinase blockade effectively suppresses malignant transformation.
Oncotarget 5, 2947 (2014)), suppressed the proliferative response
of T-cells in alloMLRs at 625 nM (32.5% inhibition) with an
IC.sub.50 of 10 NM (FIG. 1A). Synergistic suppression over T-cells
allostimulated by dendritic cells (DC) was achieved when TG101348
and alisertib were added together at a ratio of 1:5, respectively,
with a calculated combination index (CI) of <1 per the Chou and
Talalay method (T. C. Chou, Theoretical basis, experimental design,
and computerized simulation of synergism and antagonism in drug
combination studies. Pharmacol. Rev. 58, 621 (2006)) (FIG. 1A). The
observed IC.sub.50 of the combination correlated with 350 nM of
TG101348 and 1.75 .mu.M of alisertib (FIG. 1A).
[0242] Bisanilinopyrimidine (I) is a potent inhibitor of Aurora
kinase A and JAK2 that was designed and synthesized at Moffitt
Cancer Center (H. R. Lawrence et al., Development of o-chlorophenyl
substituted pyrimidines as exceptionally potent aurora kinase
inhibitors. J. Med. Chem. 55, 7392 (2012)). Bisanilinopyrimidine
(I) exerted significant suppression over Tcells in alloMLRs, with
single agent efficacy at nanomolar concentrations (FIG. 1B).
Bisanilinopyrimidine (I) significantly decreased the activity of
Aurora kinase A and JAK2 in DC-allostimulated T-cells, and reduced
phosphorylation of histone 3 serine 10 (pH3 Ser10) and signal
transducer and activator of transcription 3 (pSTAT3 Y705)
respectively (FIGS. 1C-1D). The viability of AJI-214 (750 nM) or
DMSO treated T-cells was similar after 5 days of co-culture (FIG.
1E).
[0243] Concurrent blockade of Aurora kinase A and JAK2 selectively
impairs alloreactive T.sub.conv, while sparing responder T.sub.regs
bisanilinopyrimidine (I) exerted dose-dependent inhibition of
alloreactive CD4.sup.+ T.sub.conv (CD25.sup.+, CD127.sup.+) (A. K.
Heninger et al., IL-7 abrogates suppressive activity of human
CD4.sup.+CD25.sup.+FOXP3.sup.+ regulatory T cells and allows
expansion of alloreactive and autoreactive T cells. J. Immunol.
189, 5649 (2012); S. Samarasinghe et al., Functional
characterization of alloreactive T cells identifies CD25 and CD71
as optimal targets for a clinically applicable allodepletion
strategy. Blood 115, 396 (2010); S. Touil et al., Depletion of T
regulatory cells through selection of CD127-positive cells results
in a population enriched in memory T cells: implications for
anti-tumor cell therapy. Haematologica 97, 1678 (2012)) in alloMLRs
(FIG. 2A). The CD4.sup.+ T.sub.reg (CD25.sup.+, CD127.sup.-) (W.
Liu et al., CD127 expression inversely correlates with FoxP3 and
suppressive function of human CD4.sup.+ T.sub.reg cells. J. Experi.
Med. 203, 1701 (2006); N. Seddiki et al., Expression of interleukin
(IL)-2 and IL-7 receptors discriminates between human regulatory
and activated T cells. J. Experi. Med. 203, 1693 (2006)) population
was preserved even when co-cultures were exposed to 750 nM of
bisanilinopyrimidine (I) (FIG. 2B). The high degree of selectivity
demonstrated by dual inhibition of Aurora kinase A and JAK2
resulted in a significant increase in the T.sub.reg:allo T.sub.conv
ratio (FIG. 2C-2D). Cell Trace Violet dilution was used to study
the effect of dual Aurora kinase A/JAK2 blockade on proliferation
among the individual T.sub.reg and T.sub.conv compartments. This
verified the observed shifts in T.sub.reg:T.sub.conv populations by
bisanilinopyrimidine (I) exposure, where a significant decrease in
T.sub.conv proliferation occurred with bisanilinopyrimidine (I) at
750 nM (FIGS. 2E-2F). T.sub.reg proliferation was similar between
bisanilinopyrimidine (I) and control (FIGS. 2E-2F). STAT5
activation remained functional in IL-2 stimulated CD4.sup.+ T-cells
exposed to either DMSO or bisanilinopyrimidine (I) compared with
unstimulated baseline controls, though the effect was blunted by
bisanilinopyrimidine (I) (FIG. 2G). Moreover, CD4.sup.+,
CD25.sup.+, CD127.sup.- T.sub.reg expression of Foxp3 was
maintained in the presence of bisanilinopyrimidine (I) or DMSO
(FIG. 2H).
[0244] To confirm the T.sub.reg-sparing effects were related to
concurrent inhibition of JAK2 and Aurora kinase A, as opposed to a
phenomenon of bisanilinopyrimidine (I) alone, similar allogeneic
co-cultures were treated with either TG101348 (JAK2 inhibitor, 350
nM), alisertib (Aurora kinase A inhibitor, 1.75 NM), a combination
of TG101348 and alisertib, or DMSO. These doses of TG101348 and
alisertib were based on the synergy studies, where a 1:5 ratio of
each respective compound was used concurrently. As observed with
bisanilinopyrimidine (I), dual blockade of JAK2 and Aurora kinase A
reduced the alloreactive T.sub.conv population and spared the
responder T.sub.regs (FIG. 2I).
[0245] Dual inhibition of Aurora kinase A and JAK2 favors
iT.sub.reg development and potency. The effects of dual Aurora
A/JAK2 blockade on inducible T.sub.reg (iT.sub.reg) differentiation
was studied. Naive CD4.sup.+ T-cells (>99% pure) were depleted
of natural T.sub.regs and stimulated with allogeneic DCs for 5 days
in the presence of bisanilinopyrimidine (I) or DMSO (FIG. 3A).
Bisanilinopyrimidine (I) permitted iT.sub.reg differentiation, and
significantly decreased the frequency of alloreactive T.sub.conv
(FIGS. 3B-3C). Given that iT.sub.regs are derived from
phenotypically plastic naive CD4.sup.+ T-cells, it was confirmed
that demethylated Foxp3 TSDR was similar among bisanilinopyrimidine
(I)- and DMSO-exposed iT.sub.reg (FIG. 3D). To determine the
influence of Aurora A versus JAK2 inhibition on iT.sub.reg
differentiation, co-cultures were treated with bisanilinopyrimidine
(I) 750 nM, alisertib 1.75 .mu.M, TG101348 350 nM, a combination of
both, or DMSO. While alisertib, TG101348, and the combination of
each all had less absolute numbers of iT.sub.regs and allo
T.sub.conv compared to DMSO, the suppressive effect on T.sub.conv
was greater resulting in incrementally larger T.sub.reg:allo
T.sub.conv ratios (FIG. 3E).
[0246] Bisanilinopyrimidine (I) demonstrated minimal loss of
iT.sub.regs and significantly reduced allo T.sub.conv compared to
DMSO, increasing the iT.sub.reg:allo T.sub.conv ratio (FIG. 3E). To
study the influence of dual pathway inhibition on iT.sub.reg
suppressive function, bisanilinopyrimidine (I)- or DMSO-treated
iT.sub.reg were cultured with self alloresponders targeting fresh
allogeneic DCs. The bisanilinopyrimidine (I)-treated iT.sub.reg not
only demonstrated intact suppressive function, their potency was
significantly increased by approximately 30% compared to
DMSO-treated iT.sub.reg(FIG. 4A). It was then explored how Aurora
kinase A versus JAK2 blockade contributed to this enhanced
suppression by the iT.sub.regs. iT.sub.regs were generated as
described in the presence of alisertib, TG101348, a combination of
both, or DMSO. Interestingly, Aurora kinase A inhibition with
alisertib demonstrated superior suppressive capacity compared with
either DMSO- or TG101348-exposed iT.sub.reg (FIG. 4B). The
combination of alisertib with TG101348 was similar to alisertib
alone (FIG. 4B).
[0247] Also investigated was the mechanism supporting the increased
iT.sub.reg function observed with bisanilinopyrimidine (I).
Identified was a significant increase in the relative surface
density of CD39, an ectonucleotidase that hydrolyzes ATP, among the
bisanilinopyrimidine (I)-exposed iT.sub.reg compared with DMSO
controls (FIGS. 5A-5D). CD39 expression on non-T.sub.reg CD4.sup.+
T-cells was minimal (M. Mandapathil et al., Generation and
accumulation of immunosuppressive adenosine by human
CD4.sup.+CD25highFOXP3.sup.+ regulatory T cells. J. Biol. Chem.
285, 7176 (2010)) (FIG. 5B). It was confirmed that the higher CD39
expression among the bisanilinopyrimidine (I) treated iT.sub.reg
resulted in improved scavenging of extracellular ATP, compared to
DMSO-treated iT.sub.reg (FIG. 5E). The enhanced hydrolysis of ATP
by the AJI-treated iT.sub.regs was also significantly impaired by
blocking the CD39 enzyme with ARL67156 (FIG. 5E). To determine the
influence of CD39.sup.+ iT.sub.reg in the overall efficacy of dual
Aurora A/JAK2 blockade, ARL67156 was added to alloMLRs consisting
of natural T.sub.reg-depleted CD4.sup.+ T-cell responders with
bisanilinopyrimidine (I) or DMSO. This eliminated potential
interference from CD39.sup.+ natural T.sub.reg within the
allogeneic co-culture. CD39 blockade significantly weakened the
T-cell inhibition by bisanilinopyrimidine (I) (FIG. 5F), supporting
that CD39+iT.sub.regs contribute to the immune suppressive effects
of bisanilinopyrimidine (I). With regard to other modes of
iT.sub.reg suppression, no difference in their expression of LAG3,
CTLA4, or production of IL-10 or TGF-beta after exposure to
bisanilinopyrimidine (I) or DMSO (FIGS. 5G-5J).
[0248] As previously reported JAK2 inhibition with TG101348 impairs
T.sub.H17 production of IL-17, it was investigated how Aurora
kinase A inhibition influenced RORgammaT expression. Alisertib
exerted no effect on RORgammaT levels among alloresponder CD4.sup.+
T-cells, while JI-214 significantly decreased the expression of
this key T.sub.H17-differentiation transcription factor compared
with DMSO (FIG. 6A). It was confirmed that TG101348 significantly
decreases RORgammaT expression in CD4.sup.+ alloresponders (FIG.
6B). These data suggest that bisanilinopyrimidine (I) supports
enhanced iT.sub.reg potency primarily through Aurora kinase A
blockade, while its suppression of T.sub.H17 polarization is a
function of JAK2 blockade.
[0249] Targeting Aurora kinase A and JAK2 reduces xenogeneic GVHD
and preserves GVL A xenogeneic GVHD model was used to investigate
the in vivo efficacy of dual Aurora kinase A/JAK2 blockade while
maintaining a focus on human immune responses. Recipient NSG mice
were transplanted with 30.times.10.sup.6 human PBMCs i.p. once on
day 0. Bisanilinopyrimidine (I) is not suited for in vivo use, due
to limited bioavailability. Bisanilinopyrimidine (II) is an Aurora
kinase A/JAK2 inhibitor (Moffitt Cancer Center) that differs from
bisanilinopyrimidine (I) by only a chlorine to hydrogen
substitution at the ortho position of its phenyl ring enhancing its
solubility. The bisanilinopyrimidine (II) and bisanilinopyrimidine
(I) analogues both inhibit Aurora kinase A and JAK2 with similar
potency. As observed with bisanilinopyrimidine (I),
bisanilinopyrimidine (II) reduced responder T-cell proliferation in
alloMLRs at nanomolar concentrations (IC.sub.50 200 nM, FIG.
7A).
[0250] To study the concept of dual Aurora kinase A/JAK2 inhibition
as GVHD prevention, mice were treated with bisanilinopyrimidine
(II) at 50 mg/kg daily i.p. or vehicle control from day 0 to day
+14. JAK2 inhibition was confirmed in vitro, where human T-cells
stimulated with IL-6 expressed less STAT3 phosphorylation with
bisanilinopyrimidine (II) exposure compared with DMSO (FIG. 7B).
Harvested human T-cells from the mice at day +14 showed less H3
ser10 phosphorylation with bisanilinopyrimidine (II) compared to
vehicle, confirming Aurora inhibition (FIG. 7C). The vehicle
control treated mice developed acute xenogeneic GVHD (including fur
loss, skin changes, weight loss, and kyphosis) by the third week of
the transplant with a median survival of 34 days (FIGS. 7D-7E). The
overall survival of the bisanilinopyrimidine (II)-treated mice was
71.4% during the 56 days of observation, while none of the
vehicle-treated mice survived past day +45 (FIG. 7F). The average
GVHD clinical scores were <2 among the surviving
bisanilinopyrimidine (II)-treated mice at day +56 (FIG. 7E), where
60% showed limited fur/skin changes without significant weight loss
(FIGS. 7D-7E). Conversely, bisanilinopyrimidine (II) and vehicle
treated mice both facilitated the generation of U937-specific CTL
in vivo and retained similar anti-tumor killing in vitro (FIG. 7G).
It was confirmed that the bisanilinopyrimidine (I) analogue
similarly allowed for CTL generation in vitro, and that CD8.sup.+
CTL remained functional in tumor lysis assays against U937 targets
(FIG. 7H).
[0251] The absolute number of total spleen cells and human
CD3.sup.+ T-cells within the spleens were similar among vehicle-
versus bisanilinopyrimidine (II)-treated mice (FIGS. 8A-8B).
bisanilinopyrimidine (II) significantly increased the relative
amount of human T.sub.regs in the spleen, while the percentage of
CD4.sup.+ alloreactive T.sub.conv was similar among both groups of
transplanted mice (FIGS. 8C-8G). As such, bisanilinopyrimidine (II)
significantly increased the ratio of human T.sub.reg to CD4.sup.+
alloreactive T.sub.conv in the recipient spleens, compared with
vehicle (FIG. 8E). GVHD pathology at day +14 was limited to the
liver, where bisanilinopyrimidine (II) dramatically reduced human
T-cell invasion (FIGS. 8H-8J). T.sub.reg frequency in the liver was
similar among both treatment groups, however (FIG. 8K).
[0252] T-cell costimulation and cytokine activation independently
contribute to GVHD, but control of donor alloresponses is
incomplete when targeting either pathway alone. It has been
disclosed herein that GVHD prevention with intact GVL can be
accomplished by dual inhibition of Aurora kinase A and JAK2,
respectively attenuating CD28 costimulation and IL-6-mediated
signal transduction. Concurrent blockade of Aurora kinase A and
JAK2 yields synergistic immune suppression over human allogeneic
T-cells in vitro, preserves iT.sub.reg differentiation, and
significantly enhances iT.sub.reg suppressive potency. These
characteristics are distinct from CNI-based GVHD prophylaxis, which
abrogates TCR function and indiscriminately suppresses donor
T-cells. The lack of selectivity by CNIs results in a failure to
achieve donor immune tolerance toward the host and negates the GVL
potential of the allograft. Selective targeting of Aurora kinase A
and JAK2 signal transduction controls fundamental aspects of T-cell
allo-activation, without ceding TCR function required by T.sub.regs
and anti-tumor CTL.
[0253] Blockade of Aurora kinase A or JAK2 induces pathway-specific
effects on developing iT.sub.reg and T.sub.H17. It was observed
that the dual pathway inhibitor, bisanilinopyrimidine (I),
significantly increased the suppressive potency of allo-antigen
specific iT.sub.reg. The data supports that the enhanced iT.sub.reg
potency is largely a function of Aurora kinase A inhibition. While
JAK2 blockade with TG101348 improved iT.sub.reg function compared
with DMSO, iT.sub.regs previously exposed to alisertib profoundly
eliminated T.sub.conv proliferation. On the other hand, alisertib
was unable to prevent RORgammaT expression in naive CD4.sup.+
T-cells responding to allo-antigen unlike AJI-214 or TG101348.
Given that IL-6 receptor signal transduction facilitates T.sub.H17
development, the data confirm that JAK2 blockade with
bisanilinopyrimidine (I) or TG101348 significantly restrains
T.sub.H17 differentiation.
[0254] Modes of T.sub.reg suppression to understand how
bisanilinopyrimidine (I) improved iT.sub.reg suppressive potency
was investigated. iT.sub.reg production of the anti-inflammatory
cytokines, IL-10 and TGF-beta, where comparable among
bisanilinopyrimidine (I)- and DMSO-treated iT.sub.regs. Moreover,
iT.sub.reg expression of CTLA4 and LAG3 was similar among each
experimental condition. Interestingly, it was identified that
bisanilinopyrimidine (I) significantly increased the surface
density of CD39 expressed on the iT.sub.regs. CD39 is an
ectonucleotidase that hydrolyzes extracellular ATP and reduces
T-cell activation (M. Vukmanovic-Stejic et al., The kinetics of
CD4.sup.+Foxp3.sup.+ T cell accumulation during a human cutaneous
antigen-specific memory response in vivo. The J. Clinical Invest.
118, 3639 (2008)).
[0255] CD39.sup.+ T.sub.regs correlate with clinical outcomes in
autoimmune diseases (R. S. Peres et al., Low expression of CD39 on
regulatory T cells as a biomarker for resistance to methotrexate
therapy in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 112,
2509 (2015); A. Thiolat et al., Interleukin-6 receptor blockade
enhances CD39.sup.+ regulatory T cell development in rheumatoid
arthritis and in experimental arthritis. Arthritis & Rheumatol.
66, 273 (2014)). A decrease in CD39.sup.+ T.sub.regs is associated
with methotrexate-failure among rheumatoid arthritis (RA) patients.
Alternatively, IL-6 neutralization with tocilizumab increases
CD39.sup.+ T.sub.regs in a similar group of RA patients (38). It
was verified that bisanilinopyrimidine (I)-treated iT.sub.reg
degraded extracellular ATP more efficiently than those exposed to
DMSO. This response was impaired by blocking CD39 with the
inhibitor ARL67156. Additionally, neutralization of CD39 activity
significantly attenuated the immune suppressive effect of dual
Aurora kinase A/JAK2 inhibition in nT.sub.reg depleted alloMLRs.
This data supports that increased CD39 expression is relevant to
the enhanced iT.sub.reg function mediated by bisanilinopyrimidine
(I).
[0256] While inhibition of Aurora kinase A or JAK2 activity
individually suppressed human Tcell proliferation in alloMLRs,
synergy was achieved with simultaneous blockade of both signal
transduction pathways. The bi-specific inhibitors,
bisanilinopyrimidine (II) and bisanilinopyrimidine (I),
demonstrated potent single agent activity at nanomolar
concentrations. As published, bisanilinopyrimidine (II) and
bisanilinopyrimidine (I) exhibit similar activity against Aurora
kinase A and JAK2. Bisanilinopyrimidine (II) differs from
bisanilinopyrimidine (I) by a single chlorine to hydrogen
substitution on its phenyl ring to facilitate in vivo solubility
for mouse studies, but the compounds are otherwise chemically and
functionally similar.
[0257] Target inhibition was confirmed as bisanilinopyrimidine (II)
and bisanilinopyrimidine (I) significantly reduced the
phosphorylation of both STAT3 and H3 ser10 in human T-cells.
Conversely, combined Aurora kinase A/JAK2 blockade permitted
IL-2-induced STAT5 activation in T-cells, compared with resting,
unstimulated controls. The selective inhibition of Aurora kinase A
and JAK2 paired with preserved common gamma chain cytokine
signaling establishes a platform to control alloreactivity while
maintaining antigen-specific T.sub.reg and CTL responses.
Accordingly, it was observed that bisanilinopyrimidine (II)
significantly reduces GVHD, increases the proportion of T.sub.reg
to allo T.sub.conv, and preserves CTL generation and anti-tumor
activity. CNI-free GVHD prophylaxis is an important concept in
improving patient outcomes after clinical transplantation. The
challenges of CNI-based GVHD prevention are clear; as CNIs offer
incomplete protection from severe GVHD and render the donor immune
system poorly equipped to counter post-transplant relapse. Given
that targeting Aurora kinase A and JAK2 selectively eliminates
alloreactive T.sub.conv while sparing T.sub.regs and tumor-specific
CTL, the novel concept described here may represent a translatable
CNI-free approach at GVHD prevention. A limited number of CNI-free
GVHD prophylaxis strategies currently exist, and include T-cell
depletion of the allograft (M. C. Pasquini et al., Comparative
outcomes of donor graft CD34.sup.+ selection and immune suppressive
therapy as graft-versus-host disease prophylaxis for patients with
acute myeloid leukemia in complete remission undergoing HLA-matched
sibling allogeneic hematopoietic cell transplantation. J. Clinical
Oncol. 30, 3194 (2012)) or the use of post-transplant
cyclophosphamide (C. G. Kanakry et al., Single-agent GVHD
prophylaxis with posttransplantation cyclophosphamide after
myeloablative, HLA-matched BMT for AML, ALL, and MDS. Blood 124,
3817 (2014)). The bispecific inhibitor, bisanilinopyrimidine (II),
is an attractive alternative as it does not require ex vivo
allograft modification or the need to expose freshly infused donor
stem cells to potent alkylators. As such, further investigation of
dual Aurora kinase A/JAK2 inhibition is merited to promote
selective control over donor immune responses after alloHCT.
Study 2:
[0258] Synergistic Immunosuppression is Attainable with Combined
Inhibition of Aurora Kinase a and JAK2.
[0259] Allogeneic mixed leukocyte reactions (alloMLRs) are standard
assays used to assess human T cell proliferation against polyclonal
or antigen-specific stimuli. In alloMLRs consisting of human T
cells and allogeneicmonocyte-derived dendritic cells (DCs), the
JAK2 inhibitor TG101348 reduced alloreactive T cell proliferation
at concentrations of 350 nM and greater as previously reported
(FIG. 9A). The Aurora kinase A inhibitor alisertib suppressed the
proliferative response of T cells in alloMLRs with a median
inhibitory concentration (IC.sub.50) of 10 mM (FIG. 9A).
Synergistic suppression of T cells allostimulated by DCs was
achieved when TG101348 and alisertib were added together at a ratio
of 1:5, respectively, with a calculated combination index (CI) of
<1 using the Chou-Talalay method (FIG. 9A). The observed
IC.sub.50 of the combination correlated with 350 nM TG101348 and
1.75 mM alisertib (FIG. 9A). The chemical analogs AJI-214 and
AJI-100 were designed and synthesized at the Moffitt Cancer Center
and shown to inhibit Aurora kinase A and JAK2 with similar potency.
AJI-100 differs from AJI-214 by a single chlorine to hydrogen
substitution at the ortho position of its phenyl ring, enhancing
its solubility, hence its preferred use in vivo (FIGS. 9B-9C).
Because AJI-100 is tolerated in mouse models, a kinase target
screen was performed on AJI-100 to verify its activity against
Aurora kinase A and JAK2 among a panel of 140 kinases. Aurora
kinase A and JAK2 were among the top three kinases inhibited by
AJI-100. It was found that AJI-100 also inhibits 5' AMP activated
protein kinase (AMPK) and exhibits slightly more potent suppression
of Aurora kinase B than alisertib. AJI-214 and AJI-100 exerted
significant suppression of T cells in alloMLRs, with single-agent
efficacy at nanomolar concentrations (P<0.05; FIGS. 9B-9C).
Moreover, the AJI analogs suppressed alloreactive T cell
proliferation similar to the potency of alisertib (1.75 mM) and
TG101348 (350 nM) combined (FIG. 9D). AJI-214 and AJI-100 (750 nM
for each) also exhibited similar target inhibition of Aurora kinase
A and JAK2 signal transduction in human T cells, reducing the
phosphorylation of histone 3 serine 10 (pH3Ser.sup.10) and STAT3
(pSTAT3) Y705, respectively (FIGS. 9E-9H). As expected, alisertib
only inhibited pH3Ser.sup.10 (FIGS. 9E-9F), and TG101348 only
inhibited pSTAT3 (FIGS. 9G-9H).
[0260] Concurrent Blockade of Aurora Kinase a and JAK2 is
Immunosuppressive and Permits the Differentiation of Inducible
T.sub.reg
[0261] DMSO, alisertib, TG101348, a combination of alisertib and
TG101348, AJI-214, or AJI-100 was added to allogeneic cocultures of
DC-stimulated T cells. Activated CD4.sup.+ T.sub.conv were
identified as coexpressing CD25 and CD127, and the latter assisted
in excluding T.sub.regs from the analysis. CD25 expression alone
was used to identify activated CD8.sup.+ T.sub.conv. Although all
inhibitors suppressed the activated T.sub.conv compared to DMSO,
combined inhibition of Aurora A and JAK2 offered greater
immunosuppression than either alisertib (P=0.007, FIG. 10A;
P=0.002, FIG. 10B) or TG101348 alone (P=0.02, FIG. 10A; P=0.02,
FIG. 10B). To quantify the effect of Aurora A/JAK2 blockade on
T.sub.H17, IL-17 ELISPOTs was performed using DC-stimulated,
purified CD4.sup.+ T cells in the presence of the compounds or
DMSO. As predicted by the effect of each inhibitor on STAT3
phosphorylation, the JAK2-targeting compounds significantly reduced
T.sub.H17, whereas alisertib had no effect (P<0.05; FIG. 10C).
All of the inhibitors significantly decreased the frequency of
interferon-.gamma..sup.+ (IFN-.gamma..sup.+) T.sub.H1 T cells among
treated allogeneic cocultures (P<0.05; FIG. 10D). However, dual
blockade of Aurora A and JAK2 did not offer increased suppression
of T.sub.H1 compared to either inhibitor alone (FIG. 10D). Given
that inhibition of Aurora A and JAK2 significantly reduced
alloreactive T.sub.conv, T.sub.H1, and T.sub.H17 cells, the effects
of dual blockade on inducible T.sub.reg (iT.sub.reg)
differentiation was then studied. Naive CD4.sup.+ T cells (>99%
pure; FIG. 10E) were depleted of natural T.sub.regs and stimulated
with allogeneic DCs for 5 days in the presence of DMSO, alisertib,
TG101348, a combination of alisertib and TG101348, AJI-214, or
AJI-100. The iT.sub.regs were identified as CD4.sup.+, CD127.sup.-,
CD25.sup.+, and Foxp3.sup.+. iT.sub.reg conversion from naive
CD4.sup.+ precursors was variably reduced by all of the compounds
compared to DMSO (FIGS. 10F-10G), and Aurora A inhibition appeared
to exert greater iT.sub.reg impairment than JAK2 inhibition (FIG.
10G). In contrast, IL-2-induced STAT5 phosphorylation, which is
required for T.sub.reg development, remained intact among T cells
treated with alisertib, TG101348, a combination of alisertib and
TG101348, or the AJI analogs compared to DMSO (FIG. 10H).
[0262] Dual Inhibition of Aurora Kinase A and JAK2 Supports Potent
CD39+iT.sub.reg
[0263] AJI-214 and AJI-100 were confirmed to exhibit identical
suppressive potency in regard to Aurora A and JAK2 signal
transduction and human T cell proliferation assays. Therefore,
AJI-214 was used as the representative bispecific analog for
additional iT.sub.reg-based in vitro mechanistic tests. Given that
iT.sub.regs are derived from phenotypically plastic naive CD4.sup.+
T cells, it was confirmed that demethylated Foxp3
T.sub.reg-specific demethylated region (TSDR) was similar among
AJI-214-exposed and DMSO-exposed iT.sub.regs (FIG. 11A). To study
the influence of dual pathway inhibition on iT.sub.reg-suppressive
function, AJI-214-treated or DMSO-treated iT.sub.reg were cultured
with autologous T cells targeting fresh allogeneic DCs. The
AJI-214-treated iT.sub.regs demonstrated intact suppressive
function, and its potency was significantly increased by about 30%
compared to DMSO-treated iT.sub.regs (P=0.018; FIG. 11B). How
Aurora kinase A versus JAK2 blockade contributed to this enhanced
suppression by the iT.sub.regs was explored. Antigen-specific
iT.sub.regs were generated from CD25-depleted CD4.sup.+ T cells in
the presence of alisertib, TG101348, a combination of alisertib and
TG101348, or DMSO. Aurora kinase A inhibition with alisertib
demonstrated superior suppressive capacity compared with either
DMSO-exposed (P=0.03) or TG101348-exposed (P=0.04) iT.sub.reg (FIG.
11C). The combination of alisertib with TG101348 was similar to
alisertib alone (FIG. 11C). The mechanism supporting the increased
iT.sub.reg function observed with AJI-214 was investigated. A
significant increase in the cell surface density of CD39, an
ectonucleotidase that hydrolyzes adenosine triphosphate (ATP), was
identified among the AJI-214-exposed iT.sub.reg compared with DMSO
controls (P=0.045; FIGS. 12A-12D). As reported by others, CD39
expression on non-T.sub.reg CD4.sup.+ T cells was minimal (FIGS.
12B-12D). The higher CD39 cell surface density was confirmed among
the AJI-214-treated iT.sub.regs resulted in improved scavenging of
extracellular ATP, compared to DMSO-treated iT.sub.regs (FIG. 12E).
The enhanced hydrolysis of ATP by the AJI-214-treated iT.sub.regs
was also significantly impaired by blocking the CD39 enzyme with
ARL67156 (P<0.0001; FIG. 12E). To determine the influence of
CD39+ iT.sub.reg in the overall efficacy of dual Aurora A/JAK2
blockade, ARL67156 was added to alloMLRs consisting of natural
T.sub.reg-depleted CD4.sup.+ T cell responders with AJI-214 or
DMSO. This eliminated potential interference from CD39.sup.+
natural T.sub.reg within the allogeneic coculture and ensured that
the only T.sub.regs present in the system were induced. Moreover,
ARL67156 would primarily affect the iT.sub.reg as T.sub.conv
express negligible amounts of CD39. CD39 blockade significantly
weakened the T cell inhibition by AJI-214 (P=0.037; FIG. 12F),
supporting that CD39.sup.+ iT.sub.regs contribute to the
immunosuppressive effects of AJI-214. With regard to other modes of
iT.sub.reg suppression, no difference in their expression of LAG3
and CTLA4 or production of IL-10 or transforming growth
factor-.beta. (TGF-.beta.) was found after exposure to AJI-214 or
DMSO (FIG. 12G-12J).
[0264] Targeting Aurora Kinase a and JAK2 Reduces Xenogeneic GVHD
and Preserves the Generation Antitumor CTL
[0265] A xenogeneic GVHD model was used to investigate the in vivo
efficacy of dual Aurora kinase A/JAK2 blockade to specifically
evaluate effects on human immune responses. Recipient NOD (nonobese
diabetic) scid g (NSG) mice were transplanted with human peripheral
blood mononuclear cells (PBMCs) (30.times.10.sup.6 cells)
intraperitoneally (ip) on day 0. Independent donors were used for
each experiment. Whether combining individual inhibitors of Aurora
A and JAK2 prevented acute xenogeneic GVHD was tested. Mice
received alisertib (30 mg/kg daily), TG101348 (45 mg/kg twice a
day), a combination of alisertib and TG101348, or methylcellulose
vehicle from days 0 to +14 by oral gavage. The drug combination
significantly delayed the onset and severity of GVHD, compared to
vehicle or TG101348 alone (P<0.0001 and P=0.0001, respectively;
FIGS. 13A-13B). There was also a suggestion toward an improved
median survival with the drug combination compared to alisertib
(50.5 versus 41 days; P=not significant). The bispecific inhibitor
AJI-100 was used to test the in vivo efficacy of single agent
blockade of Aurora A and JAK2 as GVHD prevention. As demonstrated,
AJI-100 offers identical on-target inhibition and immunosuppressive
properties as AJI-214 but exhibits superior bioavailability.
Compared to using the combination of alisertib and TG101348,
AJI-100 had the advantage of being given once daily by
intraperitoneal injection and avoided the need for sustained gavage
dosing. Additionally, the single bispecific compound provided a
pharmacologically cleaner approach by eliminating the variability
in pharmacokinetics between the two drugs in combination. The
recipient mice were transplanted with human cell as described.
AJI-100 (50 mg/kg) or vehicle control was administered daily by
intraperitoneal injection from days 0 to +14. AJI-100 significantly
improved the overall survival of the mice and reduced the severity
of GVHD, compared to vehicle control (P=0.003; FIGS. 13C-13D). On
target inhibition of Aurora A and JAK2 was confirmed among human T
cells harvested from recipient spleens at day +14. AJI-100
significantly reduced the amount of pH3Ser10.sup.+ and pSTAT3.sup.+
T cells, respectively (P=0.027 and P=0.0098, respectively; FIGS.
13E-13F). An established method was used to generate human
antitumor CTL in vivo and then test their specific killing.
CD8.sup.+ CTLs were generated in xenotransplanted mice receiving
AJI-100 or vehicle control, where an inoculum of irradiated U937
cells was administered on day 0 and day +7. Unvaccinated,
xenotransplanted mice served as negative control. Despite its
immunosuppressive activity, AJI-100 did not inhibit CTL generation
because CD8.sup.+ CTL from AJI-100-treated and vehicle-treated mice
demonstrated similarly enhanced killing capacity against U937
targets in vitro, compared to unvaccinated controls (FIG. 13G).
These data support that although AJI-100 significantly reduces
GVHD, it also preserves antitumor CTL responses.
[0266] AJI-100 Significantly Increases the Ratio of T.sub.reg to
Activated T.sub.conv while Eliminating T.sub.H17 and T.sub.H1 T
Cells
[0267] Similar to its activity in vitro, AJI-100 suppressed the in
vivo expansion of human T cells in the xenotransplanted mice. The
absolute number of total CD4.sup.+ T cells (P<0.0001),
T.sub.regs (P=0.001), and activated CD4.sup.+ T.sub.conv
(P<0.0001) from recipient spleens at day +14 was all
significantly reduced by AJI-100 compared to vehicle (FIGS. 14A-E).
Activated T.sub.conv were proportionally more reduced by AJI-100
compared to T.sub.reg (FIGS. 14B-14C). Therefore, the ratio of
T.sub.reg to activated T.sub.conv was significantly increased among
mice treated with AJI-100 compared to vehicle (P=0.034; FIG. 14D).
AJI-100 also significantly reduced the amount of spleen-resident
human T.sub.H17 and T.sub.H1 T cells, compared to vehicle (P=0.002
for both; FIGS. 14F-14H). AJI-100 also exerted a suppressive effect
on CD8.sup.+ T cell and CD19.sup.+ B cell reconstitution as
determined by absolute numbers compared to vehicle. However, the
frequencies of CD4.sup.+ and CD8.sup.+ T cells and CD19.sup.+ B
cells were similar among AJI-100-treated and vehicle-treated mice.
The primary host target organs affected by GVHD at day +14 in this
xenogeneic model were liver and lung. GVHD severity within these
organs was significantly reduced by AJI-100, compared to vehicle
(P=0.043 and P=0.002, respectively; FIGS. 14I-14K).
Immunohistochemistry demonstrated that the number of
tissue-infiltrating, human CD3.sup.+ T cells in recipient liver and
lung was also significantly decreased by AJI-100 treatment (P=0.006
and P=0.002, respectively; FIGS. 14L-14M).
[0268] AJI-100 Reduces Xenograft Rejection.
[0269] Using the nondiagnostic mastectomy skin from consenting
donors, a 1.times.1 cm skin graft was transplanted onto
immunodeficient NSG mice dorsally. After 30 days of healing,
5.times.10.sup.6 peripheral blood mononuclear cells (PBMC) from an
HLA-disparate random donor was given by i.p. injection. A cohort of
mice only received a skin graft without PBMCs, as negative
rejection controls. The transplanted mice were treated with AJI-100
(50 mg/kg), a dual JAK2/Aurora A kinase inhibitor, or vehicle daily
by i.p. injection. Mice were humanely euthanized on day +21 to
assess human anti-human skin rejection pathology. Representative
H&E staining of the skin grafts show normal cutaneous histology
in the no PBMC control (per the Bejarano scoring system, Am J Surg
Pathol 28:670-675, 2004); demonstrated by an obvious
dermal-epidermal junction, lack of perivascular or dermal
infiltrates, and no lymphocytic exocytosis or hyperkeratosis (grade
0, FIG. 15C). Conversely, skin grafts from the mice who received
Vehicle+PBMCs show diffuse lymphocytic infiltration of the dermis
with exocytosis, a disrupted dermal-epidermal junction, and
hyperkeratosis (grade III, FIG. 15B). Mice that received
AJI-100+PBMCs exhibited very little graft damage, with mild dermal
infiltration, a normal dermal-epidermal junction, and limited
hyperkeratosis (grade I, FIG. 15C).
[0270] Results Summary
[0271] T cell costimulation and cytokine activation independently
contribute to GVHD, but control of donor alloresponses is
incomplete when targeting either pathway alone. Here, it is
demonstrated that GVHD prevention can be accomplished by dual
inhibition of Aurora kinase A and JAK2, attenuating CD28
costimulation and IL-6-mediated signal transduction, respectively,
without ablating potential antitumor CTL responses. Concurrent
blockade of Aurora kinase A and JAK2 yields synergistic
immunosuppression of human allogeneic T cells in vitro,
significantly enhances iT.sub.reg-suppressive potency, and enhances
the ratio of T.sub.regs to activated T.sub.conv in vivo. These
characteristics are distinct from CNI-based GVHD prophylaxis, which
inhibits TCR function and indiscriminately suppresses donor T
cells. The lack of selectivity by CNIs results in a failure to
achieve donor immune tolerance toward the host and mitigates the
graft-versus-leukemia (GVL) potential of the allograft. Inhibition
of Aurora kinase A or JAK2 activity individually suppressed human T
cell proliferation in alloMLRs, and synergy was achieved in vitro
with simultaneous blockade of both signal transduction pathways. A
xenogeneic model was used to study human T cell responses in vivo
after Aurora kinase A and JAK2 blockade, understanding that the
lack of recipient conditioning does differ from clinical practice
in allogeneic HCT. It was shown that alisertib combined with
TG101348 significantly delays GVHD. However, the combination of
inhibitors appears additive at best in vivo and does not completely
eliminate GVHD. The bispecific inhibitor AJI-100 significantly
reduced GVHD and improved survival compared to vehicle control. It
was surmised that the apparent enhanced in vivo activity of AJI-100
compared to alisertib plus TG101348 may be due to inherent kinase
selectivity. The ratio of Aurora kinase A to kinase B inhibition by
AJI-100 is greater than alisertib. This could contribute to
enhanced impairment of T cell costimulation by AJI-100 and
secondarily enhance efficacy. Although the role of AMPK in GVHD is
unknown, mouse models of inflammatory colitis using AMPK-deficient
T cells suggest that AMPK neutralization may have immunosuppressive
properties as well. Therefore, off-target inhibition of AMPK by
AJI-100 could be beneficial in controlling GVHD. However, the
immunosuppressive effects of the combination of the JAK2 inhibitor
TG101348 and the Aurora kinase A inhibitor alisertib coupled with
the potent activity of AJI-100 suggests that the ability of AJI-100
to prevent GVHD is likely due to its dual JAK2/Aurora kinase A
inhibitory activity. Blockade of Aurora kinase A or JAK2 induces
pathway-specific effects on developing iT.sub.reg and T.sub.H17.
First, blockade of Aurora kinase A and JAK2 permits the
differentiation of highly suppressive, alloantigen-specific,
CD39.sup.+ iT.sub.reg. Patients with rheumatoid arthritis lacking
sufficient CD39.sup.+ T.sub.regs experience greater rates of
methotrexate failure and poor clinical outcomes, suggesting that
dual Aurora kinase A/JAK2 inhibition may benefit other inflammatory
conditions. Our data support that the enhanced iT.sub.reg potency
is largely a function of Aurora kinase A inhibition because
iT.sub.regs exposed to alisertib, an Aurora kinase A-specific
inhibitor, eliminated T.sub.conv proliferation. On the other hand,
alisertib was unable to prevent T.sub.H17 differentiation among
naive CD4.sup.+ T cells responding to alloantigen. Given that IL-6
receptor signal transduction facilitates T.sub.H17 development, our
data confirm that JAK2 blockade is capable of restraining STAT3
phosphorylation and resultant T.sub.H17 differentiation.
Additionally, JAK2 inhibition appears to exhibit less inhibition of
iT.sub.regs compared to Aurora kinase A blockade. Last, inhibition
of JAK2, Aurora kinase A, or both JAK2 and Aurora kinase A equally
impaired the T.sub.H1 response in vitro. Selective inhibition of
Aurora kinase A and JAK2 paired with preserved common gamma-chain
cytokine signaling establishes a platform to control alloreactivity
while permitting antigen-specific T.sub.reg and CTL responses.
However, there are several limitations of this study that deserve
further consideration. Although the xenogeneic model is well suited
to test whether concurrent Aurora kinase A/JAK2 inhibition can
prevent GVHD mediated by human cells in vivo, it does not entirely
replicate human GVHD pathogenesis. The recipient mice do not
receive transplant conditioning, unlike human patients, and this
may affect GVHD target-organ injury, host antigen presentation, and
the production of relevant cytokines such as IL-6. Our work
demonstrates that AJI-100 permits the generation and function of
antitumor CTL, but it is important to recognize that such
experiments are supportive and not definitive in assessing whether
the bispecific inhibitor preserves GVL in vivo. Last,
small-molecule inhibitors can exhibit off-target inhibition, as
observed with AJI-100 and its suppression of AMPK. Unlike molecular
knockout strategies, off-target effects by pharmacologic inhibitors
may be immunologically relevant and should be considered when
interpreting such data. CNI-free GVHD prophylaxis is an important
concept in improving patient outcomes after clinical
transplantation. The challenges of CNI-based GVHD prevention are
clear because CNIs offer incomplete protection from severe GVHD and
render the donor immune system poorly equipped to counter
posttransplant relapse. Given that targeting Aurora kinase A and
JAK2 significantly reduces activated T.sub.conv while permitting
T.sub.regs and tumor-specific CTL, the concept described here may
represent a translatable CNI free approach at GVHD prevention. A
limited number of CNI-free GVHD prophylaxis strategies currently
exist and include T cell depletion of the allograft or the use of
posttransplant cyclophosphamide. The bispecific inhibitor AJI-100
is an attractive alternative because it does not require ex vivo
allograft modification or the need to expose freshly infused donor
stem cells to potent alkylators. Hence, further investigation of
dual Aurora kinase A/JAK2 inhibition is merited to promote
selective control of donor immune responses after alloHCT.
[0272] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
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