U.S. patent application number 13/003562 was filed with the patent office on 2011-09-15 for mtor modulators and uses thereof.
This patent application is currently assigned to The Regents of the University of California, a California corporation. Invention is credited to Beth Apsel, Katrina Chan, Morris Feldman, David Fruman, Andrew Hsieh, Matthew Janes, Liansheng Li, Yi Liu, David Pearce, Pingda Ren, Christian Rommel, Davide Ruggero, Kevan M. Shokat, Troy Edward Wilson.
Application Number | 20110224223 13/003562 |
Document ID | / |
Family ID | 41507712 |
Filed Date | 2011-09-15 |
United States Patent
Application |
20110224223 |
Kind Code |
A1 |
Shokat; Kevan M. ; et
al. |
September 15, 2011 |
MTOR Modulators and Uses Thereof
Abstract
The present invention provides methods and compositions for
selective modulation of certain protein kinases, and especially
mTor complexes. The methods and compositions are particularly
useful in inhibiting mTor selectively for therapeutic
applications.
Inventors: |
Shokat; Kevan M.; (San
Francisco, CA) ; Fruman; David; (Irvine, CA) ;
Ren; Pingda; (San Diego, CA) ; Wilson; Troy
Edward; (San Marino, CA) ; Li; Liansheng; (San
Diego, CA) ; Hsieh; Andrew; (San Francisco, CA)
; Feldman; Morris; (San Francisco, CA) ; Apsel;
Beth; (San Francisco, CA) ; Liu; Yi; (San
Diego, CA) ; Rommel; Christian; (La Jolla, CA)
; Chan; Katrina; (San Diego, CA) ; Ruggero;
Davide; (San Francisco, CA) ; Pearce; David;
(San Francisco, CA) ; Janes; Matthew; (Altadena,
CA) |
Assignee: |
The Regents of the University of
California, a California corporation
Oakland
CA
Intellikine, Inc.
La Jolla
CA
|
Family ID: |
41507712 |
Appl. No.: |
13/003562 |
Filed: |
July 8, 2009 |
PCT Filed: |
July 8, 2009 |
PCT NO: |
PCT/US2009/049969 |
371 Date: |
May 6, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61079103 |
Jul 8, 2008 |
|
|
|
Current U.S.
Class: |
514/252.18 ;
435/184; 435/375; 514/262.1; 514/291 |
Current CPC
Class: |
G01N 2510/00 20130101;
A61P 13/12 20180101; A61P 35/00 20180101; G01N 2500/04 20130101;
A61K 31/519 20130101; C12Q 1/485 20130101; A61P 1/16 20180101; A61P
9/12 20180101; A61P 9/04 20180101 |
Class at
Publication: |
514/252.18 ;
435/375; 435/184; 514/262.1; 514/291 |
International
Class: |
A61K 31/506 20060101
A61K031/506; C12N 5/09 20100101 C12N005/09; C12N 9/99 20060101
C12N009/99; A61K 31/519 20060101 A61K031/519; A61K 31/436 20060101
A61K031/436; A61P 35/00 20060101 A61P035/00; A61P 13/12 20060101
A61P013/12; A61P 9/12 20060101 A61P009/12; A61P 9/04 20060101
A61P009/04; A61P 1/16 20060101 A61P001/16 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0002] This invention was made at least in part with government
support under R01-DK56695, AI44009, and DK007636 awarded by the
National Institutes of Health. The Government has certain rights in
the invention.
Claims
1. A method for inhibiting cell proliferation comprising contacting
a cell with a biologically active agent that selectively inhibits
mTorC1 and/or mTorC2 activity relative to one or more type I
phosphatidylinositol 3-kinases (PI3-kinase) ascertained by an in
vitro kinase assay, wherein the one or more type I PI3-kinase is
selected from the group consisting of PI3-kinase .alpha.,
PI3-kinase .beta., PI3-kinase .gamma., and PI3-kinase .delta..
2. A method of inhibiting phosphorylation of both Akt (S473) and
Akt (T308) in a cell, comprising contacting a cell with an
effective amount of biologically active agent that selectively
inhibits both mTorC1 and mTorC2 activity relative to one or more
type I phosphatidylinositol 3-kinases (PI3-kinase) as ascertained
by a cell-based assay or an in vitro kinase assay, wherein the one
or more type I PI3-kinase is selected from the group consisting of
PI3-kinase .alpha., PI3-kinase .beta., PI3-kinase .gamma., and
PI3-kinase .delta., and thereby Akt phosphorylation at residues
S473 and T308 is simultaneously inhibited.
3. The method of claim 1 or 2, wherein the biologically active
agent selectively inhibits both mTorC1 and mTorC2 activity relative
to all type I phosphatidylinositol 3-kinases (PI3-kinase)
consisting of PI3-kinase .alpha., PI3-kinase .beta., PI3-kinase
.gamma., and PI3-kinase .delta..
4. The method of claim 1 or 2, wherein the biologically active
agent inhibits mTor activity with an IC50 value of about 100 nM or
less as ascertained in an in vitro kinase assay.
5.-6. (canceled)
7. The method of claim 1 or 2, wherein said biologically active
agent inhibits phosphorylation of Akt (S473) and Akt (T308) more
effectively than rapamycin when tested at a comparable molar
concentration in an in vitro kinase assay.
8.-12. (canceled)
13. The method of claim 1 or 2, wherein the biological active agent
competes with ATP for binding to ATP-binding site on mTorC1 and/or
mTorC2.
14.-17. (canceled)
18. A method of substantially inhibiting proliferation of a
neoplastic cell comprising contacting the cell with an effective
amount of an antagonist that inhibits full activation of Akt in a
cell and an anti-cancer agent, wherein said inhibition of cell
proliferation is enhanced through a synergistic effect of said
antagonist and said anti-cancer agent.
19. (canceled)
20. A combination treatment for a subject diagnosed with or at risk
of a neoplastic condition, comprising administering to said subject
a therapeutically effective amount of an antagonist that
substantially inhibits full activation of Akt in a cell and an
anti-cancer agent, wherein the efficacy of said treatment is
enhanced through a synergistic effect of said antagonist and said
anti-cancer agent.
21. (canceled)
22. The method of claim 18 or 20, wherein the anti-cancer agent is
selected from the group consisting of rapamycine, Gleevac, or
derivative thereof that inhibits a mammalian target of rapamycine
or Gleevac.
23.-28. (canceled)
29. A method of treating a condition caused by aberrant ion
transport across epithelial cells in a patient in need thereof,
said method comprising administering to said patient a
therapeutically effective amount of a biologically active agent
that selectively inhibits mTorC1 and/or mTorC2 activity relative to
one or more type I phosphatidylinositol 3-kinases (PI3-kinase)
ascertained by an in vitro kinase assay, wherein the one or more
type I PI3-kinase is selected from the group consisting of
PI3-kinase .alpha., PI3-kinase .beta., PI3-kinase .gamma., and
PI3-kinase .delta..
30. The method of claim 29, wherein said condition caused by
aberrant ion transport across epithelial cells is polycystic kidney
disease, hypertension, congestive heart failure, nephrotic syndrome
or liver cirrhosis.
31. The method of claim 1 or 2 or 29 wherein said biologically
active agent is a compound, or pharmaceutically acceptable salt
thereof, having one of the formulae: ##STR00017## wherein, n is an
integer from 1 to 5; z is an integer from 1 to 2; R.sup.1, R.sup.3,
and R.sup.4 are independently hydrogen, halogen, --CN, --CF.sub.3,
--OH, --NH.sub.2, --SO.sub.2, --COOH, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.2 and R.sup.6 are independently
hydrogen, halogen, --CN, --CF.sub.3, --OR.sup.5, --NH.sub.2,
--SO.sub.2, --COOH, substituted or unsubstituted alkyl, substituted
or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted
heteroaryl; and R.sup.5 is independently hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
32.-36. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/079,103, filed Jul. 8, 2008, which is hereby
incorporated by reference in its entirety and for all purposes.
BACKGROUND OF THE INVENTION
[0003] Abnormal cellular proliferation, a hallmark of cancer, can
result from a wide range of cellular phenomena. Proliferative
signals are transmitted into and within a cell via a process known
as signal transduction. Over the past decades, cascades of signal
transduction pathways have been elucidated and found to play a
central role in a variety of biological responses. Defects in
various components of signal transduction pathways have been found
to account for a vast number of diseases, including numerous forms
of cancer, inflammatory disorders, metabolic disorders, vascular
and neuronal diseases (Gaestel et al. Current Medicinal Chemistry
(2007) 14:2214-2234).
[0004] Kinases constitute a large family of important signaling
molecules. Kinases can generally be classified into protein kinases
and lipid kinases, and certain kinases exhibit dual specificities.
Protein kinases are enzymes that phosphorylate other proteins
and/or themselves (i.e., autophosphorylation). Protein kinases can
be generally classified into three major groups based upon their
substrate utilization: tyrosine kinases which predominantly
phosphorylate substrates on tyrosine residues (e.g., erb2, PDGF
receptor, EGF receptor, VEGF receptor, src, abl), serine/threonine
kinases which predominantly phosphorylate substrates on serine
and/or threonine residues (e.g., mTorC1, mTorC2, ATM, ATR, DNA-PK),
and dual-specificity kinases which phosphorylate substrates on
tyrosine, serine and/or threonine residues.
[0005] The mammalian target of rapamycin (mTor) is a
serine-threonine kinase related to the lipid kinases of the
phosphatidylinositol 3 kinase (PI3K) family. mTor has been
implicated in a wide range of biological processes including cell
growth/proliferation, cell motility and survival. Dysregulation of
the mTor pathway has been reported in various types of cancer. mTor
is a multifunctional kinase that integrates growth factor and
nutrient signals to regulate protein translation, nutrient uptake,
autophagy and mitochondrial function.
[0006] mTor exists in two complexes, mTorC1 and mTorC2. mTorC1
contains the raptor subunit and mTorC2 contains rictor. These
complexes are differentially regulated, and have distinct substrate
specificities and rapamycin sensitivity. For example, mTorC1
phosphorylates S6 kinase (S6K) and 4EBP1 (eIF4E-binding protein 1,
also known as also known as EIF4EBP1), promoting increased
translation and ribosome biogenesis to facilitate cell growth and
cell cycle progression. S6K also acts in a feedback pathway to
attenuate PI3K/Akt activation. mTorC2 is generally insensitive to
rapamycin. mTorC2 is thought to modulate growth factor signaling by
phosphorylating the C-terminal hydrophobic motif of some AGC
kinases such as Akt. In many cellular contexts, mTorC2 is required
for phosphorylation of the S473 site of Akt.
[0007] The serine/threonine kinase Akt (also known as protein
kinase B) possesses a pleckstrin homology (PH) domain that binds
PIP3, leading to Akt kinase activation. Akt phosphorylates many
substrates and is a central downstream effector of PI3K for diverse
cellular responses (FIG. 1). Full activation of Akt typically
requires phosphorylation of T308 in the activation loop and S473 in
a hydrophobic motif. One important function of Akt is to augment
the activity of mTor, through phosphorylation of TSC2 and other
mechanisms.
[0008] Phosphoinositide 3-kinases (PI3Ks) are a family of lipid
kinase enzymes whose products mediate reversible membrane
localization of cytoplasmic proteins. PI3K activation in most cells
correlates with proliferation and suppression of apoptosis. PI3K
and its downstream effectors also control cell polarity, motility,
metabolism and other physiological processes. This signaling
pathway also plays a prominent role in cancer: the PI3K pathway
activity is enhanced in nearly all human tumors. This can occur by
gain-of-function mutations or amplifications of PI3K genes, loss of
the tumor suppressor PTEN (the major lipid phosphatase that opposes
PI3K signaling), or expression of oncogenes that activate PI3K
(FIG. 1). In mouse models, enhanced PI3K signaling in lymphocytes
leads to lymphoproliferation, susceptibility to leukemia, and
spontaneous autoimmunity. Conversely, deletion of PI3K genes causes
immunodeficiency and resistance to malignant transformation.
Pharmacological suppression of immune responses and cancer cell
proliferation can also be achieved using rapamycin, which inhibits
mTor (mammalian target of rapamycin) downstream of PI3K (FIG.
1).
[0009] The PI3K/Akt/mTor signaling axis has been extensively
studied with small molecule inhibitors. Wortmannin and LY294002 are
two broad-spectrum PI3K inhibitors that have potent
anti-proliferative effects; however, these agents have broad
inhibition activity towards most PI3K isozymes as well as other
cellular targets. A key example of these off-target effects is the
direct inhibition of mTor by LY294002 (and wortmannin at high
concentrations).
[0010] Conventional mTor selective inhibitors also suffer from
several profound drawbacks. For example, the mTor inhibitors,
namely rapamycin and analogs, also termed "rapalogs" are potent
immunosuppressants. They have also been used in clinical trials for
various types of cancer. Unfortunately, the results of these
clinical trials to date have been mixed, with few malignancies
showing consistent response to rapalogs. Rapamycin (RAP) has a
mechanistic limitation: it is an allosteric, noncompetitive
inhibitor of mTorC1 that does not acutely inhibit mTorC2 in most
cells. Hence, cells treated with RAP usually display increased Akt
phosphorylation on both T308 and S473, due to loss of the feedback
inhibitory circuit mediated by S6K (FIG. 1). This can lead to
chemoresistance of cancer cells treated with rapalogs. Although RAP
does inhibit mTorC2 in some cell types by disrupting assembly of
the complex, the phenomenon of rapamycin-induced stimulation of Akt
has been observed in many settings. It is also worth noting that
mTorC2 might have additional functions in tumor cells, other than
Akt-S473 phosphorylation, which remain unaffected by RAP.
BRIEF SUMMARY OF THE INVENTION
[0011] Given these concerns as well as the emerging evidence for
PI3K-independent mTorC1 activity, there exists a considerable need
for alternative methods and biological agents that can selectively
inhibit mTorC1 and/or mTorC2. In some embodiments, the methods and
compositions disclosed herein yield selective inhibition of
mTor-mediated signal transduction without affecting upstream PI3K.
In some other embodiments, the methods and compositions provided
herein can inhibit mTor-mediated activity more effectively than
rapamycin, hence providing an alternative treatment for
rapamycin-resistant conditions.
[0012] In one embodiment, the present invention provides a method
for inhibiting cell proliferation comprising contacting a cell with
a biologically active agent that selectively inhibits mTorC1 and/or
mTorC2 activity relative to one or more type I phosphatidylinositol
3-kinases (PI3-kinase), wherein the one or more type I PI3-kinase
is selected from the group consisting of PI3-kinase .alpha.,
PI3-kinase .beta., PI3-kinase .gamma., and PI3-kinase .delta.. In
some embodiment, the selective inhibition is ascertained by an in
vitro kinase assay. In some aspect, the in vitro kinase assay is a
cell-based assay.
[0013] In separate embodiment, the present invention provides a
method of inhibiting phosphorylation of both Akt (S473) and Akt
(T308) in a cell, comprising contacting a cell with an effective
amount of biologically active agent that selectively inhibits both
mTorC1 and mTorC2 activity relative to one or more type I
phosphatidylinositol 3-kinases (PI3-kinase), thereby Akt
phosphorylation at residues S473 and T308 is simultaneously
inhibited. In some embodiments, the selective inhibition is
ascertained by an in vitro kinase including but not limited to a
cell-based assay The subject inhibition methods can take place in
vitro or in vitro. The inhibition methods can cause apoptosis or
cell cycle arrest.
[0014] In one aspect, the biologically active agent used in the
subject methods selectively inhibits both mTorC1 and mTORC2
activity relative to all type I phosphatidylinositol 3-kinases
(PI3-kinase). In another aspect, the biologically active agent
inhibits mTor activity with an IC.sub.50 value of about 100 nM or
less, preferably about 50 nM, about 25 nM, about 10 nM, 5 nM, about
1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM, or less, as ascertained in
an in vitro kinase or a cell based assay. In another aspect, the
biologically active agent inhibits mTor activity with an IC.sub.50
value of about 10 nM or less as ascertained in an in vitro cell
proliferation assay. Preferably, the biologically active agent is
substantially ineffective in inhibiting a type I PI3-kinase at a
concentration of 100 nM, 150 nM, 250 nM, 500 nM, 1 uM, 5 uM, 10 uM,
100 uM or even higher, when assayed in an in vitro kinase or a
cell-based assay. In yet another aspect, the biologically active
agent is substantially ineffective in inhibiting one or more
enzymes of the group consisting of PI4K.beta., DNA-PK, and JAK2 at
a concentration of 100 nM, 150 nM, 250 nM, 500 nM, 1 uM, 5 uM, 10
uM, 100 uM or even higher, when assayed in an in vitro kinase assay
including but not limited to a cell-based assay. In still yet
another aspect, the biologically active agent inhibits
phosphorylation of Akt (S473) and Akt (T308) more effectively than
rapamycin when tested at a comparable molar concentration in vitro
kinase assay including but not limited to a cell-based assay. Where
desired, a biologically active agent that inhibits phosphorylation
of Akt (S473) and Akt (T308) with an ED50 value of 1 uM or less can
be employed in practicing the subject methods. In some aspects, the
biological active agent competes with ATP for binding to the
ATP-binding site on mTorC1 and/or mTorC2
[0015] The subject inhibitory methods apply to any cell types,
preferably eukaryotic cells such as mammalian cells (e.g., human
cells) and especially those that exhibit neoplastic phenotype or
propensity.
[0016] The present invention further provides a method of
inhibiting proliferation of a neoplastic cell. The method comprises
the step of contacting the cell with an effective amount of an
antagonist that inhibits full activation of Akt in a cell and an
anti-cancer agent, wherein said inhibition of cell proliferation is
enhanced through a synergistic effect of said antagonist and said
anti-cancer agent.
[0017] Also provided is a method of ameliorating a medical
condition mediated by mTorC1 and/or mTorC2. The method involves the
step of administering to a subject in need thereof a
therapeutically effective amount of a compound that selectively
inhibits mTorC1 and/or mTorC2 activity relative to one or more type
I phosphatidylinositol 3-kinases (PI3-kinase) as ascertained in an
in vitro kinase including but not limited to a cell-based assay,
wherein the one or more type I PI3-kinase is selected from the
group consisting of PI3-kinase .alpha., PI3-kinase .beta.,
PI3-kinase .gamma., and PI3-kinase .delta.. In some embodiments,
the selective inhibition of mTorC1 and/or mTorC2 activity relative
to one or more type I PI3-kinases is ascertained by an in vitro
kinase assay including but not limited to a cell-based assay.
[0018] Further provided is another combination treatment for a
subject diagnosed with or at risk of a neoplastic condition. The
method involves the step of administering to said subject a
therapeutically effective amount of an antagonist that inhibits
full activation of Akt in a cell and an anti-cancer agent, wherein
the efficacy of said treatment is enhanced through a synergistic
effect of said antagonist and said anti-cancer agent.
[0019] In one aspect, the antagonist used in the subject treatment
methods selectively inhibits both mTorC1 and mTORC2 activity
relative to all type I phosphatidylinositol 3-kinases (PI3-kinase)
consisting of PI3-kinase .alpha., PI3-kinase .beta., PI3-kinase
.gamma., and PI3-kinase .delta.. The anti-cancer agent is selected
from the group consisting of rapamycin, Gleevac, or derivative
thereof that inhibits a mammalian target of rapamycin or Gleevac.
The antagonist and/or the anti-cancer agent is administered
parenterally, orally, intraperitoneally, intravenously,
intraarterially, transdermally, intramuscularly, liposomally, via
local delivery by catheter or stent, subcutaneously,
intraadiposally, or intrathecally. A variety of conditions can be
treated by the subject methods. They include but are not limited to
neoplastic condition such as restenosis, various types of
cancer.
[0020] The present invention further provides a method of
developing a biologically active agent that inhibits cell
proliferation. The method comprises: (a) contacting a candidate
agent with a cell of interest; (b) detecting a selective inhibition
of mTorC1 and/or mTorC2 activity relative to one or more type I
phosphatidylinositol 3-kinases (PI3-kinase), wherein the one or
more type I PI3-kinase is selected from the group consisting of
PI3-kinase .alpha., PI3-kinase .beta., PI3-kinase .gamma., and
PI3-kinase .delta., wherein said selective inhibition is
ascertained by an in vitro kinase or a cell-based assay.
[0021] In another aspect, there is provided a method of treating a
condition caused by aberrant ion transport across epithelial cells
in a patient in need thereof. The method includes administering to
the patient a therapeutically effective amount of a biologically
active agent that selectively inhibits mTorC1 and/or mTorC2
activity relative to one or more type I phosphatidylinositol
3-kinases (PI3-kinase) ascertained by an in vitro kinase assay. The
one or more type I PI3-kinase is selected from the group consisting
of PI3-kinase .alpha., PI3-kinase .beta., PI3-kinase .gamma., and
PI3-kinase .delta..
[0022] In another aspect, there is provided a method of treating T
cell lymphoma in a patient in need thereof. The method includes
administering to the patient a therapeutically effective amount of
a biologically active agent that selectively inhibits mTorC1 and/or
mTorC2 activity relative to one or more type I phosphatidylinositol
3-kinases (PI3-kinase) ascertained by an in vitro kinase assay,
wherein the one or more type I PI3-kinase is selected from the
group consisting of PI3-kinase .alpha., PI3-kinase .beta.,
PI3-kinase .gamma., and PI3-kinase .delta..
[0023] In another aspect, there is provided a method of inhibiting
phosphorylation of 4EBP1 in a cell. The method includes contacting
a cell with an effective amount of biologically active agent that
selectively inhibits both mTorC1 and mTorC2 activity relative to
one or more type I phosphatidylinositol 3-kinases (PI3-kinase) as
ascertained by a cell-based assay or an in vitro kinase assay,
wherein the one or more type I PI3-kinase is selected from the
group consisting of PI3-kinase .alpha., PI3-kinase .beta.,
PI3-kinase .gamma., and PI3-kinase .delta., thereby Akt
phosphorylation at residues S473 and T308 is simultaneously
inhibited.
[0024] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0026] FIG. 1 is a schematic overview of the PI3K/Akt/mTor
signaling pathway. Oncongenes are underlined.
[0027] FIG. 2 is a table of in vitro IC.sub.50 values in micromolar
for inhibition of mTor or PI3K family members by PP242 (Torkinib)
and PP30 (TORKinib2). The results suggest that PP242 and PP30 can
selectively inhibit mTor as compared to type I PI3-kinases and
other protein kinases.
[0028] FIG. 3 is a western blot illustrating the dose dependent
effect of treatment with TORKinibs on Akt phosphorylation of
insulin stimulated L6 myotubes. The TORKinibs demonstrate a
differential ability to inhibit phosphorylation of Akt S473 at
lower concentrations than Akt T308. The differential effect is not
seen in the control treatment with the PI3K inhibitor PIK-90.
[0029] FIG. 4 is a western blot depicting a time course for Akt
phosphorylation of insulin stimulated L6 myotubes treated with
TORKinib at high (156 nM) and low (625 nM) concentration. The
enhanced effect of TORKinib on Akt S473 phosphorylation is not
likely due to kinetic differences between S473 and T308
phosphorylation.
[0030] FIG. 5 is a western blot illustrating the effect of
treatment of wild-type and SIN1-/- mouse embryonic fibroblasts
(MEFs) with kinase inhibitors. The differential effect of TORKinib
treatment on Akt S473 and T308 phosphorylation seen in wild-type
MEFs is compared to rapamycin and PIK-90 which do not show a
differential effect under the conditions tested. In contrast
TORKinib has no significant effect on Akt phosphorylation in
SIN1-/- MEFs. This suggests that TORKinib may block T308
phosphorylation of Akt indirectly by directly inhibiting
mTor-dependent phosphorylation at S473.
[0031] FIG. 6 is a western blot depicting the dose dependent effect
of treatment of insulin stimulated L6 myotubes with the indicated
kinase inhibitors on downstream kinase substrates. For all affected
substrates, the extent of inhibition parallels the loss of
phosphorylation at Akt T308. This indicates that loss of S473
phosphorylation alone may be unable to prevent phosphorylation of
Akt substrates under the condition tested. In contrast, inhibition
of PI3K or Akt inhibited the phosphorylation of the tested
substrates under the conditions tested.
[0032] FIG. 7 illustrates the dose dependent inhibition of
proliferation of wild-type and SIN1-/- mouse embryonic fibroblast
by TORKinibs in comparison to rapamycin. TORKinib exhibits a more
complete inhibition of proliferation than rapamycin under the
conditions tested.
[0033] FIG. 8 is a western blot depicting a comparison of Akt,
70S6K, S6, 4EBP1, and MAPK phosphorylation in insulin treated L6
myotubes in response to TORKinib or rapamycin treatments. TORKinib
is a more effective inhibitor of phosphorylation of Akt and 4EBP1
than rapamycin under the conditions tested.
[0034] FIG. 9 is a western blot illustrating inhibition of
rapamycin resistant phosphorylation of 4EBP1 by TORKinibs but not
PIK-90 in insulin treated L6 myotubes.
[0035] FIG. 10 is a western blot of co-precipitated proteins bound
to m7GTP Sepharose.TM. beads. TORKinib demonstrates increased
efficacy over rapamycin in inducing 4EBP1 dephosphorylation and
binding to the m7GTP binding protein eIF-4E in insulin treated L6
myotubes. Binding of 4EBP1 to eIF-4E results in release of eIF-4G
bound to eIF-4E.
[0036] FIG. 11 is a western blot illustrating the effect of
TORKinib treatment of wild-type and SIN1-/- MEFs on phosphorylation
of the indicated kinase substrates. TORKinib is a more complete
inhibitor of 4EBP1 phosphorylation than rapamycin on both wild-type
and SIN1-/- MEFs.
[0037] FIG. 12 is a western blot illustrating the in vitro effect
of TORKinib treatment of insulin stimulated mice on PI3K signaling
in fat, skeletal muscle, and liver. Under the conditions tested, in
fat and liver TORKinib completely inhibits Akt S473 and T308
phosphorylation. In skeletal muscle, TORKinib partially inhibits
Akt phosphorylation at the concentration tested.
[0038] FIG. 13 illustrates the relative phosphorylation levels of
Akt and S6 in T cells stimulated with anti-CD3 antibody and treated
with the indicated kinase inhibitors. TORKinib (PP242) is a potent
inhibitor of S6 and Akt phosphorylation in stimulated T-cells.
[0039] FIG. 14 illustrates the effect of the indicated kinase
inhibitors on cellular division of T cells and B cells stimulated
with antibodies to cell-surface markers (anti CD3/anti CD3+anti
CD28 for T cells and anti IgM/anti IgM+anti IL-4 for B cells).
TORKinib (PP242) is a potent inhibitor of division of stimulated T
and B-cells.
[0040] FIG. 15 illustrates the in vitro effect of the indicated
inhibitors on cellular proliferation of p190 transduced cells (bone
marrow cells expressing the p190 isoform of the oncoprotein
BCR-ABL, also known as p190-transduced cells) as measured by
reduction of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium salt (MTS assay).
[0041] FIG. 16 illustrates the synergistic activity of treatment
with the inhibitors PP242 and BEZ235 in combination with imatinib
on p190 transduced cells as determined by MTS assay.
[0042] FIG. 17 illustrates the effect of the indicated kinase
inhibitors alone or in combination with imatinib on the colony
forming activity of p190 transduced bone marrow cells. Combination
therapy shows increased efficacy in inhibiting colony forming
activity at the IC.sub.50 of the individual compounds under the
conditions tested.
[0043] FIG. 18 illustrates the differential effect of the indicated
kinase inhibitors alone or in combination with dasatinib on the
colony forming activity of primary human B-cell acute lymphoblastic
leukemia cells that are positive (Ph+) or negative (Ph-) for the
philladelphia chromosome. Combination treatment shows increased
efficacy against Ph+acute lymphoblastic leukemia cells under the
conditions tested.
[0044] FIG. 19 illustrates the effect of 48 hr treatment with the
indicated inhibitors on viability of cells as measured by MTS
assay. Combination treatment with imatinib and either rapamycin or
PI-103 shows increased inhibition of cellular viability in
comparison to treatment with a single inhibitor under the
conditions tested.
[0045] FIG. 20 illustrates the synergistic or additive effect of
rapamycin and PI-103 in combination with imatinib on viability of
cells as measured by MTS assay.
[0046] FIG. 21 illustrates the effect of the indicated inhibitors
on splenomegaly in a syngenic mouse leukemia transplant model.
[0047] FIG. 22 illustrates the reduction of leukemic blood cell
counts in mice treated with imatinib in combination with rapamycin
or PI-103 in a syngenic mouse leukemia transplant model.
[0048] FIG. 23 illustrates a significant reduction of cycling
leukemic blast cells in bone marrow (BM) and spleen in a syngenic
mouse leukemia transplant model using combination therapy.
[0049] FIG. 24 illustrates significant increase in induction of
apoptosis in leukemic blast cells in the bone marrow (BM) and
spleen of a syngenic mouse leukemia transplant model in response to
combination therapy.
[0050] FIG. 25 illustrates the effect of rapamycin and PI3K
deletion mutations on CD98 expression.
[0051] FIG. 26 illustrates that PP242 is more efficacious than
rapamycin in the first in vivo preclinical trial in an Akt-mTOR
addicted tumor model. a) 2.times.10.sup.6 Akt transgenic thymocytes
were injected subcutaneously and allowed to form tumors for 20
days. 45 tumor bearing mice were randomized into three groups of
15: vehicle, rapamycin 5 mg/kg and PP242 100 mg/kg. Treatments were
given by gavage once daily, seven days a week for 20 days. b)
Representative mice from each cohort after 20 days of therapy.
Dotted lines about the midsection indicate primary tumor; dotted
distal lines indicate multiple sites of lymph node metastasis;
arrows (inset) indicate areas of significant cervical
lymphadenopathy. c) Tumors areas: square=vehicle, triangle tip
up=rapamycin, triangle tip down .dbd.PP242. d) Mouse weights in
grams during the course of treatment; legend as in FIG. 26c.
[0052] FIG. 27 illustrates that PP242 in vivo superiority over
rapamycin is mediated by incomplete blockade of eIF4E. a) Percent
live cells by PI/Annexin exclusion (n=10 mice/cohort), * p<0.01,
** p<0.05, ANOVA with Bonferroni's post test. b) Percent
annexin+ cells (n=10 mice/cohort), * p=0.002, ** p=0.03, non-paired
t-test. c) In vivo analysis of tumor proliferation by BrDU
incorporation (n=5 mice/cohort), * p<0.01, ** p<0.05, ANOVA
with Bonferroni's post test. d) Cell cycle analysis of
post-treatment tumor samples (n=10 mice/cohort). e) Pharmacodynamic
analysis of mTOR targets through western blot analysis. Each lane
represents 1 post-treatment tumor 20 days after initiation of
therapy. .beta.-actin=loading control. f) G1 inhibition induced by
rapamycin versus PP242 in wild type (WT) and 4EBP1/2 double
knockout (DKO) mouse embryonic fibroblasts.
[0053] FIG. 28 illustrates rapamycin-resistant modulation of SGK1
phosphorylation by mTOR. a) Immunoblots of mpkCCD cell lysates
detected by holo-SGK1, anti-phosphohydrophobic motif (pHM),
anti-Akt p-S473 and anti-phospho-p70S6K antibodies. Cells were
grown on Transwell.TM. filters and treated with aldosterone and
insulin for 4 h, followed by treatment with inhibitors as shown for
1 h. .alpha.-tubulin is shown as loading control. b) For
phosphatase treatment, cells were grown on Transwell.TM. filters
and treated with aldosterone and insulin as above. Whole cell
lysates were treated with .lamda.-phosphatase (lambda-PPase) prior
to Western blotting analysis with antibodies. c) Immunodetection of
HM phosphorylated SGK1. HEK293 cells were transfected with a
flag-tagged SGK1 or vector control, incubated with insulin and
treated with inhibitors.
[0054] FIG. 29 illustrates rapamycin-resistant modulation of
ENaC-dependent Na+ current by mTOR. a) Inhibition of ENaC-dependent
Na+ current by PP242. mpkCCD cells were grown on Transwell.TM.
filters, incubated with aldosterone and insulin for 4 h, and
treated with PP242 at various concentrations for 1 h. b) A time
course showing the effects of PP242 on ENaC-dependent Na+ current
in mpkCCD cells. The cells were grown on Transwell.TM. filters,
incubated with aldosterone (Aldo) and insulin, and treated with
inhibitors at various concentrations. Legend: LY, LY294002; Rap,
rapamycin.
[0055] FIG. 30 illustrates inhibition of SGK1 phosphorylation by
knockdown of rictor expression. A) A flag-tagged SGK1 plasmid was
transfected into HEK 293 cells. 24 hrs. after transfection,
recombinant lentiviruses harboring the rictor shRNA or an
irrelevant shRNA were used to infect the transfected cells. After
another 24 hrs., cells were lysed and analyzed by Western blotting
using antibodies against rictor to assay for expression knockdown.
b) Quantitative analysis of knockdown of rictor expression. c) The
cell lysates were incubated with an anti-flag antibody cross-linked
to beads. After washing, SGK1 protein bound to the beads was
recovered and analyzed by immunoblotting using antibodies against
phosphorylated SGK1. d) Quantitative analysis of inhibition of SGK1
phosphorylation by rictor shRNA. Legend: LY, LY294002.
[0056] FIG. 31 illustrates inhibition of ENaC-dependent Na+ current
by knockdown of rictor expression. A) Recombinant lentiviruses
harboring the rictor shRNA or an irrelevant shRNA were used to
infect mpkCCD cells. The infected cells were plated on
collagen-coated Transwell.TM. polycarbonate membranes.
ENaC-dependent Na+ currents were measured and quantified. b) The
infected cells were lysed on the Transwell.TM. membranes. Cell
lysates were recovered and analyzed by Western blotting using
antibodies against rictor to assay for expression knockdown. c)
Quantitative analysis of knockdown of rictor expression in mpkCCD
cells.
[0057] FIG. 32 illustrates the effect of raptor knockdown on SGK1
phosphorylation. A) The flag-tagged SGK1 plasmid was transfected
into HEK 293 cells. 24 hrs. after transfection, recombinant
lentiviruses harboring the raptor shRNA or an irrelevant shRNA were
used to infect the transfected cells. After another 24 hrs., cells
were lysed and analyzed by Western blotting using antibodies
against raptor to assay for expression knockdown. b) Quantitative
analysis of knockdown of raptor expression. c) The cell lysates
were incubated with an anti-flag antibody cross-linked to beads.
After washing, SGK1 protein bound to the beads was recovered and
analyzed by immunoblotting using antibodies against phosphorylated
SGK1. d) quantitative analysis of SGK1 phosphorylation in cells
expressing raptor shRNA.
[0058] FIG. 33 illustrates the effect of raptor knockdown on
ENaC-dependent Na+ current. A) Recombinant lentiviruses harboring
the raptor shRNA or an irrelevant shRNA were used to infect mpkCCD
cells. The infected cells were plated on collagen-coated
Transwell.TM. polycarbonate membranes. ENaC-dependent Na+ currents
were measured and quantified. b) The infected cells were lysed on
the Transwell.TM. membranes. Cell lysates were recovered and
analyzed by Western blotting using antibodies against pS6K. c) The
lysates were analyzed by Western blotting using antibodies against
raptor to assay for expression knockdown. d) Quantitative analysis
of knockdown of raptor expression in mpkCCD cells.
[0059] FIG. 34 illustrates the association of SGK1 with the mTOR
complexes. A) A flag-tagged SGK1 plasmid was transfected into
HEK293 cells. 48 hrs. post-transfection, the cells were lysed and
incubated with anti-flag antibody cross-linked to beads. After
washing, SGK1 protein bound to the beads was recovered and analyzed
by Western blotting using an antibody against rictor. b) The same
blot of SGK1 immunoprecipitates was stripped and analyzed by
Western blotting using an antibody against mTOR. Note that a
residual rictor band was detected due to incomplete stripping of
the blot. c) The same SGK1 immunoprecipitates were analyzed by
Western blotting using an antibody against raptor. Note that a
residual rictor band was detected due to incomplete stripping of
the blot.
[0060] FIG. 35 illustrates the effect of rapamycin concentration on
cellular proliferation. The experimental conditions were generally
as described in Example 6 (FIG. 7) with the exception that after 28
hours of treatment about 10 .mu.l of approximately 440 .mu.M
Resazurin sodium salt (Sigma) was added to each well. Data are
presented as %-proliferation, as measured by fluorescence intensity
relative to the control having no rapamycin present.
[0061] FIG. 36 illustrates the effect of PP242 in a companion
experiment to that described for FIG. 35. In the figure, the data
for cellular proliferation depict raw fluorescence intensity. PP242
blunts but does not eliminate proliferation in normal collecting
duct cells. Inhibition is significant at P<0.05 for 0.3 uM and
higher concentrations of compound. PP242 had a somewhat greater
effect than rapamycin. See FIG. 35.
[0062] FIG. 37 illustrates that PP242 induces apoptosis of p190
BCR-ABL-transformed murine hematopoietic progenitors and human
Ph.sup.+ B-ALL cells in vitro. a) Mouse p190 cells (upper) and
human SUP-B15 cells (lower) were cultured with inhibitors at the
concentrations indicated for 48 hr. b) p190 cells were cultured for
24 hr with the inhibitors indicated, then DNA content was measured
by flow cytometry. c) p190 cells were cultured for 48 hr with the
indicated combinations of compounds and assessed for survival using
the median effect method. d) CD19+CD34+ magnetically sorted cells
from five different patients were assessed for colony formation
potential in cultures with DAD (5 nM) alone or in combination with
increasing concentrations [10 or 100 nM] of RAP, PP242, or BEZ-235
(*P<0.05, **P <0.01, #P<0.001. e) Schematic model of
BCR-ABL driven mechanisms of oncogenic survival (left) and a new
model of incomplete mTOR inhibition (middle) versus complete mTOR
inhibition (right) in B-ALL.
[0063] FIG. 38 illustrates that PP242 completely inhibits
mTORC2/AKT and mTORC1 signaling in B-ALL whereas rapamycin
suppresses mTORC1 driving a PI3K/AKT surge. a)-b) Western blots of
p190 cells treated for 1.5 hr (a) or 3 hr (b) with indicated
inhibitors. c) Activation of PI3K was quantified in cells by signal
pixel intensity and localized area of PIP3 accumulation by confocal
microscopy. d) PP242 and high concentrations of IM (5 .mu.M) both
inhibit cap-dependent translation whereas RAP does not. e) p190
cells expressing LC3-GFP were cultured for 8 hr in chamber wells
with DA (10 nM), PP242 (250 nM), BEZ-235 (250 nM), RAP (250 nM),
and pulsed with EdU 1 hr prior to fixation. Autophagy (LC3 puncta
accumulation), loss of proliferation (EdU accumulation), and
distinct localization patterns of Foxo1 were assessed by confocal
microscopy and representative cells were magnified for clarity.
[0064] FIG. 39 illustrates that PP242 selectively suppresses
leukemic expansion in vivo. a) Mice injected with p190 cells
(i.v.), were treated daily (q24) starting on D7 post-transplant.
Imatinib ("IM," 150 mg kg.sup.-1, i.p.), rapamycin ("RAP," 7 mg
kg.sup.-1, i.p.) and PP242 (30 and 60 mg kg.sup.-1, p.o.) were
administered to mice as the mice were followed daily for overall
survival (median.+-.interquartile range) in groups of 5 mice. b)
Schematic of treatment design. c) Leukemic burden (mean %.+-.s.d.)
was assessed by flow cytometry in the corresponding bone marrow and
peripheral blood of treated mice. d) The abundance of leukemic
cells actively cycling (EdU+) following treatment was measured by
flow cytometry (mean %.+-.s.d.). e) Pharmacodynamic activity of
PP242 using intracellular phospho-staining of bone marrow and
peripheral blood cells. f) Schematic of treatment design for
primary human Ph+ B-ALL whole bone marrow xenografts. g) Leukemic
burden and cells actively cycling (mean %.+-.s.d.) was assessed by
flow cytometry in the corresponding bone marrow of treated
mice.
[0065] FIG. 40 illustrates that mTOR kinase inhibitor inhibits
mTORC1 and mTORC2 substrates in human Ph+SUP-B15 cells. a) Western
blot analysis of SUP-B15 cells treated with the PI3K/mTOR inhibitor
BEZ-235 [600 nM], RAP [20 nM], in comparison to a low dose
titration of PP242 [5, 15, 45, 135, 405 nM] for 3 hours. b) Western
blot of SUP-B15 cells treated with PI3K/mTOR inhibitor PI-103 [2000
nM] and the ABL/Src kinase inhibitor dasatinib [DA; 10, 100 nM]
alone, or in combination [DA at 100 nM] with RAP [RAP 50, 400 nM],
PP242 [50, 400 nM], or BEZ-235 [50, 400 nM] as indicated.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
Compounds Designations:
[0066] The terms "PP242" and "TORKinib" refer to the same
pyrazolopyrimidine compound and are interchangeable.
[0067] The terms "TORKinib2" and "PP30" refer to the same compound,
and are interchangeable.
[0068] The terms "IC87114" (ICOS corporation) and IC refer to the
same compound and are interchangeable.
[0069] The terms "DAS" and dasatinib, refer to the same compound
and are interchangeable.
[0070] The terms "IM" and "imatinib", refer to the same compound
and are interchangeable.
[0071] The terms and "RAP" and "rapamycin", refer to the same
compound and are interchangeable.
[0072] The term "B-ALL" as used herein refers to B-cell Acute
Lymphoblastic Leukemia.
[0073] The term "AML" as used herein refers to Acute Myelogenous
Leukemia.
[0074] The term "IP" or "i.p." as used herein refers to
intraperitoneal administration.
[0075] The term "p.o." as used herein refers to oral administration
or oral lavage.
[0076] As used herein, "agent" or "biologically active agent"
refers to a biological, pharmaceutical, or chemical compound or
other moiety. Non-limiting examples include simple or complex
organic or inorganic molecule, a peptide, a protein, an
oligonucleotide, an antibody, an antibody derivative, antibody
fragment, a vitamin derivative, a carbohydrate, a toxin, or a
chemotherapeutic compound. Various compounds can be synthesized,
for example, small molecules and oligomers (e.g., oligopeptides and
oligonucleotides), and synthetic organic compounds based on various
core structures. In addition, various natural sources can provide
compounds for screening, such as plant or animal extracts, and the
like. A skilled artisan can readily recognize that there is no
limit as to the structural nature of the agents of the present
invention.
[0077] The term "antagonist" or "inhibitor" as used herein refers
to a molecule having the ability to inhibit a biological function
of a target polypeptide. Accordingly, the term "antagonist" is
defined in the context of the biological role of the target
polypeptide. While preferred antagonists herein specifically
interact with (e.g. bind to) the target, molecules that inhibit a
biological activity of the target polypeptide by interacting with
other members of the signal transduction pathway of which the
target polypeptide is a member are also specifically included
within this definition. A preferred biological activity inhibited
by an antagonist is associated with the development, growth, or
spread of a tumor. Antagonists, as defined herein, without
limitation, include antibodies and immunoglobulin variants,
peptides, peptidomimetics, non-peptide small molecules, antisense
molecules, and oligonucleotide decoys.
[0078] The term "agonist" as used herein refers to a molecule
having the ability to initiate or enhance a biological function of
a target polypeptide. Accordingly, the term "agonist" is defined in
the context of the biological role of the target polypeptide. While
preferred agonists herein specifically interact with (e.g. bind to)
the target, molecules that inhibit a biological activity of the
target polypeptide by interacting with other members of the signal
transduction pathway of which the target polypeptide is a member
are also specifically included within this definition. A preferred
biological activity inhibited by an agonist is associated with the
prevention or inhibition of the development, growth, or spread of a
tumor or other diseased or damaged cell or tissue. For example,
agonist ligand binding can stimulate the expression of a biological
response modifier such as a phosphatase that inhibits cell growth
or accumulation of a factor useful for the development of a tumor,
such as by way of example and without limitation, phosphorylated
4EBP1. Agonists, as defined herein, without limitation, include
antibodies and immunoglobulin variants, peptides, peptidomimetics,
non-peptide small molecules, antisense molecules, and
oligonucleotide decoys.
[0079] The term "effective amount" or "therapeutically effective
amount" refers to that amount of an antagonist or biological agent
that is sufficient to effect the intended applications, including
with out limitation, clinical results as shrinking the size of the
tumor (in the cancer context, for example, B-ALL), retardation of
cancerous cell growth, delaying the development of metastasis,
inducing apoptosis or cell cycle arrest of cancer cells, decreasing
symptoms resulting from the disease, increasing the quality of life
of those suffering from the disease, decreasing the dose of other
medications required to treat the disease, enhancing the effect of
another medication, delaying the progression of the disease, and/or
prolonging survival of individuals. The therapeutically effective
amount will vary depending upon the subject and disease condition
being treated, the weight and age of the subject, the severity of
the disease condition, the manner of administration and the like,
which can readily be determined by one of ordinary skill in the
art. The term also applies to a dose that will provide an image for
detection by any one of the imaging methods described herein. The
specific dose will vary depending on the particular antagonist
chosen, the dosing regimen to be followed, whether is administered
in combination with other compounds, timing of administration, the
tissue to be imaged, and the physical delivery system in which it
is carried.
[0080] As used herein, "treatment" or "treating," or "palliating"
or "ameliorating" are used interchangeably herein. These terms
refer to an approach for obtaining beneficial or desired results
including but not limited to therapeutic benefit and/or a
prophylactic benefit. By therapeutic benefit is meant eradication
or amelioration of the underlying disorder being treated. Also, a
therapeutic benefit is achieved with the eradication or
amelioration of one or more of the physiological symptoms
associated with the underlying disorder such that an improvement is
observed in the patient, notwithstanding that the patient may still
be afflicted with the underlying disorder. For prophylactic
benefit, the compositions may be administered to a patient at risk
of developing a particular disease, or to a patient reporting one
or more of the physiological symptoms of a disease, even though a
diagnosis of this disease may not have been made. For purposes of
this invention, beneficial or desired clinical results include, but
are not limited to, one or more of the following: reducing the
proliferation of (or destroying) cancerous cells or other diseased,
reducing metastasis of cancerous cells found in cancers, shrinking
the size of the tumor, decreasing symptoms resulting from the
disease, increasing the quality of life of those suffering from the
disease, palliating the pain resulting from the disease, decreasing
the dose of other medications required to treat the disease,
delaying the progression of the disease, and/or prolonging survival
of individuals. Treatment includes preventing the disease, that is,
causing the clinical symptoms of the disease not to develop by
administration of a protective composition prior to the induction
of the disease; suppressing the disease, that is, causing the
clinical symptoms of the disease not to develop by administration
of a protective composition after the inductive event but prior to
the clinical appearance or reappearance of the disease; inhibiting
the disease, that is, arresting the development of clinical
symptoms by administration of a protective composition after their
initial appearance; preventing re-occurring of the disease and/or
relieving the disease, that is, causing the regression of clinical
symptoms by administration of a protective composition after their
initial appearance.
[0081] The term "pharmaceutically acceptable salt" refers to salts
derived from a variety of organic and inorganic counter ions well
known in the art and include, by way of example only, sodium,
potassium, calcium, magnesium, ammonium, tetraalkylammonium, and
the like; and when the molecule contains a basic functionality,
salts of organic or inorganic acids, such as hydrochloride,
hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the
like.
[0082] "Radiation therapy" means exposing a patient, using routine
methods and compositions known to the practitioner, to radiation
emitters such as alpha-particle emitting radionuclides (e.g.,
actinium and thorium radionuclides), low linear energy transfer
(LET) radiation emitters (i.e. beta emitters), conversion electron
emitters (e.g. strontium-89 and samarium-153-EDTMP, or high-energy
radiation, including without limitation x-rays, gamma rays, and
neutrons.
[0083] An "anti-cancer agent", "anti-tumor agent" or
"chemotherapeutic agent" refers to any agent useful in the
treatment of a neoplastic condition. One class of anti-cancer
agents comprises chemotherapeutic agents. "Chemotherapy" means the
administration of one or more chemotherapeutic drugs and/or other
agents to a cancer patient by various methods, including
intravenous, oral, intramuscular, intraperitoneal, intravesical,
subcutaneous, transdermal, buccal, or inhalation or in the form of
a suppository.
[0084] A "subject," "individual" or "patient" is used
interchangeably herein, which refers to a vertebrate, preferably a
mammal, more preferably a human. Mammals include, but are not
limited to, murines, simians, humans, farm animals, sport animals,
and pets. Tissues, cells and their progeny of a biological entity
obtained in vitro or cultured in vitro are also encompassed.
[0085] "Signal transduction" is a process during which stimulatory
or inhibitory signals are transmitted into and within a cell to
elicit an intracellular response. A modulator of a signal
transduction pathway refers to a compound which modulates the
activity of one or more cellular proteins mapped to the same
specific signal transduction pathway. A modulator may augment
(agonist) or suppress (antagonist) the activity of a signaling
molecule.
[0086] The term "cell proliferation" refers to a phenomenon by
which the cell number has changed as a result of division. This
term also encompasses cell growth by which the cell morphology has
changed (e.g., increased in size) consistent with a proliferative
signal.
[0087] The term "selective inhibition" or "selectively inhibit" as
referred to a biologically active agent refers to the agent's
ability to preferentially reduce the target signaling activity as
compared to off-target signaling activity, via direct or interact
interaction with the target.
[0088] "mTorC1 and/or mTorC2 activity" as applied to a biologically
active agent refers to the agent's ability to modulate signal
transduction mediated by mTorC1 and/or mTorC2. For example,
modulation of mTorC1 and/or mTorC2 activity is evidenced by
alteration in signaling output from the PI3K/Akt/mTor pathway.
[0089] A "therapeutic effect," as that term is used herein,
encompasses a therapeutic benefit and/or a prophylactic benefit as
described above. A prophylactic effect includes delaying or
eliminating the appearance of a disease or condition, delaying or
eliminating the onset of symptoms of a disease or condition,
slowing, halting, or reversing the progression of a disease or
condition, or any combination thereof.
[0090] The term "co-administration," "administered in combination
with," and their grammatical equivalents, as used herein,
encompasses administration of two or more agents to an animal so
that both agents and/or their metabolites are present in the animal
at the same time. Co-administration includes simultaneous
administration in separate compositions, administration at
different times in separate compositions, or administration in a
composition in which both agents are present.
[0091] The term "in vivo" refers to an event that takes place in a
subject's body.
[0092] The term "in vitro" refers to an event that takes places
outside of a subject's body. For example, an in vitro assay
encompasses any assay run outside of a subject assay. In vitro
assays encompass cell-based assays in which cells alive or dead are
employed. In vitro assays also encompass a cell-free assay in which
no intact cells are employed.
[0093] Unless indicated differently, the abbreviations used herein
have their conventional meaning within the chemical and biological
arts. The chemical structures and formulae set forth herein are
constructed according to the standard rules of chemical valency
known in the chemical arts.
[0094] Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents that would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is equivalent to --OCH.sub.2--.
[0095] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight (i.e.,
unbranched) or branched chain, or combination thereof, which may be
fully saturated, mono- or polyunsaturated and can include di- and
multivalent radicals, having the number of carbon atoms designated
(i.e., C.sub.1-C.sub.10 means one to ten carbons). Examples of
saturated hydrocarbon radicals include, but are not limited to,
groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,
t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and
isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and
the like. An unsaturated alkyl group is one having one or more
double bonds or triple bonds. Examples of unsaturated alkyl groups
include, but are not limited to, vinyl, 2-propenyl, crotyl,
2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,
3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the
higher homologs and isomers. An alkoxy is an alkyl attached to the
remainder of the molecule via an oxygen linker (--O--).
[0096] The term "alkylene," by itself or as part of another
substituent, means, unless otherwise stated, a divalent radical
derived from an alkyl, as exemplified, but not limited by,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer
carbon atoms.
[0097] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or combinations thereof, consisting of at least one
carbon atom and at least one heteroatom selected from the group
consisting of O, N, P, Si, and S, and wherein the nitrogen and
sulfur atoms may optionally be oxidized, and the nitrogen
heteroatom may optionally be quaternized. The heteroatom(s) O, N,
P, S, and Si may be placed at any interior position of the
heteroalkyl group or at the position at which the alkyl group is
attached to the remainder of the molecule. Examples include, but
are not limited to: --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3,
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3, --O--CH.sub.3,
--O--CH.sub.2--CH.sub.3, and --CN. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3.
[0098] Similarly, the term "heteroalkylene," by itself or as part
of another substituent, means, unless otherwise stated, a divalent
radical derived from heteroalkyl, as exemplified, but not limited
by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--. As described above, heteroalkyl groups, as used
herein, include those groups that are attached to the remainder of
the molecule through a heteroatom, such as --C(O)R', --C(O)NR',
--NR'R'', --OR', --SR', and/or --SO.sub.2R'. Where "heteroalkyl" is
recited, followed by recitations of specific heteroalkyl groups,
such as --NR'R'' or the like, it will be understood that the terms
heteroalkyl and --NR'R'' are not redundant or mutually exclusive.
Rather, the specific heteroalkyl groups are recited to add clarity.
Thus, the term "heteroalkyl" should not be interpreted herein as
excluding specific heteroalkyl groups, such as --NR'R'' or the
like.
[0099] The terms "cycloalkyl" and "heterocycloalkyl," by themselves
or in combination with other terms, mean, unless otherwise stated,
cyclic versions of "alkyl" and "heteroalkyl," respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples
of heterocycloalkyl include, but are not limited to,
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a
"heterocycloalkylene," alone or as part of another substituent,
means a divalent radical derived from a cycloalkyl and
heterocycloalkyl, respectively.
[0100] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" includes, but is
not limited to, fluoromethyl, difluoromethyl, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0101] The term "acyl" means, unless otherwise stated, --C(O)R
where R is a substituted or unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0102] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic, hydrocarbon substituent, which can be a
single ring or multiple rings (preferably from 1 to 3 rings) that
are fused together (i.e., a fused ring aryl) or linked covalently.
A fused ring aryl refers to multiple rings fused together wherein
at least one of the fused rings is an aryl ring. The term
"heteroaryl" refers to aryl groups (or rings) that contain from one
to four heteroatoms selected from N, O, and S, wherein the nitrogen
and sulfur atoms are optionally oxidized, and the nitrogen atom(s)
are optionally quaternized. Thus, the term "heteroaryl" includes
fused ring heteroaryl groups (i.e., multiple rings fused together
wherein at least one of the fused rings is a heteroaromatic ring).
A 5,6-fused ring heteroarylene refers to two rings fused together,
wherein one ring has 5 members and the other ring has 6 members,
and wherein at least one ring is a heteroaryl ring. Likewise, a
6,6-fused ring heteroarylene refers to two rings fused together,
wherein one ring has 6 members and the other ring has 6 members,
and wherein at least one ring is a heteroaryl ring. And a 6,5-fused
ring heteroarylene refers to two rings fused together, wherein one
ring has 6 members and the other ring has 5 members, and wherein at
least one ring is a heteroaryl ring. A heteroaryl group can be
attached to the remainder of the molecule through a carbon or
heteroatom. Non-limiting examples of aryl and heteroaryl groups
include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,
2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,
pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,
3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,
5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,
3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,
purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below. An "arylene" and a "heteroarylene," alone or as
part of another substituent, mean a divalent radical derived from
an aryl and heteroaryl, respectively.
[0103] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl, and the like) including those alkyl groups in which
a carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0104] The term "oxo," as used herein, means an oxygen that is
double bonded to a carbon atom.
[0105] The term "alkylsulfonyl," as used herein, means a moiety
having the formula --S(O.sub.2)--R', where R' is an alkyl group as
defined above. R' may have a specified number of carbons (e.g.,
"C.sub.1-C.sub.4 alkylsulfonyl").
[0106] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl," and "heteroaryl") includes both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0107] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to,
--OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN, and --NO.sub.2 in a
number ranging from zero to (2 m'+1), where m' is the total number
of carbon atoms in such radical. R', R'', R''', and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl (e.g., aryl substituted with 1-3 halogens),
substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups,
or arylalkyl groups. When a compound of the invention includes more
than one R group, for example, each of the R groups is
independently selected as are each R', R'', R''', and R'''' group
when more than one of these groups is present. When R' and R'' are
attached to the same nitrogen atom, they can be combined with the
nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For
example, --NR'R'' includes, but is not limited to, 1-pyrrolidinyl
and 4-morpholinyl. From the above discussion of substituents, one
of skill in the art will understand that the term "alkyl" is meant
to include groups including carbon atoms bound to groups other than
hydrogen groups, such as haloalkyl (e.g., --CF.sub.3 and
--CH.sub.2CF.sub.3) and acyl (e.g., --C(O)CH.sub.3, --C(O)CF.sub.3,
--C(O)CH.sub.2OCH.sub.3, and the like).
[0108] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are varied and are
selected from, for example: --OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN, --NO.sub.2, --R',
--N.sub.3, --CH(Ph).sub.2, fluoro(C.sub.1-C.sub.4)alkoxy, and
fluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to the
total number of open valences on the aromatic ring system; and
where R', R'', R''', and R'''' are preferably independently
selected from hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl. When a compound of the
invention includes more than one R group, for example, each of the
R groups is independently selected as are each R', R'', R''', and
R'''' groups when more than one of these groups is present.
[0109] Two or more substituents may optionally be joined to form
aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such
so-called ring-forming substituents are typically, though not
necessarily, found attached to a cyclic base structure. In one
embodiment, the ring-forming substituents are attached to adjacent
members of the base structure. For example, two ring-forming
substituents attached to adjacent members of a cyclic base
structure create a fused ring structure. In another embodiment, the
ring-forming substituents are attached to a single member of the
base structure. For example, two ring-forming substituents attached
to a single member of a cyclic base structure create a spirocyclic
structure. In yet another embodiment, the ring-forming substituents
are attached to non-adjacent members of the base structure.
[0110] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally form a ring of the formula
-T-C(O)--(CRR').sub.q--U--, wherein T and U are independently
--NR--, --O--, --CRR'--, or a single bond, and q is an integer of
from 0 to 3. Alternatively, two of the substituents on adjacent
atoms of the aryl or heteroaryl ring may optionally be replaced
with a substituent of the formula -A-(CH.sub.2).sub.r--B--, wherein
A and B are independently --CRR'--, --O--, --NR--, --S--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2NR'--, or a single bond, and r is an
integer of from 1 to 4. One of the single bonds of the new ring so
formed may optionally be replaced with a double bond.
Alternatively, two of the substituents on adjacent atoms of the
aryl or heteroaryl ring may optionally be replaced with a
substituent of the formula --(CRR').sub.s--X'--(C''R''').sub.d--,
where s and d are independently integers of from 0 to 3, and X' is
--O--, --NR'--, --S--, --S(O)--, --S(O).sub.2--, or
--S(O).sub.2NR'--. The substituents R, R', R'', and R''' are
preferably independently selected from hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, and substituted or unsubstituted
heteroaryl.
[0111] As used herein, the terms "heteroatom" or "ring heteroatom"
are meant to include oxygen (O), nitrogen (N), sulfur (S),
phosphorus (P), and silicon (Si).
[0112] A "substituent group," as used herein, means a group
selected from the following moieties: [0113] (A)-OH, --NH.sub.2,
--SH, --CN, --CF.sub.3, --NO.sub.2, oxo, halogen, unsubstituted
alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,
unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted
heteroaryl, and [0114] (B) alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, and heteroaryl, substituted with at least
one substituent selected from: [0115] (i) oxo, --OH, --NH.sub.2,
--SH, --CN, --CF.sub.3, --NO.sub.2, halogen, unsubstituted alkyl,
unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
[0116] (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
and heteroaryl, substituted with at least one substituent selected
from: [0117] (a) oxo, --OH, --NH.sub.2, --SH, --CN, --CF.sub.3,
--NO.sub.2, halogen, unsubstituted alkyl, unsubstituted
heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
[0118] (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
or heteroaryl, substituted with at least one substituent selected
from: oxo, --OH, --NH.sub.2, --SH, --CN, --CF.sub.3, --NO.sub.2,
halogen, unsubstituted alkyl, unsubstituted heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,
unsubstituted aryl, and unsubstituted heteroaryl.
[0119] A "size-limited substituent" or "size-limited substituent
group," as used herein, means a group selected from all of the
substituents described herein for a "substituent group," wherein
each substituted or unsubstituted alkyl is a substituted or
unsubstituted C.sub.1-C.sub.20 alkyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20
membered heteroalkyl, each substituted or unsubstituted cycloalkyl
is a substituted or unsubstituted C.sub.4-C.sub.8 cycloalkyl, and
each substituted or unsubstituted heterocycloalkyl is a substituted
or unsubstituted 4 to 8 membered heterocycloalkyl.
[0120] A "lower substituent" or "lower substituent group," as used
herein, means a group selected from all of the substituents
described above for a "substituent group," wherein each substituted
or unsubstituted alkyl is a substituted or unsubstituted
C.sub.1-C.sub.8 alkyl, each substituted or unsubstituted
heteroalkyl is a substituted or unsubstituted 2 to 8 membered
heteroalkyl, each substituted or unsubstituted cycloalkyl is a
substituted or unsubstituted C.sub.5-C.sub.7 cycloalkyl, and each
substituted or unsubstituted heterocycloalkyl is a substituted or
unsubstituted 5 to 7 membered heterocycloalkyl.
[0121] Where a substituent is "R-substituted", it is to be
understood that the substituent is substituted with one or more of
the recited R groups and that each R group attached to the
substituent is optionally different. For example, an
R.sup.7-substituted alkyl is substituted with one or more R.sup.7
groups wherein each of the R.sup.7 groups are optionally
different.
I. Embodiments of the Invention
[0122] This invention pertains to the discovery of a distinct class
of biologically active agents that exhibit selective inhibition of
certain protein kinases, and the uses of these agents for treatment
of diseases mediated by such protein kinases. In one embodiment,
the present invention provides a method for inhibiting cell
proliferation comprising contacting a cell with a biologically
active agent that selectively inhibits mTorC1 and/or mTorC2
activity relative to one or more type I phosphatidylinositol
3-kinases (PI3-kinase), wherein the one or more type I PI3-kinase
is selected from the group consisting of PI3-kinase .alpha.,
PI3-kinase .beta., PI3-kinase .gamma., and PI3-kinase .delta..
[0123] It is generally recognized that there are four types of
PI3K: IA, IB, II and III. Type IA enzymes act downstream of
tyrosine kinases to generate
phosphatidylinositol-3,4,5-trisphosphate (PIP3), a crucial second
messenger that promotes proliferation and transformation. Class IA
enzymes typically exist as dimers of a 110 kDa catalytic subunit
(p110.alpha., p110.beta. or p110.delta.) and a regulatory subunit
of varying size. The single class IB PI3K enzyme, p110.gamma., is
activated downstream of G protein-coupled receptors.
[0124] Any agents that selectively and negatively regulate mTorC1
and/or mTor2C expression or activity can be used as selective mTor
inhibitors in the methods of the invention. The relative efficacies
of agents as inhibitors of mTorC1 or mTorC2 can be established by
determining the concentrations at which each agent inhibits the
activity to a predefined extent.
[0125] In one aspect, a determination is the concentration that
inhibits 50% of the activity in a cell-based assay or in an in
vitro kinase assay. IC.sub.50 determinations can be accomplished
using any conventional techniques known in the art. In general, an
IC.sub.50 can be determined by measuring the activity of a given
enzyme in the presence of a range of concentrations of the
inhibitor under study. The experimentally obtained values of enzyme
activity then are plotted against the inhibitor concentrations
used. The concentration of the inhibitor that shows 50% enzyme
activity (as compared to the activity in the absence of any
inhibitor) is taken as the "IC.sub.50" value. Analogously, other
inhibitory concentrations can be defined through appropriate
determinations of activity. For example, in some settings it can be
desirable to establish a 90% inhibitory concentration, i.e.,
IC.sub.90, etc.
[0126] Alternatively, IC.sub.50 determinations can be accomplished
by measuring the phosphorylation level of substrate proteins of the
target in a cell-based assay. For example, one substrate of mTOR is
AKT, which may be phosphorylated at T308 or S473. Cells, for
example, may be contacted with the inhibitor under study under
conditions, such as 100 nM insulin, which would normally yield
phosphorylation of mTOR substrates including but not limited to AKT
at S473 and T308. Cells may then be prepared by various methods
known to the art including fixation or lysis, and analyzed for the
phosphorylation levels of mTOR substrates. Optionally, specificity
or selectivity may be determined by examining the effect of the
inhibitor under study on the phosphorylation of substrates of other
kinases. Phosphorylation levels may be analyzed using any methods
known to the art including but not limited to the use of antibodies
specific for the phosphorylated forms of the substrates to be
assayed via immunoblot or flow cytometry.
[0127] In another aspect, a selective mTor inhibitor alternatively
can be understood to refer to an agent that exhibits a 50%
inhibitory concentration (IC.sub.50) with respect to mTorC1 and/or
mTorC2, that is at least at least 10-fold, at least 20-fold, at
least 50-fold, at least 100-fold, at least 1000-fold, at least
10,100-fold, or more, lower than the inhibitor's IC.sub.50 with
respect to one, two, three, or more type I PI3-kinases. In some
embodiment, a selective mTor inhibitor alternatively can be
understood to refer to an agent that exhibits a 50% inhibitory
concentration (IC.sub.50) with respect to mTorC1 and/or mTorC2,
that is at least at least 10-fold, at least 20-fold, at least
50-fold, at least 100-fold, at least 1000-fold, at least
10,100-fold, or more, lower than the inhibitor's IC.sub.50 with
respect to all of type I PI3-kinases.
[0128] In yet another aspect, a selective mTor inhibitor, or an
inhibitor that selectively inhibits mTor mediated signaling,
alternatively can be understood to refer to a compound that
exhibits a 50% inhibitory concentration (IC.sub.50) with respect to
mTor, that is at least at least 10-fold, at least 20-fold, at least
50-fold, at least 100-fold, at least 1000-fold, at least
10,100-fold, or lower, than the inhibitor's IC.sub.50 with respect
to one or more protein kinases selected from the group consisting
of PKC.beta.I, PKC.beta.II, and RET, PI4K.beta., DNA-PK, and
JAK2.
[0129] The subject biologically active agent may inhibit both
mTorC1 and mTorC2 activity with an IC.sub.50 value of about 100 nM
or less, preferably about 50 nM, about 25 nM, about 10 nM, about 5
nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM, or less, as
ascertained in a cell-based assay or an in vitro kinase assay.
[0130] Inhibition of mTorC1 and/or mTorC2 activity can be
determined by a reduction in signal transduction of the
PI3K/Akt/mTor pathway. A wide variety of readouts can be utilized
to establish a reduction of the output of such signaling pathway.
Some non-limiting exemplary readouts include (1) a decrease in
phosphorylation of Akt at residues, including but not limited to
S473 and T308; (2) a decrease in activation of Akt as evidenced by
a reduction of phosphorylation of Akt substrates including but not
limited to FoxO1/O3a T24/32, GSK3.alpha./.beta. S21/9, and TSC2
T1462; (3) a decrease in phosphorylation of signaling molecules
downstream of mTor, including but not limited to ribosomal S6
S240/244, 70S6K T389, and 4EBP1 T37/46; (4) inhibition of
proliferation of cells including but not limited to normal or
neoplastic cells, mouse embryonic fibroblasts, leukemic blast
cells, cancer stem cells, and cells that mediate autoimmune
reactions; (5) induction of apoptosis of cells or cell cycle
arrest; (6) reduction of cell chemotaxis; and (7) an increase in
binding of 4EBP1 to eIF4E. The term "eIF4E" refers to a 24-kD
eukaryotic translation initiation factor involved in directing
ribosomes to the cap structure of mRNAs, having human gene locus
4q21-q25.
[0131] mTor exists in two types of complexes, mTorC1 containing the
raptor subunit and mTorC2 containing rictor. As known in the art,
"rictor" refers to a cell growth regulatory protein having human
gene locus 5p13.1. These complexes are regulated differently and
have a different spectrum of substrates. For instance, mTorC1
phosphorylates S6 kinase (S6K) and 4EBP1, promoting increased
translation and ribosome biogenesis to facilitate cell growth and
cell cycle progression. S6K also acts in a feedback pathway to
attenuate PI3K/Akt activation. Thus, inhibition of mTorC1 (e.g. by
a biologically active agent as discussed herein) results in
activation of 4EBP1, resulting in inhibition of (e.g. a decrease
in) RNA translation.
[0132] mTorC2 is generally insensitive to rapamycin and selective
inhibitors. mTorC2 is thought to modulate growth factor signaling
by phosphorylating the C-terminal hydrophobic motif of some AGC
kinases such as Akt. In many cellular contexts, mTorC2 is required
for phosphorylation of the S473 site of Akt. Thus, mTorC1 activity
is partly controlled by Akt whereas Akt itself is partly controlled
by mTorC2.
[0133] Growth factor stimulation of PI3K causes activation of Akt
by phosphorylation at the two key sites, S473 and T308. It has been
reported that full activation of Akt requires phosphorylation of
both S473 and T308Active. Akt promotes cell survival and
proliferation in many ways including suppressing apoptosis,
promoting glucose uptake, and modifying cellular metabolism. Of the
two phosphorylation sites on Akt, activation loop phosphorylation
at T308, mediated by PDK1, is believed to be indispensable for
kinase activity, while hydrophobic motif phosphorylation at S473
enhances Akt kinase activity.
[0134] Selective mTor inhibition may also be determined by
expression levels of the mTor genes, its downstream signaling genes
(for example by RT-PCR), or expression levels of the proteins (for
example by immunocytochemistry, immunohistochemistry, Western
blots) as compared to other PI3-Kinases or protein kinases.
[0135] Cell-based assays for establishing selective inhibition of
mTorC1 and/or mTorC2 can take a variety of formats. This generally
will depend on the biological activity and/or the signal
transduction readout that is under investigation. For example, the
ability of the agent to inhibit mTorC1 and/or mTorC2 to
phosphorylate the downstream substrate(s) can be determined by
various types of kinase assays known in the art. Representative
assays include but are not limited to immunoblotting and
immunoprecipitation with antibodies such as anti-phosphotyrosine,
anti-phosphoserine or anti-phosphothreonine antibodies that
recognize phosphorylated proteins. Alternatively, antibodies that
specifically recognize a particular phosphorylated form of a kinase
substrate (e.g. anti-phospho AKT S473 or anti-phospho AKT T308) can
be used. In addition, kinase activity can be detected by high
throughput chemiluminescent assays such as AlphaScreen.TM.
(available from Perkin Elmer) and eTag.TM. assay (Chan-Hui, et al.
(2003) Clinical Immunology 111: 162-174). In another aspect, single
cell assays such as flow cytometry as described in the phosflow
experiment can be used to measure phosphorylation of multiple
downstream mTOR substrates in mixed cell populations.
[0136] One advantage of the immunoblotting and phosflow methods is
that the phosphorylation of multiple kinase substrates can be
measured simultaneously. This provides the advantage that efficacy
and selectivity can be measured at the same time. For example,
cells may be contacted with an mTOR inhibitor at various
concentrations and the phosphorylation levels of substrates of both
mTOR and other kinases can be measured. In one aspect, a large
number of kinase substrates are assayed in what is termed a
"comprehensive kinase survey." Selective mTOR inhibitors are
expected to inhibit phosphorylation of mTOR substrates without
inhibiting phosphorylation of the substrates of other kinases.
Alternatively, selective mTOR inhibitors may inhibit
phosphorylation of substrates of other kinases through anticipated
or unanticipated mechanisms such as feedback loops or
redundancy.
[0137] Effect of inhibition of mTorC1 and/or mTorC2 can be
established by cell colony formation assay or other forms of cell
proliferation assay. A wide range of cell proliferation assays are
available in the art, and many of which are available as kits.
Non-limiting examples of cell proliferation assays include testing
for tritiated thymidine uptake assays, BrdU
(5'-bromo-2'-deoxyuridine) uptake (kit marketed by Calibochem), MTS
uptake (kit marketed by Promega), MTT uptake (kit marketed by
Cayman Chemical), CyQUANT.RTM. dye uptake (marketed by
Invitrogen).
[0138] Apoptosis and cell cycle arrest analysis can be performed
with any methods exemplified herein as well other methods known in
the art. Many different methods have been devised to detect
apoptosis. Exemplary assays include but are not limited to the
TUNEL (TdT-mediated dUTP Nick-End Labeling) analysis, ISEL (in situ
end labeling), and DNA laddering analysis for the detection of
fragmentation of DNA in populations of cells or in individual
cells, Annexin-V analysis that measures alterations in plasma
membranes, detection of apoptosis related proteins such p53 and
Fas.
[0139] A cell-based assay typically proceeds with exposing the
target cells (e.g., in a culture medium) to a candidate mTorC1
and/or mTorC2 selective inhibitor, and then assaying for readout
under investigation. Depending on the nature of the candidate mTor
inhibitors, they can directly be added to the cells or in
conjunction with carriers. For instance, when the agent is nucleic
acid, it can be added to the cell culture by methods well known in
the art, which include without limitation calcium phosphate
precipitation, microinjection or electroporation. Alternatively,
the nucleic acid can be incorporated into an expression or
insertion vector for incorporation into the cells. Vectors that
contain both a promoter and a cloning site into which a
polynucleotide can be operatively linked are well known in the art.
Such vectors are capable of transcribing RNA in vitro or in vitro,
and are commercially available from sources such as Stratagene (La
Jolla, Calif.) and Promega Biotech (Madison, Wis.). In order to
optimize expression and/or in vitro transcription, it may be
necessary to remove, add or alter 5' and/or 3' untranslated
portions of the clones to eliminate extra, potential inappropriate
alternative translation initiation codons or other sequences that
may interfere with or reduce expression, either at the level of
transcription or translation. Alternatively, consensus ribosome
binding sites can be inserted immediately 5' of the start codon to
enhance expression. Examples of vectors are viruses, such as
baculovirus and retrovirus, bacteriophage, adenovirus,
adeno-associated virus, cosmid, plasmid, fungal vectors and other
recombination vehicles typically used in the art which have been
described for expression in a variety of eukaryotic and prokaryotic
hosts, and may be used for gene therapy as well as for simple
protein expression. Among these are several non-viral vectors,
including DNA/liposome complexes, and targeted viral protein DNA
complexes. To enhance delivery to a cell, the nucleic acid or
proteins of this invention can be conjugated to antibodies or
binding fragments thereof which bind cell surface antigens.
Liposomes that also comprise a targeting antibody or fragment
thereof can be used in the methods of this invention. Other
biologically acceptable carriers can be utilized, including those
described in, for example, REMINGTON'S PHARMACEUTICAL SCIENCES,
19th Ed. (2000), in conjunction with the subject compounds.
[0140] The subject agents can also be utilized to inhibit
phosphorylation of both Akt (S473) and Akt (T308) in a cell.
Accordingly, the present invention provides a method comprises the
step of contacting a cell with an effective amount of such
biologically active agent such that Akt phosphorylation at residues
S473 and T308 is simultaneously inhibited. In one aspect, the
biologically active agent inhibits phosphorylation of S473 of Akt
more effectively than phosphorylation of T308 of Akt when tested at
a comparable molar concentration, preferably at an identical molar
concentration.
[0141] Inhibition of Akt phosphorylation can be determined using
any methods known in the art or described herein. Representative
assays include but are not limited to immunoblotting and
immunoprecipitation with antibodies such as anti-phosphotyrosine
antibodies that recognize the specific phosphorylated proteins.
Cell-based ELISA kit quantifies the amount of activated
(phosphorylated at S473) Akt relative to total Akt protein is also
available (SuperArray Biosciences).
[0142] In practicing the subject methods, any cells that express
mTorC1, mTorC2 and/or Akt can be utilized. Non-limiting examples of
specific cell types whose proliferation can be inhibited include
fibroblast, cells of skeletal tissue (bone and cartilage), cells of
epithelial tissues (e.g. liver, lung, breast, skin, bladder and
kidney), cardiac and smooth muscle cells, neural cells (glia and
neurones), endocrine cells (adrenal, pituitary, pancreatic islet
cells), melanocytes, and many different types of haemopoietic cells
(e.g., cells of B-cell or T-cell lineage, and their corresponding
stem cells, lymphoblasts). Also of interest are cells exhibiting a
neoplastic propensity or phenotype. Of particular interest is the
type of cells that differentially expresses (over-expresses or
under-expresses) a disease-causing gene. The types of diseases
involving abnormal functioning of genes include but are not limited
to autoimmune diseases, cancer, obesity, hypertension, diabetes,
neuronal and/or muscular degenerative diseases, cardiac diseases,
endocrine disorders, and any combinations thereof.
[0143] A. Active Agents
[0144] The subject methods can employ any biologically active
agents that exhibit selective inhibitory activities towards mTorC1
and/or mTorC2 described herein. One class of biologically active
agent for use in the subject methods encompasses compounds, e.g.,
pyrazolopyrimidine derivatives, including but not limited to
compounds having a structure of the following Formula (I) and
Formula (II).
##STR00001##
[0145] In some embodiments of the compounds of Formulae (I) or
(II), R.sup.1, R.sup.3, and R.sup.4 are independently hydrogen,
halogen, --CN, --CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl. In some
embodiments, at least one of R.sup.3 or R.sup.4 is hydrogen. In
some embodiments, R.sup.1, R.sup.3, and R.sup.4 are independently
hydrogen or substituted or unsubstituted C.sub.1-C.sub.10 alkyl
(e.g. C.sub.1-C.sub.5 alkyl or C.sub.1-C.sub.3 alkyl). R', R.sup.3,
and R.sup.4 may also independently be hydrogen or unsubstituted
C.sub.1-C.sub.10 alkyl (e.g. C.sub.1-C.sub.5 alkyl or
C.sub.1-C.sub.3 alkyl).
[0146] In some embodiments of Formulae (I) or (II), R.sup.1,
R.sup.3, and R.sup.4 are independently hydrogen, halogen, --CN,
--CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
R.sup.7-substituted or unsubstituted alkyl, R.sup.7-substituted or
unsubstituted heteroalkyl, R.sup.7-substituted or unsubstituted
cycloalkyl, R.sup.7-substituted or unsubstituted heterocycloalkyl,
R.sup.7-substituted or unsubstituted aryl, or R.sup.7-substituted
or unsubstituted heteroaryl. R.sup.7 is independently oxo, halogen,
--CN, --CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
R.sup.8-substituted or unsubstituted alkyl, R.sup.8-substituted or
unsubstituted heteroalkyl, R.sup.8-substituted or unsubstituted
cycloalkyl, R.sup.8-substituted or unsubstituted heterocycloalkyl,
R.sup.8-substituted or unsubstituted aryl, or R.sup.8-substituted
or unsubstituted heteroaryl. R.sup.8 is independently halogen, oxo,
--CN, --CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted
cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or
unsubstituted heteroaryl. In some embodiments, R.sup.1 is
substituted or unsubstituted alkyl or substituted or unsubstituted
heterocycloalkyl (e.g. morpholino). In some embodiments, R.sup.1 is
substituted with --C(O)R.sup.8A, wherein R.sup.8A is unsubstituted
alkyl.
[0147] In some embodiments of Formula (I), R.sup.2 is independently
hydrogen, halogen, --CN, --CF.sub.3, --OR.sup.5, --NH.sub.2,
--SO.sub.2, --COOH, substituted or unsubstituted alkyl, substituted
or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted
heteroaryl. In some embodiments, R.sup.2 is independently hydrogen,
--OR.sup.5, --CN, --NH.sub.2, --SH, --CN, --CF.sub.3, NO.sub.2, or
substituted or unsubstituted alkyl. R.sup.5 is independently
hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl. In
some embodiments, R.sup.5 is hydrogen or substituted or
unsubstituted alkyl (e.g. unsubstituted C.sub.1-C.sub.5 alkyl).
[0148] R.sup.2 may independently be hydrogen, halogen, --OR.sup.5,
--CN, --NH.sub.2, --SH, --CN, --CF.sub.3, --NO.sub.2,
R.sup.9-substituted or unsubstituted alkyl, R.sup.9-substituted or
unsubstituted heteroalkyl, R.sup.9-substituted or unsubstituted
cycloalkyl, R.sup.9-substituted or unsubstituted heterocycloalkyl,
R.sup.9-substituted or unsubstituted aryl, or R.sup.9-substituted
or unsubstituted heteroaryl. R.sup.9 is independently halogen, oxo,
--CN, --CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
R.sup.10-substituted or unsubstituted alkyl, R.sup.10-substituted
or unsubstituted heteroalkyl, R.sup.10-substituted or unsubstituted
cycloalkyl, R.sup.10-substituted or unsubstituted heterocycloalkyl,
R.sup.10-substituted or unsubstituted aryl, or R.sup.10-substituted
or unsubstituted heteroaryl. R.sup.10 is independently halogen,
oxo, --CN, --CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted
cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or
unsubstituted heteroaryl. In other embodiments, R.sup.2 is
--OR.sup.5. In some related embodiments, R.sup.5 is hydrogen or
unsubstituted C.sub.1-C.sub.5 alkyl (e.g. hydrogen).
[0149] In some embodiments of Formulae (I) or (II), R.sup.5 is
independently hydrogen, R.sup.11-substituted or unsubstituted
alkyl, R.sup.11-substituted or unsubstituted heteroalkyl,
R.sup.11-substituted or unsubstituted cycloalkyl,
R.sup.11-substituted or unsubstituted heterocycloalkyl,
R.sup.11-substituted or unsubstituted aryl, or R.sup.11-substituted
or unsubstituted heteroaryl. R.sup.11 is independently halogen,
oxo, --CN, --CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
R.sup.12-substituted or unsubstituted alkyl, R.sup.12-substituted
or unsubstituted heteroalkyl, R.sup.12-substituted or unsubstituted
cycloalkyl, R.sup.12-substituted or unsubstituted heterocycloalkyl,
R.sup.12-substituted or unsubstituted aryl, or R.sup.12-substituted
or unsubstituted heteroaryl. R.sup.12 is independently halogen,
oxo, --CN, --CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted
cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or
unsubstituted heteroaryl.
[0150] In some embodiments of Formula (II), R.sup.6 is
independently hydrogen, halogen, --CN, --CF.sub.3, --OR.sup.5,
--NH.sub.2, --SO.sub.2, --COOH, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl. R.sup.6 may also independently be
hydrogen, --OR.sup.5, --CN, halogen, --NH.sub.2, --SH, --CN,
--CF.sub.3, --NO.sub.2, halogen, or substituted or unsubstituted
alkyl (e.g. unsubstituted C.sub.1-C.sub.5 alkyl). R.sup.5 is as
defined above in the description of Formula (I). In some
embodiments, R.sup.6 is independently hydrogen, --OR.sup.5, --CN,
--NH.sub.2, --SH, --CN, --CF.sub.3, --NO.sub.2,
R.sup.13-substituted or unsubstituted alkyl, R.sup.13-substituted
or unsubstituted heteroalkyl, R.sup.13-substituted or unsubstituted
cycloalkyl, R.sup.13-substituted or unsubstituted heterocycloalkyl,
R.sup.13-substituted or unsubstituted aryl, or R.sup.13-substituted
or unsubstituted heteroaryl. R.sup.13 is independently halogen,
oxo, --CN, --CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
R.sup.14-substituted or unsubstituted alkyl, R.sup.13-substituted
or unsubstituted heteroalkyl, R.sup.14-substituted or unsubstituted
cycloalkyl, R.sup.14-substituted or unsubstituted heterocycloalkyl,
R.sup.14-substituted or unsubstituted aryl, or R.sup.14-substituted
or unsubstituted heteroaryl. R.sup.14 is independently halogen,
oxo, --CN, --CF.sub.3, --OH, --NH.sub.2, --SO.sub.2, --COOH,
unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted
cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or
unsubstituted heteroaryl. R.sup.6 may also independently be
hydrogen, --OR.sup.5, --CN, halogen, --NH.sub.2, --SH, --CN,
--CF.sub.3, --NO.sub.2, halogen, or unsubstituted C.sub.1-C.sub.5
alkyl. In some embodiments, R.sup.6 is hydrogen.
[0151] In some embodiments of Formula (I) or (II), R.sup.3 and
R.sup.4 are hydrogen. In some embodiments of Formula (I), n is 1 or
2. In some related embodiments of Formula (I), n is 1. In other
related embodiments, R.sup.2 is --OR.sup.5 and n is 1. In still
other related embodiments, R.sup.5 is hydrogen. In some embodiments
of Formula (II), z is an integer from 1 to 2. In some embodiments,
z is 1.
[0152] In some embodiments, R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11,
R.sup.12, R.sup.13 and/or R.sup.14 are size-limited substituents.
In some embodiments, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
R.sup.13 and/or R.sup.14 are C.sub.1-C.sub.10, C.sub.1-C.sub.5
alkyl or C.sub.1-C.sub.3 alkyl, for example methyl, ethyl, propyl,
isopropyl, butyl and the like, optionally substituted as described
herein. In some embodiments, R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11,
R.sup.12, R.sup.13 and/or R.sup.14 are 2-10 membered, 2-5 membered,
or 2-3-membered heteroalkyl, optionally substituted as described
herein. In some embodiments, R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11,
R.sup.12, R.sup.13 and/or R.sup.14 are C.sub.3-C.sub.10,
C.sub.3-C.sub.8, C.sub.3-C.sub.6 or C.sub.3-C.sub.5 cycloalkyl,
including but not limited to cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl and the like, optionally
substituted as described herein. In some embodiments, R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13 and/or R.sup.14 are
3-membered, 4-membered, 5-membered, 6-membered, 7-membered,
8-membered, 9-membered or 10-membered heterocycloalkyl, including
but not limited to aziridine, oxirane, thiirane, azetidine,
oxetane, thietane, pyrrolidine, dihydrofuran, tetrahydropyran,
dihydrothiophene, tetrahydrothiophene, piperidine, dihydropyran,
tetrahydropyran, dihydrothiopyran, tetrahydrothiopyran, optionally
substituted as described herein. In some embodiments, R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13 and/or R.sup.14 are
C.sub.6-C.sub.10 aryl, including but not limited to phenyl or
naphthyl, optionally substituted as described herein. In some
embodiments, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13
and/or R.sup.14 are 5-10-membered, 5-6-membered heteroaryl as
described herein, optionally substituted as described herein.
[0153] In other embodiments, the biologically active agent (e.g.
the compounds of Formula (I) or (II)) is selected from the
following compounds:
##STR00002##
[0154] The table below summarizes the IC.sub.50 values of these
compounds when tested against mTor complexes and type I
PI3-kinases. The ability of these compounds to inhibit
proliferation of PC3 cells is also indicated.
TABLE-US-00001 mTor PI3K .alpha. PI3K .beta. PI3K .gamma. PI3K
.delta. PC3 Compound IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50
IC.sub.50 proliferation Structure (nM) (nM) (nM) (nM) (nM) (nM)
##STR00003## 6.7 22500 18400 400 120 100 ##STR00004## 79 11189 2062
##STR00005## 0.7 3465 3580 450 330 38 ##STR00006## 26 279 2160 115
395 1000 ##STR00007## 80 3000 5800 680 990 ##STR00008## 14 5750
25000 -- 9800 11800
[0155] Additional anti-cell proliferation agents can be screened
based on the ability of selective inhibition of mTorC1 and/or
mTorC2. Accordingly, the present invention provides, in one
embodiment, a method of developing a biologically active agent that
inhibits cell proliferation. The method comprises: (a) contacting a
candidate agent with a population of cells of interest; (b)
detecting selective inhibition of mTorC1 and/or mTorC2 activity
relative to one or more type I phosphatidylinositol 3-kinases
(PI3-kinase), wherein the one or more type I PI3-kinase is selected
from the group consisting of PI3-kinase .alpha., PI3-kinase .beta.,
PI3-kinase .gamma., and PI3-kinase .delta..
[0156] For the purposes of this invention, a candidate agent
effective to selectively inhibit mTorC1 and/or mTorC2 activity is
intended to include, but not be limited to a biological or chemical
compound such as a simple or complex organic or inorganic
compounds, peptide, peptide mimetic, protein (e.g. antibody),
liposome, small interfering RNA, or a polynucleotide (e.g.
anti-sense). A class of preferred agents include those that block
the downstream signaling effect of mTorC1 and/or mTorC2. Any of the
methods and assays disclosed herein that evidence modulation of
mTorC1 and/or mTorC2 activity can be employed in developing such
agent.
[0157] In some embodiments, the biologically active agent (e.g.
compound) is capable of selectively inhibiting mTorC2 activity (or
mTorC2 mediated effects) relative to mTorC1 activity (or mTorC2
mediated effects). The biologically active agent may be capable of
decreasing phosphorylation of Akt at residues, including but not
limited to, S473 and T308 relative to the amount of phosphorylation
in the absence of the biologically active agent. In other
embodiments, the biologically active agent is capable of decreasing
phosphorylation of Akt substrates including but not limited to
FoxO1/O3a T24/32, GSK3.alpha./.beta. S21/9, and TSC2 T1462 relative
to the amount of phosphorylation in the absence of the biologically
active agent.
[0158] In some embodiments, the biologically active agent (e.g.
compound) is capable of selectively inhibiting mTorC1 activity (or
mTorC2 mediated effects) relative to mTorC2 activity (or mTorC2
mediated effects). In some embodiments, the biologically active
agent is capable of decreasing phosphorylation of signaling
molecules downstream of mTor, including but not limited to
ribosomal S6 S240/244, 70S6K T389, and/or 4EBP1 (e.g. 4EBP1 T37/46)
relative to the amount of phosphorylation in the absence of the
biologically active agent. The biologically active agent may also
be capable of increasing binding of 4EPB1 to eIF4E relative to the
amount of binding in the absence of the biologically active agent.
This increased binding results in a decrease in RNA translation.
Therefore, in some embodiments, where the biologically active agent
(e.g. compound) is capable of selectively inhibiting mTorC1
activity (or mTorC2 mediated effects) relative to mTorC2 activity
(or mTorC2 mediated effects), the biologically active agent is
useful in treating cancer (e.g. solid tumors, lymphomas and
leukemia).
[0159] In other embodiments, the biologically active agent may
increase inhibition of proliferation of cells including but not
limited to normal or neoplastic cells, mouse embryonic fibroblasts,
leukemic blast cells, cancer stem cells, and cells that mediate
autoimmune reactions and/or increase induction of apoptosis of
cells or cell cycle arrest relative to the amount of increase in
the absence of the biologically active agent.
[0160] A vast array of compounds can be synthesized, for example
polymers, such as polypeptides and polynucleotides, and synthetic
inorganic and organic compounds based on various core structures,
all of which are also contemplated herein. In addition, various
natural sources can provide compounds for screening, such as plant
or animal extracts, and the like. It should be understood, although
not always explicitly stated that the active agent can be used
alone or in combination with another modulator, having the same or
different biological activity as the agents identified by the
subject screening method. A subject agent can assert its selective
inhibitory effect by directly binding to or directly interacting
with the target. An agent can also assert its inhibitory effect
indirectly by first interacting with a molecule in the same
signaling pathway. The mTor selective inhibitors of the present
invention encompasses simple or complex organic or inorganic
molecule, peptide, peptide mimetic, protein (e.g. antibody),
liposome, small interfering RNA, or a polynucleotide (e.g.
anti-sense) that can reduce the deleterious effect of mTor in
vitro.
[0161] The compounds described above, including the compounds of
Formula (I) or (II), or pharmaceutically acceptable salts thereof,
may be used in the methods of the present invention.
[0162] B. Methods
[0163] The invention also relates to a method of ameliorating a
medical condition mediated by mTorC1 and/or mTorC2. In one aspect,
the present invention encompasses a method of treating a
hyperproliferative disorder in a mammal that comprises
administering to said mammal a therapeutically effective amount of
a biologically active agent of the present invention, or a
pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate
or derivative thereof. In some embodiments, said method relates to
the treatment of cancer such as acute myeloid leukemia, thymus,
brain, lung, squamous cell, skin, eye, retinoblastoma, intraocular
melanoma, oral cavity and oropharyngeal, bladder, gastric, stomach,
pancreatic, bladder, breast, cervical, head, neck, renal, kidney,
liver, ovarian, prostate, colorectal, esophageal, testicular,
gynecological, thyroid, CNS, PNS, AIDS related AIDS-related (e.g.
Lymphoma and Kaposi's Sarcoma) or viral-induced cancer. In some
embodiments, said method relates to the treatment of a
non-cancerous hyperproliferative disorder such as benign
hyperplasia of the skin (e.g., psoriasis), restenosis, or prostate
(e.g., benign prostatic hypertrophy (BPH)).
[0164] In some embodiments, the medical condition mediated by
mTorC1 and/or mTorC2 is polycystic kidney disease (PKD), such as
autosomal dominant PKD. In other embodiments, the medical condition
mediated by mTorC1 and/or mTorC2 is organ rejection derived from an
organ transplantation procedure, such as kidney transplantation.
Thus, in some embodiments methods are provided for prophylaxis of
organ rejection an organ transplant recipient patient (e.g. kidney
transplant). The method includes administering to the organ
transplant recipient patient a prophylactic amount of a
biologically active agent that selectively inhibits mTorC1 and/or
mTorC2 activity relative to one or more type I phosphatidylinositol
3-kinases (PI3-kinase), wherein the one or more type I PI3-kinase
is selected from the group consisting of PI3-kinase .alpha.,
PI3-kinase .beta., PI3-kinase .gamma., and PI3-kinase .delta.. In
some related embodiments, the biologically active agent is a
compound of Formula (I) or (II).
[0165] The invention also relates to a method for the treatment of
a hyperproliferative disorder in a mammal that comprises
administering to said mammal a therapeutically effective amount of
a biologically active agent of the present invention, or a
pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate
or derivative thereof, in combination with an anti-tumor agent. In
some embodiments, the anti-tumor agent is selected from the group
consisting of mitotic inhibitors, alkylating agents,
anti-metabolites, intercalating antibiotics, growth factor
inhibitors, cell cycle inhibitors, enzyme inhibitors, topoisomerase
inhibitors, biological response modifiers, anti-hormones,
angiogenesis inhibitors, and anti-androgens.
[0166] The invention also relates to a method of treating diseases
related to vasculogenesis or angiogenesis in a mammal that
comprises administering to said mammal a therapeutically effective
amount of a biologically active agent of the present invention, or
a pharmaceutically acceptable salt, ester, prodrug, solvate,
hydrate or derivative thereof. In some embodiments, said method is
for treating a disease selected from the group consisting of tumor
angiogenesis, chronic inflammatory disease such as rheumatoid
arthritis, atherosclerosis, inflammatory bowel disease, skin
diseases such as psoriasis, eczema, and scleroderma, diabetes,
diabetic retinopathy, retinopathy of prematurity, age-related
macular degeneration, hemangioma, glioma, melanoma, Kaposi's
sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and
epidermoid cancer.
[0167] Patients that can be treated with biologically active agents
of the present invention, or pharmaceutically acceptable salt,
ester, prodrug, solvate, hydrate or derivative of said biologically
active agents, according to the methods of this invention include,
for example, patients that have been diagnosed as having psoriasis;
restenosis; atherosclerosis; BPH; breast cancer such as a ductal
carcinoma in duct tissue in a mammary gland, medullary carcinomas,
colloid carcinomas, tubular carcinomas, and inflammatory breast
cancer; ovarian cancer, including epithelial ovarian tumors such as
adenocarcinoma in the ovary and an adenocarcinoma that has migrated
from the ovary into the abdominal cavity; uterine cancer; cervical
cancer such as adenocarcinoma in the cervix epithelial including
squamous cell carcinoma and adenocarcinomas; prostate cancer, such
as a prostate cancer selected from the following: an adenocarcinoma
or an adenocarcinoma that has migrated to the bone; pancreatic
cancer such as epithelioid carcinoma in the pancreatic duct tissue
and an adenocarcinoma in a pancreatic duct; bladder cancer such as
a transitional cell carcinoma in urinary bladder, urothelial
carcinomas (transitional cell carcinomas), tumors in the urothelial
cells that line the bladder, squamous cell carcinomas,
adenocarcinomas, and small cell cancers; leukemia such as acute
myeloid leukemia (AML), acute lymphocytic leukemia, chronic
lymphocytic leukemia, chronic myeloid leukemia, hairy cell
leukemia, myelodysplasia, myeloproliferative disorders, acute
myelogenous leukemia (AML), chronic myelogenous leukemia (CML),
mastocytosis, chronic lymphocytic leukemia (CLL), multiple myeloma
(MM), and myelodysplastic syndrome (MDS); bone cancer; lung cancer
such as non-small cell lung cancer (NSCLC), which is divided into
squamous cell carcinomas, adenocarcinomas, and large cell
undifferentiated carcinomas, and small cell lung cancer; skin
cancer such as basal cell carcinoma, melanoma, squamous cell
carcinoma and actinic keratosis, which is a skin condition that
sometimes develops into squamous cell carcinoma; eye
retinoblastoma; cutaneous or intraocular (eye) melanoma; primary
liver cancer (cancer that begins in the liver); kidney cancer;
thyroid cancer such as papillary, follicular, medullary and
anaplastic; AIDS-related lymphoma such as diffuse large B-cell
lymphoma, B-cell immunoblastic lymphoma and small non-cleaved cell
lymphoma; Kaposi's Sarcoma; viral-induced cancers including
hepatitis B virus (HBV), hepatitis C virus (HCV), and
hepatocellular carcinoma; human lymphotropic virus-type 1 (HTLV-1)
and adult T-cell leukemia/lymphoma; and human papilloma virus (HPV)
and cervical cancer; central nervous system cancers (CNS) such as
primary brain tumor, which includes gliomas (astrocytoma,
anaplastic astrocytoma, or glioblastoma multiforme),
Oligodendroglioma, Ependymoma, Meningioma, Lymphoma, Schwannoma,
and Medulloblastoma; peripheral nervous system (PNS) cancers such
as acoustic neuromas and malignant peripheral nerve sheath tumor
(MPNST) including neurofibromas and schwannomas, malignant fibrous
cytoma, malignant fibrous histiocytoma, malignant meningioma,
malignant mesothelioma, and malignant mixed Mullerian tumor; oral
cavity and oropharyngeal cancer such as, hypopharyngeal cancer,
laryngeal cancer, nasopharyngeal cancer, and oropharyngeal cancer;
stomach cancer such as lymphomas, gastric stromal tumors, and
carcinoid tumors; testicular cancer such as germ cell tumors
(GCTs), which include seminomas and nonseminomas, and gonadal
stromal tumors, which include Leydig cell tumors and Sertoli cell
tumors; thymus cancer such as to thymomas, thymic carcinomas,
Hodgkin disease, non-Hodgkin lymphomas carcinoids or carcinoid
tumors; rectal cancer; and colon cancer.
[0168] The invention also relates to a method of treating diabetes
in a mammal that comprises administering to said mammal a
therapeutically effective amount of a biologically active agent of
the present invention, or a pharmaceutically acceptable salt,
ester, prodrug, solvate, hydrate or derivative thereof.
[0169] The invention also relates to a method of treating an
inflammation disorder, including autoimmune diseases, in a mammal
that comprises administering to said mammal a therapeutically
effective amount of a biologically active agent of the present
invention, or a pharmaceutically acceptable salt, ester, prodrug,
solvate, hydrate or derivative thereof. Examples of autoimmune
diseases includes but is not limited to acute disseminated
encephalomyelitis (ADEM), Addison's disease, antiphospholipid
antibody syndrome (APS), aplastic anemia, autoimmune hepatitis,
coeliac disease, Crohn's disease, Diabetes mellitus (type 1),
Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome
(GBS), Hashimoto's disease, lupus erythematosus, multiple
sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome (OMS),
optic neuritis, Ord's thyroiditis, oemphigus, polyarthritis,
primary biliary cirrhosis, psoriasis, rheumatoid arthritis,
Reiter's syndrome, Takayasu's arteritis, temporal arteritis (also
known as "giant cell arteritis"), warm autoimmune hemolytic anemia,
Wegener's granulomatosis, alopecia universalis, Chagas' disease,
chronic fatigue syndrome, dysautonomia, endometriosis, hidradenitis
suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis,
scleroderma, ulcerative colitis, vitiligo, and vulvodynia. Other
disorders include bone-resorption disorders and thromobsis.
[0170] For instance, the biologically active agents described
herein can be used to treat encephalomyelitis. In other embodiments
the biologically active agents described herein are used for the
treatment of obstructive pulmonary disease. Chronic obstructive
pulmonary disease (COPD) is an umbrella term for a group of
respiratory tract diseases that are characterized by airflow
obstruction or limitation. Conditions included in this umbrella
term are: chronic bronchitis, emphysema, and bronchiectasis.
[0171] In another embodiment, the biologically active agents
described herein are used for the treatment of asthma. Also, the
biologically active agents described herein may be used for the
treatment of endotoxemia and sepsis. In one embodiment, the
biologically active agents described herein are used to for the
treatment of rheumatoid arthritis (RA). In yet another embodiment,
the biologically active agents described herein is used for the
treatment of contact or atopic dermatitis. Contact dermatitis
includes irritant dermatitis, phototoxic dermatitis, allergic
dermatitis, photoallergic dermatitis, contact urticaria, systemic
contact-type dermatitis and the like. Irritant dermatitis can occur
when too much of a substance is used on the skin of when the skin
is sensitive to certain substance. Atopic dermatitis, sometimes
called eczema, is a kind of dermatitis, an atopic skin disease.
[0172] In addition, the biologically active agents described herein
may be used to treat acne.
[0173] In addition, the biologically active agents described herein
may be used for the treatment of arteriosclerosis, including
atherosclerosis. Arteriosclerosis is a general term describing any
hardening of medium or large arteries. Atherosclerosis is a
hardening of an artery specifically due to an atheromatous
plaque.
[0174] Further the biologically active agents described herein may
be used for the treatment of glomerulonephritis. Glomerulonephritis
is a primary or secondary autoimmune renal disease characterized by
inflammation of the glomeruli. It may be asymptomatic, or present
with hematuria and/or proteinuria. There are many recognized types,
divided in acute, subacute or chronic glomerulonephritis. Causes
are infectious (bacterial, viral or parasitic pathogens),
autoimmune or paraneoplastic.
[0175] Additionally, the biologically active agents described
herein may be used for the treatment of bursitis, lupus, acute
disseminated encephalomyelitis (ADEM), Addison's disease,
antiphospholipid antibody syndrome (APS), aplastic anemia,
autoimmune hepatitis, coeliac disease, Crohn's disease, diabetes
mellitus (type 1), Goodpasture's syndrome, graves' disease,
Guillain-Barre syndrome (GBS), Hashimoto's disease, inflammatory
bowel disease, lupus erythematosus, myasthenia gravis, opsoclonus
myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis,
ostheoarthritis, uveoretinitis, pemphigus, polyarthritis, primary
biliary cirrhosis, Reiter's syndrome, Takayasu's arteritis,
temporal arteritis, warm autoimmune hemolytic anemia, Wegener's
granulomatosis, alopecia universalis, Chagas' disease, chronic
fatigue syndrome, dysautonomia, endometriosis, hidradenitis
suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis,
scleroderma, ulcerative colitis, vitiligo, vulvodynia,
appendicitis, arteritis, arthritis, blepharitis, bronchiolitis,
bronchitis, cervicitis, cholangitis, cholecystitis,
chorioamnionitis, colitis, conjunctivitis, cystitis,
dacryoadenitis, dermatomyositis, endocarditis, endometritis,
enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis,
fibrositis, gastritis, gastroenteritis, gingivitis, hepatitis,
hidradenitis, ileitis, iritis, laryngitis, mastitis, meningitis,
myelitis, myocarditis, myositis, nephritis, omphalitis, oophoritis,
orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis,
peritonitis, pharyngitis, pleuritis, phlebitis, pneumonitis,
proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis,
sinusitis, stomatitis, synovitis, tendonitis, tonsillitis, uveitis,
vaginitis, vasculitis, or vulvitis.
[0176] The invention also relates to a method of treating a
cardiovascular disease in a mammal that comprises administering to
said mammal a therapeutically effective amount of a biologically
active agent of the present invention, or a pharmaceutically
acceptable salt, ester, prodrug, solvate, hydrate or derivative
thereof. Examples of cardiovascular conditions include, but are not
limited to, atherosclerosis, restenosis, vascular occlusion and
carotid obstructive disease.
[0177] In another aspect, the present invention provides methods of
disrupting the function of a leukocyte or disrupting a function of
an osteoclast. The method includes contacting the leukocyte or the
osteoclast with a function disrupting amount of a biologically
active agent of the invention.
[0178] In some embodiments, a method of treating a condition caused
by aberrant ion transport across epithelial cells in a patient in
need thereof is provided. The method includes administering to the
patient a therapeutically effective amount of a biologically active
agent that selectively inhibits mTorC1 and/or mTorC2 activity
relative to one or more type I phosphatidylinositol 3-kinases
(PI3-kinase) ascertained by an in vitro kinase assay. The one or
more type I PI3-kinase is selected from the group consisting of
PI3-kinase .alpha., PI3-kinase .beta., PI3-kinase .gamma., and
PI3-kinase .delta..
[0179] A condition caused by aberrant ion (e.g. sodium ion, proton,
lithium ion, potassium ion) transport across epithelial cells is a
condition that would not occur but for the presence of aberrant ion
transport across at least some epithelial cells in the patient. The
epithelial cells typically form at least part of glands, connective
tissue (e.g. the outer layer of connective tissues) and/or tissues
lining the cavities of surfaces of structures (e.g. organs)
throughout the body. In some embodiments, the epithelial cells are
renal, lung, or colon epithelial cells.
[0180] The epithelial cells may include an epithelial sodium
channel (ENaC) (also commonly referred to as sodium channel
non-neuronal 1 (SCNN1) or amiloride sensitive sodium channel
(ASSC)), a membrane-bound ion-channel that is permeable to
Li.sup.+-ions, protons and Na.sup.+-ions. Thus, in some
embodiments, the condition caused by aberrant ion transport across
epithelial cells is a condition caused by aberrant ion transport
across ENaC channels, including for example, cystic fibrosis and
Liddle's syndrome.
[0181] In other embodiments, the condition caused by aberrant ion
transport across epithelial cells is a condition caused by aberrant
ion transport across kidney epithelial cells, such as kidney
collecting duct cells.
[0182] The condition caused by aberrant ion transport across
epithelial cells may also be a disease caused by aberrant sodium
ion transport across epithelial cells, such as ENaC-dependent Na+
transport in renal epithelial cells. The collecting duct is the
major site for cyst generation in the autosomal dominant and
autosomal recessive forms of human polycystic kidney disease (PKD).
Cysts may form due to abnormal cellular proliferation, and abnormal
ion and fluid transport, which fills the cysts. Therefore, in some
embodiments, the condition caused by aberrant ion transport across
epithelial cells is PKD, a disease of collecting duct cell
proliferation kidney (e.g. cyst formation), a blood pressure
disease, a kidney electrolyte disorders, hypertension, congestive
heart failure, nephrotic syndrome and/or cirrhosis of the
liver.
[0183] In some embodiments, the biologically active agent useful in
methods of treating a condition caused by aberrant ion transport
across epithelial cells is an active agent capable of selectively
inhibiting mTorC2 activity (or mTorC2 mediated effects) relative to
mTorC1 activity (or mTorC2 mediated effects). In other embodiments,
the biologically active agent is capable of inhibiting cyst
progression in animal models of PKD to a greater degree than
rapamycin. In other embodiments, the biologically active agent
inhibits (e.g. decreases) ion transport processes in kidney tubule
cells relative to the amount of ion transport in the absence of the
biologically active agent. In other embodiments, the biologically
active agent is excreted in the kidney. In other embodiments, the
biologically active agent inhibits (e.g. decreases) phosphorylation
and/or activation of SGK1, a key mediator of hormone-regulated Na+
transport, relative to the amount of phosphorylation and/or
activation of SGK1 in the absence of the biologically active agent.
In other embodiments, the biologically active agent is a compound
of Formula (I) or (II) as described above. In some embodiments, the
biologically active agent is PP242.
[0184] In another embodiment, a method of treating T cell lymphoma,
or a cancer arising out of thymocyte proliferation such as thymic
lymphoma, in a patient in need thereof is provided. The method
includes administering to the patient a therapeutically effective
amount of a biologically active agent that selectively inhibits
mTorC1 and/or mTorC2 activity relative to one or more type I
phosphatidylinositol 3-kinases (PI3-kinase) ascertained by an in
vitro kinase assay, wherein the one or more type I PI3-kinase is
selected from the group consisting of PI3-kinase .alpha.,
PI3-kinase .beta., PI3-kinase .gamma., and PI3-kinase .delta.. In
some embodiments, the biologically active agent is a compound of
Formula (I) or (II) as described above. In some embodiments, the
biologically active agent is PP242.
[0185] In another embodiment, a method of treating a tumor arising
from oncogenic Akt-mTOR signaling in a patient in need thereof is
provided. The method includes administering to the patient a
therapeutically effective amount of a biologically active agent
that selectively inhibits mTorC1 and/or mTorC2 activity relative to
one or more type I phosphatidylinositol 3-kinases (PI3-kinase)
ascertained by an in vitro kinase assay, wherein the one or more
type I PI3-kinase is selected from the group consisting of
PI3-kinase .alpha., PI3-kinase .beta., PI3-kinase .gamma., and
PI3-kinase .delta.. In some embodiments, the biologically active
agent is a compound of Formula (I) or (II) as described above. In
some embodiments, the biologically active agent is PP242.
[0186] C. Combination Treatments
[0187] The present invention also provides methods for combination
therapies in which an agent known to modulate other pathways, or
other components of the same pathway, or even overlapping sets of
target enzymes are used in combination with a biologically active
agent of the present invention, or a pharmaceutically acceptable
salt, ester, prodrug, solvate, hydrate or derivative thereof.
[0188] In one embodiment, the present invention provides a method
of inhibiting proliferation of a neoplastic cell comprising
contacting the cell with an effective amount of an antagonist that
inhibits full activation of Akt in a cell and an anti-cancer agent,
wherein said inhibition of cell proliferation is enhanced through a
synergistic effect of said antagonist and said anti-cancer
agent.
[0189] In another embodiment, provided is a combination treatment
for a subject diagnosed with or at risk of a neoplastic condition.
The combination treatment involves administering to said subject a
therapeutically effective amount of an antagonist that inhibits
full activation of Akt in a cell and an anti-cancer agent, wherein
the efficacy of said treatment is enhanced through a synergistic
effect of said antagonist and said anti-cancer agent.
[0190] In one aspect, such therapy includes but is not limited to
the combination of the subject biologically active agent with
chemotherapeutic agents, therapeutic antibodies, and radiation
treatment, to provide a synergistic therapeutic effect.
[0191] Specifically, in one aspect, this invention also relates to
a pharmaceutical composition for inhibiting abnormal cell growth in
a mammal which comprises an amount of a biologically active agent
of the present invention, or a pharmaceutically acceptable salt,
ester, prodrug, solvate, hydrate or derivative thereof, in
combination with an amount of an anti-cancer agent (e.g. a
chemotherapeutic agent), wherein the amounts of the biologically
active agent, salt, ester, prodrug, solvate, hydrate or derivative,
and of the chemotherapeutic are together effective in inhibiting
abnormal cell growth. Many chemotherapeutics are presently known in
the art and can be used in combination with the biologically active
agents of the invention.
[0192] In some embodiments, the chemotherapeutic is selected from
the group consisting of mitotic inhibitors, alkylating agents,
anti-metabolites, intercalating antibiotics, growth factor
inhibitors, cell cycle inhibitors, enzymes, topoisomerase
inhibitors, biological response modifiers, anti-hormones,
angiogenesis inhibitors, and anti-androgens.
[0193] A wide variety of anti-cancer agents can be employed in
combination. Of particular interest is the combination of the
subject mTor selective inhibitors with PI3kinase inhibitors, other
non-Tor protein kinase inhibitors, and dual kinase inhibitors
(e.g., those that inhibit both protein kinase and lipid kinases).
Non limiting examples of other kinase inhibitors that can be
combined with the subject agent include rapamycin, TORKinhibs,
PI-103, BEZ235, Akt i, IC87114, and PIK-90.
[0194] Additional examples of therapeutic agents for a combined
treatment are chemotherapeutic agents, cytotoxic agents, and
non-peptide small molecules such as Gleevec (Imatinib Mesylate),
Velcade (bortezomib), Casodex (bicalutamide), Iressa (gefitinib),
and Adriamycin as well as a host of chemotherapeutic agents.
Non-limiting examples of chemotherapeutic agents include alkylating
agents such as thiotepa and cyclosphosphamide (CYTOXAN.TM.); alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen
mustards such as chlorambucil, chlornaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;
antibiotics such as aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,
caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine, androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfomithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.R.TM.; razoxane; sizofuran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL.TM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel
(TAXOTERE.TM., Rhone-Poulenc Rorer, Antony, France); retinoic acid;
esperamicins; capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included as suitable
chemotherapeutic cell conditioners are anti-hormonal agents that
act to regulate or inhibit hormone action on tumors such as
anti-estrogens including for example tamoxifen, raloxifene,
aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY 117018, onapristone, and toremifene
(Fareston); and anti-androgens such as flutamide, nilutamide,
bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine;
6-thioguanine; mercaptopurine; methotrexate; platinum analogs such
as cisplatin and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;
vinorelbine; navelbine; novantrone; teniposide; daunomycin;
aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-11);
topoisomerase inhibitor RFS 2000; difluoromethylornithine
(DMFO).
[0195] This invention further relates to a method for inhibiting
abnormal cell growth in a mammal or treating a hyperproliferative
disorder which method comprises administering to the mammal an
amount of a biologically active agent of the present invention, or
a pharmaceutically acceptable salt, ester, prodrug, solvate,
hydrate or derivative thereof, in combination with radiation
therapy, wherein the amounts of the biologically active agent,
salt, ester, prodrug, solvate, hydrate or derivative, is in
combination with the radiation therapy effective in inhibiting
abnormal cell growth or treating the hyperproliferative disorder in
the mammal. Techniques for administering radiation therapy are
known in the art, and these techniques can be used in the
combination therapy described herein. The administration of the
biologically active agent of the invention in this combination
therapy can be determined as described herein.
[0196] Radiation therapy can be administered through one of several
methods, or a combination of methods, including without limitation
external-beam therapy, internal radiation therapy, implant
radiation, stereotactic radiosurgery, systemic radiation therapy,
radiotherapy and permanent or temporary interstitial brachytherapy.
The term "brachytherapy," as used herein, refers to radiation
therapy delivered by a spatially confined radioactive material
inserted into the body at or near a tumor or other proliferative
tissue disease site. The term is intended without limitation to
include exposure to radioactive isotopes (e.g. At -211, I-131,
I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and radioactive
isotopes of Lu), Suitable radiation sources for use as a cell
conditioner of the present invention include both solids and
liquids. By way of non-limiting example, the radiation source can
be a radionuclide, such as I-125, I-131, Yb-169, Ir-192 as a solid
source, I-125 as a solid source, or other radionuclides that emit
photons, beta particles, gamma radiation, or other therapeutic
rays. The radioactive material can also be a fluid made from any
solution of radionuclide(s), e.g., a solution of I-125 or I-131, or
a radioactive fluid can be produced using a slurry of a suitable
fluid containing small particles of solid radionuclides, such as
Au-198, Y-90. Moreover, the radionuclide(s) can be embodied in a
gel or radioactive micro spheres.
[0197] Without be limiting to any theory, the biologically active
agents of the present invention can render abnormal cells more
sensitive to treatment with radiation for purposes of killing
and/or inhibiting the growth of such cells. Accordingly, this
invention further relates to a method for sensitizing abnormal
cells in a mammal to treatment with radiation which comprises
administering to the mammal an amount of a biologically active
agent of the present invention or pharmaceutically acceptable salt,
ester, prodrug, solvate, hydrate or derivative thereof, which
amount is effective is sensitizing abnormal cells to treatment with
radiation. The amount of the biologically active agent, salt, or
solvate in this method can be determined according to the means for
ascertaining effective amounts of such biologically active agents
described herein.
[0198] The invention also relates to a method of and to a
pharmaceutical composition of inhibiting abnormal cell growth in a
mammal which comprises an amount of a biologically active agent of
the present invention, or a pharmaceutically acceptable salt,
ester, prodrug, solvate, hydrate or derivative thereof, or an
isotopically-labeled derivative thereof, and an amount of one or
more substances selected from anti-angiogenesis agents, signal
transduction inhibitors, and antiproliferative agents.
[0199] Anti-angiogenesis agents, such as MMP-2
(matrix-metalloproteinase 2) inhibitors, MMP-9
(matrix-metalloprotienase 9) inhibitors, and COX-11 (cyclooxygenase
11) inhibitors, can be used in conjunction with a biologically
active agent of the present invention and pharmaceutical
compositions described herein. Examples of useful COX-II inhibitors
include CELEBREX.TM. (alecoxib), valdecoxib, and rofecoxib.
Examples of useful matrix metalloproteinase inhibitors are
described in WO 96/33172 (published Oct. 24, 1996), WO 96/27583
(published Mar. 7, 1996), European Patent Application No.
97304971.1 (filed Jul. 8, 1997), European Patent Application No.
99308617.2 (filed Oct. 29, 1999), WO 98/07697 (published Feb. 26,
1998), WO 98/03516 (published Jan. 29, 1998), WO 98/34918
(published Aug. 13, 1998), WO 98/34915 (published Aug. 13, 1998),
WO 98/33768 (published Aug. 6, 1998), WO 98/30566 (published Jul.
16, 1998), European Patent Publication 606,046 (published Jul. 13,
1994), European Patent Publication 931, 788 (published Jul. 28,
1999), WO 90/05719 (published May 31, 1990), WO 99/52910 (published
Oct. 21, 1999), WO 99/52889 (published Oct. 21, 1999), WO 99/29667
(published Jun. 17, 1999), PCT International Application No.
PCT/IB98/01113 (filed Jul. 21, 1998), European Patent Application
No. 99302232.1 (filed Mar. 25, 1999), Great Britain Patent
Application No. 9912961.1 (filed Jun. 3, 1999), U.S. Provisional
Application No. 60/148,464 (filed Aug. 12, 1999), U.S. Pat. No.
5,863,949 (issued Jan. 26, 1999), U.S. Pat. No. 5,861,510 (issued
Jan. 19, 1999), and European Patent Publication 780,386 (published
Jun. 25, 1997), all of which are incorporated herein in their
entireties by reference. Preferred MMP-2 and MMP-9 inhibitors are
those that have little or no activity inhibiting MMP-1. More
preferred, are those that selectively inhibit MMP-2 and/or AMP-9
relative to the other matrix-metalloproteinases (i.e., MAP-1,
MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12,
and MMP-13). Some specific examples of MMP inhibitors useful in the
present invention are AG-3340, RO 32-3555, and RS 13-0830.
[0200] The invention also relates to a method of and to a
pharmaceutical composition of treating a cardiovascular disease in
a mammal which comprises an amount of a biologically active agent
of the present invention, or a pharmaceutically acceptable salt,
ester, prodrug, solvate, hydrate or derivative thereof, or an
isotopically-labeled derivative thereof, and an amount of one or
more therapeutic agents use for the treatment of cardiovascular
diseases.
[0201] Examples for use in cardiovascular disease applications are
anti-thrombotic agents, e.g., prostacyclin and salicylates,
thrombolytic agents, e.g., streptokinase, urokinase, tissue
plasminogen activator (TPA) and anisoylated
plasminogen-streptokinase activator complex (APSAC), anti-platelets
agents, e.g., acetyl-salicylic acid (ASA) and clopidrogel,
vasodilating agents, e.g., nitrates, calcium channel blocking
drugs, anti-proliferative agents, e.g., colchicine and alkylating
agents, intercalating agents, growth modulating factors such as
interleukins, transformation growth factor-beta and congeners of
platelet derived growth factor, monoclonal antibodies directed
against growth factors, anti-inflammatory agents, both steroidal
and non-steroidal, and other agents that can modulate vessel tone,
function, arteriosclerosis, and the healing response to vessel or
organ injury post intervention. Antibiotics can also be included in
combinations or coatings comprised by the invention. Moreover, a
coating can be used to effect therapeutic delivery focally within
the vessel wall. By incorporation of the active agent in a
swellable polymer, the active agent will be released upon swelling
of the polymer.
[0202] The biologically active agents of the invention may be
formulated or administered in conjunction with other agents that
act to relieve the symptoms of inflammatory conditions such as
encephalomyelitis, asthma, and the other diseases described herein.
These agents include non-steroidal anti-inflammatory drugs
(NSAIDs), e.g. acetylsalicylic acid; ibuprofen; naproxen;
indomethacin; nabumetone; tolmetin; etc. Corticosteroids are used
to reduce inflammation and suppress activity of the immune system.
The most commonly prescribed drug of this type is Prednisone.
Chloroquine (Aralen) or hydroxychloroquine (Plaquenil) may also be
very useful in some individuals with lupus. They are most often
prescribed for skin and joint symptoms of lupus. Azathioprine
(Imuran) and cyclophosphamide (Cytoxan) suppress inflammation and
tend to suppress the immune system. Other agents, e.g. methotrexate
and cyclosporin are used to control the symptoms of lupus.
Anticoagulants are employed to prevent blood from clotting rapidly.
They range from aspirin at very low dose which prevents platelets
from sticking, to heparin/coumadin.
[0203] The biologically active agents describe herein may be
formulated or administered in conjunction with liquid or solid
tissue barriers also known as lubricants. Examples of tissue
barriers include, but are not limited to, polysaccharides,
polyglycans, seprafilm, interceed and hyaluronic acid.
[0204] Medicaments which may be administered in conjunction with
the biologically active agents described herein include any
suitable drugs usefully delivered by inhalation for example,
analgesics, e.g. codeine, dihydromorphine, ergotamine, fentanyl or
morphine; anginal preparations, e.g. diltiazem; antiallergics, e.g.
cromoglycate, ketotifen or nedocromil; anti-infectives, e.g.
cephalosporins, penicillins, streptomycin, sulphonamides,
tetracyclines or pentamidine; antihistamines, e.g. methapyrilene;
anti-inflammatories, e.g. beclomethasone, flunisolide, budesonide,
tipredane, triamcinolone acetonide or fluticasone; antitussives,
e.g. noscapine; bronchodilators, e.g. ephedrine, adrenaline,
fenoterol, formoterol, isoprenaline, metaproterenol, phenylephrine,
phenylpropanolamine, pirbuterol, reproterol, rimiterol, salbutamol,
salmeterol, terbutalin, isoetharine, tulobuterol, orciprenaline or
(-)-4-amino-3,5-dichloro-.alpha.-[[[6-[2-(2-pyridinyl)ethoxy]hexyl]-amino-
]methyl]benzenemethanol; diuretics, e.g. amiloride;
anticholinergics e.g. ipratropium, atropine or oxitropium;
hormones, e.g. cortisone, hydrocortisone or prednisolone; xanthines
e.g. aminophylline, choline theophyllinate, lysine theophyllinate
or theophylline; and therapeutic proteins and peptides, e.g.
insulin or glucagon. It will be clear to a person skilled in the
art that, where appropriate, the medicaments may be used in the
form of salts (e.g. as alkali metal or amine salts or as acid
addition salts) or as esters (e.g. lower alkyl esters) or as
solvates (e.g. hydrates) to optimize the activity and/or stability
of the medicament.
[0205] Other exemplary therapeutic agents useful for a combination
therapy include but are not limited to agents as described above,
radiation therapy, hormone antagonists, hormones and their
releasing factors, thyroid and antithyroid drugs, estrogens and
progestins, androgens, adrenocorticotropic hormone; adrenocortical
steroids and their synthetic analogs; inhibitors of the synthesis
and actions of adrenocortical hormones, insulin, oral hypoglycemic
agents, and the pharmacology of the endocrine pancreas, agents
affecting calcification and bone turnover: calcium, phosphate,
parathyroid hormone, vitamin D, calcitonin, vitamins such as
water-soluble vitamins, vitamin B complex, ascorbic acid,
fat-soluble vitamins, vitamins A, K, and E, growth factors,
cytokines, chemokines, muscarinic receptor agonists and
antagonists; anticholinesterase agents; agents acting at the
neuromuscular junction and/or autonomic ganglia; catecholamines,
sympathomimetic drugs, and adrenergic receptor agonists or
antagonists; and 5-hydroxytryptamine (5-HT, serotonin) receptor
agonists and antagonists.
[0206] Therapeutic agents can also include agents for pain and
inflammation such as histamine and histamine antagonists,
bradykinin and bradykinin antagonists, 5-hydroxytryptamine
(serotonin), lipid substances that are generated by
biotransformation of the products of the selective hydrolysis of
membrane phospholipids, eicosanoids, prostaglandins, thromboxanes,
leukotrienes, aspirin, nonsteroidal anti-inflammatory agents,
analgesic-antipyretic agents, agents that inhibit the synthesis of
prostaglandins and thromboxanes, selective inhibitors of the
inducible cyclooxygenase, selective inhibitors of the inducible
cyclooxygenase-2, autacoids, paracrine hormones, somatostatin,
gastrin, cytokines that mediate interactions involved in humoral
and cellular immune responses, lipid-derived autacoids,
eicosanoids, .beta.-adrenergic agonists, ipratropium,
glucocorticoids, methylxanthines, sodium channel blockers, opioid
receptor agonists, calcium channel blockers, membrane stabilizers
and leukotriene inhibitors.
[0207] Additional therapeutic agents contemplated herein include
diuretics, vasopressin, agents affecting the renal conservation of
water, rennin, angiotensin, agents useful in the treatment of
myocardial ischemia, anti-hypertensive agents, angiotensin
converting enzyme inhibitors, .beta.-adrenergic receptor
antagonists, agents for the treatment of hypercholesterolemia, and
agents for the treatment of dyslipidemia.
[0208] Other therapeutic agents contemplated include drugs used for
control of gastric acidity, agents for the treatment of peptic
ulcers, agents for the treatment of gastroesophageal reflux
disease, prokinetic agents, antiemetics, agents used in irritable
bowel syndrome, agents used for diarrhea, agents used for
constipation, agents used for inflammatory bowel disease, agents
used for biliary disease, agents used for pancreatic disease.
Therapeutic agents used to treat protozoan infections, drugs used
to treat Malaria, Amebiasis, Giardiasis, Trichomoniasis,
Trypanosomiasis, and/or Leishmaniasis, and/or drugs used in the
chemotherapy of helminthiasis. Other therapeutic agents include
antimicrobial agents, sulfonamides, trimethoprim-sulfamethoxazole
quinolones, and agents for urinary tract infections, penicillins,
cephalosporins, and other, .beta.-Lactam antibiotics, an agent
comprising an aminoglycoside, protein synthesis inhibitors, drugs
used in the chemotherapy of tuberculosis, mycobacterium avium
complex disease, and leprosy, antifungal agents, antiviral agents
including nonretroviral agents and antiretroviral agents.
[0209] Examples of therapeutic antibodies that can be combined with
a subject biologically active agent include but are not limited to
anti-receptor tyrosine kinase antibodies (cetuximab, panitumumab,
trastuzumab), anti CD20 antibodies (rituximab, tositumomab), and
other antibodies such as alemtuzumab, bevacizumab, and
gemtuzumab.
[0210] Moreover, therapeutic agents used for immunomodulation, such
as immunomodulators, immunosuppressive agents, tolerogens, and
immunostimulants are contemplated by the methods herein. In
addition, therapeutic agents acting on the blood and the
blood-forming organs, hematopoietic agents, growth factors,
minerals, and vitamins, anticoagulant, thrombolytic, and
antiplatelet drugs.
[0211] Further therapeutic agents that can be combined with a
subject biologically active agent may be found in Goodman and
Gilman's "The Pharmacological Basis of Therapeutics" Tenth Edition
edited by Hardman, Limbird and Gilman or the Physician's Desk
Reference, both of which are incorporated herein by reference in
their entirety.
[0212] The biologically active agents described herein can be used
in combination with the agents disclosed herein or other suitable
agents, depending on the condition being treated. Hence, in some
embodiments the biologically active agents of the invention will be
co-administered with other agents as described above. When used in
combination therapy, the biologically active agents described
herein may be administered with the second agent simultaneously or
separately. This administration in combination can include
simultaneous administration of the two agents in the same dosage
form, simultaneous administration in separate dosage forms, and
separate administration. That is, a biologically active agent
described herein and any of the agents described above can be
formulated together in the same dosage form and administered
simultaneously. Alternatively, a biologically active agent of the
present invention and any of the agents described above can be
simultaneously administered, wherein both the agents are present in
separate formulations. In another alternative, a biologically
active agent of the present invention can be administered just
followed by and any of the agents described above, or vice versa.
In the separate administration protocol, a biologically active
agent of the present invention and any of the agents described
above may be administered a few minutes apart, or a few hours
apart, or a few days apart.
Administration
[0213] Administration of the biologically active agents of the
present invention can be effected by any method that enables
delivery of the biologically active agents to the site of action.
These methods include oral routes, intraduodenal routes, parenteral
injection (including intravenous, intraarterial, subcutaneous,
intramuscular, intravascular, intraperitoneal or infusion), topical
(e.g. transdermal application), rectal administration, via local
delivery by catheter or stent. Biologically active agents can also
be administered intraadiposally or intrathecally.
[0214] The amount of the biologically active agent administered
will be dependent on the mammal being treated, the severity of the
disorder or condition, the rate of administration, the disposition
of the biologically active agent and the discretion of the
prescribing physician. However, an effective dosage is in the range
of about 0.001 to about 100 mg per kg body weight per day,
preferably about 1 to about 35 mg/kg/day, in single or divided
doses. For a 70 kg human, this would amount to about 0.05 to 7
g/day, preferably about 0.05 to about 2.5 g/day. In some instances,
dosage levels below the lower limit of the aforesaid range may be
more than adequate, while in other cases still larger doses may be
employed without causing any harmful side effect, e.g. by dividing
such larger doses into several small doses for administration
throughout the day.
[0215] The biologically active agent may be applied as a sole
therapy or may involve one or more other anti-tumor substances, for
example those selected from, mitotic inhibitors, for example
vinblastine; alkylating agents, for example cis-platin, carboplatin
and cyclophosphamide; anti-metabolites, for example 5-fluorouracil,
cytosine arabinside and hydroxyurea, or, for example, one of the
preferred anti-metabolites disclosed in European Patent Application
No. 239362 such as
N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]--
2-thenoyl)-L-glutamic acid; growth factor inhibitors; cell cycle
inhibitors; intercalating antibiotics, for example adriamycin and
bleomycin; enzymes, for example, interferon; and anti-hormones, for
example anti-estrogens such as Nolvadex.TM. (tamoxifen) or, for
example anti-androgens such as Casodex.TM.
(4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluoromet-
hyl) propionanilide). Such conjoint treatment may be achieved by
way of the simultaneous, sequential or separate dosing of the
individual components of treatment.
[0216] In some embodiments, a biologically active agent of the
invention is administered in a single dose. Typically, such
administration will be by injection, e.g., intravenous injection,
in order to introduce the agent quickly. However, other routes may
be used as appropriate. A single dose of a biologically active
agent of the invention may also be used for treatment of an acute
condition.
[0217] In some embodiments, a biologically active agent of the
invention is administered in multiple doses. Dosing may be about
once, twice, three times, four times, five times, six times, or
more than six times per day. Dosing may be about once a month, once
every two weeks, once a week, or once every other day. In another
embodiment a biologically active agent of the invention and another
agent are administered together about once per day to about 6 times
per day. In another embodiment the administration of a biologically
active agent of the invention and an agent continues for less than
about 7 days. In yet another embodiment the administration
continues for more than about 6, 10, 14, 28 days, two months, six
months, or one year. In some cases, continuous dosing is achieved
and maintained as long as necessary.
[0218] Administration of the agents of the invention may continue
as long as necessary. In some embodiments, an agent of the
invention is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or
28 days. In some embodiments, an agent of the invention is
administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In
some embodiments, an agent of the invention is administered
chronically on an ongoing basis, e.g., for the treatment of chronic
effects.
[0219] An effective amount of a biologically active agent of the
invention may be administered in either single or multiple doses by
any of the accepted modes of administration of agents having
similar utilities, including rectal, buccal, intranasal and
transdermal routes, by intra-arterial injection, intravenously,
intraperitoneally, parenterally, intramuscularly, subcutaneously,
orally, topically, as an inhalant.
[0220] The compositions of the invention may also be delivered via
an impregnated or coated device such as a stent, for example, or an
artery-inserted cylindrical polymer. Such a method of
administration may, for example, aid in the prevention or
amelioration of restenosis following procedures such as balloon
angioplasty. Without being bound by theory, biologically active
agents of the invention may slow or inhibit the migration and
proliferation of smooth muscle cells in the arterial wall which
contribute to restenosis. A biologically active agent of the
invention may be administered, for example, by local delivery from
the struts of a stent, from a stent graft, from grafts, or from the
cover or sheath of a stent. In some embodiments, a biologically
active agent of the invention is admixed with a matrix. Such a
matrix may be a polymeric matrix, and may serve to bond the
biologically active agent to the stent. Polymeric matrices suitable
for such use, include, for example, lactone-based polyesters or
copolyesters such as polylactide, polycaprolactonglycolide,
polyorthoesters, polyanhydrides, polyaminoacids, polysaccharides,
polyphosphazenes, poly (ether-ester) copolymers (e.g. PEO-PLLA);
polydimethylsiloxane, poly(ethylene-vinylacetate), acrylate-based
polymers or copolymers (e.g. polyhydroxyethyl methylmethacrylate,
polyvinyl pyrrolidinone), fluorinated polymers such as
polytetrafluoroethylene and cellulose esters. Suitable matrices may
be nondegrading or may degrade with time, releasing the
biologically active agent or biologically active agents.
Biologically active agents of the invention may be applied to the
surface of the stent by various methods such as dip/spin coating,
spray coating, dip-coating, and/or brush-coating. The biologically
active agents may be applied in a solvent and the solvent may be
allowed to evaporate, thus forming a layer of biologically active
agent onto the stent. Alternatively, the biologically active agent
may be located in the body of the stent or graft, for example in
microchannels or micropores. When implanted, the biologically
active agent diffuses out of the body of the stent to contact the
arterial wall. Such stents may be prepared by dipping a stent
manufactured to contain such micropores or microchannels into a
solution of the biologically active agent of the invention in a
suitable solvent, followed by evaporation of the solvent. Excess
drug on the surface of the stent may be removed via an additional
brief solvent wash. In yet other embodiments, biologically active
agents of the invention may be covalently linked to a stent or
graft. A covalent linker may be used which degrades in vitro,
leading to the release of the biologically active agent of the
invention. Any bio-labile linkage may be used for such a purpose,
such as ester, amide or anhydride linkages. Biologically active
agents of the invention may additionally be administered
intravascularly from a balloon used during angioplasty.
Extravascular administration of the biologically active agents via
the pericard or via advential application of formulations of the
invention may also be performed to decrease restenosis.
[0221] A variety of stent devices which may be used as described
are disclosed, for example, in the following references, all of
which are hereby incorporated by reference: U.S. Pat. No.
5,451,233; U.S. Pat. No. 5,040,548; U.S. Pat. No. 5,061,273; U.S.
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[0222] The biologically active agents of the invention may be
administered in dosages as described herein. It is known in the art
that due to intersubject variability in biologically active agent
pharmacokinetics, individualization of dosing regimen is necessary
for optimal therapy. Dosing for a biologically active agent of the
invention may be found by routine experimentation.
[0223] When a biologically active agent of the invention, is
administered in a composition that comprises one or more agents,
and the agent has a shorter half-life than the biologically active
agent of the invention unit dose forms of the agent and the
biologically active agent of the invention may be adjusted
accordingly.
[0224] The subject pharmaceutical composition may, for example, be
in a form suitable for oral administration as a tablet, capsule,
pill, powder, sustained release formulations, solution, suspension,
for parenteral injection as a sterile solution, suspension or
emulsion, for topical administration as an ointment or cream or for
rectal administration as a suppository. The pharmaceutical
composition may be in unit dosage forms suitable for single
administration of precise dosages. The pharmaceutical composition
will include a conventional pharmaceutical carrier or excipient and
a biologically active agent according to the invention as an active
ingredient. In addition, it may include other medicinal or
pharmaceutical agents, carriers, adjuvants, etc.
[0225] Exemplary parenteral administration forms include solutions
or suspensions of active biologically active agent in sterile
aqueous solutions, for example, aqueous propylene glycol or
dextrose solutions. Such dosage forms can be suitably buffered, if
desired.
[0226] The activity of the biologically active agents of the
present invention may be determined by the following procedure, as
well as the procedure described in the examples below. N-terminal 6
His-tagged, constitutively active kinase is expressed in E. coli
and protein is purified by conventional methods (Ahn et al. Science
1994, 265, 966-970). The activity of the kinase is assessed by
measuring the incorporation of .gamma.-.sup.33P-phosphate from
.gamma.-.sup.33P-ATP onto N-terminal His tagged substrate, which is
expressed in E. coli and is purified by conventional methods, in
the presence of the kinase. The assay is carried out in 96-well
polypropylene plate. The incubation mixture (100, .mu.L) comprises
of 25 mM HEPES, pH 7.4, 10 mM MgCl.sub.2, 5 mM
.beta.-glycerolphosphate, 100 .mu.M Na-orthovanadate, 5 mM DTT, 5
nM kinase, and 1 .mu.M substrate Inhibitors are suspended in DMSO,
and all reactions, including controls are performed at a final
concentration of 1% DMSO. Reactions are initiated by the addition
of 10 .mu.M ATP (with 0.5 .mu.Ci .gamma.-.sup.33P-ATP/well) and
incubated at ambient temperature for 45 minutes. Equal volume of
25% TCA is added to stop the reaction and precipitate the proteins.
Precipitated proteins are trapped onto glass fiber B filterplates,
and excess labeled ATP washed off using a Tomtec MACH III
harvestor. Plates are allowed to air-dry prior to adding 30
.mu.L/well of Packard Microscint 20, and plates are counted using a
Packard TopCount.
[0227] The examples and preparations provided below further
illustrate and exemplify the biologically active agents of the
present invention and methods of preparing such biologically active
agents. It is to be understood that the scope of the present
invention is not limited in any way by the scope of the following
examples and preparations. In the following examples molecules with
a single chiral center, unless otherwise noted, exist as a racemic
mixture. Those molecules with two or more chiral centers, unless
otherwise noted, exist as a racemic mixture of diastereomers.
Single enantiomers/diastereomers may be obtained by methods known
to those skilled in the art.
EXAMPLES
Example 1
Kinase Inhibition Assay
[0228] Purified kinase domains (e.g. mTor, P110.alpha., P110.beta.,
P110.gamma., P110.delta., PI4K.beta., DNA-PK, PKC.alpha.,
PKC.beta.1, PKC.beta.II, RET, and JAK2) were incubated with
inhibitors at 2- or 4-fold dilutions over a concentration range of
50-0.001 .mu.M or with vehicle (0.1% DMSO) in the presence of 10
.mu.M ATP, 2.5 .mu.Ci of .gamma.-.sup.32P-ATP and substrate.
Reactions were terminated by spotting onto nitrocellulose or
phosphocellulose membranes, depending on the substrate; this
membrane was then washed 5-6 times to remove unbound radioactivity
and dried. Transferred radioactivity was quantitated by
phosphorimaging and IC.sub.50 values were calculated by fitting the
data to a sigmoidal dose-response using Prism software.
[0229] The results as shown in FIG. 2 demonstrate that TORKinib is
a potent and specific inhibitor of mTor with an IC.sub.50 of about
8 nM. TORKinib was also relatively inactive against PKC.beta., RET,
and JAK2 (V617F), but inhibited PKC.alpha. with an IC.sub.50 of
about 50 nM. TORKinib2 likewise is an extremely potent and specific
inhibitor of mTor with an IC.sub.50 of about 80 nM. Furthermore,
TORKinib2 does not inhibit PKC.alpha. significantly at about 1
.mu.M or less. Therefore, TORKinib2 can be used to confirm that the
effects of TORKinib are due to inhibition of mTor and not
PKC.alpha..
Example 2
Effect of mTor Inhibitors on Kinase Substrate Phosphorylation
[0230] L6 myoblasts were grown typically grown in DMEM supplemented
with about 10% FBS, glutamine and penicillin/streptomycin.
Confluent L6 myoblasts were differentiated into myotubes by
culturing them for approximately 5 days in media containing about
2% FBS. L6 myotubes were maintained in media containing
approximately 2% FBS until use.
[0231] In order to compare the effect of TORKinibs and PIK-90 on
Akt phosphorylation, L6 myotubes were serum starved overnight and
incubated with inhibitors or about 0.1% DMSO for approximately 30
minutes prior to stimulation with insulin (e.g. 100 nM) for about
10 minutes. Cells were lysed by scraping into ice cold lysis buffer
(generally: 300 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 1% Triton
X-100, 0.02% NaN3, 20 nM microcystin, Sigma phosphatase inhibitor
cocktails 1 and 2, Roche protease inhibitor cocktail and 2 mM
PMSF). After contacting cells with lysis buffer, the solution was
briefly sonicated. Lysates were cleared by centrifugation, resolved
by SDS-PAGE, transferred to nitrocellulose and immunoblotted using
antibodies to phospho-Akt S473, phospho-Akt T308, Akt, and
.beta.-actin (Cell Signaling Technologies).
[0232] The results as shown in FIG. 3 demonstrate that TORKinib and
TORKinib2 inhibit insulin stimulated phosphorylation of Akt at
S473, confirming that mTor kinase activity is required for
hydrophobic motif phosphorylation under the conditions tested.
Inhibition of mTor by TORKinibs also may result in loss of Akt
phosphorylation at T308, but higher doses of the TORKinibs may be
required to inhibit T308 phosphorylation as compared to S473
phosphorylation. In contrast, the PI3K inhibitor PIK-90 may inhibit
the phosphorylation of both Akt sites equipotently under the
conditions tested.
Example 3
Kinetics of Kinase Substrate Phosphorylation
[0233] L6 myotubes were serum starved overnight and incubated with
TORKinib or 0.1% DMSO for about 30 minutes prior to stimulation
with insulin (e.g. 100 nM) for about 1, 3, 10, and 60 minutes.
Cells were lysed by scraping into ice cold lysis buffer (generally
300 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.02%
NaN3, 20 nM microcystin, Sigma phosphatase inhibitor cocktails 1
and 2, Roche protease inhibitor cocktail and 2 mM PMSF). After
contacting cells with lysis buffer, the solution was briefly
sonicated. Lysates were cleared by centrifugation, resolved by
SDS-PAGE, transferred to nitrocellulose and immunoblotted using
antibodies to phospho-Akt S473, phospho-Akt T308, and actin. The
results as shown in FIG. 4 demonstrate that differential
sensitivity of S473 and T308 to inhibition of phosphorylation by
TORKinibs may not reflect differing kinetics of
phosphorylation.
Example 4
Effect of Kinase Inhibitors on Mouse Embryonic Fibroblasts
[0234] Wild-type and SIN1-/- primary mouse embryonic fibroblasts
(MEFs) were grown in DMEM supplemented with about 10% FBS,
glutamine and penicillin/streptomycin. MEFs were treated with
TORKinib at about 2.5, 0.63, and 0.16 uM; rapamycin at
approximately 15 nM; or PIK-90 at 625 nM for 30 minutes prior to
insulin stimulation (e.g. 100 nM) for 10 minutes. Cells were lysed
by scraping into ice cold lysis buffer (typically 300 mM NaCl, 50
mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.02% NaN3, 20 nM
microcystin, Sigma phosphatase inhibitor cocktails 1 and 2, Roche
protease inhibitor cocktail and 2 mM PMSF). After contacting cells
with lysis buffer, the solution was briefly sonicated. Lysates were
cleared by centrifugation, resolved by SDS-PAGE, transferred to
nitrocellulose and immunoblotted using antibodies to phospho-Akt
S473, phospho-Akt T308, Akt, and .beta.-actin (Cell Signaling
Technologies). The results are shown in FIG. 5.
[0235] SIN1 is a component of mTorC2, and knockout of SIN1
compromises the physical integrity of mTorC2 leading to a complete
loss of Akt phosphorylation at S473 without affecting its
phosphorylation at T308. Consistent with the results of FIG. 3 and
FIG. 4, FIG. 5 shows that TORKinib inhibits the phosphorylation of
Akt at both S473 and T308 in wild-type MEFs, but has no effect on
the phosphorylation of T308 in SIN1-/- MEFs that lack mTorC2 under
the conditions tested. Furthermore, the PI3K inhibitor PIK-90
blocks phosphorylation of T308 in SIN1-/- MEFs, indicating that the
failure of TORKinib to block T308 in SIN1-/- MEFs may not reflect a
general resistance of T308 to dephosphorylation in cells that lack
mTorC2. This suggests that T308 phosphorylation caused by the
TORKinibs results from inhibition of mTor mediated phosphorylation
of S473, rather than inhibition of an unknown or off-target kinase.
Further, this indicates that TORKinib blocks T308 phosphorylation
indirectly by directly inhibiting mTor-dependent phosphorylation at
S473.
Example 5
Phosphorylation of AKT, GSK3, TSC2, FoxO1/O3a, and S6
[0236] L6 myotubes were serum starved overnight and pre-treated
with TORKinib at approximately 2.5, 0.63, 0.16, 0.04, and 0.01
.mu.M; PIK-90 at about 625 nM; Akt-allo (AKTi Calbiochem) at about
10 .mu.M; or rapamycin at approximately 15 nM followed by insulin
stimulation (e.g. 100 nM). Cells were lysed by scraping into ice
cold lysis buffer (generally: 300 mM NaCl, 50 mM Tris pH 7.5, 5 mM
EDTA, 1% Triton X-100, 0.02% NaN3, 20 nM microcystin, Sigma
phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor
cocktail and 2 mM PMSF). After contacting cells with lysis buffer,
the solution was briefly sonicated. Lysates were cleared by
centrifugation, resolved by SDS-PAGE, transferred to nitrocellulose
and immunoblotted using antibodies to phospho-Akt S473, phospho-Akt
T308, phospho GSK3.alpha./.beta. S21/9, phospho TSC2 T1462, phospho
FoxO1/O3a T24/32, FoxO3a, phospho-S6 S240/244, and .beta.-actin
(Cell Signaling Technologies).
[0237] Low concentrations of TORKinib inhibit the phosphorylation
of Akt S473 and higher concentrations inhibit Akt T308
phosphorylation under the conditions tested. Thus TORKinib may be
used as in this experiment to determine if any substrates of Akt
are especially sensitive to loss of phosphorylated S473. The
results as shown in FIG. 6 suggest that kinase substrates
downstream of mTor signaling are not sensitive to loss of
phosphorylated Akt S473 alone. TORKinib partially inhibited the
phosphorylation of both cytoplasmic and nuclear substrates of Akt
and for all substrates tested the extent of inhibition parallels
the phosphorylation of Akt at T308 under the concentrations used.
This indicates that loss of phospho-5473 alone may be unable to
prevent the phosphorylation of any of the Akt substrates tested. In
contrast upstream inhibition of PI3K with PIK-90, and direct
inhibition of Akt with Akt-allo completely inhibited the
phosphorylation of Akt and its substrates under the conditions
tested.
Example 6
Effect of Kinase Inhibitors on Cellular Proliferation
[0238] Wild-type and SIN1-/- MEFs were plated in 96 well tissue
culture plates in DMEM supplemented with approximately 10% FBS,
glutamine and penicillin/streptomycin at about 30% confluence and
left overnight at about 37.degree. C. in a humidified incubator to
adhere. The following day, cells were treated with TORKinib,
rapamycin, or vehicle (e.g. 0.1% DMSO). After 72 hours of treatment
about 10 .mu.l of approximately 440 .mu.M Resazurin sodium salt
(Sigma) was added to each well, and after about 18 hours the
fluorescence intensity in each well was measured using a
top-reading fluorescent plate reader with excitation at about 530
nM and emission at approximately 590 nM. The results are shown in
FIG. 7.
[0239] Rapamycin was tested at concentrations above its mTor
IC.sub.50 and at all concentrations tested it inhibited cell growth
to the same extent. In contrast, TORKinib had a dose dependent
effect on proliferation and at higher doses was more effective than
rapamycin at blocking cell proliferation under the conditions
tested. In SIN1-/- MEFs, rapamycin was also typically less
effective at blocking cell proliferation than TORKinib. That
TORKinib and rapamycin exhibit very different anti-proliferative
effects in SIN1-/- suggests that the two compounds may
differentially affect mTorC1. Alternatively, the ability of
TORKinib to more effectively bock MEF cellular proliferation than
rapamycin may be a result of its ability to inhibit rapamycin
resistant mTorC2.
Example 7
Phosphorylation of AKT, 70S6K, S6, 4EBP1, and MAPK
[0240] L6 myotubes were serum starved overnight and pre-treated
with TORKinib at about 2.5, 0.63, 0.16, 0.04, and 0.01 .mu.M; or
rapamycin at approximately 62, 15, 4, 1, and 0.63 nM followed by
insulin stimulation (at for example 100 nM). Cells were lysed by
scraping into ice cold lysis buffer (typically 140 mM KCl, 10 mM
Tris pH 7.5, 1 mM EDTA, 4 mM MgCl2, 1 mM DTT, 1% NP-40, 20 nM
microcystin, Sigma phosphatase inhibitor cocktails 1 and 2, Roche
protease inhibitor cocktail without EDTA and 2 mM PMSF). After
contacting cells with lysis buffer, the solution was briefly
sonicated. Lysates were cleared by centrifugation, resolved by
SDS-PAGE, transferred to nitrocellulose and immunoblotted using
antibodies to phospho-Akt S473, phospho-Akt T308, phospho p70S6K
T389, phospho S6 S235/238, phospho S6 S240/244, phospho 4EBP1
T37/46, phospho 4EBP1 S65, 4EBP1, phospho-MAPK (p44/p42), and
.beta.-actin (Cell Signaling Technologies).
[0241] The results as shown in FIG. 8 demonstrate that both
rapamycin and TORKinib may inhibit phosphorylation of S6 kinase
(S6K) and its substrate S6, and neither rapamycin nor TORKinib
affect the phosphorylation of 4EBP1 at T70 under the conditions
tested. In contrast, rapamycin weakly enhances the phosphorylation
of 4EBP1 at T37/46 and weakly inhibits phosphorylation of 4EBP1 at
S65, while TORKinib fully inhibits the phosphorylation at both
sites at the concentrations examined.
Example 8
Phosphorylation of 70S6K and 4EBP1
[0242] L6 myotubes were serum starved overnight and pre-treated
with TORKinib2 at about 10, 2.5, 0.63, 0.16, and 0.04 .mu.M;
TORKinib at approximately 2.5, 0.63, 0.16, 0.04, and 0.01 .mu.M; or
PIK-90 at about 625 nM followed by insulin stimulation (with for
example 100 nM insulin). Cells were lysed by scraping into ice cold
lysis buffer (typically 140 mM KCl, 10 mM Tris pH 7.5, 1 mM EDTA, 4
mM MgCl2, 1 mM DTT, 1% NP-40, 20 nM microcystin, Sigma phosphatase
inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail
without EDTA and 2 mM PMSF). After contacting cells with lysis
buffer, the solution was briefly sonicated. Lysates were cleared by
centrifugation, resolved by SDS-PAGE, transferred to nitrocellulose
and immunoblotted using antibodies to phospho p70S6K T389, and
phospho 4EBP1 T37/46 (Cell Signaling Technologies). The results are
shown in FIG. 9.
[0243] The results show that PIK-90 does not reduce the
phosphorylation of 4EBP1 at T37/46, whereas both TORKinib and
TORKinib2 are able to inhibit 4EBP1 phosphorylation at T37/46 under
the conditions tested. This demonstrates that inhibition of PI3K
and Akt activation alone may not be sufficient to block the
phosphorylation of 4EBP1 at T37/46. This further supports the
mechanism whereby TORKinibs are able to more fully inhibit mTor
activity than rapamycin, or PI3K/Akt inhibitors.
Example 9
Method of m7GTP Cap Pull-Down Assay
[0244] L6 myotubes were serum starved overnight and pre-treated
with TORKinib at about 2.5, 0.63, 0.16, 0.04, and 0.01 .mu.M; or
rapamycin at approximately 62, 15, 4, and 1 nM followed by insulin
stimulation (100 nM). Cells were lysed by scraping into ice cold
lysis buffer (generally 140 mM KCl, 10 mM Tris pH 7.5, 1 mM EDTA, 4
mM MgCl2, 1 mM DTT, 1% NP-40, 20 nM microcystin, Sigma phosphatase
inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail
without EDTA and 2 mM PMSF). After contacting cells with lysis
buffer, the solution was briefly sonicated. Lysates were cleared by
centrifugation, about 50 .mu.l of detergent free cap lysis buffer
and about 20 .mu.l of prewashed m7GTP sepharose beads were added to
approximately 150 .mu.l of cleared lysate and incubated at
4.degree. C. overnight with spinning. The beads were washed twice
with about 400 .mu.l of cap wash buffer (typically cap lysis buffer
with 0.5% NP-40 instead of 1% NP-40) and twice with about 500 .mu.l
PBS. The beads were boiled in SDS-PAGE sample buffer and the
retained proteins were resolved by SDS-PAGE, transferred to
nitrocellulose and immunoblotted using antibodies to eIF-4E, 4EBP1
(Cell Signaling Technologies) and eIF-4E (BD Biosciences). The
results are shown in FIG. 10.
[0245] Mammalian translation initiation factor 4F (eIF-4F) consists
of three subunits, eIF-4A, eIF-4E, and eIF-4G. eIF-4E binds tightly
to m7GTP, and the subunit eIF-4G binds tightly to the m7GTP bound
eIF-4E during translation initiation. In response to
anti-proliferative signals, PTEN activation, or kinase inhibition,
the mTor substrate 4EBP1 can become dephosphorylated. Upon
dephosphorylation, 4EBP1 binds to eIF-4E and displaces eIF-4G
inhibiting translation initiation. The phosphorylation of 4EBP1 by
mTor is complicated in that it occurs at multiple sites and not all
sites are equally effective at causing disassociation of 4EBP1 from
eIF-4E. Furthermore, a hierarchy is thought to exist whereby T37/46
require phosphorylation prior to 565/T70. Phosphorylation at S65
may cause the greatest degree of disassociation of 4EBP1 from
eIF-4E and is probably the most important site in cells, but other
phosphorylation sites of 4EBP1 play a role in regulating
translation initiation as well. eIF-4E also binds tightly to m7GTP
sepharose beads. This allows the examination of proteins bound to
eIF-4E by pull-down or co-precipitation assay, which is a proxy for
protein translation activity.
[0246] The results show that, under the conditions tested,
rapamycin causes partial inhibition of the insulin stimulated
release of 4EBP1 from eIF-4E consistent with the partial inhibition
of S65 phosphorylation seen in FIG. 8. The rapamycin induced
retention of 4EBP1 was accompanied by a loss of recovery of eIF-4G
because the binding of 4EBP1 and eIF-4G to eIF-4E are mutually
exclusive. In contrast, treatment with TORKinib caused a greater
retention of 4EBP1, raising the retention of 4EBP1 above the level
seen in unstimulated serum-starved cells which are known to have
extremely low levels of protein translation.
Example 10
Phosphorylation of 70S6K and 4EBP1 in Mouse Embryonic
Fibroblasts
[0247] Wild-type and SIN1-/- primary mouse embryonic fibroblasts
(MEFs) were grown in DMEM supplemented with about 10% FBS,
glutamine and penicillin/streptomycin. MEFs were treated with
TORKinib at approximately 2.5, 0.63, and 0.16 uM; rapamycin at
about 15 nM; or PIK-90 at about 625 nM for approximately 30 minutes
prior to insulin stimulation (e.g. 100 nM) for about 10 minutes.
Cells were lysed by scraping into ice cold lysis buffer (generally:
300 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.02%
NaN3, 20 nM microcystin, Sigma phosphatase inhibitor cocktails 1
and 2, Roche protease inhibitor cocktail and 2 mM PMSF). After
contacting cells with lysis buffer, the solution was briefly
sonicated. Lysates were cleared by centrifugation, resolved by
SDS-PAGE, transferred to nitrocellulose and immunoblotted using
antibodies to phospho-p70S6K T389, phospho-4EBP1 T37/46,
phospho-4EBP1 S65, and .beta.-actin (Cell Signaling Technologies).
The results are shown in FIG. 11.
[0248] TORKinib is a more complete inhibitor of 4EBP1
phosphorylation under the conditions tested than rapamycin in
wild-type and SIN1-/- cells as well, indicating that the presence
of mTorC2 may not be required for rapamycin and TORKinib to have
distinct effects on 4EBP1 phosphorylation, and demonstrating that
TORKinib is a more complete inhibitor of mTorC1 than rapamycin
under the conditions tested.
[0249] Rapamycin appears to be a substrate selective inhibitor of
mTorC1 as shown by its ability to completely block the
phosphorylation of p70S6K but not 4EBP1 at the concentrations
examined. Consistent with this finding, experiments with purified
proteins have shown that rapamycin/FKBP12 only partially inhibits
the in vitro phosphorylation of 4EBP1 at Ser 65 by mTor but can
fully inhibit the in vitro phosphorylation of S6K. By contrast,
LY294002, a direct inhibitor of many PI3K family members including
mTor, was equally effective at inhibiting the phosphorylation of
S6K and 4EBP1 by mTor in vitro. These results show that TORKinib,
in addition to being useful for investigating mTorC2, reveal
rapamycin-resistant components of mTorC1 function. Indeed,
proliferation of SIN1-/- MEFs is more sensitive to TORKinib than
rapamycin (FIG. 7), suggesting that rapamycin-resistant functions
of mTorC1, including aspects of translation initiation, may be key
to the anti-proliferative effects of TORKinib.
Example 11
In Vivo Effect of mTor Inhibitors on Kinase Substrate
Phosphorylation
[0250] To explore the tissue specific roles of mTorC1 and mTorC2
and confirm the pathway analysis from cell culture experiments,
mice were treated with TORKinib or rapamycin, and the acute effect
of these drugs on insulin signaling in fat, skeletal muscle and
liver tissue were examined.
[0251] Approximately 6 week old male C57BL/6 mice were starved of
food overnight. Drugs were prepared in about 100 .mu.l of vehicle
containing approximately 20% DMSO, 40% PEG-400 and 40% Saline;
TORKinib (e.g. 0.4 mg), rapamycin (e.g. 0.1 mg) or vehicle alone
was injected intraperitoneally. After about 30 minutes for the
rapamycin-treated mouse or about 10 min for the TORKinib and
vehicle treated mice, approximately 250 mU of insulin in about 100
.mu.l of saline was injected intraperitoneally. Typically 15
minutes after the insulin injection, the mice were sacrificed by
CO.sub.2 asphyxiation followed by cervical dislocation. Tissues
were harvested and frozen on liquid nitrogen in about 200 .mu.l of
cap lysis buffer. The frozen tissue was thawed on ice, manually
disrupted with a mortar and pestle, and then further processed with
a micro tissue-homogenizer (Fisher PowerGen 125 with Omni-Tip
probe). Protein concentration of the cleared lysate was measured by
Bradford assay and 5-10 .mu.g of protein was analyzed by western
blot. The results are shown in FIG. 12.
[0252] In fat and liver, TORKinib was able to completely inhibit
the phosphorylation of Akt at S473 and T308, under the conditions
tested, which is consistent with its effect on these
phosphorylation sites observed in cell culture. TORKinib was only
partially able to inhibit the phosphorylation of Akt in skeletal
muscle. Consistent with this finding, a muscle specific knockout of
the integral mTorC2 component rictor also resulted in only a
partial loss of Akt phosphorylation at S473. These results suggest
that a kinase other than mTor may contribute to phosphorylation of
Akt in muscle.
[0253] Rapamycin often stimulates the phosphorylation of Akt,
probably by relieving feedback inhibition from S6K to the insulin
receptor substrate 1 (IRS1), a key signaling molecule that links
activation of the insulin receptor to PI3K activation. In all
tissues examined, and especially in fat and muscle, acute rapamycin
treatment activated the phosphorylation of Akt at S473 and T308
(FIG. 12). In contrast to rapamycin, by inhibiting both mTorC2 and
mTorC1, TORKinib suppresses rather than enhances Akt activation
under the conditions tested.
[0254] As was seen in cell culture, rapamycin and TORKinib
differentially affect the mTorC1 substrates S6K and 4EBP1 in vitro.
S6 phosphorylation was equally inhibited by rapamycin and TORKinib
in all tissues examined. TORKinib was effective at blocking the
phosphorylation of 4EBP1 on both T37/46 and S65 in all tissues
examined. While rapamycin was more effective at inhibiting the
phosphorylation of 4EBP1 in vitro than in cell culture experiments,
rapamycin did not block 4EBP1 phosphorylation as completely as
TORKinib under the conditions tested.
[0255] Rapamycin has been a powerful pharmacological tool allowing
the discovery of mTor's central role in the control of protein
synthesis. Since the discovery of a rapamycin-insensitive mTor
complex there has been a significant effort to develop
pharmacological tools for studying this complex. Here two
structurally distinct compounds were used to chemically dissect the
effects of mTor kinase inhibition toward mTorC1 and mTorC2
activity. The results of the present invention has shown through
the use of these inhibitors that the inhibition of mTor kinase
activity is sufficient to prevent the phosphorylation of Akt at
S473, under the conditions tested, providing further evidence that
mTorC2 may be the kinase responsible for Akt hydrophobic motif
phosphorylation. The results disclosed herein further provide that
phosphorylation at T308 is probably linked to phosphorylation at
S473, as had been observed in experiments where mTorC2 was disabled
by RNAi, but not homologous recombination. However, inhibition of
mTorC2 does not result in a complete block of Akt signaling, as
T308P is partially maintained and Akt substrate phosphorylation is
only modestly affected when S473 is not phosphorylated under the
conditions tested. Despite its modest effect on Akt substrates,
TORKinib was a more effective anti-proliferative agent than
rapamycin. These results were reproduced even in cells lacking
mTorC2 (SIN1-/-), suggesting that downstream mTorC1 substrates
might be responsible for TORKinib's strong anti-proliferative
effects. The results disclosed herein provide that phosphorylation
of the mTorC1 substrate 4EBP1 was partially resistant to rapamycin
treatment at concentrations that fully inhibit p70S6K while
TORKinib completely inhibits both p70S6K and 4EBP1. Consequently,
the enhanced block of cell proliferation by TORKinib compared with
rapamycin may reflect in part its ability to more efficiently
inhibit eIF4E-dependent translation control.
Example 12
Kinase Signaling in Blood
[0256] PI3K/Akt/mTor signaling was measured in blood cells using
the phosflow method. The advantage of this method is that it is by
nature a single cell assay so that cellular heterogeneity can be
detected rather than population averages. This allows concurrent
distinction of signaling states in different populations defined by
other markers. Phosflow is also highly quantitative. To test the
effects of PP242 (TORKinib), unfractionated murine splenocytes were
stimulated with anti-CD3 to initiate T-cell receptor signaling. The
cells were then fixed and stained for surface markers and
intracellular phosphoproteins. The results showed that PP242
inhibits anti-CD3 mediated phosphorylation of Akt-S473 and S6,
whereas rapamycin inhibits S6 phosphorylation and enhances Akt
phosphorylation under the conditions tested.
[0257] Aliquots of whole blood were incubated for 15 minutes with
vehicle (e.g. 0.1% DMSO) or kinase inhibitors at various
concentrations, before addition of stimuli to crosslink the T cell
receptor (TCR) (anti-CD3 with secondary antibody) or the B cell
receptor (BCR) using anti-kappa light chain antibody (Fab'2
fragments). After approximately 5 and 15 minutes, samples were
fixed (e.g. with cold 4% paraformaldehyde) and used for phosflow.
Surface staining was used to distinguish T and B cells using
antibodies directed to cell surface markers that are known to the
art. Akt and S6 phosphorylation levels were then measured by
incubating the fixed cells with alexa fluor labeled antibodies
specific to the phosphorylated isoforms of these proteins. The
population of cells was then analyzed by flow cytometry. The
results are shown on FIG. 13.
Example 13
Effect of Kinase Inhibitors on Lymphocyte Proliferation
[0258] To determine the effects of PP242 on lymphocyte function,
purified murine T cells from lymph nodes and B cells from spleen
were measured for their proliferative response to antigen receptor
engagement in the presence or absence of various inhibitors. The
results are shown in FIG. 14.
[0259] Compared to rapamycin, TORKinib caused a more complete
suppression of lymphocyte proliferation under all conditions
tested, with IC.sub.50 values of approximately 100-300 nM. These
effects were similar to treatment with LY294002 (10 .mu.M), a non
specific PI3K/mTor inhibitor.
Example 14
Effect of Kinase Inhibitors on Leukemic Cell Viability
[0260] Murine B lymphoid progenitor cells transformed by a
retrovirus encoding human p190-BCR-ABL (p190 transduced cells) were
used to study the effect of kinase inhibitors on cell viability,
proliferation, and colony forming activity. P190 transduced cells
were cultured in duplicate for about 48 hrs in the presence of the
indicated drugs, and reduction of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium salt (MTS) was measured over the approximately the
last 2 hr. FIG. 15a shows mean IC.sub.50 values and the 95%
confidence interval. FIG. 15b shows the results of a representative
experiment.
[0261] FIG. 15 illustrates the effect of kinase inhibitors on p190
transduced cells on cell viability. The plateau effect seen at high
concentrations of IC87114 and rapamycin is due to the fact that
under the conditions tested, these two inhibitors are primarily
cytostatic, while the others tested appear to induce both cell
cycle arrest and apoptosis. These findings show that single
inhibition of PI3K or mTorC1 signaling nodes may be insufficient to
eradicate leukemia cells, whereas dual inhibition of both nodes is
sufficient to cause massive cell death.
Example 15
Effect of Combination Therapy
[0262] P190 transduced cells are highly sensitive to imatinib and
dasatinib. To test for drug synergism at submaximal doses of
imatinib, p190 transduced cells were treated with a range of drug
concentrations alone and in combination at fixed rations. Calcusyn
software was then used to determine the combination index according
to the method of Chou 2006 Pharmacological reviews 58:621-681. The
results as shown in FIG. 16 show that PP242 and BEZ235 both display
a moderate to strong synergy with imatinib over a broad dose
range.
Example 16
Method of Colony Forming Assay on Kinase Inhibitors with Mouse
Cells
[0263] Murine bone marrow cells freshly transformed with p190
BCR-Abl were plated in the presence of various drug combinations in
M3630 methylcellulose media for about 7 days with recombinant human
IL-7 in about 30% serum, and the number of colonies formed was
counted by visual examination under a microscope. The results as
shown in FIG. 17 show that, compared to rapamycin, PP242 and BEZ235
potentiate the effects of a half maximal concentration of imatinib
at the concentrations examined.
Example 17
Colony Forming Assay on Kinase Inhibitors with Human Cells
[0264] Human peripheral blood mononuclear cells were obtained from
philladelphia chromosome positive (Ph+) and negative (Ph-) patients
upon initial diagnosis or relapse. Live cells were isolated and
enriched for CD19+CD34+ B cell progenitors. After overnight liquid
culture, cells were plated in methocult GF+H4435, Stem Cell
Technologies) supplemented with cytokines (IL-3, IL-6, IL-7, G-CSF,
GM-CSF, CF, Flt3 ligand, and erythropoietin) and about 50 nM
dasatinib in combination with either rapamycin or PI-103. Colonies
were counted by microscopy 12-14 days later. The results as shown
in FIG. 18 show a stronger effect of dasatinib-PI-103 combination
as compared to dasatinib-rapamycin combination, although both
combinations show evidence of additive or synergistic activity.
Example 18
Effect of Combination Therapy
[0265] P190 Transduced cells were treated with various
concentrations of the drugs or drug combinations as indicated for
approximately 48 hrs, and reduction of MTS was measured over about
the last two hours. The results as shown in FIG. 19 show that both
rapamycin and PI-103 potentiate the anti-proliferative effects of
imatinib under the conditions tested. FIG. 20 shows that
combination index of imatinib in combination with rapamycin or
PI-103. This suggests that in combination with rapamycin, PI-103 is
more synergistic than rapamycin over a broad dose range under the
conditions tested.
Example 19
In Vivo Effect of Kinase Inhibitors, Spleen
[0266] The murine bone marrow infection/transplantation method was
used to determine the effect of kinase inhibitors on disregulated
cellular proliferation in vitro. The MSCV-p190-BCR-Abl-Ires-GFP
(MIG-p190) or MSCV-p190-BCR-Abl-Ires-hCD4.DELTA.tail (MIC-p190)
retroviral vectors were assembled in 293T cells and used to
transduce bone marrow cells. The GFP and CD4.DELTA.tail markers
allow quantitation by FACS analysis and/or enrichment by magnetic
sorting of transduced cells.
[0267] Female Balb/c recipient mice were lethally irradiated from a
.gamma. source in two doses about 4 hr apart, with approximately 5
Gy each. About 1 hr after the second radiation dose, mice were
injected i.v. with about 1.times.10.sup.6 leukemic cells from early
passage MIG-p190 or MIC p190 cultures. These cells were
administered together with a radioprotective dose of about
5.times.10.sup.6 normal bone marrow cells from 3-5 week old donor
Balb/c mice. Recipients were given antibiotics in the water and
monitored daily. Mice who became sick after about 14 days were
euthanized and lymphoid organs were harvested for analysis. Kinase
inhibitor treatment began about 10 days after leukemic cell
injection and continued daily until the mice became sick or a
maximum of approximately 35 days post-transplant. Inhibitors were
given by oral lavage.
[0268] FIG. 21 shows the results of treatment with imatinib,
rapamycin, imatinib in combination with rapamycin and imatinib in
combination with PI-103. The results show a significant decrease in
splenomegaly with kinase inhibitor. The results also show the
enhanced beneficial effect of combination therapy under the
conditions tested.
Example 20
In Vivo Effect of Kinase Inhibitors, Peripheral Blood
[0269] Female Balb/c recipient mice were lethally irradiated from a
y source in two doses about 4 hr apart, with approximately 5 Gy
each. About 1 hr after the second radiation dose, mice were
injected i.v. with about 1.times.10.sup.6 leukemic cells from early
passage MIG-p190 or MIC p190 cultures. These cells were
administered together with a radioprotective dose of about
5.times.10.sup.6 normal bone marrow cells from 3-5 week old donor
Balb/c mice. Recipients were given antibiotics in the water and
monitored daily. Mice who became sick after about 14 days were
euthanized and lymphoid organs were harvested for analysis. Kinase
inhibitor treatment began about 10 days after leukemic cell
injection and continued daily until the mice became sick or a
maximum of approximately 35 days post-transplant. Inhibitors were
given by oral lavage.
[0270] Peripheral blood cells were collected approximately on day
10 (pre-treatment) and upon euthanization (post treatment),
contacted with labeled anti-hCD4 antibodies and counted by flow
cytometry. The results as shown in FIG. 22 show that combination
therapy significantly reduced leukemic blood cell counts as
compared to treatment with rapamycin, imatinib, or PI-103 alone
under the conditions tested.
Example 21
In Vivo Effect of Kinase Inhibitors, Bone Marrow and Spleen
[0271] Female Balb/c recipient mice were lethally irradiated from a
y source in two doses about 4 hr apart, with approximately 5 Gy
each. About 1 hr after the second radiation dose, mice were
injected i.v. with about 1.times.10.sup.6 leukemic cells from early
passage MIG-p190 or MIC p190 cultures. These cells were
administered together with a radioprotective dose of about
5.times.10.sup.6 normal bone marrow cells from 3-5 week old donor
Balb/c mice. Recipients were given antibiotics in the water and
monitored daily. Mice who became sick after about 14 days were
euthanized and lymphoid organs were harvested for analysis. Kinase
inhibitor treatment began about 10 days after leukemic cell
injection and continued daily until the mice became sick or a
maximum of approximately 35 days post-transplant. Inhibitors were
given by oral lavage.
[0272] Mice were injected with the BrdU analogue EdU and
sacrificed. Bone marrow and spleen were collected, blast cells were
harvested from the tissue, and stained with antibodies to hCD4, and
analyzed by flow cytometry. The results as shown in FIG. 23 show
that under the conditions tested in both bone marrow and spleen,
imatinib, rapamycin, and PI-103 significantly reduced the number of
cycling leukemic blasts as compared to control. Furthermore,
combination therapy of imatinib plus rapamycin or PI-103
significantly reduced the number of cycling leukemic blasts as
compared to single drug inhibition at the concentrations
examined.
Example 22
In Vivo Effect of Kinase Inhibitors on Apoptosis of Leukemic
Cells
[0273] Female Balb/c recipient mice were lethally irradiated from a
y source in two doses about 4 hr apart, with approximately 5 Gy
each. About 1 hr after the second radiation dose, mice were
injected i.v. with about 1.times.10.sup.6 leukemic cells from early
passage MIG-p190 or MIC p190 cultures. These cells were
administered together with a radioprotective dose of about
5.times.10.sup.6 normal bone marrow cells from 3-5 week old donor
Balb/c mice. Recipients were given antibiotics in the water and
monitored daily. Mice who became sick after about 14 days were
euthanized and lymphoid organs were harvested for analysis. Kinase
inhibitor treatment began about 10 days after leukemic cell
injection and continued daily until the mice became sick or a
maximum of approximately 35 days post-transplant. Inhibitors were
given by oral lavage.
[0274] Bone marrow and spleen were collected, blast cells were
harvested from the tissue, and stained with antibodies to hCD4,
Annexin V, and 7AAD, and analyzed by flow cytometry. The results as
shown in FIG. 24 show induction of apoptosis in leukemic blast
cells of the bone marrow and spleen by imatinib, rapamycin, and
PI-103 under the conditions tested. The results also show
significant increase at the concentrations examined in induction of
apoptosis at the concentrations examined of imatinib in combination
with rapamycin or PI-103.
Example 23
Measurement of CD98 Surface Expression
[0275] Flow cytometry was used to measure the surface expression of
CD98, a component of an essential amino acid transporter. Rapamycin
treatment, or genetic deletion of class IA PI3K, each reduced CD98
expression and these effects were additive FIG. 25. Although the
effects were quantitatively modest, small changes in nutrient
availability can have a major impact on cellular bioenergetics
(imagine reducing your food intake by 20-25%). The observation that
PI3K and mTorC1 contribute independently to CD98 expression
provides strong rationale for studying further the regulation of
nutrient uptake by PI3K and different mTor complexes.
Example 24
Treatment of Leukemic Mice with Kinase Inhibitors
[0276] Female Balb/c recipient mice are lethally irradiated from a
y source in two doses about 4 hr apart, with approximately 5 Gy
each. About 1 hr after the second radiation dose, mice are injected
i.v. with about 1.times.10.sup.6 leukemic cells from early passage
MIG-p190 or MIC p190 cultures. Alternatively, NOG mice (Jackson
labs) are irradiated and injected with human Ph+acute lymphocytic
leukemia cells. These cells are administered together with a
radioprotective dose of about 5.times.10.sup.6 normal bone marrow
cells from 3-5 week old donor Balb/c mice. Recipients are given
antibiotics in the water and monitored daily. Mice that become sick
after about 14 days are euthanized and lymphoid organs are
harvested for analysis. Kinase inhibitor (e.g. mTor inhibitor,
dasatinib, imatinib etc.) or chemotherapeutic agent treatment
begins about 10 days after leukemic cell injection and continues
daily until the mice become sick or a maximum of approximately 35
days post-transplant Inhibitors are given by oral lavage.
[0277] Mice are optionally injected with EdU, and sacrificed.
Peripheral blood, bone marrow and spleen are harvested for analysis
and hCD4+ cells are optionally enriched for. Analysis includes
phosflow analysis, western blot, flow cytometry, analysis of CD98
expression, or Annexin V or 7-AAD staining. It is expected that the
results of treatment of the leukemic mice with mTor inhibitors show
decreased leukemic cell counts in peripheral blood and lymphoid
tissues, decreased CD98 expression, increased Annexin V or 7-AAD
staining, decreased phosphorylation of PI3K pathway targets, and
increased apoptosis of leukemic cells in response to the drugs
administered. It is also expected that the results of treatment of
the leukemic mice with a combination of mTor inhibitors and other
therapeutic agents provide increased efficacy above that achieved
with a single agent.
Example 25
Treatment of Lupus Disease Model Mice
[0278] Mice lacking the inhibitory receptor Fc.gamma.RIIb that
opposes PI3K signaling in B cells develop lupus with high
penetrance. Fc.gamma.RIIb knockout mice (R2KO, Jackson Labs) are
considered a valid model of the human disease as some lupus
patients show decreased expression or function of
Fc.gamma.RIIb.
[0279] The R2KO mice develop lupus-like disease with anti-nuclear
antibodies, glomerulonephritis and proteinurea within about 4-6
months of age. For these experiments, the rapamycin analogue RAD001
(available from LC Laboratories) is used as a benchmark compound,
and administered p.o. This compound has been shown to ameliorate
lupus symptoms in the B6.Sle1z.Sle3z model.
[0280] Lupus disease model mice such as R2KO, BXSB or MLR/lpr are
treated (groups of 10) at about 2 months old, approximately 4 times
per week for a period of about two months. Mice are given p.o.
doses of: (1) vehicle (2) RAD001, about 10 mg/kg (3) PP242 (or
analog), approximately 10 mg/kg (4) PP242, about 50 mg/kg. Blood
and urine samples are obtained at approximately day 0, 30 and 60,
and tested for antinuclear antibodies (in dilutions of serum) or
protein concentration (in urine) as described. Serum is also tested
for anti-ssDNA and anti-dsDNA antibodies by ELISA. One set of about
5 animals is euthanized at day 60 and tissues harvested for
measuring spleen weight and kidney disease. Glomerulonephritis is
assessed in kidney sections stained with H&E, scoring on a
scale of 1-4. Another set of about 5 mice from each group is
studied at 6 months of age, 60 days after cessation of treatment,
using the same endpoints.
[0281] The results of this experiment demonstrate that mTor
inhibitors can suppress or delay the onset of lupus symptoms in
lupus disease model mice. In comparison to RAD001, it is expected
that kinase inhibitors disclosed herein (e.g. PP242, PP30, PI-103,
PIK-90) perform as well or better to suppress autoimmunity than an
established mTorC1 inhibitor.
Example 26
Effect of mTor Inhibitors on In Vivo T and B Cell Function
[0282] Groups of about 5 WT mice (generally 6 wk old) are given
daily i.p. injections of rapamycin (about 7 mg/kg) or vehicle, or
daily oral administration of kinase inhibitors disclosed herein
(e.g. PP242, PP30, PI-103, PIK-90, or an analogue of the compounds
herein) or vehicle. Lymphoid tissues (BM, thymus, LN, spleen) are
analyzed with standard panels of antibodies to quantify percentages
of different B and T cell progenitor and mature subsets. Typically
2 hr before euthanasia, mice are given an i.p. injection of EdU to
allow measurement of DNA synthesis rates in different subsets by
FACS.
[0283] The same drug treatment regime is used to analyze effects on
the following model immune responses. For B cell function, mice are
immunized with a T-independent antigen (NP-Ficoll) and serum
collected about 7 days later for quantification of NP-specific
antibodies by ELISA. For T and B cell collaboration, mice are
immunized with NP-ovalbumin and serum tested for NP-specific
antibodies on about day 14, with germinal center formation analyzed
in spleen sections by immunofluorescence. For CD4 T cell function,
DO11.10 TCR-Tg cells are purified, labeled with CFSE, and
approximately 2.5.times.10.sup.6 cells injected into wild-type
recipients before immunization with about 100 .mu.g ovalbumin
protein in adjuvant. About 3 days later (+/-drug treatment), spleen
and LN are harvested and in vitro expansion assessed by CFSE
dilution. It is expected that the results of this experiment
provide a reasonably comprehensive survey of drug effects on
lymphocyte development, proliferation, signaling and immune
responsiveness.
Example 27
Treatment of Leukemic Cells with Kinase Inhibitors
[0284] p190 cells in 96-well plates are treated with 2-fold
dilutions of dasatinib or imatinib over a range above and below its
IC.sub.50 (e.g. 0, 5, 10, 20, 40, 80, 160, 320 nM) with one
concentration per row. Columns of the plates contain 2-fold
dilutions of either rapamycin, PP242, IC or BEZ235 around their
respective IC.sub.50 values. Controls include wells with untreated
cells, or with vehicle. MTS reagent is added to all wells during
about the last 2 hr of an approximately 48 hr incubation, then
absorbance quantified using a microplate reader. Synergy studies
are also done using both wild-type (WT) p190 and the mutants
p190-Y253F, p190-E255K and p190-T3151. Calcusyn software is used to
calculate IC.sub.50 values for individual compounds and to compute
combination indeces to distinguish synergy, additivity or
antagonism.
[0285] The results of the synergy experiments are expected to
provide a limited set of single drug and combination treatments to
measure cell proliferation and apoptosis by flow cytometry
(FACS).
Example 28
Effect of mTor Inhibitors on BCR-Abl+ Cells
[0286] WT p190 cells are treated with about IC.sub.50 or
approximately 5.times.IC.sub.50 concentrations of individual drugs
or vehicle for about 18 hr, with or without IC.sub.50 concentration
of dasatinib, imatinib, or other chemotherapeutic agent (e.g.
PP242, PI-103, PIK-90, or analogue of the compounds herein). DNA
content analysis is used to quantify the fraction of cells in G0/G1
vs. S and G2/M phases, and to estimate apoptotic fraction using the
sub-diploid peak. As a more precise and temporal measurement of
apoptosis, caspase-3 cleavage at about 6 hr and about 18 hr using
intracellular staining and FACS is assayed. Immunoblots are also
used to measure expression of selected proteins as correlates of
cell cycle progression vs. arrest. p190 cells are treated for about
2, 6 and 18 hrs and then lysates prepared and probed for expression
of c-Myc (known mitogenic function in BCR-ABL-transformed cells),
D-type cyclins (whose expression is regulated by PI3K and mTor) and
p27kip (inhibitor of cyclin-dependent kinases whose expression is
reduced by PI3K/Akt through FOXO inhibition).
[0287] It is expected that patterns of drug potency in p190 cells
can be generalized to other BCR-ABL+ cells. Two additional cell
lines are used to confirm this. BaF3-p190 cells are generated by
infecting the IL-3-dependent BaF3 pro-B cell line with p190
retrovirus. This renders cells IL-3-independent but completely
dependent on BCR-ABL kinase activity. K562 cells are derived from a
human patient in Ph+ CML blast crisis. Cell cycle and MTS assays of
these cell lines in the presence of the inhibitors disclosed herein
are used to monitor drug potency.
[0288] It is expected that the data generated establish the potency
of mTor inhibitor in suppressing BCR-ABL-driven proliferation and
survival in three cellular models, including cells expressing WT
BCR-ABL or mutants resistant to dasatinib and/or imatinib. The
formal synergy studies provide a rigorous comparison of the
efficacy of PP242 (or analogue) vs. a mTorC1 inhibitor (rapamycin)
or a dual PI3K/mTor inhibitor (BEZ235), when given in combination
with a clinically used ABL kinase inhibitor.
Example 29
Comprehensive Survey of Kinase Substrate Phosphorylation of Treated
Cells
[0289] To obtain a more comprehensive and quantitative survey of
cellular protein phosphorylation at different concentrations of
PP242, a Typhoon molecular imager is used to analyze immunoblots of
treated cells. Using different dyes coupled to antibodies (Abs)
raised in different species, specific phosphorylation and total
target protein is measured on the same blot. Similarly,
immunofluorescent staining with anti-PIP3 monoclonal antibodies
allows measurement of inositol phosphorylation. All of the required
Abs are available from Cell Signaling Technologies and other
vendors. p190 cells are treated with vehicle alone or with
different agents for about 2 hr prior to preparation of cell
lysates. Each gel typically contain the following samples: vehicle
control; rapamycin (about 40 nM=5.times.IC.sub.50); PP242 (about 13
nM=IC.sub.50, 65 nM, 325 nm); BEZ235 (about 5 nM=IC.sub.50, about
30 nM, about 150 nM); IC (about 10 .mu.M=5.times.IC.sub.50);
dasatinib (about IC.sub.50); dasatinib+rapamycin; dasatinib+PP242
(about 65 nM); dasatinib+BEZ (30 nM); dasatinib+IC87114.
[0290] It is expected that PP242 inhibits Akt phosphorylation at
S473, and the "turn motif" in Akt (T450) and PKC isoforms as well
as inhibits phosphorylation of Akt substrates such as for example
FOXO1/FOXO3 (on residues T24/T32), TSC2 (T1462) and GSK3 (S9/21).
It is also expected that the results show diminished
phosphorylation of mTor substrates 4EBP1 phosphorylationT37/45 and
S65. It is further expected that the results demonstrate enhanced
phosphorylation of ERK1/ERK2 (T202/Y204) and its upstream kinase
MEK (S217/S221) in treated cells.
[0291] A particular goal of this experiment is to define
intermediate concentrations of dasatinib and PP242 that synergize
to suppress mTor activity and kill p190 cells without strongly
diminishing PIP3 production and p Akt-T308. Accordingly, a
dasatinib-PP242 combination may have anti-leukemic effects with
limited toxicity to normal cells whose signaling is not driven by
BCR-ABL.
[0292] It is further expected that the status of MAP kinase and
JAK/STAT pathways defines potential compensatory changes that occur
in response to mTorC1/mTorC2 inhibition. Again, the results of this
experiment define drug combinations that achieve cell killing
without augmenting other mitogenic signals. Significant changes in
flux through other signaling pathways may reflect off-target
effects of PP242 rather than compensatory rewiring. In this case,
comparison of structural analogs of PP242 may provide a related
compound with better selectivity.
Example 30
Effect of Inhibitors on Cellular Metabolism
[0293] p190 cells are treated for about 6 and about 18 hr with
vehicle alone or the following inhibitor treatments: rapamycin
(about 40 nM); PP242 (about 65 nM); BEZ235 (about 30 nM); IC (about
10 .mu.M); dasatinib (approximately IC.sub.50); dasatinib+rapamycin
(about 40 nM); dasatinib+PP242 (about 65 nM); dasatinib+BEZ (about
30 nM); dasatinib+IC (about 10 .mu.M). Cells are analyzed by flow
cytometry for surface expression of CD98 and CD71 (transferrin
receptor), CD19 and B220 as negative controls whose expression
should be unaltered. Cell samples are also fixed, permeabilized and
stained for immunofluorescence microscopy (IFM) using antibodies to
the glucose transporter Glut1 (compared to isotype control Ab).
Available antibodies to Glut1 distinguish intracellular vs. surface
expression by IFM but are not suitable for flow cytometry. Glucose
uptake is measured based on incorporation of 3H-2-deoxyglucose.
Glycolytic rate is measured by two assays: conversion of
5-3H-glucose to 3H2O, and lactate accumulation in the media. AMP
kinase phosphorylation is assayed by immunoblot.
[0294] It is expected that the results of this experiment provide
effective concentrations at which cell metabolism is adversely
affected in leukemic cells. The results of this experiment and
example 30 further help determine which signaling events are
disabled at drug concentrations that induce cell cycle arrest
and/or apoptosis. It is expected that the anti-proliferative and
pro-apoptotic effects of PP242 correlate with reduced p Akt-S473
and stronger suppression of p4EBP1 relative to rapamycin.
Alternatively the results may provide that the greater potency of
PP242 vs. rapamycin in leukemia cells correlates with differential
effects on p Akt-S473.
[0295] Another important outcome of examples 30 and 31 is the
definition of biomarkers that can be used for pharmacodynamic
assays in animal models and in human clinical studies. Phospho-Akt
and phospho-S6 are commonly used as biomarkers of PI3K and mTorC1
activity for cancer studies in animals and humans. It is expected
that the results of examples 30 and 31 establish a set of
phosphorylation sites whose dephosphorylation correlates best with
cellular potency of PP242 compared to rapamycin. Having a set of
candidate biomarkers facilitates the development of the most
sensitive and simple techniques for pharmacodynamic monitoring, for
example by FACS instead of immunoblot. It is further expected that
p4EBP1 and p Akt-S473 are useful biomarkers, and possibly some
other Akt substrates. Studies of metabolic effects might provide
other biomarkers such as altered nutrient transport expression
(e.g. CD98).
Example 31
Effect of Dasatinib and mTor Inhibitors on Leukemic Cells
[0296] The experiments described in example 16, are performed with
two important differences. In one set of experiments, dasatinib
instead of imatinib as the ABL kinase inhibitor is used. p190
transduced cells are treated with a range of dasatinib
concentrations (e.g. 4, 12, 40, 120 nM)+/-PP242 (about 13 nM and
about 65 nM) or rapamycin (e.g. at 40 nM) to determine an
approximate IC.sub.50 for dasatinib. These compounds are then
tested alone or together in various combinations at a dasatinib
concentration of about the IC.sub.50 and about 5.times.IC.sub.50.
It is expected that the results of the experiment provide
therapeutic concentrations at which leukemic cell colony formation
is inhibited. It is further expected that the combination of
dasatinib with mTor inhibitors provide enhanced inhibition of
colony formation at lower dasatinib doses. This suggests that
combination therapy is an advantageous strategy for treating
B-ALL.
Example 32
Ph+B-ALL Colony Formation Assays
[0297] Peripheral blood mononuclear cells (PBMC) are obtained from
human leukemia patients. Patients who have pathologically confirmed
B-ALL or CML with B lymphoid blast crisis (CML-BC) are asked to
participate in the study. Consented patients provide up to seven
extra tubes containing a total of about 30 ml of blood. Live PBMC
are isolated by density gradient centrifugation, resuspended in
media (typically IMDM/30% FCS/1.times. pen-strep) and frozen in
approximately 1 ml aliquots of about 5-10.times.106 cells
(typically in 15% DMSO/85% medium). After cytogenetic diagnosis,
experiments are performed with Ph+ cells. Ph- cells are saved and
studied in further experiments described herein. Colony assays are
performed in sets of 3-4 patient samples, using the protocol
described for example 17. In each assay, duplicate wells of 48-well
plates are seeded with about 5.times.104 cells (sorted CD19+CD34+)
and scored after 12-14 days of growth. Treatment groups are vehicle
alone, dasatinib (IC.sub.50 and 5.times.IC.sub.50), rapamycin
(about 40 nM), PP242 (about 13 nM and about 65 nM), dasatinib
(IC.sub.50)+rapamycin (about 40 nM), dasatinib (IC.sub.50)+PP242
(about 13 nM), dasatinib (IC.sub.50)+PP242 (about 65 nM). About
9-12 Ph+ALL patient samples are studied. It is expected that the
results of the experiment provide therapeutic concentrations at
which leukemic cell colony formation is inhibited. It is further
expected that the combination of dasatinib with mTor inhibitors
provide enhanced inhibition of colony formation at lower dasatinib
doses. This suggests that combination therapy is an advantageous
strategy for treating B-ALL.
Example 33
Normal Human Progenitor Assays
[0298] Normal human CD34+ bone marrow cells (Lonza) are purchased.
Cells are plated in Methocult H4434 that contains complete
cytokines to support growth of myeloid and erythroid cells. The
effects of drugs and combinations, using the same concentrations of
dasatinib, rapamycin and PP242 listed in Example 32. After 14-16
days, the number of colonies of different type (CFU-E, BFU-E,
CFU-GM, CFU-G, CFU-M and CFU-GEMM) is scored by light microscopy
using instructions supplied by the manufacturer (Stem Cell). In
these experiments samples treated with BEZ235 or dasatinib+BEZ235
are included to determine if mechanistic differences identified in
examples 30 and 31 correlate with differences in toxicity to
nonleukemic cells.
Example 34
Colony Assay Using Ph- B-ALL and AML
[0299] Human Ph- B-ALL and AML specimens are obtained. Ph- B-ALL
and AML are cytogenetically diverse, with diverse therapeutic
regimens currently in use depending on patient risk factors. The
majority of Ph- B-ALL patients are treated initially with a
combination of three drugs: glucocorticoid (e.g. dexamethasone),
vincristine, and asparaginase or anthracycline (e.g. daunorubicin).
Most AML patients receive an anthracycline only. Pilot experiments
with 3 patient samples are conducted to determine concentrations of
vincristine (Ph- ALL) or daunorubicin (AML) that partially reduce
colony formation in methylcellulose. It is expected that these
drugs are effective in the range of 0.1-1 .mu.M (vincristine) and
3-30 nM (daunorubicin). Optionally, the Ph- ALL assays may be
supplemented with dexamethasone and/or daunorubicin. 9-12
independent leukemia specimens of each time in colony assays using
chemotherapeutic agent alone or together with PP242 (13 nM and 65
nM) or rapamycin (40 nM) are then tested at optimized vincristine
or daunorubicin concentrations.
[0300] It is expected that PP242 shows greater efficacy than
rapamycin in suppressing colony formation, although a considerable
degree of variability among patients is anticipated. However, the
chosen sample size (9-12) is sufficient to establish whether any
trends are statistically significant. It is further expected that
mTor inhibitor may have anti-leukemic effects in acute leukemias
that are not driven by BCR-ABL (e.g. AML).
Example 35
Murine Bone Marrow Transplant Assay
[0301] Female Balb/c recipient mice are lethally irradiated from a
y ray source in two doses about 4 hr apart, at approximately 5 Gy
each. About 1 hr after the second radiation dose, mice are injected
i.v. with about 1.times.106 leukemic cells from early passage
MIG-p190 or MIC-p190 cultures. These cells are administered
together with a radioprotective dose of approximately 5.times.106
normal BM cells from 3-5 wk old donor Balb/c mice. Recipients are
given antibiotics in the water and monitored daily. On rare
occasions mice become moribund in the first 14 days due to failed
engraftment or infection, and are euthanized. Mice who become sick
after about 14 days are euthanized and lymphoid organs harvested
for FACS analysis and/or magnetic enrichment. Treatment begins on
approximately day 10 and continues daily until mice become sick, or
after a maximum of about 35 days post-transplant. Drugs are given
by oral gavage (p.o.). In a pilot experiment a dose of dasatinib
that is not curative but delays leukemia onset by about one week or
less is identified; controls are vehicle-treated or treated with
imatinib (about 70 mg/kg twice daily), previously shown by us and
others to delay but not cure leukemogenesis in this model. For the
PP242 experiment groups of about 8 mice per treatment are studied.
These groups are: (1) vehicle (2) dasatinib (3) rapamycin, about 7
mg/kg/day i.p. (4-6) PP242 at about 10 mg/kg, about 30 mg/kg, about
60 mg/kg p.o., twice daily (7) dasatinib+rapamycin (8-10)
dasatinib+PP242. For feasibility, the experiment is carried out in
two phases, with about 4 mice per group in each phase. For the
first phase MIG-p190 cells are used, and postmortem analysis is
limited to enumeration of the percentage of leukemic cells in BM,
spleen and lymph node (LN) by flow cytometry. In the second phase,
MIC-p190 cells are used and the postmortem analysis includes
magnetic sorting of hCD4+ cells from spleen followed by immunoblot
analysis of key signaling endpoints: p Akt-T308 and S473; pS6 and
p4EBP-1. As controls for immunoblot detection, sorted cells are
incubated in the presence or absence of pathway inhibitors before
lysis. Optionally, "phosflow" is used to detect p Akt-S473 and
pS6-S235/236 in hCD4-gated cells without prior sorting. These
signaling studies are particularly useful if, for example,
drug-treated mice have not developed clinical leukemia at the 35
day time point. Kaplan-Meier plots of survival are generated and
statistical analysis done according to methods known in the art.
Results from MIG-p190 and MIC-p190 cells are analyzed separated as
well as cumulatively.
[0302] Samples of peripheral blood (100-200 .mu.l) are obtained
weekly from all mice, starting on day 10 immediately prior to
commencing treatment. Plasma is used for measuring drug
concentrations, and cells are analyzed for leukemia markers (eGFP
or hCD4) and signaling biomarkers as described herein.
[0303] It is expected that the results of the analysis demonstrate
effective therapeutic doses of the biological agents disclosed
herein for inhibiting the proliferation of leukemic cells. It is
further expected that combination therapy of the mTor inhibitors
disclosed herein with other chemotherapeutic agents (e.g.
dasatinib) exhibit a greater degree of efficacy or decreased
toxicity in comparison to the use of a single chemotherapeutic
agent.
Example 36
Murine Xenograft Model
[0304] Xenografts of human leukemia cells can expand and cause
lethal disease when injected into immunocompromised mice. This
provides a useful preclinical system for studying whether in vitro
drug delivery can affect the expansion of leukemic cells in human
patient material. The NOG mouse strain is used as recipients. NOG
mice (available from Jackson labs) are derived from crosses between
non-obese diabetic (NOD), severe combined immunodeficient (SCID)
and common gamma-chain knockout mice. The strain has more complete
ablation of mature T and B cells and NK cell activity compared to
NOD-SCID, and is now used commonly for human xenograft studies.
These mice are radiation-sensitive due to the SCID mutation, but
require only 3 Gy of irradiation to create bone marrow space for
transplant engraftment. Groups of 2 NOG mice are injected with
approximately 1.times.106 Ph+B-ALL cells (sorted CD19+CD34+) from
at least 5 different patients. Mice are monitored daily for signs
of illness, and bled twice weekly (about 50 .mu.l, from tail vein)
to monitor the percentage of hCD19+ cells in peripheral blood. It
is expected that this experiment, will provide at least 2 human
Ph+B-ALL samples that cause progressive leukemia in NOG mice, to
use in a drug treatment study as described herein.
[0305] Recipient mice are bled twice weekly (about 50 .mu.l, from
for example the tail vein) until more than about 5% of peripheral
WBC are hCD19+. At that time daily drug treatments are initiated
for about 14 days. Blood samples are obtained twice weekly to
quantify the % hCD19+ over time. Mice are monitored daily for signs
of illness and euthanized if necessary. NOG mice are studied in
groups of about 5 animals per human donor cell sample: (1) vehicle
(2) dasatinib only (3) rapamycin only (4) PP242 at a dose that
shows efficacy in the p190 model (5) dasatinib+rapamycin (6)
dasatinib+PP242. At the end of the 14 day treatment period, or when
mice become sick, mice are injected i.p. with EdU (BrdU analog with
improved detection) 2 hr before sacrifice. Lymphoid organs are
harvested and FACS analysis is used to quantify % hCD19+ along with
% dividing (EdU+) and % apoptotic (7AAD-/AnnexinV+). p Akt-S473 and
pS6 in splenic hCD19+ cells is also assessed, and other biomarkers
of signaling and metabolism as developed in example 30.
[0306] It is expected that the experiments in examples 35 and 36
provide in-depth and complementary information about the
anti-leukemic effects of PP242 in comparison to rapamycin, and in
combination with dasatinib. A key aspect of example 35 is the
leukemia survival endpoint, it is expected that PP242 can achieve
the ultimate goal of delaying or preventing mortality. By comparing
different PP242 doses in the p190 system the results disclosed
herein define a minimum effective dose while also obtaining useful
information about pharmacokinetics (PK) and pharmacodynamics (PD)
from blood samples as described. The studies in example 36 have the
advantage of assessing efficacy on primary human, rather than
mouse, leukemia cells. In addition, the modified treatment and
analysis scheme provide information about leukemia cell
proliferation and survival to correlate with data on total leukemic
burden. In both types of experiment we expect to obtain information
about signaling status of leukemia cells postmortem.
Example 37
Non Invasive Sampling
[0307] Clinical trials and patient management benefit from
noninvasive sampling procedures to determine drug efficacy and
distinguish on-target and off-target effects. Aliquots of whole
blood (WB) are incubated for 15 min with vehicle or drugs at
various concentrations, before addition of stimuli to crosslink the
TCR (anti-CD3 with secondary Ab) or the B cell receptor (BCR) using
anti-kappa light chain Ab (F(ab')2 fragments). After about 5 and
about 15 min, samples are typically fixed with cold 4%
paraformaldehyde and used for phosflow. Surface staining is used to
distinguish T and B cells, with the nonresponding population as an
internal control. The signaling endpoints include p Akt and pS6
along with a control response that is not affected by mTor
inhibition (i.e. pSTAT5).
[0308] Whole blood assays for mouse lymphocytes are then used to
measure pharmacodynamic (PD) activity of selected compounds in
mice. As described above, blood samples are obtained weekly from
mice in the p190 model. Using 4-color staining, mouse T cells, B
cells, leukemia cells (GFP or hCD4) and one intracellular
phosphoprotein per sample are distinguished in flow cytometry
analysis. At early time points the blood contains too few leukemia
cells to quantify signaling, so the signaling response of host T or
B cells acts as a surrogate for PD of the drugs.
[0309] It is expected that the experiment allows convenient
monitoring of signaling states in peripheral lymphocytes and
leukemia cells over the course of in vitro drug treatments,
providing a powerful tool for correlating therapeutic efficacy with
on-target vs. off-target molecular effects. Mice treated with
dasatinib (+/-rapamycin or PP242) might exhibit greater suppression
of signaling in the leukemia compartment (BCR-ABL-dependent)
compared to the normal lymphocytes. On the other hand, both
imatinib and dasatinib have been shown to suppress signaling and
proliferation of normal lymphocytes due to effects on cellular
kinases of the ABL and SRC families. These experiments also provide
proof-of-principle and a reference dataset for the incorporation of
blood sampling approaches in clinical trials.
Example 38
Non Invasive Imaging
[0310] The practice of medicine, and oncology in particular, has
been revolutionized by noninvasive imaging approaches. The use of
PET scanning with 18-fluorodeoxyglucose (18FDG) as a positron
emitter is commonly used for cancer detection, based on the high
glucose uptake rates of cancer cells originally observed by
Warburg. This approach has been used to monitor treatment responses
in Ph+CML. Increased glucose uptake is driven in part by elevated
PI3K/Akt and mTor signaling in cancer cells. Indeed, rapmycin
analogs diminished 18FDG in a glioma model, which correlated with
suppression of tumor growth. A miniature version of the PET scanner
apparatus ("microPET") can be used to image leukemia development in
mice. Thus, microPET is an excellent preclinical tool for
monitoring leukemia responses to mTor inhibitors and for
establishing protocols that are useful in human clinical
trials.
[0311] The p190 leukemia model is used to allow stable expression
of genetically encoded enzymes for imaging applications. A
concentration of mTor inhibitor (e.g. PP242) that augments the
anti-leukemic effect of dasatinib and, optionally, has some effect
on its own is used. 2 replicate experiments of 2 mice per
treatment: (1) vehicle (2) dasatinib (3) rapamycin (4) PP242 (5)
dasatinib+rapamycin (6) dasatinib+PP242 are performed. Drug
treatments start on day 10 post-transplantation, and mice are
imaged on about day 9 and 14, then weekly (e.g. day 21, etc) for
surviving healthy animals until about day 35. Mice are fasted
overnight, then injected with 18FDG i.v. about 30 min prior to
anesthetization (typically with ketamine/xylazine). Serial microPET
scans are obtained, and the data normalized and used for
3-dimensional reconstructions using methods known in the art.
[0312] It is expected that microPET scanning data correlates with
other measures of disease development, with lower 18FDG uptake in
animals treated with anti-leukemic drug combinations. One concern
with 18FDG is that uptake could be diminished in cancer cells that
are nonetheless expanding due to an altered metabolic profile. This
is controlled for by analyzing a separate cohort of mice
transplanted with leukemia cells expressing a PET reporter gene,
e.g. Herpes Simplex Virus Thymidine Kinase (sr39 mutant). This TK
mutant has a high affinity for acycloguanosines such as the PET
radiotracer 9-(4-[18F]-fluoro-3 hydroxymethylbutyl)guanine (FHBG).
Alternatively, imaging is performed via the use of p190 cells
superinfected with a virus expressing luciferase to monitor disease
progression noninvasively by bioluminescence.
Example 39
Pharmacokinetic and Toxicology Studies of mTor Inhibitors
[0313] The present invention provides PP242 ADME properties
(adsorption, distribution, metabolism and excretion) and a
preliminary toxicity profile. The compound exhibits good drug-like
properties: oral bioavailability (F %30-70) with moderate clearance
and good volume of distribution; tolerated at 10, 30 or 100 mg/kg
in mice and rats (four times or twice daily dosing, p.o.);
metabolically stable in microsome and cellular assays; minimal
activity in ligand binding receptor profiling screen at 10 .mu.M
(CEREP Lead Profiling Screen.RTM.); highly selective in screen of
220 protein and lipid kinases. PP242 has a clean profile in genetic
toxicology (mini-Ames test) and hERG (potassium channel) test.
[0314] Prior to toxicology (TOX) studies a PK dose escalation study
is performed to prove increasing plasma concentration upon higher
dosages by the preferred route of administration (p.o.). Also, a
formulation allowing high exposures is tested, optimized and
confirmed to be non toxic. Prior to or in parallel to the first
rodent in vitro TOX study, a compound metabolite identification
across species (mice/rat/dog/pig/non-human primate (monkey) and
human) is initiated to select the appropriate non-rodent species
for subsequent in vitro TOX. The maximum tolerated dose of PP242
and a biologically active analog is determined, in a rodent species
(e.g. rats etc). About 3 rats/sex/group receive one of
approximately 5 dose strengths for about 4 days, and are monitored
for clinical signs, body weight changes, and gross necropsy
findings. Generally, a 10 day repeat-dose experiment using about 5
rats/sex/group, with toxicokinetic monitoring of plasma, and
necropsy on day about 11 is then performed. Finally, an
approximately 28-day repeat dose with about a 14-day recovery
experiment is performed, using about 3 dose strengths plus vehicle
control (N=10/sex/dose+5/sex for high-dose recovery). Clinical
signs, body weight changes, food consumption, gross necropsy
findings, clinical pathology (hematology, coagulation, clinical
chemistry, & urinalysis), histopathology (full tissues for high
dose and controls), and toxicokinetic monitoring are included. It
is expected that the results of this experiment will define the
severely toxic dose to 10% of the rodents. Following the rodent
studies, a similar progression of 3 TOX protocols is carried out in
a non-rodent species (e.g. dog, possibly pig or non-human primate)
although with smaller group size (for 4-day and 10-day studies,
N=1/sex/dose; for 28-day study, N=3/sex/dose+N=2/sex for high dose
recovery). Electrocardiogram and ophthalmological examinations are
performed on the large animal species in the 28-day study.
[0315] The results of the present experiment are expected to
provide pharmacokinetic and pharmacodynamic data on mTor inhibitors
(e.g. PP242) and their analogues.
Example 40
Effect of mTor Inhibitors on In Vitro Lymphocyte Function
[0316] Balb/c mice (WT), or DO11.10 TCR transgenic (specific for
ovalbumin (OVA) peptide presented by I-Ad) mice in the Balb/c
background are used for all experiments of the example herein. T
cells are purified from LN and B cells from spleen by magnetic
sorting. For all experiments, cells are preincubated with test
compounds (e.g. rapamycin or PP242 in 3-fold dilutions, or vehicle)
for about 15 min at 37.degree. C. before plating in wells
containing prewarmed stimuli. For proliferation assays, cells
generally are labeled with a cell division tracker dye (e.g. CFSE)
and stimulated in 96-well plates for about 48 and 72 hr.
Supernatants are saved for cytokine measurements, and cell division
history determined by FACS. Co-staining with AnnexinV provides a
readout for cell death. WT T cells are stimulated with titrations
of anti-CD3 alone, or together with anti-CD28. Similar experiments
are conducted using human peripheral blood T cells (PBT) stimulated
with anti-human CD3+/-CD28. To measure T cell responses to antigen
presenting cells (APC) bearing cognate peptide, TCR-Tg T cells are
cultured with APC (irradiated syngeneic splenocytes, T-depleted)
and a concentration range of OVA peptide. A large panel of
cytokines are measured using a Luminex multiplex system (reagents
from Millipore). WT B cell proliferation is assessed following
stimulation with concentration ranges of anti-IgM,
lipopolysaccharide (LPS), or anti-CD40, each +/-IL-4.
[0317] Differentiation of activated T and B cells is also assessed.
Purified mouse CD4+ T cells are labeled with CFSE before
stimulation with syngeneic APC and soluble anti-CD3. Appropriate
cytokines and blocking antibodies are included in different samples
to skew differentiation towards Th1, Th2, Th17 or induced
regulatory T cell (iTreg) subsets. After 3 days, cells are washed
and replated in IL-2 for two days, then analyzed by FACS for cell
division (CFSE) vs. cytokine production (IFN.gamma., IL-4, IL-10,
IL-17) and/or the Treg marker FoxP3. For B cell differentiation,
splenic B cells are labeled with CFSE, and stimulated for 3-5 days
with LPS or CD40-ligand, +/-IL-4. Supernatants are collected for
measurement of IgM and IgG secretion by ELISA, and cells analyzed
by FACS for cell division (CFSE) vs. isotype-switching (IgG1).
Plasma cell differentiation is monitored by staining for expression
of CD138 and downregulation of B220.
[0318] To define signaling defects, T or B cells are treated with
drug concentrations that impair functional responses to TCR or BCR
engagement, and then stimulated with anti-CD3 or anti-IgM for about
5 min and 15 min. Phosphorylation (p Akt, pS6, p4EBP-1, pFOXO,
pERK, pI.kappa.B) is measured by phosflow or immunoblot as
described. As a readout of PI3K-dependent, mTorC1-independent
signaling Ca2+ flux is measured. FACS-based assays include surface
staining to distinguish subsets of T (CD4, CD8) and B cells
(transitional, follicular, marginal zone).
[0319] Rapamycin (Sirolimus) is a clinically approved and widely
used immunosuppressive agent. Although the molecular target of
rapamycin (mTor) has been known for about 15 years, there is still
much to learn about how mTor signaling is wired in different cell
types and the possible therapeutic and toxic effects of novel mTor
inhibitors in the immune system. PP242 is, a selective and
competitive inhibitor of both mTor complexes, and a potent
inhibitor of T and B cell proliferation. The results of the present
experiment are expected to provide a thorough evaluation of how
PP242 (or a structural analog) affects lymphocyte development and
function in vitro. This experiment is also expected to provide
novel information about mTor function in immunity and methods for
therapeutic immunosuppression by mTor inhibition.
Example 41
Synthetic Preparation and Examples for Indolyl Pyrazolopyrimidine
mTor Inhibitor Methods
[0320] The compounds disclosed herein as useful in the methods of
the invention, are synthesized as illustrated in the following
schemes.
[0321] Scheme 1. Synthesis of
2-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl) iodide (Compound
2-4).
##STR00009##
[0322] Scheme 1 depicts the synthesis of
2-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl) iodide. Cyano
substituted aminopyrazole 1-1 is heated with formamide at
160.degree. C. for 5 hours to yield
2-(4-amino-1H-pyrazolo[3,4-d]pyrimidine (compound 1-2) in 90%
yield. This intermediate is reacted with N-iodosuccinimide in
dimethylformamide at 80.degree. C. for 16 hours, to produce
2-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl) iodide (Cpd. 1-3) in
90% yield.
[0323] Synthesis of
2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol
(Compound 2-4): Compound 2-4 is synthesized as shown in Scheme 2.
Compound 1-3 is reacted with isopropyl bromide in dimethylformamide
with potassium carbonate at 80.degree. C., to provide the
1-isopropyl pyrazolopyrimidine intermediate, compound 2-1. This
intermediate with the protected indolyl boronic acid species 2-2,
using tetrakistriphenylphosphine palladium catalysis in DME-water
solvent at 80.degree. C. for 4-5 hours, to produce the Suzuki
coupling product, compound 2-3. Removal of the protecting groups
with acid in dioxane yields the product,
2-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl) iodide (Cpd. 2-4).
[0324] Scheme 2. Synthesis of
2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol
(Compound 2-4).
##STR00010##
[0325]
2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole
(Compound 3-2): Synthesis of
2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole
(Compound 3-3) is accomplished via the same reactions except that
boronic acid 3-1 is used, as shown in Scheme 3.
[0326] Scheme 3. Synthesis of
2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole
(Compound 3-3).
##STR00011##
[0327] The synthesis of
2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-7-ol
(Compound 3-4) is accomplished via the same reactions as in Schemes
1 and 2, using a 7-tert-butyldimethylsilyloxy (TBS) indolyl boronic
acid instead of the 5-TBSO indolyl species illustrated.
Alternatively, Compound 3-4 is synthesized via methoxy protected
intermediates as shown in Scheme 3-B. 5-Methoxy indolyl boronic
acid, compound 3-1 is coupled to pyrazolopyrimidine iodide
(compound 2-1) using palladium acetate and triphenylphosphine in
the presence of sodium carbonate base to provide intermediate 3-6.
Along with the desired product, some partially deprotected product
is also formed. The crude mixture is taken into the next step for
complete Boc deprotection. Deprotection is accomplished with
aqueous HCl in ethanol solution and compound 3-7 is isolated as the
HCl salt. In the last step, the salt is brought to pH 8 in aqueous
potassium carbonate to obtain the free base. This material is
treated with boron tribromide to remove the methyl ether protection
and yield the final product, Compound 3-4.
[0328] Scheme 3-B. Synthesis of
2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-7-ol
(Compound 3-4).
##STR00012##
[0329] The synthesis of
2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-6-ol
(Compound 3-5) is accomplished via the same reactions as in Schemes
1 and 2, using a 6-tert-butyldimethylsilyloxy (TBS) indolyl boronic
acid instead of the 5-TBSO indolyl species illustrated or it is
synthesized via reactions as shown in Scheme 3-B, using a 6-methoxy
indolyl boronic acid instead of the 5-methoxy indolyl boronic acid
illustrated.
##STR00013##
[0330] The synthesis of PP-30 is shown in Scheme 4.
[0331] Scheme 4. The synthesis of PP-30 is accomplished via the
same reactions as in Schemes 1 and 2, employing palladium catalyzed
Suzuki coupling between the pyrazolopyrimidine intermediate 2-1 and
a boronic acid 4-1 to obtain the product, PP-30.
##STR00014##
[0332] The synthesis of
2-(4-amino-1-(4-N-acetyl-piperidin-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl-
)-1H-indol-5-ol (Compound 5-6) is accomplished as illustrated in
Scheme 5. Acetic anhydride is used to protect the nitrogen of
4-hydroxy piperidine to obtain compound 5-2. Tosyl chloride, with
triethylamine and dimethylaminopyridine (DMAP) in methylene
chloride is used to produce the tosylate 5-3. The
iodopyrazolopyrimidine intermediate 1-3 is reacted with tosylate
5-3 in dimethylformamide in the presence of cesium carbonate at
80.degree. C. to couple the piperidinyl moiety to the
pyrazolopyrimidine molecule, yielding intermediate 5-4. Compound
5-4 is transformed via a Suzuki coupling with boronic acid 2-2
using dichloro[1,1'-bis(diphenylphosphino)ferrocene] palladium II
(PdCl.sub.2(dppf)) in aqueous DME, to obtain compound 5-5, which is
deprotected under acidic conditions to yield compound 5-6.
[0333] Scheme 5. Synthesis of
2-(4-amino-1-(4-N-acetyl-piperidin-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl-
)-1H-indol-5-ol (Compound 5-6).
##STR00015## ##STR00016##
[0334] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Example 42
Comparison of Therapeutic Effectiveness of Direct Active-Site mTor
Kinase Inhibition and Rapamycin in Oncogenic Akt-mTOR Driven
Tumors
[0335] The therapeutic efficacy of PP242 in vivo was assessed by
conducting a randomized preclinical trial of PP242 in an Akt murine
allograft model. See FIG. 26. Using a murine allograft model
addicted to oncogenic Akt-mTOR signaling, the outstanding question
of whether or not more stringent inhibition of mTOR (via PP242) has
a significant biological effect on in vivo tumor growth was
addressed. Mice were randomized three groups: vehicle control,
rapamycin 5 mg/kg and PP242 100 mg/kg. After 20 days of treatment,
all groups maintained body weight demonstrating drug tolerability
and lack of noticeable toxicity. Out of the three cohorts, only
mice treated with PP242 had no tumor growth. Mice treated with
rapamycin mirrored the vehicle treated cohort. See FIG. 27. This
demonstrates for the first time that tumors that arise from
oncogenic Akt-mTOR signaling are selectively responsive to PP242,
but not rapamycin.
Materials and Methods
[0336] Cell line. Immortalized wild type and 4EBP1/2 double
knockout mouse embryonic fibroblasts were kindly provided by Dr.
Nahum Sonenberg (Department of Biochemistry, McGill University,
3655 Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6). Cells
were propagated in DMEM, 10% FBS, 2 mM L-glutamine and
penicillin/streptomycin.
[0337] Mice and allograft model preparation. Transgenic Lck-Akt2
mice where kindly provided by J. R. Testa (Human Genetics Program,
Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, Pa.
19111, USA). Thymi were dissociated in PBS supplemented with 3%
fetal bovine serum (PBS-FBS) and filtered through a 40 .mu.M nylon
mesh (BD Biosciences). Thymocytes were then washed in PBS-FBS and
pelleted at 300.times.g for 5 min. 8-12 week old NOD/SCID mice were
injected with 2.times.10 6 total myristolated-Akt transgenic
thymocytes mixed in a 1:1 ratio of RPMI and matrigel. A volume was
200 .mu.l of the cell mileu was injected per mouse in the right
subscapular region subcutaneously. Tumors were allowed to form for
20 days. Length and width caliper measurements were taken every 2-3
days. UCSF IACUC approval was obtained for all murine
experimentation.
[0338] Treatment and randomization. 45 allograft mice were
generated. 43 of the 45 mice developed tumors and were randomized
on day 21 to receive either vehicle, rapamycin (5 mg/kg), or PP242
(100 mg/kg) by gavage 7 days a week.
[0339] Tumor preparation. Whole tumor samples were dissociated
using a 40 .mu.m nylon cell strainer (BD Biosciences) and
resuspended in 3% FBS in PBS.
[0340] Western blot analysis. Tumors were removed from euthanized
mice on day 45. Single cell suspension was generated a described
herein. Cells were pelleted and lysed in protein lysis buffer (150
mM NaCl, 50 mM Tris, 4 mM KCl, 1 mM MgCl2, 1 mM Na3VO4, 10%
glycerol, 1% Nonidet P-40, Complete Protease inhibitor (Roche),
Phos-stop (Roche) and 20 nM microcysteine (Calbiochem)). Total
protein was quantified using the Bradford method (Bio-Rad),
resolved on 4-20% gradient SDS-PAGE and transferred onto
nitrocellulose membranes (Bio-Rad). Blots were blocked in non-fat
dry milk (4% w/v in TBS and 0.1% Tween 20) for 1 hr at room
temperature. The following primary antibodies were used according
to the manufacturer's instructions: rabbit monoclonal
anti-phospho-Akt (Ser473), anti-phospho-rpS6 (Ser240/244),
anti-phospho-4EBP1 (Thr37/46), Akt, rpS6 and 4EBP1 (all from Cell
Signaling) as well as mouse monoclonal .beta.-actin (Sigma). The
appropriate secondary antibodies (Amersham) were used at 1:10,000
(v/v) in TBS+0.1% Tween 20 for 1 hr at room temperature and blots
were developed using PICO (Pierce Biotechnology).
[0341] Annexin/PI analysis of apoptosis. To characterize in vivo
apoptosis, equal numbers of freshly isolated tumor cells were
labeled with APC-Cy7-conjugated anti-CD4 (BD Biosciences) and
pacific blue-conjugated anti-CD8 (Caltag) in PBS-FBS (all
antibodies diluted 1:100 v/v). Apoptotic cells were labeled with
Annexin V-FITC (BD Biosciences) as well as propidium iodide,
following manufacturer's instructions. Samples (1.times.10 6 cells)
were acquired on a BD LSRII flow cytometer. Analysis and
quantification were done using FlowJo (version 8.7.1).
[0342] In vivo quantification of cell proliferation. Mice were
injected with 300 .mu.g of BrDU three hours before tumors were
collected. Cells were labeled with APC-Cy7-conjugated anti-CD4 (BD
Biosciences) and pacific blue-conjugated anti-CD8 (Caltag) in
PBS-FBS (all antibodies diluted 1:100 v/v). Stained cells were
washed, fixed and processed for flow cytometry using the FITC BrdU
Flow kit (BD Biosciences), following manufacturer's instructions.
Cells were analyzed on a BD LSRII flow cytometer and percentage of
BrdU positive cells was determined using the FlowJo software
(version 8.7.1).
[0343] Cell cycle analysis. Tumor cells and mouse embryonic
fibroblasts were fixed in 70% ethanol overnight in -20.degree. C.
Cells were subsequently washed with PBS twice and treated with 100
.mu.g/mL DNase free RNase (Roche) for 30 minutes at room
temperature. Following another PBS wash, the cells were
permeabilized and treated with propidium iodide (PI) using a
mixture of 10 mg/mL PI, 0.1% Tween, 0.1% sodium citrate. Cell cycle
data was acquired using a BD FACS Caliber (BD Biosciences).
Example 43
mTOR Complex-2 Modulation of SGK1 Phosphorylation and
ENaC-Dependent Na+ Transport
Summary
[0344] The present experiments were designed to investigate which
mTOR complex was involved in the phosphorylation of SGK1 and
ENaC-dependent Na+ transport in renal epithelial cells. By using a
specific inhibitor of mTOR, reduction was observed in SGK1
phosphorylation and amiloride-sensitive Na+ current. In contrast,
rapamycin treatment showed minimum effect on the phosphorylation of
SGK1 and ENaC-dependent Na+ transport, suggesting involvement of
mTORC2. Furthermore, shRNA-mediated knockdown of the expression of
rictor inhibited both SGK1 phosphorylation and amiloride-sensitive
Na+ current. In contrast, the phosphorylation of SGK1 and
ENaC-dependent Na+ transport in renal epithelial cells remained
unchanged upon knockdown of the expression of raptor. Finally, SGK1
was found to be associated mainly with mTORC2 and marginally with
mTORC1.
Materials and Methods
[0345] Cell culture and recombinant plasmid transfection Human
Embryonic Kidney (HEK 293) cells were regularly maintained in
plastic tissue culture flasks at 37.degree. C. in Dulbecco's
Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine
serum and 100 units/ml penicillin/streptomycin. Cells were seeded
on 10 cm dishes (3.times.106 cells/dish) and allowed to grow
overnight. They were then transfected with 5 .mu.g pMO/Flag/mSGK1
(Flag-epitope at N-terminal of mouse SGK1) or the empty vector
using lipofectamine according to manufacturer's instructions
(Invitrogen, Carlsbad, Calif.). Cells were maintained in serum-free
DMEM supplemented with 10% Hyclone Cell Boost 1 supplement
(Hyclone, Logan, Utah, USA) for 24 h prior to treatment with 100 nM
Insulin for 1 h and then treated with 1 .mu.M PP242, 20 .mu.M
LY294002 or 0.1 .mu.M rapamycin for 1 h.
[0346] Immunoprecipitation and immunoblotting. Transfected cells
were lysed in binding buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol,
1 mM EDTA, 2 mM DTT, 150 mM NaCl, 1% Triton X-100 or 0.3% CHAPS)
for 15 min. After centrifugation, the supernatants were collected
and incubated with the anti-flag M2 affinity beads (Sigma). The
immunoprecipitates were collected by centrifugation, washed three
times and boiled for 5 min in 50 .mu.l of cracking buffer (50 mM
Tris-HCl, pH 7.0, 10% glycerol, 2% SDS, 2% .beta.-mercaptoethanol).
Immunoblotting was carried out by separating the immunoprecipitates
on 10% polyacrylamide gels as described using a Bio-Rad minigel
apparatus, and transferred electrophoretically to Hybond-C Extra
membranes (GE Healthcare) using a Trans-Blot apparatus (Bio-Rad).
The membranes were incubated to block non-specific binding in 5%
non-fat dry milk in T-PBS (1.5 mM KH2PO4, 8 mM Na2HPO4, 2.7 mM KCl,
130 mM NaCl and 0.1% Tween 20) with gentle agitation for 1 hr at
room temperature. Rabbit antisera against rictor, raptor or mTOR
were diluted 1:1000 in T-PBS, respectively, and applied to the
membranes for overnight. After washing with T-PBS, the membranes
were incubated with peroxidase-conjugated goat anti-rabbit IgG in
T-PBS for 1 hr, washed three times in T-PBS, and incubated with ECL
Plus Western Blotting Detection System working solution (GE
Healthcare) according to the manufacturer's instructions.
[0347] Generation of recombinant lentiviruses harboring rictor or
raptor shRNAs. Synthesized sense and antisense oligos were annealed
using a touchdown protocol on a PTC-200 thermal cycler at 950 C for
30 s, 600 C for 10 min, then cooled to 200 C at 1 degree every 15
s. The annealed shRNAs were ligated with the pLentiLox 3.7 vector
digested with XhoI/HpaI and treated with calf intestinal alkaline
phosphatase. The ligated DNA was transformed into DH5.alpha.
competent bacterial cells. Ampicillin-resistant colonies were
picked and grown in LB broth for 16 hrs. Plasmid DNA was isolated
using mini-prep columns (Fermentas). Positive recombinants were
identified by restriction enzyme digestion and verified by DNA
sequencing. Recombinant lentiviruses were generated by
co-transfection of plasmids harboring the shRNAs and a mixture of
packaging plasmids into HEK 293T packaging cells. Viral
supernatants were harvested 48 hr after transfection. To determine
the viral titer, a 10-fold dilution series of viruses was made and
used to infect fresh HEK 293T cells. A viral titer of
5.times.105/ml was routinely observed by visualizing cells for EGFP
fluorescence.
[0348] Measurement of ENaC-dependent Na+ transport. Renal
epithelial cells, mpkCCDc14, were maintained in plastic tissue
culture flasks in modified DMEM/Ham's F12 (1:1) medium ("Regular
medium") as described previously (Wang et al., 2008, Id.). For
electrophysiological measurements, cells were seeded on type VI
collagen (Sigma, St. Louis, Mo.) coated filters (Transwell.TM.,
pore-size 0.4 .mu.m, Corning Costar) and grown at least 24 h prior
to treatment with aldosterone at a concentration of 1 .mu.M in the
presence and absence of PP242, LY294002, and rapamycin.
Transepithelial resistance and potential difference across the cell
monolayer were measured using a mini-volt-ohmmeter (MilliCell ERS,
Milipore) at specified time points following treatment. The
equivalent short-circuit current was calculated using Ohm's
law.
Results
[0349] mTOR modulates SGK-1 phosphorylation in mammalian kidney
epithelial cells. By taking advantage of a recently developed,
highly selective, ATP-competitive inhibitor of mTOR to acutely
inhibit mTOR (Feldman et al., 2009, PLoS Biol. 7:e38), it was
sought to determine whether SGK1 phosphorylation and its
physiologic function are dependent on mTOR activity. Unlike
rapamycin, this compound, PP242, binds to the active site of mTOR,
irrespective of whether it is associated with components of complex
1 or 2, and specifically inhibits both TORC1 and TORC2 outputs
(Feldman et al., 2009, Id.). Inhibition occurs at concentrations
that do not inhibit 219 other kinases, including PDK1 as well as
all isoforms of PI3K, SGK and Akt (Feldman et al., 2009, Id.). We
first asked whether mTOR inhibition affects SGK1 phosphorylation in
mpkCCD cells, a cell line which is derived from the kidney's
cortical collecting duct (CCD) and retains the molecular machinery
required for hormone-regulated transepithelial sodium transport,
when grown on ion-permeable filters (Bens et al., 1999, J. Am. Soc.
Nephrol. 10:923-934). In these cells, SGK1 phosphorylation is
stimulated by insulin, and its expression is markedly increased by
aldosterone, through effects on SGK1 gene transcription (Pearce,
2003, Id.) Attempts to detect HM phosphorylation of endogenous SGK1
using a commercially available antibody (anti-SGK1-pSer422) were
unsuccessful, consistent with a previous report (Garcia-Martinez
and Alessi, 2008, Id.). Therefore a highly sensitive and specific
holo-SGK1 antibody was used, which recognizes both the
phosphorylated and unphosphorylated forms of SGK1 (Webster et al.,
1993, Mol. Cell. Biol. 13:203120-40.; Wang et al., 2001, Am. J.
Physiol. 280:F303-F313; Wang et al., 2008, Id.), to detect
phosphorylated forms of SGK1 by mobility shift. In the presence of
aldosterone and insulin, multiple SGK1 bands were detected; the
upper bands were eliminated by the pan-PI3K family inhibitor
LY294002 (FIG. 28a). Previous work strongly supports the
identification of these upper bands as the phosphorylated forms of
SGK1, phosphorylated in both the HM and activation loop. Treatment
of extracts with lambda phosphatase eliminated the upper bands,
further supporting the conclusion that they represent
phosphorylated SGK1 (FIG. 28b). Rapamycin, which inhibits mTORC1
but not mTORC2, had no effect on the upper bands (FIG. 28a), while
PP242 blocked their appearance at concentrations of 0.2-0.3 .mu.M,
which do not inhibit the other relevant kinases, notably PI3K and
PDK1 (Feldman et al., 2009, Id.). As confirmation that mTORC1 is
expressed in these cells and inhibited by rapamycin, we examined
the phosphorylation state of the prototypical mTORC1 substrate,
p70S6K. Rapamycin, LY294002 and PP242 all eliminated the band
detected by anti-phospho p70S6K antibody, indicative of an
mTORC1-mediated effect. Importantly, p70S6K phosphorylation was
fully abrogated at a concentration at which rapamycin treatment
failed to alter appearance of the phosphorylated forms of SGK1.
Together, these data suggest that SGK1 phosphorylation is dependent
upon mTORC2, but not mTORC1.
[0350] In order to look directly at the role of mTOR in SGK1 HM
phosphorylation, conditions were established in which an anti-SGK1
pS422 antibody would give a specific SGK1-pS422-dependent signal.
In a previous report, Garcia-Martinez and Alessi (2008, Id.) were
unable to detect a specific pS422-SGK1 signal, and that the
endogenous species detected with this antibody in murine embryo
fibroblasts co-migrated with a species detected by a well
characterized antibody recognizing the HM phosphorylated form of
p70-S6K. The current data (for example, FIG. 28) are consistent
with this observation. In contrast, Hong et al. (2008, Id.)
reported detecting endogenous HM phosphorylated SGK1 using the same
antibody; it is not clear why these findings are not consistent
with either the current findings, or those of Garcia-Martinez and
Alessi (2008, Id.). The latter were able to detect phospho-SGK1
when it was expressed as a GST-fusion protein in murine embryo
fibroblasts and enriched on glutathione beads. A similar strategy
was pursued, that is expressing a flag epitope-tagged SGK1 in
HEK293 cells, which were treated with insulin followed by PP242,
LY294002 or rapamycin. Whole cell lysates were subjected to
immunoprecipitation with anti-flag antibody and probed by Western
blotting. In immunoblots stained with anti-pS422, a band was
detected, which was consistent with pS422-SGK1: it depended on the
presence of SGK1 expression vector, and co-migrated precisely with
the upper band detected with either anti-holo-SGK1 or anti-flag
antibody (not shown). This band was abrogated by PP242 and LY294002
(LY), but not by rapamycin (FIG. 28c). These data support the
notion that phosphorylation of transfected SGK1 is mTORC2--and not
mTORC1--dependent in HEK293 cells, and further support the
identification of the shifted species detected by holo-SGK1
antibody in CCD cell lysates as HM-phosphorylated SGK1 (and not
SGK1 phosphorylated only at the activation loop by PDK1). Taken
together, these data support the notion that a rapamycin-resistant
output of mTOR is required for SGK1 phosphorylation, and that
although mTORC1 is expressed in CCD cells, it does not
phosphorylate SGK1 at the HM motif site.
[0351] mTOR activity is required for Na+ transport in mammalian
kidney epithelial cells. The best-characterized function of SGK1 in
mammals is to mediate aldosterone-induced ENaC-dependent Na+
transport. The fundamental role of SGK1 in ENaC regulation has been
demonstrated in numerous different systems, including a variety of
cultured cells, Xenopus oocytes, and knockout mice. In order to
determine if mTOR activity is important for Na+ transport, we
examined the effect of PP242 on Na+ currents in mpkCCD cells grown
on Transwell.TM. filters. As shown in FIG. 29a and FIG. 29b, PP242
completely blocked aldosterone-induced current with an IC-50 of
approximately 0.2 .mu.M, while rapamycin, at concentrations that
fully blocked mTORC1 had no effect (FIG. 29a). Although a precise
IC-50 for PP242 inhibition of SGK1 HM phosphorylation could not be
determined, it was of the same order of magnitude (compare FIG. 28A
and FIG. 29a), and well below the IC-50 for the other relevant
kinases, including PI3K (Feldman et al., 2009, Id.). The effect of
PP242, like that of LY294002, was rapid (T1/2 of approximately 15
minutes), reversible, and occurred without any significant drop in
electrical resistance, a sign of tight junction integrity and cell
health (FIG. 29c). It should be noted that prolonged rapamycin
treatment (>16 h) does inhibit Na+ current, an effect that has
been attributed to blockade of mineralocorticoid receptor function
(Edinger et al., 2002, Am. J. Physiol. 283:F254-F261). It also
should be noted that prolonged treatment with PP242 or LY294002
(>24 h) diminishes electrical resistance and causes
morphological changes (e.g. blebbing) in cells, consistent with a
toxic effect. Taken together, these data strongly support the idea
that mTORC2 is required for SGK1 HM phosphorylation and acute
control of transepithelial sodium current in mammalian kidney
collecting duct cells.
[0352] The role of rictor in SGK1 phosphorylation and
ENaC-dependent Na+ transport. The preceding experiments showed that
SGK1 phosphorylation and ENaC-dependent Na+ transport were reduced
by PP242 at concentrations that selectively inhibits the activity
of mTOR (FIG. 28 and FIG. 29). The inhibition on SGK1
phosphorylation and ENaC-dependent Na+ transport was, however,
largely resistant to rapamycin treatment (FIG. 28 and FIG. 29),
suggesting involvement of mTORC2, but not mTORC1. To further
investigate whether mTORC2 mediates SGK1 phosphorylation and
ENaC-dependent Na+ transport, recombinant lentiviruses were
generated harboring shRNAs directed at the mTORC2-specific
component, rictor. Since the use of transfected HEK293 cells
expressing SGK1 allows us to determine direct and unambiguous SGK1
phosphorylation at the HM motif site (FIG. 28c), we used this assay
to examine the effect of rictor shRNA on SGK1 phosphorylation.
Using lentiviral-mediated transduction, rictor expression was
decreased by 67% in HEK293 cells (FIG. 30a and FIG. 30b), and HM
phosphorylation of flag-tagged SGK1 was reduced by 53% (FIG. 30c
and FIG. 30d), whereas control shRNA exerted no discernible effect
(FIG. 30c and FIG. 30d).
[0353] To examine whether knockdown of rictor expression affects
ENaC-dependent Na+ transport, we infected mpkCCD cells with
lentiviruses harboring the rictor shRNA, seeded the cells on
Transwell.TM. filters, and determined aldosterone-induced Na+
currents (FIG. 31). Rictor shRNA reduced Na+ current by
approximately 60% (FIG. 31a), which corresponded well to the
knockdown in rictor expression (.about.72%) (FIG. 31b and FIG.
31c). Together with the small molecule inhibitor data, these data
provide strong support for the conclusion that mTORC2 mediates SGK1
phosphorylation and ENaC-dependent Na+ transport.
[0354] Raptor is not required for SGK1 phosphorylation and
ENaC-dependent Na+ transport. To further investigate whether mTORC1
is similarly required for SGK1 phosphorylation and ENaC-dependent
Na+ transport, lentiviruses were generated harboring raptor shRNA.
A knockdown of 68% in raptor expression was observed (FIG. 32b).
The reduction in raptor expression, however, showed no effect on
SGK1 phosphorylation in transfected HEK293 cells (FIG. 32c, FIG.
32d). When the raptor shRNA was expressed in mpkCCD cells,
determination of Na+ transport showed a normal increase in
aldosterone-induced currents (FIG. 33a). No inhibitory effect was
observed (FIG. 33a), suggesting that mTORC1 is not required for
ENaC-dependent Na+ transport. Since S6K is a well-known substrate
for mTROC1, the effect of S6K phosphorylation upon knockdown of
raptor expression was examined. Reduction in raptor expression
resulted in a decrease in S6K phosphorylation in mpkCCD cells (FIG.
33b), demonstrating the effectiveness of the raptor shRNA in
disrupting the function of mTORC1.
[0355] SGK1 physically associates with mTORC2. It was next examined
whether the SGK1 protein binds to the mTOR complexes using
flag-tagged SGK1. The plasmid was transfected into HEK293 cells. 48
hrs post-transfection, the cells were lysed and immunoprecipitation
was carried out using anti-flag antibodies cross-linked to agarose
beads. The immunoprecipitates were analyzed by Western blotting
using antibodies against mTOR, rictor and raptor, respectively. As
shown in FIG. 34, strong signals were detected for mTOR and rictor
in the SGK1 immunoprecipitates, while only a faint raptor band was
detected (<10% of the intensity of the mTOR and rictor signals).
These data demonstrate that the majority of SGK1 in the mTOR
complexes is associated with mTORC2 and only a minor portion of
SGK1 binds to mTORC1.
Discussion
[0356] We observed reduction in SGK1 phosphorylation (FIG. 28). In
contrast, rapamycin treatment showed minimum effect on the
phosphorylation of SGK1. This mTOR inhibitor also dramatically
reduced amiloride-sensitive Na+ current, whereas rapamycin showed
minimum effect on Na+ transport (FIG. 29).
[0357] To independently confirm a role for mTORC2 in SGK1
phosphorylation, we generated lentiviruses harboring a rictor
shRNA. When the shRNA was introduced into HEK293 cells, knockdown
of endogenous rictor expression was identified (FIG. 30). A
decrease in the phosphorylation of SGK1 was also observed (FIG.
30). Subsequently, the effect of the rictor shRNA was tested in
renal epithelial cells, and a reduction in ENaC-dependent Na+
transport was detected (FIG. 31). By using similar experimental
approaches, we made lentiviruses harboring a raptor shRNA and
examined the effect of raptor expression knockdown. Both SGK1
phosphorylation and ENaC-dependent Na+ transport were found to
remain at normal levels (FIG. 32 and FIG. 33).
[0358] It is of note that the specific inhibitor of mTOR, PP242,
appears to exert strong effects on SGK1 phosphorylation and
ENaC-dependent Na+ transport. At a concentration of 0.3 .mu.M, both
the phosphorylation of SGK1 and the amiloride-sensitive Na+ current
were completely abolished (FIG. 28 and FIG. 29). The extent of
inhibition by PP242 is similar to that observed with the PI3K
inhibitor, LY294002. On the other hand, the shRNA shows partial
inhibition on the expression of rictor (FIG. 30 and FIG. 31).
Accordingly, a partial reduction was observed in SGK1
phosphorylation and ENaC-dependent Na+ transport. To obtain direct
and unambiguous results on SGK1 phosphorylation at the HM motif
site, we developed an assay by transfection of a tagged SGK1 along
with lentiviral-mediated infection of shRNA into HEK293 cells. The
assay requires immunoprecipitation of SGK1 by an antibody against
the flag tag. The enrichment of the SGK1 protein allows detection
of its phosphorylation at the HM domain (FIG. 30).
[0359] In summary, we have carried pharmacological studies and
identified a role for mTORC2 in SGK1 phosphorylation and
ENaC-dependent Na+ transport in renal epithelial cells (FIG. 28 and
FIG. 29). We have subsequently generated recombinant lentiviruses
harboring rictor or raptor shRNAs. Knockdown of rictor expression
leads to decreases in both SGK1 phosphorylation and
amiloride-sensitive Na+ current (FIG. 30 and FIG. 31). In contrast,
the phosphorylation of SGK1 and ENaC-dependent Na+ transport appear
not to be affected upon knockdown of raptor expression (FIG. 32 and
FIG. 33). Taken together, these findings indicate mTORC2-modulates
SGK1 phosphorylation and ENaC-dependent Na+ transport in renal
epithelial cells. We have shown that SGK1 associates strongly with
the rictor-containing mTORC2 (FIG. 34).
Example 44
mpkCCD Cell Proliferation in Response to Rapamycin and PP242
[0360] In order to compare the effect of rapamycin and PP242 on
cellular proliferation in mpkCCD cells, the experiment described in
FIG. 35 and FIG. 36 was conducted. The experimental procedure
followed that described for Example 6, with the exception that
treatment lasted 28 hr prior to the introduction of Resazurin. As
shown in the figures, PP242 blunts proliferation in normal
collecting duct cells, but proliferation is not eliminated.
Inhibition is significant at P<0.05 for 0.3 uM and higher
concentrations of compound. Furthermore, PP242 has a somewhat
greater effect than rapamycin, as judged by a comparison of FIG. 35
and FIG. 36.
Example 45
PP242 Induction of Apoptosis
[0361] In order to investigate the PP242 induction of apoptosis of
p190 BCR-ABL-transformed murine hematopoietic progenitors and human
Ph.sup.+ B-ALL cells in vitro, the following experiment was
conducted. With reference to FIG. 37a, Mouse p190 cells (upper) and
human SUP-B15 cells (lower) were cultured with inhibitors at the
concentrations indicated for 48 hr. The MTS assay, as described
herein, was used to quantify viable cell number and the data were
expressed as % of control viability in untreated cells. p190 cells
were cultured for 24 hr with the inhibitors indicated, then DNA
content was measured by flow cytometry, as illustrated in FIG. 37b.
p190 cells were cultured for 48 hr with the combinations of
compounds indicated in FIG. 37c and assessed for survival using the
median effect method, as known in the art. Drug combinations were
assessed for synergy by calculating the combination index (CI)
using CalcuSyn software, as known in the art. The CI was modeled
with Monte Carlo simulation and plotted as a function of the
fraction affected by treatment. CI<1, =1, and >1 indicate
synergism, additive effect, and antagonism, respectively. The
anti-clonogenic effects of PP242 combined with DA in primary
Ph+B-ALL. CD19+CD34+ magnetically sorted cells from five different
patients were assessed for colony formation potential in cultures
with DAD (5 nM) alone or in combination with increasing
concentrations [10 or 100 nM] of RAP, PP242, or BEZ-235
(*P<0.05, **P <0.01, #P<0.001. A schematic model (FIG.
37e) of BCR-ABL driven mechanisms of oncogenic survival (left) and
a new model of incomplete mTOR inhibition (middle) versus complete
mTOR inhibition (right) in B-ALL.
Example 46
PP242 Inhibition of mTORC2/AKT and mTORC1 Signaling
[0362] In order to assess the effect of PP242 on mTORC2/AKT and
mTORC1 signaling, the experiment illustrated in FIG. 38 was
conducted. In summary, PP242 completely inhibits mTORC2/AKT and
mTORC1 signaling in B-ALL whereas rapamycin suppresses mTORC1
driving a PI3K/AKT surge. Western blots of p190 cells treated for
1.5 hr (a) or 3 hr (b) with indicated inhibitors are depicted in
FIG. 38a and FIG. 38b. Cells were treated with IM (0.5 and 1.0
.mu.M), DA (5 and 50 nM), PP242 and RAP (50 and 400 nM). Clinically
achievable concentrations of IM (1.0 .mu.M) and DA (100 nM) were
used for the combination treatments. See also FIG. 40a and FIG.
40b. Activation of PI3K was quantified in cells by signal pixel
intensity and localized area of PIP3 accumulation by confocal
microscopy, as depicted in FIG. 38c. Cells were cultured for 4 hr
with PI-103 (2 .mu.M), IC87114 (10 .mu.M), PP242 (20 and 200 nM),
and RAP (20 and 200 nM). A minimum of 250 cells was quantified from
2 separate images. RAP significantly activated PI3K signaling
(*P<0.05, #P<0.001) whereas PP242 had no significant effect.
Representative images depict PIP3 accumulation, nuclear content
(DAPI stain) merged onto DIC images (13.5 .mu.m scale bar). PP242
and high concentrations of IM (5 .mu.M) both inhibit cap-dependent
translation whereas RAP does not, as judged by the result depicted
in FIG. 38d. Cap-binding proteins in lysates were purified by
7-methyl GTP (m.sup.7GTP) affinity and analyzed by western
blotting. p190 cells expressing LC3-GFP were cultured for 8 hr in
chamber wells with DA (10 nM), PP242 (250 nM), BEZ-235 (250 nM),
RAP (250 nM), and pulsed with EdU 1 hr prior to fixation, as shown
in FIG. 38e. Autophagy (LC3 puncta accumulation), loss of
proliferation (EdU accumulation), and distinct localization
patterns of Foxo1 were assessed by confocal microscopy and
representative cells were magnified for clarity. Cytoplasmic
localization of Foxo1 corresponds to AKT activation, whereas
nuclear accumulation signifies AKT inhibition.
Example 47
PP242 Selective Suppression of Leukemic Expansion
[0363] In summary, PP242 selectively suppresses leukemic expansion
in vivo. Mice injected with p190 cells (i.v.), were treated daily
(q24) starting on D7 post-transplant, as shown in FIG. 39a.
Imatinib ("IM," 150 mg kg.sup.-1, i.p.), rapamycin ("RAP," 7 mg
kg.sup.-1, i.p.) and PP242 (30 and 60 mg kg.sup.-1, p.o.) were
administered to mice as the mice were followed daily for overall
survival (median.+-.interquartile range) in groups of 5 mice. Delay
in survival was assessed by the log rank test, as known in the art.
After 30 days, the surviving 2 mice were analyzed for leukemic
burden in the bone marrow (inset) by flow cytometry. FIGS. 39b-e
illustrate short-term pharmacodynamic profile and anti-leukemic
efficacy of PP242 in conditioned recipients (450 rad) engrafted
with mouse p190 B-ALL. A schematic depiction of the treatment
design is provided in FIG. 39b, where groups of 3 mice each were
treated twice daily (b.i.d. or q12) starting on day 7 for 4 days (7
treatments), with the indicated doses of PP242 or vehicle (PEG400).
1 hr following the last dose, and 2 hr before sacrifice, mice were
injected (i.p.) with EdU to mark cycling cells. Leukemic burden
(mean %.+-.s.d.) was assessed by flow cytometry in the
corresponding bone marrow and peripheral blood of treated mice, as
depicted in FIG. 39c. The abundance of leukemic cells actively
cycling (EdU+) following treatment was measured by flow cytometry
(mean %.+-.s.d.), as shown in FIG. 39d. FIG. 39e illustrates the
pharmacodynamic activity of PP242 using intracellular
phospho-staining of bone marrow and peripheral blood cells.
Representative gating strategy (left panel) in the bone marrow for
phospho-signature determination in leukemic (hCD4+B220+) and normal
lymphocyte populations (hCD4-B220+) from each corresponding mouse
is displayed. Quantified phospho-signatures are displayed as P-flow
scores (right panel). A schematic of treatment design for primary
human Ph+B-ALL whole bone marrow xenografts is depicted in FIG.
39f. Groups of 4-5 NSG mice each with equally engrafted disease
were treated q24 for 5 days (6 treatments), with the indicated
doses of DA, DA combined with PP242, or vehicle. 1 hr following the
last dose, and 2 hr before sacrifice, mice were injected (i.p.)
with EdU to mark cycling cells. Leukemic burden and cells actively
cycling (mean %.+-.s.d.) was assessed by flow cytometry in the
corresponding bone marrow of treated mice, as shown in FIG. 39g.
Representative residual disease in the bone marrow following
treatment is depicted (left panel) and % EdU+ cells were calculated
from normal (red gate) and leukemic (blue gate) bone marrow (middle
panel). Arrows signify leukemia-specific eradication in
non-actively cycling cells. The leukemic burden (% hCD45+) was
calculated is depicted (right panel. *P<0.05, **P<0.01,
#P<0.001. (1) Plasma concentrations of PP242 following in vivo
administration.
Example 48
mTOR Kinase Inhibitor Effects on mTORC1 and TORC2
[0364] In order to determine the effect of mTOR kinase inhibitor on
the inhibition of mTORC1 and mTORC2 substrates in human Ph+SUP-B15
cells, the experiment of FIG. 40 was conducted. Western blot
analysis of SUP-B15 cells treated with the PI3K/mTOR inhibitor
BEZ-235 [600 nM], RAP [20 nM], in comparison to a low dose
titration of PP242 [5, 15, 45, 135, 405 nM] for 3 hours, as
depicted in FIG. 40a. Western blot of SUP-B15 cells treated with
PI3K/mTOR inhibitor PI-103 [2000 nM] and the ABL/Src kinase
inhibitor dasatinib [DA; 10, 100 nM] alone, or in combination [DA
at 100 nM] with RAP [RAP 50, 400 nM], PP242 [50, 400 nM], or
BEZ-235 [50, 400 nM] as indicated, is depicted in FIG. 40b.
* * * * *