Note: Descriptions are shown in the official language in which they were submitted.
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COMBINATION THERAPY COMPRISING A DIARYL UREA COMPOUND AND A PI3, AKT KINASE OR
MTOR INHIBITORS (RAPAMYCINS) FOR CANCER TREATMENT
BACKGROUND OF THE INVENTION
Diary] urea compounds e.g. 4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-
fluorophenoxy}-
pyridine-2-carboxylic acid methylamide as desciibed e.g. in US 20050038080 are
potent anti-
cancer and anti-angiogenic agents that possess various activities, including
inhibitory activity on
the VEGFR, PDGFR, raf, p38, and/or flt-3 kinase signalling molecules. The
RAS/R.AF/MEK/ERK
pathway is involved in cellular proliferation, differentiation, and
transformation, and is implicated
in many cancers. The PI3K/AKT signalling pathway is another important
physiological pathway
in cells. It mediates extracellular stimuli, including growth factors,
cytokines, cell-cell adhesion
and cell-extracellular matrices (Vivanco and Sawyers, Nat Rev Cancer, 2: 489-
501, 2002,
Downward, Curr Opin Cell Biol, 10: 262-267, 1998). The AKT pathway appears to
be active in
many types of human cancer (Nicholson and Anderson, Cell Signal, 14: 381-395,
2002).
DESCRIPTION OF THE INVENTION
- The present invention provides drug combinations, compositions, and methods
for treating
diseases and conditions, including, but not limited to, cell proliferative
disorders (such as cancer),
inflammation, immunomodulatory disorders, and conditions associated with
abnormal or
undesirable angiogenesis. The drug combinations comprise a compound of formula
I and at least
one second compound that is_an inhibitor of the PI3K/AKT signalling pathway.
The methods can
comprise, e.g., administering a diaryl urea compound as described below and a
signalling pathway
inhibitor, pharmaceutically-acceptable salts thereof, and derivatives thereof,
etc.
The phosphatidylinositol-3-kinase (P13K) and AKT (Protein Kinase B) signalling
pathway
regulates a variety of biological processes including cell survival, cell
proliferation, cell,growth,
and cell motility. Abnormalities in PI3K-AKT signalling contribute to the
pathogenesis of a
number of diseases and conditions, including cell proliferative disorders
(such as cancer),
inflammation, and immunomodulatory disorders.
Many growth and survival factors activate P13K family members to specifically
convert one lipid
signalling molecule, PIP2, into another, PI(3,4,5)P3. The phosphorylated
product recruits Akt
family members to the inner plasma membrane, stimulating their protein kinase
activity. To date,
many Akt effectors involved in several biological processes have been
identified. For example, the
Akt kinases mediate cell survival though phosphorylation and inactivation of
apoptotic machinery
components. The PI3K/AKT signalling pathway includes any members or components
that
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participate in the signal transduction cascade. These include, but are not
limited to, e.g., P13-
kinase, Akt-kinase, FKBP12, mTOR (mammalian target of rapamycin; also known as
FRAP,
RAFT1, or RAPTI), RAPTOR (regulatory associated protein if mTOR), TSC
(tuberous sclerosis
complex), PTEN, (phosphatase and tensin homolog) and downstream effectors
thereof.
Combinations of the present invention can be used to treat and/or prevent any
condition and/or
diseases associated with any of the aforementioned activities.
An inhibitor of the PI3K/AKT signalling pathway is a compound that inhibits
one or more
members of the aforementioned signal transduction cascade. While such
compounds may be
referred to as pathway inhibitors, the present invention includes the use of
these inhibitors to treat
any of the mentioned diseases or conditions, regardless of the mechanism of
action or how the
therapeutic effect is achieved. Indeed, it is recognized that such compounds
may have more than
one target, and the initial activity recognized for a compound may not be the
activity that it
possesses in vivo when administered to a subject, or whereby it achieves its
therapeutic efficacy.
Thus, the description of a compound as a pathway or protein target (e.g., Akt
or mTOR) inhibitor
indicates that a compound possesses such activity, but in no way restricts a
compound to having
that activity when used as a therapeutic or prophylactic agent.
Examples of AKT family members include: Aktl, Akt2 (commonly over-expressed in
tumors;
Bellacosa et al., Int. J. Cancer, 64:280-285, 1995), and Akt3.
Examples of P13K family members include: p110-alpha, p110-beta, p110-delta,
and p110-gamma
(catalytic).
Examples of PI3K/AKT signalling pathway inhibitors include, but are not
limited to, e.g., FTY720
(e.g., Lee et al., Carcinogenesis, 25(12):2397-2405, 2004);
UCN-01 (e.g., Amornphimoltham et al., Clin Cancer Res., 10(12 Pt 1):4029-37,
2004).
Examples of phosphatidylinositol-3-kinase (P13-kinase) inhibitors, include,
but are not limited to,
e.g.,
celecoxib and analogs thereof, such as OSU-03012 and OSU-03013 (e.g., Zhu et
al., Cancer Res.,
64(12):4309-18, 2004);
3-deoxy-D-myo-inositol analogs (e.g., U.S. Application No. 20040192770;
Meuillet et al., Oncol.
Res., 14:513-27, 2004), such as PX-316;
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2'-substituted, 3'-deoxy-phosphatidyl-myo-inositol analogs (e.g., Tabellini et
al., Br. J. Haematol.,
126(4):574-82, 2004);
fused heteroaryl derivatives (U.S. Pat. No. 6,608,056);
3-(imidazo[1,2-a]pyridin-3-yl) derivatives (e.g., U.S. Pat. Nos. 6,403,588 and
6,653,320);
Ly294002 (e.g., Vlahos, et al., J. Biol., Chem., 269(7) 5241-5248, 1994);
quinazoline-4-one derivatives, such as IC486068 (e.g., U.S. Application No.
20020161014; Geng
et al., Cancer Res., 64:4893-99, 2004);
3-(hetero)aryloxy substituted benzo(b)thiophene derivatives (e.g., WO 04
108715; also WO 04
108713);
viridins, including semi-synthetic viridins such as such as PX-866 (acetic
acid
(1 S,4E, l OR,11 R, 13 S,14R)-[4-diallylaminomethylene-6-hydroxy-l-
methoxymethyl-10,13-dime-
thyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-
cyclopenta[a]phenanthren-
11-y1 ester) (e.g., Ihle et al., Mol Cancer Ther., 3(7):763-72, 2004; U.S.
Application No.
20020037276; U.S. Pat. 5,726,167); and
wortmannin and derivatives thereof (e.g., U.S. Pat. Nos. 5,504,103; 5;480;906,
5,468,773;
5,441,947; 5,378,725; 3,668,222).
Examples of Akt-kinase (also known as protein kinase B) inhibitors, include,
but are not limited to,
e.g.,
Akt-1-1 (inhibits Aktl) (Barnett et al., Biochem. J., 385 (Pt.2):399-408,
2005);
Akt-1-1,2 (inhibits Akl and 2) (Barnett et al., Biochem. J., 385 (Pt.2):399-
408, 2005);
API-59CJ-Ome (e.g., Jin et al., Br. J. Cancer., 91:1808-12, 2004);
1-H-imidazo[4,5-c]pyridinyl compounds (e.g., W005011700);
indole-3-carbinol and derivatives thereof (e.g., U.S. Pat. Nos. 6,656,963;
Sarkar and Li, J Nutr.,
134(12 Suppl):3493S-3498S, 2004);
perifosine (e.g., interferes with Akt membrane localization; Dasmahapatra et
al., Clin. Cancer
Res., 10(15):5242-52, 2004);
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phosphatidylinositol ether lipid analogues (e.g., Gills and Dennis, Expert.
Opin. Investig. Drugs,
13:787-97, 2004);
triciribine (TCN or API-2 or NCI identifier: NSC 154020; Yang et al., Cancer
Res., 64:4394-9,
2004).
Examples of mTOR inhibitors include, but are not limited to, e.g.,
FKBP12 enhancer;
rapamycins and derivatives thereof, including: CCI-779 (temsirolimus), RAD001
(Everolimus;
WO 9409010), TAFA93 and AP23573; rapalogs, e.g. as disclosed in WO 98/02441
and WO
01/14387, e.g. AP23573, AP23464, AP23675, or AP23841; 40-(2-
hydroxyethyl)rapamycin, 40-[3-
hydroxy(hydroxymethyl) methylpropanoate]-rapamycin (also called CC1779), 40-
epi-(tetrazolyt)-
rapamycin (also called ABT578), 32-deoxorapamycin, 16-pentynyloxy-32(S)-
dihydrorapamycin,
and other derivatives disclosed in WO 05005434; derivatives disclosed in USP
5,258,389, WO
94/090101, WO 92/05179, USP 5,118,677, USP 5,118,678, USP 5,100,883, USP
5,151,413, USP
5,120,842, WO 93/111130, WO 94/02136, WO 94/02485, WO 95/14023, WO 94/02136,
WO
95/16691 (e.g. SAR 943), EP 509795, WO 96/41807, WO 96/41807 and USP 5,256,
790;
phosphorus-containing rapamycin derivatives (e.g., WO 05016252);
4H-1-benzopyran-4-one derivatives (e.g., U.S. Provisional Application No.
60/528,340).
Examples of compounds in preclinical or clinical use, include, e.g., AP23573,
AP23841, CCI-779,
and RAD001.
Examples of phosphatidylinositol-3-kinase (P13-kinase) inhibitors of interest
are wortmannin and
the derivatives or analogs thereof and the pharmaceutically acceptable salts
of wortmannin and its
derivatives and analogs. Consequently, methods of this invention include the
use of the P13-kinase
inhibitors of formula W:
CH3
O CH3 O
CH30- CH,
0 0 0
wortmannin
0
(W)
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derivatives or analogs of the compound of formula W, pharmaceutically
acceptable salts of the
compound of formula W, and pharmaceutically acceptable salts of the
derivatives or analogs of the
compound of formula W.
Reference to the derivatives and analogs of wortmannin or the compound of
"formula W" herein is
intended to include the derivatives and analogs identified in U.S. Pat. Nos.
5,504,103; 5,480,906,
5,468,773; 5,441,947; 5,378,725; 3,668,222. Suitable derivatives and analogs
of the compound of
formula W include:
a) compounds of formula W 1
R c"'O
R'OCH2,, CH3
O ~
O I o
o W1
._ where R is H(11-desacetoxywortmannin) or acetoxy and R' is CI-C6 alkyl,
b) 09,11- dehydrodesacetoxywortmannin compounds of formula W2
CH3 0
R'OCig 3 I O O
W2
where R' is C
I -C6 alkyl,
c) 17( a-dihydro-wortmannin compounds of formula W3
CH30R"
R
R'OCH2,,, CH3
~
O-
O I O
O W3
where R is H or acetoxy and R' is CI-C6 alkyl, and R" is H, CI-C6 alkyl,
-C(O)OH or -C(O)O- CI-C6 alkyl;
d) open A-ring acid or ester of wortmannin compounds of formula W4
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CH3 O
H3C
O ~
RiO 0 OR2
W4
where R, is H, methyl or ethyl and R2 is H or methyl or
e) 11-substituted and 17- substituted derivatives of wortmannin of formula W5
R4 CH3 R3
CH30CH2,,, CH3
O ~
O I ~ O
O W5
where R4 is =0 or -O(CO)R6, R3 is =0, -OH or -O(CO)R6, each Rb is
independently phenyl,
CI-C6 alkyl or substituted Cl-C6 alkyl, where R4 is =0 or -0H, R3 is not =0.
The compound with the structure of formula (I) wich corresponds to 4{4-[3-(4-
chloro-3-
trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylic acid
methylamide,
pharmaceutically acceptable salts, polymorphs, solvates, hydrates, metabolites
and prodrugs
thereof, are collectively referred to herein as the "compounds of formula I".
formula (I) is as
follows:
CF3 O
CI O N,CH3
O
I
1~ H
110!~ N
N N
H H
F
Where the plural form of the word compounds, salts, and the like, is used
herein, this is taken to
mean also a single compound, salt, or the like.
The term CI-6 alkyl, unless indicated otherwise, means straight, branched
chain or cyclic alkyl
groups having from one to six carbon atoms, which may be cyclic, linear or
branched with single
or multiple branching. Such groups include for example methyl, ethyl, n-
propyl, isopropyl, n-
butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclobutyl and the like.
The present invention
also relates to useful forms of the compounds as disclosed herein, such as
pharmaceutically
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acceptable salts and metabolites. The present invention also relatesto
prodrugs of the compound of
formula (I). The term "pharmaceutically acceptable salt" refers to a
relatively non-toxic, inorganic
or organic acid addition salt of a compound of the present invention. For
example, see S. M.
Berge, et al. "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19.
Pharmaceutically acceptable
salts include those obtained by reacting the main compound, functioning as a
base, with an
inorganic or organic acid to form a salt, for example, salts of hydrochloric
acid, sulfuric acid,
phosphoric acid, methane sulfonic acid, camphor sulfonic acid, oxalic acid,
maleic acid, succinic
acid and citric acid. Pharmaceutically acceptable salts also include those in
which the main
compound functions as an acid and is reacted with an appropriate base to form,
e.g., sodium,
potassium, calcium, mangnesium, ammonium, and choline salts. Those skilled in
the art will
further recognize that acid addition salts of the claimed compounds may be
prepared by reaction of
the compounds with the appropriate inorganic or organic acid via any of a
number of known
methods. Alternatively, alkali and alkaline earth metal salts are prepared by
reacting the
compounds of the invention with the appropriate base via a variety of known
methods.
Representative salts of the compounds of this invention include the
conventional non-toxic salts
and the quaternary ammonium salts which are formed, for example, from
inorganic or organic
acids or bases by means well known in the art. For example, such acid addition
salts include
acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate,
citrate, camphorate, camphorsulfonate, cinnamate, - cyclopentanepropionate,
digluconate,
dodecylsulfate, ethanesulfonate, furriarate, glucoheptanoate,
glycerophosphate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-
hydroxyethanesulfonate,
itaconate, lactate, maleate, mandelate, methanesulfonate, 2-
naphthalenesulfonate, nicotinate,
nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate,
pivalate, propionate,
succinate, sulfonate, tartrate, thiocyanate, tosylate,
trifluoromethanesulfonate, and undecanoate.
Base salts include alkali metal salts such as potassium and sodium salts,
alkaline earth metal salts
such as calcium and magnesium salts, and ammonium salts with organic bases
such as
dicyclohexylamine and N-methyl-D-glucamine. Additionally, basic nitrogen
containing groups
may be quaternized with such agents as lower alkyl halides such as methyl,
ethyl, propyl, and butyl
chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, and
dibutyl sulfate; and
diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and
strearyl chlorides, bromides
and iodides, aryl or aralkyl halides like benzyl and phenethyl bromides and
others monosubstituted
aralkyl halides or polysubstituted aralkyl halides.
Solvates for the purposes of the invention are those forms of the compounds
where solvent
molecules form a complex in the solid state and include, but are not limited
to for example ethanol
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and methanol. Hydrates are a specific form of solvates, where the solvent
molecule is water.
Certain pharmacologically active agents can be further modified with labile
functional groups that
are cleaved after in vivo administration to furnish the parent active agent
and the
pharmacologically inactive derivatizing group. These derivatives, commonly
referred to as
prodrugs, can be used, for example, to alter the physicochemical properties of
the active agent, to
target the active agent to a specific tissue, to alter the pharmacokinetic and
pharmacodynamic
properties of the active agent, and to reduce undesirable side effects.
Prodrugs of the invention
include, e.g., the esters of appropriate compounds of this invention that are
well-tolerated,
pharmaceutically acceptable esters such as alkyl esters including methyl,
ethyl, propyl, isopropyl,
butyl, isobutyl or pentyl esters. Additional esters such as phenyl-CI-C5 alkyl
may be used,
although methyl ester is preferred.
Methods which can be used to synthesize other prodrugs are described in the
following reviews on
the subject, which are incorporated herein by reference for their description
of these synthesis
methods:
= Higuchi, T.; Stella, V. eds. Prodrugs As Novel Drug Delivery Systems. ACS
Symposium
Series. American Chemical Society: Washington, DC (1975).
= Roche, E. B. Design of Biopharmaceutical Properties through Prodrugs and
Analogs.
American Pharmaceutical Association: Washington, DC (1977).
= Sinkula, A. A.; Yalkowsky, S. H. JPharm Sci. 1975, 64, 181-210.
= Stella, V. J.; Charman, W. N. Naringrekar, V. H. Drugs 1985, 29, 455-473.
= Bundgaard, H., ed. Design ofProdrugs. Elsevier: New York (1985).
= Stella, V. J.; Himmelstein, K. J. J. Med. Chem. 1980, 23, 1275-1282.
= Han, H-K; Amidon, G. L. AAPS Pharmsci 2000, 2, 1- 11.
= Denny, W. A. Eur. J. Med. Chem. 2001, 36, 577-595.
= Wermuth, C. G. in Wermuth, C. G. ed. The Practice of Medicinal Chemistry
Academic Press:
San Diego (1996), 697-715.
= Balant, L. P.; Doelker, E. in Wolff, M. E. ed. Burgers Medicinal Chemistry
And Drug
Discovery John Wiley & Sons: New York (1997), 949-982.
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The metabolites of the compounds of this invention include oxidized
derivatives of the compounds
of formula I, wherein one or more of the nitrogens are substituted with a
hydroxy group; which
includes derivatives where the nitrogen atom of the pyridine group is in the
oxide form, referred to
in the art as 1-oxo-pyridine or has a hydroxy substituent, referred to in the
art as 1-hydroxy-
pyridine.
General Preparative Methods
The compounds of the invention may be prepared by use of known chemical
reactions and
procedures as described e.g. in the following published international
application WO
2005/009961.
The compounds of formula I have been previously characterized as having
various activities,
including for inhibiting the Raf/MEK/ERK pathway, raf kinase, p38 kinase,
VEGFR kinase,
PDGFR kinase. These activities and their use in treating various diseases and
conditions are
disclosed in, e.g., WO 2005/009961, which are hereby incorporated by reference
in their entirety.
Indications
Drug combinations of the present invention can be utilized to treat any_
diseases or conditions that
are associated with, or mediated by, the cellular pathways modulated by the
compounds
comprising the combinations. These pathways, include, but are not limited to
signalling pathways
which comprise, e.g., VEGFR, VEGFR2, Raf/Mek/Erk, Akt/PI3K, MTOR, PTEN, etc.
(see also
above). The drug combinations can be useful to treat diseases that are
associated with, or
mediated by, mutations in one of more genes present in these pathways,
including cancer-
associated mutations in PTEN, ras, Raf, Akt, PI3K, etc.
As mentioned above, although the compounds may be known as specific
inhibitors, the present
invention includes any ameliorative or therapeutic effect, regardless of the
mechanism of action or
how it is achieved.
The drug combination can have one or more of the following activities,
including, anti-
proliferative; anti-tumor; anti-angiogenic; inhibiting the proliferation of
endothelial or tumor cells;
anti-neoplastic; immunosuppressive; immunomodulatory; apoptosis-promoting,
etc.
Conditions or diseases that can be treated in accordance with the present
invention include
proliferative disorders (such as cancer), inflammatory disorders, immuno-
modulatory disorders,
allergy, autoimmune diseases, (such as rheumatoid arthritis, or multiple
sclerosis), abnormal or
excessive angiogenesis, etc.
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Any tumor or cancer can be treated, including, but not limited to, cancers
having one or more
mutations in raf, VEGFR-2, VEGFR-3, PDGFR-beta, Flt-3, ras, PTEN, Akt, PI3K,
mTOR, as well
as any upstream or downstream member of the signalling pathways of which they
are a part. A
tumor or cancer can be treated with a drug combination of the present
invention irrespective of the
mechanism that is responsible for it. Cancers of any organ can be treated,
including cancers of, but
are not limited to, e.g., colon, pancreas, breast, prostate, bone, liver,
kidney, lung, testes, skin,
pancreas, stomach, prostate, ovary, uterus, head and neck, blood cell, lymph,
etc.
Cancers that can be treated in accordance with the present invention include,
especially, but not
limited to, brain tumors, breast cancer, bone sarcoma (e.g., osteosarcoma and
Ewings sarcoma),
bronchial premalignancy, endometrial cancer, glioblastoma, hematologic
malignancies,
hepatocellular carcinoma, Hodgkin's disease, kidney neoplasms, leukemia,
leimyosarcoma,
liposarcoma, lymphoma, Lhermitte-Duclose disease, malignant glioma, melanoma,
malignant
melanoma, metastases, multiple myeloma, myeloid metaplasia, myeloplastic
syndromes, non-small
cell lung cancer, pancreatic cancer, prostate cancer, renal cell carcinoma
(e.g., advanced, advanced
refractory), rhabdomyosarcoma, soft tissue sarcoma, squamous epithelial
carcinoma of the skin,
cancers associated with loss of function of PTEN; activated Akt (e.g. PTEN
null tumors and
tumors with ras mutations).
Examples of breast cancer include, but are not limited to, invasive ductal
carcinoma, invasive
lobular carcinoma, ductal carcinoma in.situ, and lobular carcinoma in situ.
Examples of cancers of the respiratory tract include, but are not limited to,
small-cell, non-small-
cell lung carcinoma, bronchial adenoma, and pleuropulmonary blastoma.
Examples of brain cancers include, but are not limited to, brain stem and
hypophtalmic glioma,
cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and
neuroectodermal and
pineal tumor.
Tumors of the male reproductive organs include, but are not limited to,
prostate and testicular
cancer. Tumors of the female reproductive organs include, but are not limited
to, endometrial,
cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the
uterus.
Tumors of the digestive tract include, but are not limited to, anal, colon,
colorectal, esophageal,
gallbladder, gastric, pancreatic, rectal, small intestine, and salivary gland
cancers.
Tumors of the urinary tract include, but are not limited to, bladder, penile,
kidney, renal pelvis,
ureter, and urethral cancers.
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Eye cancers include, but are not limited to, intraocular melanoma and
retinoblastoma.
Examples of liver cancers include, but are not limited to, hepatocellular
carcinoma (liver cell
carcinomas with or without fibrolamellar variant), cholangiocarcinoma
(intrahepatic bile duct
carcinoma), and mixed hepatocellular cholangiocarcinoma.
Skin cancers include, but are not limited to, squamous cell carcinoma,
Kaposi's sarcoma,
malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
Head-and-neck cancers include, but are not limited to, laryngeal,
hypopharyngeal, nasopharyngeal,
and/or oropharyngeal cancers, and lip and oral cavity cancer.
Lymphomas include, but are not limited to, AIDS-related lymphoma, non-
Hodgkin's lymphoma,
cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of the central
nervous system.
Sarcomas include, but are not limited to, sarcoma of the soft tissue,
osteosarcoma, malignant
fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.
Leukemias include, but are not limited to, acute myeloid leukemia, acute
lymphoblastic leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell
leukemia.
In addition to inhibiting the proliferation of tumor cells, drug combinations
of the present
invention can also cause tumor regression, e.g., a decrease in the size of a
tumor, or in the extent of
cancer in the body.
Preference is given to the treatment of melanoma, renal cancer, hepatocellular
cancer, non small
lung cancer, ovarian cancer, prostate cancer, colorectal cancer, breast cancer
or pancreatic cancer.
Angiogenesis-related conditions and disorders can also be treated with drug
combinations of the
present invention. Inappropriate and ectopic expression of angiogenesis can be
deleterious to an
organism. A number of pathological conditions are associated with the growth
of extraneous
blood vessels. These include, e.g., diabetic retinopathy, neovascular
glaucoma, psoriasis,
retrolental fibroplasias, angiofibroma, inflammation, restenosis, etc. In
addition, the increased
blood supply associated with cancerous and neoplastic tissue, encourages
growth, leading to rapid
tumor enlargement and metastasis. Moreover, the growth of new blood vessels in
a tumor provides
an escape route for renegade cells, encouraging metastasis and the consequence
spread of the
cancer.
Useful systems for modulating angiogenesis, include, e.g., neovascularization
of tumor explants
(e.g., U.S. Pat. Nos. 5,192,744; 6,024,688), chicken chorioallantoic membrane
(CAM) assay (e.g.,
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Taylor and Folkman, Nature, 297:307-312, 1982; Eliceiri et al., J. Cell Biol.,
140, 1255-1263,
1998), bovine capillary endothelial (BCE) cell assay (e.g., U.S. Pat. No.
6,024,688; Polverini, P. J.
et al., Methods Enzymol., 198: 440-450, 1991), migration assays, and HUVEC
(human umbilical
cord vascular endothelial cell) growth inhibition assay (e.g., U.S. Pat. No.
6,060,449). In addition,
useful systems for modulating lymphangiogenesis, include, e.g., rabbit ear
model (e.g., Szuba et
al., FASEB J., 16(14):1985-7, 2002).
Modulation of angiogenesis can be determined by any suitable method. For
example, the degree
of tissue vascularity is typically determined by assessing the number and
density of vesssels
present in a given sample. For example, microvessel density (MVD) can be
estimated by counting
the number of endothelial clusters in a high-power microscopic field, or
detecting a marker
specific for microvascular endothelium or other markers of growing or
established blood vessels,
such as CD31 (also known as platelet-endothelial cell adhesion molecule or
PECAM). A CD31
antibody can be employed in conventional immunohistological methods to
immunostain tissue
sections as described by, e.g., Penfold et al., Br. J. Oral and Maxill. Surg.,
34: 37-41; U.S. Pat. No.
6,017,949; Dellas et al., Gyn. Oncol., 67:27-33, 1997; and others. Other
markers for angiogenesis,
include, e.g., Vezfl (e.g., Xiang et al., Dev. Bio., 206:123-141, 1999),
angiopoietin, Tie-1, and
Tie-2 (e.g., Sato et al., Nature, 376:70-74, 1995).
The drug combinations of this invention also have a broad therapeutic activity
to treat or prevent
the progression of a broad array of diseases, such as inflammatory conditions,
coronary restenosis,
tumor-associated angiogenesis, atherosclerosis, autoimmune diseases,
inflammation, certain
kidney diseases associated with proliferation of glomerular or mesangial
cells, and ocular diseases
associated with retinal vessel proliferation. psoriasis, hepatic cirrhosis,
diabetes, atherosclerosis, =
restenosis, vascular graft restenosis, in-stent stenosis, angiogenesis,
ocurlar diseases, pulmonary
fibrosis, obliterative brorichiolitis, glomerular nephritis, rheumatoid
arthritis.
The present invention also provides for treating, preventing, modulating,
etc., one or more of the
following conditions in humans and/or other mammals: retinopathy, including
diabetic
retinopathy, ischemic retinal-vein occlusion, retinopathy of prematurity and
age related macular
degeneration; rheumatoid arthritis, psoriasis, or bullous disorder associated
with subepidermal
blister formation, including bullous pemphigoid, erythema multiforme, or
dermatitis herpetiformis,
rheumatic fever, bone resorption, postmenopausal osteoperosis, sepsis, gram
negative sepsis, septic
shock, endotoxic shock, toxic shock syndrome, systemic inflammatory response
syndrome,
inflammatory bowel disease (Crohn's disease and ulcerative colitis), Jarisch-
Herxheimer reaction,
asthma, adult respiratory distress syndrome, acute pulmonary fibrotic disease,
pulmonary
sarcoidosis, allergic respiratory disease, silicosis, coal worker's
pneumoconiosis, alveolar injury,
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hepatic failure, liver disease during acute inflammation, severe alcoholic
hepatitis, malaria
(Plasmodium falciparum malaria and cerebral malaria), non-insulin-dependent
diabetes mellitus
(NIDDM), congestive heart failure, damage following heart disease,
atherosclerosis, Alzheimer's
disease, acute encephalitis, brain injury, multiple sclerosis (demyelation and
oligiodendrocyte loss
5. in multiple sclerosis), advanced cancer, lymphoid malignancy, pancreatitis,
impaired wound
healing in infection, inflammation and cancer, myelodysplastic syndromes,
systemic lupus
erythematosus, biliary cirrhosis, bowel necrosis, radiation injury/ toxicity
following administration
of monoclonal antibodies, host-versus-graft reaction (ischemia reperfusion
injury and allograft
rejections of kidney, liver, heart, and skin), lung allograft rejection
(obliterative bronchitis), or
complications due to total hip replacement, ad an infectious disease selected
from tuberculosis,
Helicobacter pylori infection during peptic ulcer disease, Chaga's disease
resulting from
Trypanosoma cruzi infection, effects of Shiga-like toxin resulting from E.
coli infection, effects of
enterotoxin A resulting from Staphylococcus infection, meningococcal
infection, and infections
from Borrelia burgdorferi, Treponema pallidum, cytomegalovirus, influenza
virus, Theiler's
encephalomyelitis virus, and the human immunodeficiency virus (HIV),
papilloma, blastoglioma,
Kaposi's sarcoma, melanoma, lung cancer, ovarian cancer, prostate cancer,
squamous cell
carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast
cancer, colorectal cancer,
thyroid cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma,
leukemia, lymphoma,
Hodgkin's disease, Burkitt's disease, arthritis, rheumatoid arthritis,
diabetic retinopathy,
angiogenesis, restenosis, in-stent restenosis, vascular graft restenosis,
pulmonary fibrosis, hepatic
cirrhosis, atherosclerosis, glomerulonophritis, diabetic nephropathy, thrombic
micoangiopathy
syndromes, transplant rejection, psoriasis, diabetes, wound healing,
inflammation, and
neurodegenerative diseases. hyperimmune disorders, hemangioma, myocardial
angiogenesis,
coronary and cerebral collateral vascularization, ischemia, corneal disease,
rubeosis, neovascular
glaucoma, macular degeneration retinopathy of prematurity, wound healing,
ulcer HelicQbacter
related diseases, fractures, endometriosis, a diabetic condition, cat scratch
fever, thyroid
hyperplasia, asthma or edema following bums, trauma, chronic lung disease,
stroke, polyps, cysts,
synovitis, chronic and allergic inflammation, ovarian hyperstimulation
syndrome, pulmonary and
cerebral edema, keloid, fibrosis, cirrhosis, carpal tunnel syndrome, adult
respiratory distress
syndrome, ascites, an ocular condition, a cardiovascular condition, Crow-
Fukase (POEMS)
disease, Crohn's disease, glomerulonophritis, osteoarthritis, multiple
sclerosis, graft rejection,
Lyme disease, sepsis, von Hippel Lindau disease, pemphigoid, Paget's disease,
polycystic kidney
disease, sarcoidosis, throiditis, hyperviscosity syndrome, Osler-Weber-Rendu
disease, chronic
occlusive pulmonary disease, radiation, hypoxia, preeclampsia,
menometrorrhagia, endometriosis,
infection by Herpes simplex, ischemic retinopathy, corneal angiogenisis,
Herpes Zoster, human
immunodeficiency virus, parapoxvirus, protozoa, toxoplasmosis,
spondylarthritis, ankylosing
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spondylitis, Morbus Bechterew, avian influenza including e.g. serotype H5N1,
and tumor-
associated effusions and edema.
The present invention provides methods of treating any of the aforementioned
diseases and/or
conditions (including those mentioned in any of the cited references),
comprising administering
effective amounts of a compound of formula I and at least one second compound
that is an
inhibitor of the PI3K/AKT signalling pathway (e.g. rapamycin or a derivative
or analog of
rapamycin, or wortmannin or a derivative or analog of wortmannin). An
"effective amount" is the
quantity of the compound that is useful to achieve the desired result, e.g.,
to treat the disease or
condition.
The present invention also relates to methods of inhibiting angiogenesis in a
system comprising
cells, comprising administering to the system a combination of effective
amounts of compounds
described herein. A system comprising cells can be an in vivo system, such as
a tumor in a patient,
isolated organs, tissues, or cells, in vitro assays systems (CAM, BCE, etc),
animal models (e.g., in
vivo, subcutaneous, cancer models), hosts in need of treatment (e.g., hosts
suffering from diseases
having an angiogenic component, such as cancer; experiencing restenosis), etc.
In addition, the drug combinations can be administered to modulate one or more
the following
processes, cell growth (e.g., proliferation), tumor cell growth (including,
e.g., differentiation, cell
survival, and/or proliferation), tumor regression, endothelial cell growth
(including, e.g.,
differentiation, cell survival, and/or proliferation), angiogenesis (blood
vessel growth),
angiogenesis, and/or hematopoiesis (e.g., proliferation, T-cell development,
etc.).
Compounds or drug combinations of the present invention can be administered in
any form by any
effective route, including, e.g., oral, parenteral, enteral, intravenous,
intraperitoneal, topical,
transdermal (e.g., using any standard patch), ophthalmic, nasally, local, non-
oral, such as aerosal,
inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal,
intra-arterial, and
intrathecal, etc. They can be administered alone, or in combination with any
ingredient(s), active
or inactive. They can be administered in any effective dosage, e.g., from
about 0.1 to about 200
mg/kg of total body weight.
The combinations of the present invention can be administered at any time and
in any effective
form. For example, the compounds can be administered simultaneously, e.g., as
a single
composition or dosage unit (e.g., a pill or liquid containing both
compositions), or they can be
administered as separate compositions, but at the same time (e.g., where one
drug is administered
intravenously and the other is administered orally or intramuscularly. The
drugs can also be
administered sequentially at different times. Agents can be formulated
conventionally to achieve
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the desired rates of release over extended period of times, e.g., 12-hours, 24-
hours. This can be
achieved by using agents and/or their derivatives which have suitable
metabolic half-lives, and/or
by using controlled release formulations.
The drug combinations can be synergistic, e.g., where the joint action of the
drugs is such that the
combined effect is greater than the algebraic sum of their individual effects.
Thus, reduced
amounts of the drugs can be administered, e.g., reducing toxicity or other
deleterious or unwanted
effects, and/or using the same amounts as used when the agents are
administered alone, but
achieving greater efficacy, e.g., in having more potent antiproliferative and
pro-apoptotic action.
Compounds or drug combinations of the present invention can be further
combined with any other
suitable additive or pharmaceutically acceptable carrier. Such additives
include any of the
substances already mentioned, as well as any of those used conventionally,
such as those described
in Remington: The Science and Practice of Pharmacy (Gennaro and Gennaro, eds,
20th edition,
Lippincott Williams & Wilkins, 2000); Theory and Practice of Industrial
Pharmacy (Lachman et
al., eds., 3rd edition, Lippincott Williams & Wilkins, 1986); Encyclopedia of
Pharmaceutical
Technology (Swarbrick and Boylan, eds., 2nd edition, Marcel Dekker, 2002).
These can be
referred to herein as "pharmaceutically acceptable carriers" to indicate they
are combined with the
active drug and can be administered safely to a subject for therapeutic-
purposes.
In addition, compounds or drug combinations of the present invention can be
administered with
other active agents or therapies (e.g., radiation) that are utilized to treat
any of the above-
mentioned diseases and/or conditions.
The present invention provides combinations of at least one compound of
Formula I and at least
one second compound which is a PI3K/AKT signalling pathway inhibitor useful in
treating a
disease or disorder. "Combinations" for the purposes of the invention include:
-single compositions or dosage forms which contain at least one compound of
Formula I
and at least one second compound which is an PI3K/AKT signalling pathway
inhibitor;
-combination packs containing at least one compound of Formula I and at least
one second
compound which is an PI3K/AKT signalling pathway inhibitor, to be administered
concurrently or sequentially;
-kits which comprise at least one compound of Formula I and at least one
second
compound which is an PI3K/AKT signalling pathway inhibitor packaged separate
from
one another as unit dosages or as independent unit dosages, with or without
instructions
that they be administered concurrently or sequentially; and
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-separate independent dosage forms of at least one compound of Formula I and
at least one
second compound which is an PI3K/AKT signalling pathway inhibitor which
cooperate to
achieve a therapeutic effect, e.g., prophylaxis or treatment of the same
disease, when
administered concurrently or sequentially.
The dosage of each agent of the combination can be selected with reference to
the other and/or the
type of disease and/or the disease status in order to provide the desired
therapeutic activity. For
example, the active agents in the combination can be present and administered
in a fixed
combination. "Fixed combination" is intended here to mean pharmaceutical forms
in which the
components are present in a fixed ratio that provides the desired efficacy.
These amounts can be
determined routinely for a particular patient, where various parameters are
utilized to select the
appropriate dosage (e.g., type of cancer, age of patient, disease status,
patient health, weight, etc.),
or the amounts can be relatively standard.
The combination can comprise effective amounts of at least one compound of
Formula I and at
least one second compound which is a PI3K/AKT signalling pathway inhibitor,
which achieves a
greater therapeutic efficacy than when either compound is used alone. The
combination can be
useful to produce tumor regression, to produce disease stability, to prevent
or reduce metastasis, or
other therapeutic endpoints, where the therapeutic effect is not observed when
the agents are used
alone, or where an enhanced effect is observed when the combination is
administered.
The relative ratios of each compound in the combination can also be selected
based on their
respective mechanisms of action and the disease biology. For example,
activating mutations of
the B-RAF gene are observed in more than 60% of human melanomas and a
composition for
treatment of melanoma may advantageously comprise a formula I compound in a
more potent
amount than the compound which is a P13K/AKT signalling pathway inhibitor. In
comparison,
where a cancer is associated with a mutation in the PI3K/AKT signalling
pathway (e.g., ovarian
and breast cancers), an agent which has activity in this signalling pathway
can be present in more
potent amounts relative to the Ref/MEK/ERK pathway inhibitor. The relative
ratios of each
compound can vary widely and this invention includes combinations for treating
cancer where the
amounts of the formula I compound and the second active agent can be adjusted
routinely such that
either is present in higher amounts.
The release of one or more agents of the combination can also be controlled,
where appropriate, to
provide the desired therapeutic activity when in a single dosage form,
combination pack, kit or
when in separate independent dosage forms.
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Assays
Activity of combinations of the present invention can be determined according
to any effective in
vitro or in vivo method.
Kinase activity
Kinase activity can be determined routinely using conventional assay methods.
Kinase assays
typically comprise the kinase enzyme, substrates, buffers, and components of a
detection system.
A typical kinase assay involves the reaction of a protein kinase with a
peptide substrate and an
ATP, such as 32P-ATP, to produce a phosphorylated end-product (for instance, a
phosphoprotein
when a peptide substrate is used). The resulting end-product can be detected
using any suitable
method. When radioactive ATP is utilized, a radioactively labeled
phosphoprotein can be
separated from the unreacted gamma-32P-ATP using an affinity membrane or gel
electrophoresis,
and then visualized on the gel using autoradiography or detected with a
scintillation counter. Non-
radioactive methods can also be used. Methods can utilize an antibody which
recognizes the
phosphorylated substrate, e.g., an anti-phosphotyrosine antibody. For
instance, kinase enzyme can
be incubated with a substrate in the presence of ATP and kinase buffer under
conditions which are
effective for the enzyme to phosphorylate the substrate. _ The reaction
mixture can be separated,
e.g., electrophoretically, and then phosphorylation of the substrate can be
measured, e.g., by
Western blotting using an anti-phosphotyrosine antibody. The antibody can be
labeled with a
detectable label, e.g., an enzyme, such as HRP, avidin or biotin,
chemiluminescent reagents, etc.
Other methods can utilize ELISA formats, affinity membrane separation,
fluorescence polarization
assays, luminescent assays, etc.
An alternative to a radioactive format is time-resolved fluorescence resonance
energy transfer (TR-
FRET). This method follows the standard kinase reaction, where a substrate,
e.g., biotinylated
poly(GluTyr), is phosphorylated by a protein kinase in the presence of ATP.
The end-product can
then detected with a europium chelate phosphospecific antibody (anti-
phosphotyrosine or
phosphoserine/threonine), and streptavidin-APC, which binds the biotinylated
substrate. These two
components are brought together spatially upon binding, and energy transfer
from the
phosphospecific antibody to the acceptor (SA-APC) produces fluorescent readout
in the
homogeneous format.
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Raf/MEK/ERK activity
A c-Raf kinase assay can be performed with a c-Raf enzyme activated
(phosphorylated) by Lck
kinase. Lck-activated c-Raf (Lck/c-Raf) is produced in Sf9 insect cells by co-
infecting cells with
baculoviruses expressing, under the control of the polyhedrin promoter, GST-c-
Raf (from amino
acid 302 to amino acid 648) and Lck (full-length). Both baculoviruses are used
at the multiplicity
of infection of 2.5 and the cells are harvested 48 hours post infection.
MEK-1 protein is produced in Sf9 insect cells by infecting cells with the
baculovirus expressing
GST-MEK-1 (full-length) fusion protein at the multiplicity of infection of 5
and harvesting the
cells 48 hours post infection. Similar purification procedure is used for GST-
c-Raf 302-648 and
GST-MEK-1.
Transfected cells are suspended at 100 mg of wet cell biomass per mL in a
buffer containing 10
mM sodium phosphate, 140 mM sodium chloride pH 7.3, 0.5% Triton X-100 and the
protease
inhibitor cocktail. The cells are disrupted with a Polytron homogenizer and
centrifuged 30,000g
for 30 minutes. The 30,000g supernatant is applied applied onto GSH-Sepharose.
The resin is
washed with a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCI and 0.01%
Triton X-100.
The GST-tagged proteins are eluted with a solution containing 100 mM
Glutathione, 50 mM Tris,
pH 8.0, 150 mM NaCI and 0.01% Triton X-100. The purified proteins are dialyzed
into a buffer
containing 20 mM Tris, pH 7.5, 150 mM NaCI and 20% Glycerol.
Test compounds are serially diluted in DMSO using three-fold dilutions to
stock concentrations
ranging typically from 50 M to 20 nM (e.g., final concentrations in the assay
can range from 1
M to 0.4 nM). The c-Raf biochemical assay is performed as a radioactive
filtermat assay in 96-
well Costar polypropylene plates (Costar 3365). The plates are loaded with 75
L solution
containing 50 mM HEPES pH 7.5, 70 mM NaCl, 80 ng of Lck/c-Raf and 1 g MEK-1.
Subsequently, 2 L of the serially diluted individual compounds is added to
the reaction, prior to
the addition of ATP. The reaction is initiated with 25 L ATP solution
containing 5 M ATP and
0.3 Ci [33P]-ATP. The plates were sealed and incubated at 32 C for 1 hour.
The reaction is
quenched with the addition of 50 l of 4 % Phosphoric Acid and harvested onto
P30 filtermats
(PerkinElmer) using a Wallac Tomtec Harvester. Filtermats are washed with 1%
Phosphoric Acid
first and deinonized H20 second. The filters are dried in a microwave, soaked
in scintillation fluid
and read in a Wallac 1205 Betaplate Counter (Wallac Inc., Atlanta, GA,
U.S.A.). The results are
expressed as percent inhibition.
% Inhibition = [100-(Tib/Ti)] x 100 where
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Tib = (counts per minute with inhibitor)-(background)
Ti = (counts per minute without inhibitor)-(background)
Raf activity can also be monitored by its ability to initiate the cascade
leading to ERK
phosphorylation (i.e., raf/MEK/ERK), resulting in phospho-ERK. A Bio-Plex
Phospho-ERKI/2
immunoassay can be performed as follows:
A 96-well phospho-ERK (pERK) immunoassay, using laser flow cytometry platform
has been
established to measure inhibition of basal pERK in cell lines. MDA-MB-231
cells are plated at
50,000 cells per well in 96-well microtitre plates in complete growth media.
For effects of test
compounds on basal pERKl/2 inhibition, the next day after plating, MDA-MB-231
cells are
transferred to DMEM with 0.1% BSA and incubated with test compounds diluted
1:3 to a final
concentration of 3 mM to 12 nM in 0.1 % DMSO. Cells are incubated with test
compounds for 2 h,
washed, and lysed in Bio-Plex whole cell lysis buffer A. Samples are diluted
with buffer B 1:1
(v/v) and directly transferred to assay plate or frozen at -80 C degrees until
processed. 50 niL of
diluted MDA-MB-231 cell lysates are incubated with about 2000 of 5 micron Bio-
Plex beads
conjugated with an anti-ERK1/2 antibody overnight on a shaker at room
temperature. The next
day, biotinylated phospho-ERK1/2 sandwich immunoassay is performed, beads are
washed 3 times
during each incubation and then 50 mL of PE-strepavidin is used as a
developing reagent. The
relative fluorescence units of pERKl/2 is detected by counting 25 beads with
Bio-Plex flow cell
(probe) at high sensitivity. The IC50 is calculated by taking untreated cells
as maximum and no
cells (beads only) as background.
Phosphatidylinositol 3-kinase activity
PKI3 activity can be determined routinely, e.g., using commercially available
kits (e.g., Perkin-
Elmer, FlashPlate Platform), Frew et al., Anticancer Res., 14(6B):2425-8,
1994. See also,
publications listed under PKI3 inhibitors.
Akt activity
AKT can be isolated from insect cells expressing His-tagged AKTI (aa 136-480)
as described in
WO 05011700. Expressing cells are lysed in 25 mM HEPES, 100 mM NaCI, 20 mM
imidazole;
pH 7.5 using a polytron (5 mis lysis buffer/g cells). Cell debris is removed
by centrifuging at
28,000 x g for 30 minutes. The supernatant is filtered through a 4.5 micron
filter then loaded onto
a nickel-chelating column pre-equilibrated with lysis buffer. The column is
washed with 5 column
volumes (CV) of lysis buffer then with 5 CV of 20% buffer B, where buffer B is
25 mM HEPES,
100 mM NaCI, 300 mM imidazole; pH 7. His-tagged AKTI (aa 136-480) is eluted
with a 20-100%
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linear gradient of buffer B over 10 CV. His-tagged AKTI (136-480) eluting
fractions are pooled
and diluted three-fold with buffer C, where buffer C is 25 mM HEPES, pH 7. The
sample is then
chromatographed over a Q-Sepharose HP column pre-equilibrated with buffer C.
The column is
washed with 5 CV buffer C, then step eluted with 5 CV 10 %D, 5 CV 20%D, 5 CV
30% D, 5 CV
50% D. and 5 CV of 100% D; where buffer D is 25 mM HEPES, 1000 mM NaCI; pH
7.5.
His-tagged AKTI (aa 136-480) containing fractions are pooled and concentrated
in a 10-kDa
molecular weight cutoff concentrator. His-tagged AKT1 (aa 136-480) is
chromatographed over a
Superdex 75 gel filtration column pre-equilibrated with 25 mM HEPES, 200 mM
NaCI, 1 mM
DTT; pH 7.5. His-tagged AKTI (aa 136-480) fractions are examined using SDS-
PAGE and mass
spec. The protein is pooled, concentrated, and stored at 80 C.
His-tagged AKT2 (aa 138-481) and His-tagged AKT3 (aa 135-479) can be isolated
and purified in
a similar fashion.
AKT Enzyme Assay Compounds can be tested for AKT protein serine kinase
inhibitory activity in
substrate phosphorylation assays. This assay examines the ability of small
molecule organic
compounds to inhibit the serine phosphorylation of a peptide substrate. The
substrate
phosphorylation assays use the catalytic domains of AKT 1, 2, or 3. AKT 17 2
and 3 are also
commercially available from Upstate USA, Inc. The method measures the ability
of the isolated
enzyme to catalyze the transfer of the gamma-phosphate from ATP onto the
serine - 72 residue of a
biotinylated synthetic peptide (Biotin-ahx-ARKRERAYSFGHHA-amide). Substrate
phosphory-
lation can be detected by the following procedure described in WO 05011700.
Assays are performed in 384 well U-bottom white plates. 10 nM activated AKT
enzyme is
incubated for 40 minutes at room temperature in an assay volume of 20u1
containing 50 mM
MOPS, pH 7.5, 20 mM MgCIZ, 4uM ATP, 8uM peptide, 0.04 uCi [g- 33P] ATP/well, 1
mM
CHAPS, 2 mM DTT, and 1 l of test compound in 100% DMSO. The reaction is
stopped by the
addition of 50 l SPA bead mix (Dulbecco's PBS without Mg2+ and Ca2+, 0.1 %
Tritoin X-100, 5
mM EDTA, 50 M ATP, 2.5mg/ml Streptavidin-coated SPA beads). The plate is
sealed, the beads
are allowed to settle overnight, and then the plate was counted in a Packard
Topcount Microplate
Scintillation Counter (Packard Instrument Co., Meriden, CT).
The data for dose responses can be plotted as % Control calculated with the
data reduction formula
100*(UI-C2)/(C1-C2) versus concentration of compound where U is the unknown
value, Cl is the
average control value obtained for DIVISO, and C2 is the average control value
obtained for 0.1M
EDTA. Data are fitted to the curve described by: y=((Vmax * x) K + x)) where
Vmax is the upper
asymptote and K is the IC50.
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Cell proliferation
An example of a cell proliferation assay is described in the Examples below.
However,
proliferation assays can be performed by any suitable method. For example, a
breast carcinoma
cell proliferation assay can be performed as follows. Other cell types can be
substituted for the
MDA-MB-231 cell line.
Human breast carcinoma cells (MDA MB-23 1, NCI) are cultured in standard
growth medium
(DMEM) supplemented with 10% heat-inactivated FBS at 37 C in 5% COZ (vol/ vol)
in a
humidified incubator. Cells are plated at a density of 3000 cells per well in
90 L growth medium
in a 96 well culture dish. In order to determine Toh CTG values, 24 hours
after plating, 100 L of
CellTiter-Glo Luminescent Reagent (Promega) is added to each well and
incubated at room
temperature for 30 minutes. Luminescence is recorded on a Wallac Victor II
instrument. The
Ce1lTiter-Glo reagent results in cell lysis and generation of a luminescent
signal proportional to the
amount of ATP present, which, in turn is directly proportional to the number
of cells present.
Test compounds are dissolved in 100% DMSO to prepare 10 mM stocks. Stocks are
further diluted
1:400 in growth medium to yield working stocks of 25 M test compound in 0.25%
DMSO. Test
compounds are serially diluted in growth medium containing 0.25% DMSO to
maintain constant
DMSO concentrations for all wells. 60 L of diluted test compound are added to
each culture well
to give a final volume of 180 L. The cells with and without individual test
compounds are
incubated for 72 hours at which time ATP dependent luminescence was measured,
as described
previously, to yield T72h values. Optionally, the IC50 values can be
determined with a least squares
analysis program using compound concentration versus percent inhibition.
% Inhibition = [1-(T72n ce5t-Ton)/(Z'nn ai-Ton)] x 100, where
T72h te5t = ATP dependent luminescence at 72 hours in the presence of test
compound
T72n ,vi = ATP dependent luminescence at 72 hours in the absence of test
compound
Toh = ATP dependent luminescence at Time Zero.
Angiogenesis
One useful model to study angiogenesis is based on the observation that, when
a reconstituted
basement membrane matrix, such as Matrigel, supplemented with growth factor
(e.g., FGF-1), is
injected subcutaneously into a host animal, endothelial cells are recruited
into the matrix, forming
new blood vessels over a period of several days. See, e.g., Passaniti et al.,
Lab. Invest., 67:519-
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528, 1992. By sampling the extract at different times, angiogenesis can be
temporally dissected,
permitting the identification of genes involved in all stages of angiogenesis,
including, e.g.,
migration of endothelial cells into the matrix, commitment of endothelial
cells to angiogenesis
pathway, cell elongation and formation of sac-like spaces, and establishment
of functional
capillaries comprising connected, and linear structures containing red blood
cells. To stabilize the
growth factor and/or slow its release from the matrix, the growth factor can
be bound to heparin or
another stabilizing agent. The matrix can also be periodically re-infused with
growth factor to
enhance and extend the angiogenic process.
Other useful systems for studying angiogenesis, include, e.g.,
neovascularization of tumor explants
(e.g., U.S. Pat. Nos. 5,192,744; 6,024,688), chicken chorioallantoic membrane
(CAM) assay (e.g.,
Taylor and Folkman, Nature, 297:307-312, 1982; Eliceiri et al., J. Cell Biol.,
140, 1255-1263,
1998), bovine capillary endothelial (BCE) cell assay (e.g., U.S. Pat. No.
6,024,688; Polverini, P. J.
et al., Methods Enzymol., 198: 440-450, 1991), migration assays, HUVEC (human
umbilical cord
vascular endothelial cell) growth inhibition assay (e.g., U.S. Pat. No.
6,060,449).
The present invention provides one or more of the following features.
A method of treating ariy of the aforementioned diseases and/or conditions,
comprising
administering effective amounts of a compound of formula I and a second
compound which is an
PI3K/AKT signalling pathway inhibitor.
A method of modulating (e.g., inhibiting) one or more aforementioned
activities, comprising
administering effective amounts of a compound of formula I and a second
compound which is an
PI3K/AKT signalling pathway inhibitor.
Combinations comprising a compound of formula I and a second compound which.
is an
PI3K/AKT signalling pathway inhibitor.
Without further elaboration, it is believed that one skilled in the art can,
using the
preceding description, utilize the present invention to its fullest extent.
The following
preferred specific embodiments are, therefore, to be construed as merely
illustrative, and
not limitative. of the remainder of the disclosure in any way whatsoever. The
entire
disclosure of all patents and publications, cited above and below are hereby
incorporated
by reference in their entirety.
