Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
3-HETEROARYLIDENYL-2-INDOLINONE COMPOUNDS FOR MODULATING
PROTEIN KINASE ACTIVITY AND FOR USE IN CANCER CHEMOTHERAPY
INTRODUCTION
The present invention relates generally to chemistry,
biochemistry, pharmacology, medicine and cancer treatment.
More particularly, it relates to 3-heteroarylidenyl-2-
indolinone compounds that modulate the activity of protein
kinases (PKs) and to methods for their use in treating
disorders related to abnormal protein kinase activity
including cancer wherein combinations of the compounds with
other chemotherapeutic agents are used.
BACKGROUND OF THE INVENTION
The following is provided by way of background
information only and is not admitted to be or to describe
prior art to the present invention.
PKs are enzymes that catalyze the phosphorylation of
hydroxy groups on tyrosine, serine and threonine residues of
proteins. The consequences of this seemingly simple activity
are staggering; cell growth, differentiation and
proliferation; i.e., virtually all aspects of cell life, in
one way or another depend on PK activity. Furthermore,
abnormal PK activity has been related to a host of disorders,
ranging from relatively non-life threatening diseases such as
psoriasis to extremely virulent diseases such as glioblastoma
(brain cancer) .
The PKs can conveniently be broken down into two
classes, the protein tyrosine kinases (PTKs) and the serine-
threonine kinases (STKs).
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One of the prime aspects of PK activity is involvement
with growth factor receptors. Growth factor receptors are
cell-surface proteins. When bound by a growth factor ligand,
growth factor receptors are converted to an active form which
interacts with proteins on the inner surface of a cell
membrane. This leads to phosphorylation on tyrosine residues
of the receptor as well as other proteins and to the
formation inside the cell of complexes with a variety of
cytoplasmic signaling molecules. These complexes, in turn,
affect numerous cellular responses such as cell division
(proliferation), cell differentiation, cell growth,
expression of metabolic effects on the extracellular
microenvironment, etc. For a more complete discussion, see
Schlessinger and Ullrich, Neuron, 1992, 9:303-391 which is
incorporated by reference, including any drawings, as if
fully set forth herein.
Growth factor receptors with PK activity are known as
receptor tyrosine kinases ("RTKs"). They comprise a large
family of transmembrane receptors with diverse biological
activity. At present, at least nineteen (19) distinct
subfamilies of RTKs have been identified. An example of
these is the subfamily designated the "HER" RTKs, which
includes EGFR (epithelial growth factor receptor), HER2, HERS
and HER4. These RTKs consist of an extracellular
glycosylated ligand binding domain, a transmembrane domain
and an intracellular cytoplasmic catalytic domain that can
phosphorylate tyrosine residues on proteins.
Another RTK subfamily consists of insulin receptor (IR),
insulin-like growth factor I receptor (IGF-1R) and insulin
receptor related receptor (IRR). IR and IGF-1R interact with
insulin, IGF-I and IGF-II to form a heterotetramer composed
of two entirely extracellular glycosylated a subunits and two
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(3 subunits which cross the cell membrane and which contain
the tyrosine kinase domain.
A third RTK subfamily is referred to as the platelet
derived growth factor receptor ("PDGFR") group, which
includes PDGFRa, PDGFR(3, CSFIR, c-kit and c-fms. These
receptors consist of glycosylated extracellular domains
composed of variable numbers of immunoglobin-like loops and
an intracellular domain wherein the tyrosine kinase domain is
interrupted by unrelated amino acid sequences.
Another group which, because of its similarity to the
PDGFR subfamily, is sometimes subsumed in the later group, is
the fetus liver kinase ("flk") receptor subfamily. This
group is believed to be composed of kinase insert domain-
receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4 and
fms-like tyrosine kinase 1 (flt-1).
One further member of the tyrosine kinase growth factor
receptor family is the fibroblast growth factor
("FGF")receptor group. This group consists of four receptors,
FGFR1 - FGFR4, and seven ligands, FGF1 - FGF7. While not yet
well characterized, it appears that the receptors also
consist of a glycosylated extracellular domain containing a
variable number of immunoglobin-like loops and an
intracellular domain in which the PTK sequence is interrupted
by regions of unrelated amino acid sequences.
A more complete listing of the known RTK subfamilies is
described in Plowman et al., DN&P, 1994, 7(6):334-339 which
is incorporated by reference, including any drawings, as if
fully set forth herein.
In addition to the RTKs, there also exists a family of
entirely intracellular PTKs called "non-receptor tyrosine
kinases" or "cellular tyrosine kinases." This latter
designation, abbreviated "CTK", will be used herein. CTKs do
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not contain extracellular and transmembrane domains. At
present, over 24 CTKs in il subfamilies (Src, Frk, Btk, Csk,
Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified.
The Src subfamily appear so far to be the largest group of
CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and
Yrk. For a more detailed discussion of CTKs, see Bolen,
Oncogene, 1993, 8:2025-2031, which is incorporated by
reference, including any drawings, as if fully set forth
herein.
The serine-threonine kinases or STKs, like the CTKs, are
predominantly intracellular although there are a few STK
receptor kinases. STKs are the most common of the cytosolic
kinases; i.e., kinases which perform their function in that
part of the cytoplasm other than the cytoplasmic organelles
and cytoskelton. The cytosol is the region within the cell
where much of the cell's intermediary metabolic and
biosynthetic activity occurs; e.g., it is in the cytosol that
proteins are synthesized on ribosomes.
RTKs, CTKs and STKs have all been implicated in a host
of pathogenic conditions including, significantly, cancer.
Others pathogenic conditions which have been associated with
PTKs include, without limitation, psoriasis, hepatic
cirrhosis, diabetes, atherosclerosis, angiogenesis,
restenosis, ocular diseases, rheumatoid arthritis and other
inflammatory disorders, autoimmune disease and a variety of
renal disorders.
With regard to cancer, two of the major hypotheses
advanced to explain the excessive cellular proliferation that
drives tumor development relate to functions known to be PK
regulated. That is, it has been suggested that malignant
cell growth results from a breakdown in the mechanisms that
control cell division and/or differentiation. It has been
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shown that the protein products of a number of proto-
oncogenes are involved in the signal transduction pathways
that regulate cell growth and differentiation. These protein
products of proto-oncogenes include the extracellular growth
factors, transmembrane growth factor PTK receptors (RTKs),
cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.
Cancer continues to be one of the leading causes of death
in human beings. The majority of cancers are solid tumor
cancers such as, without limitation, ovarian cancer, colorectal
cancer, brain cancer, liver cancer, kidney cancer, stomach
cancer, prostate cancer, lung cancer, thyroid cancer, Kaposi's
sarcoma and skin cancer. Of the solid tumor cancers,
colorectal cancer is a particularly common malignancy;
adenocarcinoma of the large bowel affects about one person in
20 in the United States and in most Westernized countries. In
the United States, colorectal cancer represents about 15% of
all newly diagnosed cancers. While colorectal cancer is the
third leading cause of cancer-related death, prognosis and
outcome is highly dependent on the stage the disease at
diagnosis. If diagnosed in early stages, colorectal cancer is
highly curable using a multidisciplinary treatment regime.
Nevertheless, 20 - 25% of patients diagnosed with the disease
will present with metastases or will develop locally recurrent
or metastatic disease; the majority of these patients will
eventually die of the disease.
The primary modes of treatment of solid tumor cancers,
including colorectal cancer, are surgery, radiation therapy and
chemotherapy, separately and in combination.
Although the initial formation and growth of tumors does
not require new blood vessel formation, any further growth
does require neovascularization. That is, for tumors to grow
beyond 3 to 4 mm3 in volume, new blood vessel growth; i.e.,
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angiogenesis, the sprouting of new capillaries from existing
blood vessels, must occur. In fact, immunohistochemical
analysis of tumor sections from the margins of growing tumors
show a preponderance of blood vessels, irrespective of tumor
type. To accomplish this neovascularization, angiogenic
factors are released from hypoxic tumor cells and migrate to
nearby blood vessel endothelial cells, activating these cells
to undergo morphologic changes, to move and to divide. Tumors
that lack adequate vasculature become necrotic (Brem, S., et
al., Cancer Res., 1976, 36, 2807-12) and/or apoptotic
(Holmgren, L., et al., Nature Med., 1995, 1:149-53; Parangi,
S., et al., Cancer Res., 1995, 55:6071-6), whereas tumors
wh~;ch have_undergone neovascularization not only can enter a
phase of rapid growth but also demonstrate increased
metastatic potential. In support of the significance of
angiogenesis in human tumors, recent studies relating the
angiogenic phenotype and survival in people have shown that
the number of microvessels in a primary tumor has prognostic
significance in breast carcinoma (Gasparini, G., and Harris,
A. L., J. Clin. Oncol., 1995, 13:765-82; Toi, M., et al.,
Japan. J. Cancer Res., 1994, 85:1045-9), bladder carcinomas
(Dickinson, A.J., et al., Br. J. Urol., 1994, 74:762-6),
colon carcinomas (Ellis, L. M., et al., Surgery, 1996,
120(5):871-8) oral cavity tumors (Williams, J.K., et al., Am.
J. Surg., 1994, 168:373-80). Angiogenesis may also play a
role in the growth of hematopoietic neoplasms and multiple
myeloma (Bellamy, W.T., et al., Proc. Amer. Assoc. Cancer
Res., 1998, Abstract #2566.
At present, the central mediator of malignant tumor
angiogensis is thought to be the endothelial mitogen,
vascular endothelial growth factor (VEGF). VEGF is mitogenic
for many types of small and large vessel endothelial cells.
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It induces the production of tissue -factors, collagenase and
plasminogen activators and inhibitors. VEGF is sometimes
referred to as "vascular permeability factor" by virtue of
its permeability enhancing effects (Landriscina, M., et al.,
Brit. J. Cancer, 1998, 78(6):765-770). In fact, the
vascular permeability factor potency of VEGF is some 50,000
times higher than that of histamine which is a well-known
vascular permeabilizing molecule (Dvorak, H. F., et al., Am.
J. Path., 1995, 146:1029-39). This increased permeability
results in extravasion of macromolecules such as fibrogen
from the circulation which provides a fibrin gel meshwork or
substratum for the migration and organization of endothelial
cells as well as tumor cells (Kumar, H., et al., Clin.
Cancer Res., 1998, 4:1279-85. VEGF expression has been
demonstrated in vitro in a number of human cancer cells lines
and surgically in resected tumors of the gastrointestinal
tract, ovary, brain, breast and kidney (Thomas, K. A., J.
Biol. Chem., 1996, 271:603-6).
VEGF has also been closely associated with the
development of colorectal cancer; i.e, increased levels of
VEGF have been found in tumor tissue from patients with
colorectal cancer. In fact, a strong correlation has been
observed between the increases VEGF and the stage and depth
of intestinal wall invasion (C. Barone, et al., Brit. J.
Cancer, 1998, 78(6):765-70). Consistent with this result is
the finding that serum levels of VEGF correlate significantly
with Dukes stage and carcinoembryotic antigen levels and that
patients with hepatic and/or lymph node metastases tend to
show higher serum VEGF levels than those patients without
such metastases (Fujisaki, K., et al., Am. J.
Gastroenterology, 1998, 93(2):249-52).
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Given the necessity of neovascularization for the growth
of solid tumors and the role of VEGF as one of the most
important mediators of angiogenesis, particularly in
colorectal cancer, compounds capable of inhibiting the
angiogenic effect of VEGF would be expected to retard the
rebound effect observed with fluorouracil-based colorectal
cancer treatment and thereby increase the chemotherapeutic
efficacy of fluorouracil, with or without leucovorin. An
additional advantage to such a method might be that the use
of an angiogenic inhibitor that reduces the ability of the
tumor to develop new blood vessels and thus would be
cytostatic rather than cytotoxic may compliment standard
cytotoxic chemotherapy; that is, utilize different mechanisms
of action to increase the efficacy of the cytotoxic agent
without additional toxicity.
SUMMARY OF THE INVENTION
Our search for small organic molecules which modulate
protein kinase mediated signal transduction has resulted in
the discovery of 3-heteroarylidenyl-2-indolinones which
modulate the activity of protein kinases (PKs) such as
receptor tyrosine kinases (RTKs), cellular tyrosine kinases
(CTKs) and serine-threonine tyrosine kinases (STKs). The
RTKs include, among others, Flk-1, Flt-1, Tie-1 and Tie-2,
all of whose expression have been found to be restricted to
endothelial cells. Of particular significance with regard to
the present invention is the fact that Flk-1 is believed to
play a critical role in angiogenesis and that that role is
mediated by VEGF. This suggests that 3-heteroarylidenyl-2-
indolinones should be capable of inhibiting VEGF-mediated
vascularization, and thereby the growth, of tumors during the
period when no chemotherapeutic agent, such as, without
limitation, a fluorinated pyrimidine, is being administered
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to a patient and thus should increase the efficacy of the
chemotherapeutic agent.
Thus, in one aspect, the present invention relates to
a 3-heteroarylidenyl-2-indolinone compound that inhibits
angiogenesis or vasculogenesis in a cell, the compound
having the chemical structure:
R
R2
R
or a pharmaceutically acceptable salt or prodrug thereof,
wherein
R, i s H or alkyl ;
Rz is O or S;
R3 is hydrogen;
R4, R5, R6, and R, are each independently selected from the
group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy,
alkaryl, alkaryloxy, halogen, trihalomethyl, S(O}R, SOzNRR',
S03R, SR, NO2, NRR' , OH, CN, C (O) R, OC (O) R, (CHZ) nCOzR, and
CONRR';
A is a five membered heteroaryl ring selected from the group
consisting of thiophene, pyrrole, pyrazole, imidazole, 1,2,3-
triazole, 1,2,4-triazole, oxazole, i~soxazole, thiazole,
isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole,
1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole,
1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole,
1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole,
1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, and tetrazole,
A
R.
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optionally substituted at one or more positions with alkyl,
alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen,
trihalomethyl, S (O) R, S02NRR' , SO,R, SR, NO2, NRR' , OH, CN,
C (O) R, OC (0) R, (CHZ) nCO2R, or CONRR' ;
n is 0-3; and,
R and R' are independently selected from the group consisting
of alkyl or aryl.
"Alkyl" refers to a straight-chain, branched or cyclic
saturated aliphatic hydrocarbon. Preferably, the alkyl group
has 1 to 12 carbons. More preferably, it has from 1 to 7
carbons and most preferably, it is a lower alkyl having from
1 to 4 carbons. Typical alkyl groups include methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl,
hexyl, and the like. The alkyl group may be optionally
substituted with one or more substituents selected from the
group consisting of hydroxyl, -C(0)OR, cyano, unsubstituted
alkoxy, =O, =S, NOZ, halogen, NRR' and SR.
"Alkenyl" refers to an alkyl group containing at least
one carbon-carbon double bond.
"Alkynyl" refers to an alkyl group containing at least
one carbon-carbon triple bond.
"Alkoxy" refers to an "-Oalkyl" group wherein the alkyl
group may be optionally substituted with one or more halo
groups.
"Aryl" refers to a group having at least one aromatic
ring structure; that is, a one ring having a conjugated pi
electron system and includes carbocyclic aryl, heterocyclic
aryl and biaryl groups. The aryl group may be optionally
substituted with one or more substituents selected from the
group consisting of halogen, trihalomethyl, hydroxyl, SR,
nitro, cyano, alkoxy, alkyl and NRR'.
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"Alkaryl" refers to an alkyl that is covalently joined
to an aryl group. Preferably, the alkyl is an unsubstituted
lower alkyl.
"Carbocyclic aryl" refers to an aryl group wherein the
ring atoms are carbon.
"Heterocyclic aryl" refers to an aryl group having from
1 to 3 heteroatoms as ring atoms, the remainder of the ring
atoms being carbon. Heteroatoms include oxygen, sulfur, and
nitrogen. The ring may be five-membered or six-membered.
Examples of heterocyclic aryl groups include furanyl,
thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl,
pyrazinyl, imidazolyl and the like.
"Amide" refers to -C (O) NHRa, where Ra is alkyl, aryl,
alkylaryl or hydrogen.
"Thioamide" refers to -C(S)NHRa
"Amino" refers to an NRR' group in which both R and R'
are hydrogen.
"Thioether" refers to an -SRb group wherein Rb is alkyl,
aryl or alkylaryl.
"Halogen" refers to fluorinem chlorine, bromine or
iodine.
"Sulfonyl" refers to -S (O) ZR', where R° is aryl, -
C (CN) =C-aryl, -CHZCN, alkyaryl, -S02NRR' , -NH (alkyl) , -
NH(alkylaryl), or -NH(aryl).
Physiologically acceptable salts and prodrugs of the 3-
heteroarylidenyl-2-indolinones are also within the scope of
this invention.
A "physiologically acceptable salt" refers to a salt
that is non-deleterious to the physical well-being of a
patient to whom it is administered. The physiologically
acceptable salts which the compounds of this invention may
form include negatively or the positively charged species.
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Examples of salts in which the compound forms the positively
charged moiety include, without limitation, quaternary
ammonium (defined elsewhere herein), salts such as the
hydrochloride, sulfate, carbonate, lactate, tartrate,
maleate, succinate wherein the nitrogen atom of the
quaternary ammonium group is a nitrogen of the selected
compound of this invention which has reacted with the
appropriate acid. Salts in which a compound of this invention
forms the negatively charged species include, without
limitation, the sodium, potassium, calcium and magnesium
salts formed by the reaction of a carboxylic acid group in
the compound with an appropriate base (e. g. sodium hydroxide
(NaOH), potassium hydroxide (KOH), Calcium hydroxide
(Ca(OH)2), etc.).
A "prodrug" refers to an agent which is converted into
the parent drug in vivo. Prodrugs are often useful because,
in some situations, they may be easier to administer than the
parent drug. They may, for instance, be bioavailable by oral
administration whereas the parent drug is not. The prodrug
may also have improved solubility in pharmaceutical
compositions over the parent drug. An example, without
limitation, of a prodrug would be a compound of the present
invention which is administered as an ester (the "prodrug")
to facilitate transmittal across a cell membrane where water
solubility is detrimental to mobility but which then is
metabolically hydrolyzed to the carboxylic acid, the active
entity, once inside the cell where water solubility is
beneficial. A further example of a prodrug might be a short
polypeptide bonded to a carboxy group wherein metabolic
removal of the polypeptide group releases the active
compound.
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The 3-heteroarylidenyl-2-indolinone compounds of this
invention may exist as the E or the Z isomers of a
combination thereof. All of these configurations are within
the scope of this invention. In preferred embodiments of
this invention, the 3-heteroarylidenyl-2-indolinones are
predominantly (greater than 90~) the Z-isomer.
By "inhibit" is meant eliminate, reduce, contain,
impede, prevent, slow, retard and/or restrict. In a
presently preferred embodiment of this invention, inhibit
refers to the inhibition of angiogenesis or vasculogenesis.
By "angiogenesis" activity is meant the formation of new
blood vessels in a tissue.
By "vasculogenesis" is meant the spread of new blood
vessels through a tissue to form a vascular system.
In another aspect, the 3-heteroarylidenyl-2-indolinone
compound of this invention is 3-[4-(2-carboxyethyl-3,5-
dimethylpyrrol-2-yl)methylidenyl]-2-indolinone (Structure 1).
The 3-heteroarylidenyl-2-indolinone is 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (Structure 2)
in yet another aspect of this invention.
1_
In a further aspect of this invention, a method is
provided for treating cancer comprising administering to a
patient in need of such treatment a therapeutically effective
amount of another chemotherapeutic agent and a
therapeutically effective amount of a 3-heteroarylidenyl-2-
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indolinone wherein the 3-heteroarylidenyl-2-indolinone has
the chemical structure:
A
wherein R1, Rz, R3, R4, R5, R6, R7 and A are the same as
set forth above.
In a presently preferred embodiment, the
chemotherapeutic agent is a fluorinated pyrimidine.
Again, physiologically acceptable salts or prodrugs of
the 3-heteroarylidenyl-2-indolines are within the scope of
this combination chemotherapy aspect of the present
invention.
The term "method" refers to manners, means, techniques
and procedures for accomplishing a given task including, but
not limited to, those manners, means, techniques and
procedures either known to, or readily developed from known
manners, means, techniques and procedures by, practitioners
of the chemical, pharmacological, biological, biochemical and
medical arts.
With regard to cancer, the term "treating" simply means
that the life expectancy of an individual affected with a
cancer will be increased, that one or more of the symptoms of
the disease will be reduced and/or that quality of life will
be enhanced.
As used herein, "administer," "administering" or
"administration" refers to the delivery of a compound, salt or
prodrug of the present invention or of a pharmaceutical
R7 "~
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composition containing a compound, salt or prodrug of this
invention to a patient for the purpose of treatment of cancer
or the prevention or treatment of a PK-related disorder.
"Comprising" as used herein in connection with
"administering" is intended to mean that drugs being
administered pursuant to the present invention may be
administered as simply a combination of a 3-heteroarylidenyl-2-
indolinone compound and a chemotherapeutic agent alone or may b
expanded to include additional drugs, such as, when the
chemotherapeutic agent is a fluorinated pyrimidine, leucovorin,
which are known or expected to offer additional beneficial
characteristics to the combination.
In general, a "therapeutically effective amount" refers
to that amount of a drug or its metabolite which is effective
to prevent, alleviate, reduce or ameliorate symptoms of
disease or prolong the survival of the patient being treated.
More particularly, in reference to the treatment of cancer,
a therapeutically effective amount refers to that amount
which has the effect of (1) reducing the size of (or
preferably eliminating) the tumor; (2) inhibiting (that is,
slowing to some extent, preferably stopping) tumor
metastasis; (3) inhibiting to some extent (that is slowing to
some extent, preferably stopping) tumor growth; and/or, (4)
relieving to some extent (or preferably eliminating) one or
more symptoms associated with the cancer.
In addition to the above general definition, by a
"therapeutically effective amount" of a chemotherapeutic
agent is meant any amount administered in any manner and in
any treatment regime as may be currently recognized in the
medical arts or as may come about as the result of future
developments regarding the use of these agents. In a
presently preferred embodiment of this invention, the
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chemotherapeutic agent is a fluorinated pyrimidine, in
particular, fluorouracil, and the treatment regimes are those
known in the chemotherapeutic art for the administation of
fluorouracil.
A "treatment regime" refers to specific quantities of
selected chemotherapeutic agents (and, optionally, other
agent such as the 3-heteroarylidenly-2-indolinone compound of
this invention) administered at set times in a set manner
over an established time period. For example, without
limitation, a common treatment regime for treating colorectal
cancer with fluorouracil/leucovorin comprises administering
425 mg/mz (milligrams per square meter of body surface area,
a manner of measuring chemotherapeutic agent dosage well
known to those skilled in the art) flourouracil plus 20 mg/mz
leucovorin (specific quantities of selected agents) daily for
5 days (set times) by intravenous push (set manner) repeated
at 4 to 5 week intervals (established time period).
When referring to "set times" of administration within a
treatment regime, "consecutive days" means consectutive
calendar days; i.e., Monday, Tuesday, Wednesday, etc.
"Staggered" days means calendar days with other calendar days
between them, e.g., without limitation, Monday, Wednesday,
Saturday, etc.
Furthermore, with regard to a "therapeutically effective
amount of a 3-heteroarylidenyl-2-indolinone," the phrase
refers to an amount of the compound sufficient to inhibit the
growth, size and vascularization; i.e., angiogenesis and/or
vasculogenesis, of tumors during the "recovery" periods,
i.e., the periods in a treatment regime when no other
chemotherapeuic agent is being administered to a patient.
A "patient" refers to any higher organism which is
susceptible to a PK related disorder including in particular
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cancer. Preferentially, "patient" refers to a mammal,
especially a human being.
"Fluorinated pyrimidine chemotherapeutic agents" are
well known to those skilled in chemotherapeutic art;
examples, without limitation, of fluorinated pyrirnidines
which may be used with the compounds of this invention
include, without limitation, carmofur, doxifluridine,
fluorouracil, floxuridine, tegafur, capecitabine and uracil-
ftorafur (UFT).
In a presently preferred embodiment of this invention,
the fluorinated pyrimidine chemotherapeutic agent is
fluorouracil.
It is also a presently preferred embodiment of this
invention that, when the fluorinated pyrimidine
chemotherapeutic agent is fluorouracil, the above method for
the treatment of cancer also comprises leucovorin.
The 3-heteroarylidenyl-2-indolinone used to treat cancer
incombination with other chemotherapeutic agents is selected
from the group consisting of 5-hydroxy-3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (Structure 3),
4-methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrole-2-carboxylic acid (Structure 4), 4-methyl-5-(2-oxo-
1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2~-carboxylic
acid methyl ester (Structure 5), 3- (5-hydroxymethyl-3-methyl-
1H-pyrrol-2-ylmethylene)-1,3-dihydroindole-2-one (Structure
6) and 4-methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
1H-pyrrole-2-carbaldehyde (Structure 7) in yet another aspect
of this invention.
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H
3_ 4
COOCH3 H20H
H
,~ ø
H
7
In a further aspect of this invention, the 3-
heteroarylidenly-2-indolinone compound used to treat cancer
in combination with other chemotherapeutic agents is 3-[4-(2-
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carboxyethyl-3,5-dimethylpyrrol-2-yl)methylidenyl]-2-
indolinone (Structure 1).
In a still further aspect of this invention, the 3-
heteroarylidenyl-2-indolinone compound used to treat cancer
in combination with other chemotherpeutic agents is 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (Structure 2).
The cancer which may be treated using the above-
described method may be selected from the group consisting of
breast cancer, gastric cancer, ovarian cancer, renal cancer,
hepatic cancer, pancreatic cancer, bladder cancer, thyroid
cancer, prostate cancer and colorectal cancer.
Yet another aspect of this invention is a method for
treating cancer comprising administering to a patient in need
of such treatment a therapeutically effective amount of
fluorouracil and a therapeutically effective amount of a
compound selected from the group consisting of 3-[4-(2-
carboxyethyl-3,5-dimethylpyrrol-2-yl)methylidenyl]-2-
indolinone and 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone.
In a preferred embodiment, the cancer is colorectal
cancer.
In another aspect of this invention, the above method
for the treatment of cancer includes the use of leucovorin.
The therapeutically effective amount of fluorouracil
comprises from about 300 to about 800 mg/mz, preferably from
about 400 to about 500 mg/mz of the compound.
The therapeutically effective amount of fluoruracil may
be administered as an intravenous bolus injection or as a
continuous intravenous infusion in yet another aspect of this
invention.
The therapeutically effective amount of 3-[(2,4
dimethylpyrrol-5-yl)-methylidenyl]-2-indolinone comprises
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from about 4 to about 190 mg/m2, preferrably from about 72 to
145 mg/m~ of the compound. -
The therapeutically effective amount of leucovorin
comprises from about 20 to about 500 mg/m2, preferrably from
about 20 to about 200 mg/m2 of the compound.
A still further aspect of this invention is a treatment
regime comprising the administration of from about 400 to
about 500 mg/mz fluorouracil on one or more days, which may
be consecutive or staggered, after which from about 72 to
about 145 mg/m2 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone are administered on one or more days, which days
likewise may be consecutive or staggered.
In another aspect, 20 mg/m2 leucovorin may also be
adminstered on the days on which fluorouracil is
administered.
In a presently preferred embodiment of this invention,
the above treatment regime is a four week treatment regime,
fluorouracil (and, optionally, leucovorin) being administered
as an intravenous bolus injection on days 1, 2, 3, 4 and 5 of
the first week of the treatment regime while the 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone being
administered as an intravenous bolus injection twice a week
during weeks 2, 3 and 4 of the treatment regime.
Another aspect of this invention is a method for
treating cancer comprising administering to a patient in need
of such treatment a therapeutically effective amount of 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone and a
therapeutically effective amount of gemcitabine, another
fluoropyrimidine compound. Gemcitabine has shown particular
effectiveness in the treatment of advanced pancreatic cancer.
Furthermore, in combination with other chemotherapeutic
agents, e.g., paclitaxel, carboplatin, doxorubicin (in
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particular, liposomal doxorubicin) and topotecan, gemcitabine
has shown substantial activity against other refractory solid
tumor cancers including advanced ovarian cancer, small cell
lung cancer and kidney cancer. The combination of
gemcitabine with 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone, alone or in further combination with additional
chemotherapeutic agents such as those indentified above,
should, for the reasons discussed with regard to
fluoropyrimidines generally, provide additional solid tumor
inhibiting capacity without adding further toxicity.
Combinations of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone with nucleoside analogs other
than gemcitabine are also contemplated by the present
invention.
Another pyrimidine analog which should benefit from
combination with 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone is capecitabine which has shown effectiveness
against metastatic breast cancer; such a combination is
another aspect of this invention.
In addition, the chemotherapeutic combination of 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone with
either of the pyrimidine chemotherapeutic agents 5-FU or UFT
or derivatives, analogs or agents related thereto, is an
aspect of this invention.
A further aspect of this invention is the combination of
3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone with
carboplatin, oxaliplatin, cisplatin or related
chemotherapeutic agents. Carboplatin and cisplatin are
presently the pre-eminent drugs for the treatment of advanced
ovarian cancer while oxaliplatin is a first-line
chemotherapeutic agent in metastatic colorectal cancer. The
use of 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone
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in combination with carboplatin or cisplantin may permit a
reduction in the amount of these two very toxic
chemotherapeutic agents necessary to treat the cancer. In
addition, combination of carboplatin or cisplatin with
paclitaxel has shown promise in the treatment of ovarian
cancer. The addition of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone to this combination of
chemotherapeutic agents could result in the same advantages
discussed with regard to the above combinations. A presently
preferred chemotherapeutic combination is comprised of 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone,
cisplatin and gemcitabine.
A further aspect of this invention is the combination of
3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone with
paclitaxel (taxol), its synthetic analog docetaxel or
polyglutamated taxanes. Paclitaxel has been approved by the
FDA for the treatment of ovarian, breast, lung and AIDS-
reiated cancers. Paclitaxel/docetaxel work by a different
mechanism than the compounds of this invention, that is, they
block a cell's ability to break down the mitotic spindle
during mitosis. Thus, these drugs with their particular mode
of action, combined with the anti-angiogenetic activity of 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone, could
result in a potent tumoricidal/tumoristatic combination.
Yet another aspect of this invention is the combination
of 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone
with CPT1I (irinotecan), a derivative of campothecin that is
a topoisomerase I inhibitor and which has proven effective
against colorectal cancer.. Combination therapies with
chemotherapeutic agents related to CPT11 are also
contemplated by this invention. Again, the combination of
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modes of action could be of substantial benefit in the
treatment of this form of cancer.
A still further aspect of this invention is the
combination of 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone with thalidomide which is showing substantial
chemotherapeutic utility particularly against refractory
myelomas but also against glioblasoma multiforma,an extremely
virulent brain cancer. Other cancer which may be responsive
to this combination include prostate, breast and skin
ZO (Kaposi's sarcoma) cancers.
An aspect of this invention is a chemotherapeutic
combination of 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2
indalinone with COX-2 inhibitors. The inhibition of
cyclooxygenase-2 prevents production of factors that prompt
angiogenesis. The combination would provide a two way attack
on the vascularization essential to the vitality of cancer
cells.
A combination therapy consisting of 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone and tamoxifen
or derivatives thereof is as aspect of this invention.
Tamoxifen interferes with the activity of estrogen which has
been shown to promote the growth of breast cancer cells. The
combination of 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone, an anti-angiogenesis compound, with this "anti-
estrogen" compound could provide a potent additional
treatment for breast cancer.
Another aspect of this invention is the combination of
3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone with
leuprolide, a synthetic nonapeptide analog of naturally
ocurring gonadotropin-releasing hormone that has demonstrated
effectiveness particularly aginst testicular cancer but also
against ovarian and breast cancer. Combination therapy using
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agents related to leuprolide is also contemplated by this
invention. Again, a substantial benefit could be gained by
combining the two different mode of action compounds.
The chemotherapeutic combination of 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone with
angiostatin, endostatin or similar chemotherapeutic agents,
which inhibit angiogenesis by apoptosis, is likewise an
aspect of this invention. Apoptosis is programmed cell
death. The combination of cell-killing anti-angiogenesis
with cell stasis anti-angiogenesis could be a powerful
chemotherapeutic combination.
In addition, a chemotherapeutic combination of 3-((2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone with a matrix
metalloprotease inhibitor. MMPs having been shown to be
involved in many disease states including cancer. MMP
inhibitors, such as, without limitation, AG3340, are showing
tumoristatic efficacy againt solid tumor cancers such as non-
small.cell lung cancer and hormone-refractory prostate
cancer. The addition of an angiogenesis inhibitor could
provide a synergistic combination.
The combination of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone with an interferon is another
aspect of this invention. Interferon alpha, and its various
subtypes (e. g., without limitation, interferons alpha A/2a,
alpha/2b, alpha B2/alpha 8) are well-established
chemotherapeutic agents against such cancers as hairy-cell
leukemia, chronic myeloid leukemia, kidney cancer, melanoma,
low grade lymphomas, multiple myeloma and Kaposi~s sarcoma.
A further aspect of this invention is the
chemotherapeutic combination of 3-((2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone with doxorubicin, daunorubicin
and other anthracycline antineoplastic antibiotic, and
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derivatives and formulations thereof such as, without
limitation, liposomal doxorubicin. Doxorubicin is widely
used in the treatment of malignant lymphomas, leukemias,
sguamous cell cancer of the head and neck, breast cancer and
thyroid cancer. Liposomal doxorubicin has been approved for
the treatment of Kaposi's sarcoma. Tumor cells weakened by
the anti-angiogenesis activity of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone could be much more susceptible
to doxorubicin. Combination therapy using 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone and
metoxantrone, a related chemotherapeutic agent, is
specifically contemplated by this invention.
Another chemotherapeutic combination which is an aspect
of this invention is the combination of 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone with
estramustine and chemotherapeutic agents related thereto,
which has shown particular utility in the treatment of
refractory prostate cancer. Estramustine causes cell death
by interferring with DNA synthesis. Again the combination of
differing modes of action, DNA synthesis disruption and anti-
angiogenesis could provide a useful chemotherapeutic
combination.
Combination therapy using 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone and the vinca alkaloids
including, without limitation, vincristine and vinblastine is
also contemplated by the present invention.
A further aspect of this invention is a 3-
heteroarylidenyl-2-indolinone selected from the group
consisting of 5-hydroxy-3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone (Structure 3), 4-methyl-5-(2-
oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic
acid (Structure 4), 4-methyl-5-(2-oxo-1,2-dihydroindol-3-
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ylidenemethyl)-1H-pyrrole-2-carboxylic acid methyl ester
(Structure 5), 3-(5-hydroxymethyl-3-methyl-1H-pyrrol-2-
ylmethylene)-1,3-dihydroindole-2-one (Structure 6) and 4-
methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-
2-carbaldehyde (Structure 7).
Physiologically acceptable salts and prodrugs of the
above compounds are within the scope of this invention.
In another aspect, this present invention relates to a
method of modulating the catalytic activity of PKs comprising
contacting the PK with a compound having one of the
structures shown above.
As used herein, the term "modulation" or "modulating"
refers to the alteration of the catalytic activity of RTKs,
CTKs and STKs. In particular, modulating refers to the
activation of the catalytic activity of RTKs, CTKs and STKs,
preferably the activation or inhibition of the catalytic
activity of RTKs, CTKs and STKs, depending on the
concentration of the compound or salt to which the RTK, CTK
or STK is exposed or, more preferably, the inhibition of the
catalytic activity of RTKs, CTKs and STKs.
The term "catalytic activity" as used herein refers to
the rate of phosphorylation of tyrosine under the influence,
direct or indirect, of RTKs and/or CTKs or the
phosphorylation of serine and threonine under the influence,
direct or indirect, of STKs.
The term "contacting" as used herein refers to bringing
a compound of this invention and a target PK together in such
a manner that the compound can affect the catalytic activity
of the PK, either directly; i.e., by interacting with the
kinase itself, or indirectly; i.e., by interacting with
another molecule on which the catalytic activity of the
kinase is dependent. Such "contacting" can be accomplished
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"in vitro," i.e., in a test tube, a petri dish or the like.
In a test tube, contacting may involve only a compound and a
PK of interest or it may involve whole cells. Cells may also
be maintained or grown in cell culture dishes and contacted
with a compound in that environment. In this context, the
ability of a particular compound to affect a PK related
disorder; i.e., the ICso of the compound, defined below, can
be determined before use of the compounds in vivo with more
complex living organisms is attempted. For cells outside the
organism, multiple methods exist, and are well-known to those
skilled in the art, to get the PKs in contact with the
compounds including, but not limited to, direct cell
microinjection and numerous transmembrane carrier techniques.
The above-referenced PK is selected from the group
consisting of an RTK, a CTK or an STK in another aspect of
this invention.
Furthermore, it is an aspect of this invention that the
receptor protein kinase whose catalytic activity is modulated
by a compound of this invention is selected from the group
consisting of EGF, HER2, HERS, HER4, IR, IGF-1R, IRR, PDGFRa,,
PDGFR(3, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-l,
FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.
In addition, it is an aspect of this invention that
the cellular tyrosine kinase whose catalytic activity is
modulated by a compound of this invention is selected from
the group consisting of Src, Frk, Btk, Csk, Abl, ZAP70,
Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr
and Yrk.
Another aspect of this invention is that the serine-
threonine protein kinase whose catalytic activity is
modulated by a compound of this invention is selected from
the group consisting of CDK2 and Raf.
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In another aspect, this invention relates to a method
for treating or preventing a PK-related disorder in a patient
in need of such treatment comprising administering to the
patient a therapeutically effective amount of one or more of
the compounds described above.
As used herein, "PK related disorder," "PK driven
disorder," and "abnormal PK activity" all refer to a
condition characterized by inappropriate; i.e., under or,
more commonly, over, PK catalytic activity, where the
particular PK can be an RTK, a CTK or an STK. Inappropriate
catalytic activity can arise as the result of either: (1) PK
expression in cells which normally do not express PKs; (2)
increased PK expression leading to unwanted cell
proliferation, differentiation and/or growth; or, (3)
decreased PK expression leading to unwanted reductions in
cell proliferation, differentiation and/or growth. Over-
activity of a PK refers to either amplification of the gene
encoding a particular PK or production of a level of PK
activity which can correlate with a cell proliferation,
differentiation and/or growth disorder (that is, as the level
of the PK increases, the severity of one or more of the
symptoms of the cellular disorder increases). Under-activity
is, of course, the converse, wherein the severity of one or
more symptoms of a cellular disorder increase as the level of
the PK activity decreases.
"Treat," "treating" or "treatment" with regard to a PK-
related disorder refers to alleviating or abrogating the
cause and/or the effects of a PK-related disorder.
As used herein, the terms "prevent", "preventing" and
"prevention" refer to a method for barring an organism from
acquiring a PK related disorder in the first place.
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The PK related disorder may be selected from the group
consisting of an RTK, a CTK and an STK related disorder in a
further aspect of this invention.
In yet another aspect of this invention, the above
referenced PK related disorder may be selected from the group
consisting of an EGFR related disorder, a PDGFR related
disorder, an IGFR related disorder and a flk related
disorder.
The above referenced protein kinase related disorder is
a cancer selected from the group consisting of squamous cell
carcinoma, astrocytoma, glioblastoma, lung cancer, bladder
cancer, head and neck cancer, melanoma, ovarian cancer,
prostate cancer, breast cancer, small-cell lung cancer,
colorectal cancer, gastrointestinal cancer and glioma in a
further aspect of this invention.
The above referenced protein kinase related disorder is
selected from the group consisting of diabetes, an autoimmune
disorder, a hyperproliferation disorder, restenosis,
fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis, an
inflammatory disorder and angiogenesis in yet another aspect
of this invention.
Other disorders which might be treated with compounds of
this invention include, without limitation, immunological and
cardiolovascular disorders such as, for instance
aetherosclerosis.
Pharmaceutical compositions of the above compounds are a
further aspect of this invention.
A "pharmaceutical composition" refers to a mixture of
one or more of the compounds or drugs described herein, or
physiologically acceptable salts or prodrugs thereof, with
other chemical components, such as physiologically acceptable
carriers and excipients. The purpose of a pharmaceutical
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composition is to facilitate administration of a compound to
ari organism.
As used herein, a "physiologically acceptable carrier"
refers to a carrier or diluent that does not abrogate the
biological activity and properties of the administered
compound while facilitating administration by, for example,
stabilizing or solubilizing the compound. Preferably, the
carrier does not cause significant irritation to the
organism.
An "excipient" refers to a substance added to a
pharmaceutical composition to further facilitate
administration of a compound. Examples, without limitation,
of excipients include calcium carbonate, calcium phosphate,
various sugars and types of starch, cellulose derivatives,
gelatin, vegetable oils, surfactants and polyethylene
glycols.
Yet another aspect of this invention is a method for
inhibiting tumorigenic activity in a cell comprising
contacting the cell with a 3-heteroarylidenyl-2-indolinone of
this invention.
"Tumorigenic" activity, as used herein and as it relates
to a cell, refers to both intracellular and extracellular
biochemical activity which contributes to the formation of a
neoplasm.
A "neoplasm" is an abnormal tissue that grows by
cellular proliferation more rapidly than normal and continues
to grow even after the stimuli that initiated the new growth
cease. A neoplasm partially or completely lacks structural
organization and functional coordination with the normal
tissue and usually forms a distinct mass of tissue. Such
masses may be benign (benign tumors) or malignant (solid
tumor cancer). Malignant neoplasms are locally invasive and
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destructive and in many cases metastasize (spread to and
invade and destroy tissues in areas of the affected organism
remote fram the site of origin). The process of neoplasm
formation is generally referred to as "neoplasia"; i.e.
neoplasia is the biochemical process by which a neoplasm
forms and grows.
The terms "malignant neoplasm", "cancer", "tumor" and
"solid tumor cancer" are used interchangeably herein to refer
to the condition well known to those skilled in the art as
the life-threatening disease commonly referred to simply as
"cancer".
With regard to tumorigenic activity, "inhibit" or
"inhibiting" refers to eliminating, reducing, containing,
impeding, preventing, slowing, retarding and/or restricting
neoplasia.
A "chemotherapeutic agent" refers to a chemical
substance or drug used to treat a disease; the term is most
often applied to such substances or drugs which are used
primarily for the treatment of cancer.
DETAILED DESCRIPTION OF THE INVENTION
1. INDICATIONS/TARGET DISEASES
General
The PKs whose catalytic activity is modulated by the
compounds of this invention include protein tyrosine kinases
of which there are two types, receptor tyrosine kinases
(RTKs) and cellular tyrosine kinases (CTKs), and serine-
threonine kinases (STKs). RTK mediated signal transduction,
is initiated by extracellular interaction with a specific
growth factor (ligand), followed by receptor dimerization,
transient stimulation of the intrinsic protein tyrosine
kinase activity and phosphorylation. Binding sites are
thereby created for intracellular signal transduction
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molecules and lead to the formation of complexes with a
spectrum of cytoplasmic signaling molecules that facilitate
the appropriate cellular response (e. g., cell division,
metabolic effects on the extracellular microenvironment,
etc.). See, Schlessinger and Ullrich, 1992, Neuron 9:303-
391.
It has been shown that tyrosine phosphorylation sites on
growth factor receptors function as high-affinity binding
sites for SH2 (src homology) domains of signaling molecules.
Fantl et al., 1992, Cell 69:413-423; Songyang et al., 1994,
Mol. Cell. Biol. 14:2777-2785); Songyang et al., 1993, Cell
72:767-778; and Koch et al., 1991, Science 252:668-678.
Several intracellular substrate proteins that associate with
RTKs have been identified. They may be divided into two
principal groups: (1) substrates which have a catalytic
domain; and (2) substrates which lack such domain but which
serve as adapters and associate with catalytically active
molecules. Songyang et al., 1993, Cell 72:767-778. The
specificity of the interactions between receptors and SH2
domains of their substrates is determined by the amino acid
residues immediately surrounding the phosphorylated tyrosine
residue. Differences in the binding affinities between SH2
domains and the amino acid sequences surrounding the
phosphotyrosine residues on particular receptors are
consistent with the observed differences in their substrate
phosphorylation profiles. Songyang et al., 1993, Cell
72:767-778. These observations suggest that the function of
each RTK is determined not only by its pattern of expression
and ligand availability but also by the array of downstream
signal transduction pathways that are activated by a
particular receptor. Thus, phosphorylation provides an
important regulatory step which determines the selectivity of
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signaling pathways recruited by specific growth factor
receptors, as well as differentiation factor receptors.
STKs, being primarily cytosolic, affect the internal
biochemistry of the cell, often as a down-line response to a
PTK event. STKs have been implicated in the signaling
process which initiates DNA synthesis and subsequent mitosis
leading to cell proliferation.
Thus, PK signal transduction results in, among other
responses, cell proliferation, differentiation, growth and
metabolism. Abnormal cell proliferation may result in a wide
array of disorders and diseases, including the development of
neoplasia such as carcinoma, sarcoma, glioblastoma and
hemangioma, disorders such as leukemia, psoriasis,
arteriosclerosis, arthritis and diabetic retinopathy and
other disorders related to uncontrolled angiogenesis and/or
vasculogenesis.
A precise understanding of the mechanism by which the
compounds of this invention inhibit PKs is not required in
order to practice the present invention. However, while not
hereby being bound to any particular mechanism or theory, it
is believed that the compounds interact with the amino acids
in the catalytic region of PKs. PKs typically possess a bi-
lobate structure wherein ATP appears to bind in the cleft
between the two lobes in a region where the amino acids are
conserved among PKs. Inhibitors of PKs are believed to bind
by non-covalent interactions such as hydrogen bonding, van
der Waals forces and ionic interactions in the same general
region where the aforesaid ATP binds to the PKs. More
specifically, it is thought that the 2-indolinone component
of the compounds of this invention binds in the general space
normally occupied by the adenine ring of ATP. Specificity of
a particular molecule for a particular PK may then arise as
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the result of additional interactions between the various
substituents on the 2-indolinone care and the amino~acid
domains specific to particular PKs. Thus, different
indolinone substituents may contribute to preferential
binding to particular PKs. The ability to select compounds
active at different ATP (or other nucleotide) binding sites
makes the compounds of this invention useful for targeting
any protein with such a site. The compounds disclosed herein
may thus have utility as in vitro assays for such proteins as
well as exhibiting in vivo therapeutic effects through
interaction with such proteins.
In another aspect, the protein kinase, the catalytic
activity of which is modulated by contact with a compound of
this invention, is a protein tyrosine kinase, more
particularly, a receptor protein tyrosine kinase. Among the
receptor protein tyrosine kinases whose catalytic activity can
be modulated with a compound of this invention, or salt
thereof, are, without limitation, EGF, HER2, HER3, HER4, IR,
IGF-1R, IRR, PDGFRa, PDGFR~i, CSFIR, C-Kit, C-fms, Flk-1R,
Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.
The protein tyrosine kinase whose catalytic activity is
modulated by contact with a compound of this invention, or a
salt or a prodrug thereof, can also be a non-receptor or
cellular protein tyrosine kinase (CTK). Thus, the catalytic
activity of CTKs such as, without limitation, Src, Frk, Btk,
Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck,
Blk, Hck, Fgr and Yrk, may be modulated by contact with a
compound or salt of this invention.
Still another group of PKs which may have their
catalytic activity modulated by contact with a compound of
this invention are the serine-threonine protein kinases such
as, without limitation, CDK2 and Raf.
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In another aspect, this invention relates to a method
for treating or preventing a PK related disorder by
administering a therapeutically effective amount of a
compound of this invention, or a salt or a prodrug thereof,
to an organism.
It is also an aspect of this invention that a
pharmaceutical composition containing a compound of this
invention or a salt or prodrug thereof is administered to an
organism for the purpose of preventing or treating a PK
related disorder.
This invention is therefore directed to compounds which
modulate PK signal transduction by affecting the enzymatic
activity of RTKs, CTKs and/or STKs, thereby interfering with
the signals transduced by such proteins. More particularly,
the present invention is directed to compounds which modulate
RTK, CTK and/or STK mediated signal transduction pathways as
a therapeutic approach to cure many kinds of solid tumors,
including but not limited to carcinomas, sarcomas including
Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma,
astrocytoma, melanoma and myoblastoma. Treatment or
prevention of non-solid tumor cancers such as leukemia are
also contemplated by this invention. Indications may
include, but are not limited to brain cancers, bladder
cancers, ovarian cancers, gastric cancers, pancreatic
cancers, colon cancers, blood cancers, lung cancers and bone
cancers.
Further examples, without limitation, of the types of
disorders related to inappropriate PK activity that the
compounds described herein may be useful in preventing,
treating and studying, are cell proliferative disorders,
fibrotic disorders and metabolic disorders.
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Cell proliferative disorders, which may be prevented,
treated or further studied by the present invention~include
cancer, blood vessel proliferative disorders and mesangial
cell proliferative disorders.
S Blood vessel proliferative disorders refer to disorders
related to abnormal vasculogenesis (blood vessel formation)
and angiogenesis (spreading of blood vessels). While
vasculogenesis and angiogenesis play important roles in a
variety of normal physiological processes such as embryonic
development, corpus luteum formation, wound healing and organ
regeneration, they also play a pivotal role in cancer
development where they result in the formation of new
capillaries needed to keep a tumor alive. Other examples of
blood vessel proliferation disorders include arthritis, where
new capillary blood vessels invade the joint and destroy
cartilage, and ocular diseases, like diabetic retinopathy,
where new capillaries in the retina invade the vitreous,
bleed and cause blindness.
Conversely, disorders related to the shrinkage,
contraction or closing of blood vessels, such as restenosis,
are also implicated and may be treated or prevented by the
methods of this invention.
Fibrotic disorders refer to the abnormal formation of
extracellular matrices. Examples of fibrotic disorders
include hepatic cirrhosis and mesangial cell proliferative
disorders. Hepatic cirrhosis is characterized by the
increase in extracellular matrix constituents resulting in
the formation of a hepatic scar. An increased extracellular
matrix resulting in a hepatic scar can also be caused by a
viral infection such as hepatitis. Lipocytes appear to play
a major role in hepatic cirrhosis. Other fibrotic disorders
implicated include atherosclerosis.
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Mesangial cell proliferative disorders refer to
disorders brought about by abnormal proliferation o~
mesangial cells. Mesangial proliferative disorders include
various human renal diseases such as glomerulonephritis,
diabetic nephropathy and malignant nephrosclerosis as well as
such disorders as thrombotic microangiopathy syndromes,
transplant rejection, and glomerulopathies. The RTK PDGFR has
been implicated in the maintenance of mesangial cell
proliferation. Floege et al., 1993, Kidney International,
43:47S-54S.
Many cancers are cell proliferative disorders and, as
noted previously, PKs have been associated with cell
proliferative disorders. Thus, it is not surprising that PKs
such as, for example, members of the RTK family have been
associated with the development of cancer. Some of these
receptors, like EGFR (Tuzi et al.,Br. J. Cancer, 1992,
63:227-233; Torp et al., 1992, APMIS 100:713-719) HER2/neu
(Slamon et al., Science, 1989, 244:707-712) and PDGF-R
(Kumabe et al., Oncogene,1992, 7:627-633) are over-expressed
in many tumors and/or persistently activated by autocrine
loops. In fact, in the most common and severe cancers these
receptor over-expressions (Akbasak and Suner-Akbasak et
al.,J. Neurol. Sci., 1992, 111:119-133; Dickson et al.,
Cancer Treatment Res., 1992, 61:249-273; Korc et al., J.
Clin. Invest., 1992, 90:1352-1360) and autocrine loops (Lee
and Donoghue, J. Cell. Biol., 1992, 118:1057-1070; Korc et
al., supra; Akbasak and Suner-Akbasak et al., supra) have
been demonstrated. For example, EGFR has been associated
with squamous cell carcinoma, astrocytoma, glioblastoma, head
and neck cancer, lung cancer and bladder cancer. HER2 has
been associated with breast, ovarian, gastric, lung, pancreas
and bladder cancer. PDGFR has been associated with
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glioblastoma and melanoma as well as lung, ovarian and
prostate cancer. The RTK c-met has also been associated with
malignant tumor formation. For example, c-met has been
associated with, among other cancers, colorectal, thyroid,
pancreatic, gastric and hepatocellular carcinomas and
lymphomas. Additionally c-met has been linked to leukemia.
Over-expression of the c-met gene has also been detected in
patients with Hodgkins disease and Burkitts disease.
Flk has likewise been associated with a broad spectrum
of tumors including, without limitation, mammary, ovarian and
lung tumors as well as gliomas such as glioblastoma.
IGF-IR, in addition to being implicated in nutritional
support and in type-II diabetes, has also been associated
with several types of cancers. For example, IGF-I has been
implicated as an autocrine growth stimulator for several
tumor types, e.g. human breast cancer carcinoma cells
(Arteaga et al., J. Clin. Invest., 1989, 84:1418-1423) and
small lung tumor cells (Macauley et al., Cancer Res., 1989,
50:2511-2517). In addition, IGF-I, while integrally involved
in the normal growth and differentiation of the nervous
system, also appears to be an autocrine stimulator of human
gliomas. Sandberg-Nordqvist et al., Cancer Res., 1993,
53:2475-2478. The importance of IGF-IR and its ligands in
cell proliferation is further supported by the fact that many
cell types in culture (fibroblasts, epithelial cells, smooth
muscle cells, T-lymphocytes, myeloid cells, chondrocytes and
osteoblasts (the stem cells of the bone marrow)) are
stimulated to grow by IGF-I. Goldring and Goldring,
Eukaryotic Gene Expression, 1991, 1:301-326. In a series of
recent publications, Baserga suggests that IGF-IR plays a
central role in the mechanism of transformation and, as such,
could be a preferred target for therapeutic interventions for
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a broad spectrum of human malignancies. Baserga, Cancer
Res., 1995, 55:249-252; Baserga, Cell, 1994, 79:92'7-930;
Coppola et al., Mol. Cell. Biol., 1994, 14:4588-4595.
STKs have been implicated in many types of cancer
including, notably, breast cancer (Cance, et al., Int. J.
Cancer, 1993, 54:571-77).
The association between abnormal PK activity and disease
is not restricted to cancer. For example, RTKs have been
associated with diseases such as psoriasis, diabetes
mellitus, endometriosis, angiogenesis, atheromatous plaque
development, Alzheimer's disease, epidermal
hyperproliferation, neurodegenerative diseases, age-related
macular degeneration and hemangiomas. For example, EGFR has
been indicated in corneal and dermal wound healing. Defects
in Insulin-R and IGF-1R are indicated in type-II diabetes
mellitus. A more complete correlation between specific RTKs
and their therapeutic indications is set forth in Plowman et
al., DN&P, 1994, 7:334-339.
As noted previously, not only RTKs but CTKs including, but
not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck,
fgr and yrk (reviewed by Bolen et al., FASEB J., 1993, 6:3403-
3409) are involved in the proliferative and metabolic signal
transduction pathway and thus could be expected, and have been
shown, to be involved in many PTK-mediated disorders to which
the present invention is directed. For example, mutated src
(v-src) has been shown to be an oncoprotein (pp60°'er') in
chicken. Moreover, its cellular homolog, the proto-oncogene
pp60'~sr' transmits oncogenic signals of many receptors. Over-
expression of EGFR or HER2/neu in tumors leads to the
constitutive activation of pp60'-$r', which is characteristic of
malignant cells but absent in normal cells. On the other hand,
mice deficient in the expression of c-src exhibit an
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osteopetrotic phenotype, indicating a key participation of c-
src in osteoclast function and a possible involvement in
related disorders.
Similarly, Zap70 has been implicated in T-cell signaling
which may relate to autoimmune disorders.
STKs have been associated with inflamation, autoimmune
disease, immunoresponses, and hyperproliferation disorders such
as restenosis, fibrosis, psoriasis, osteoarthritis and rheumatoid
arthritis.
PKs have also been implicated in embryo implantation.
Thus, the compounds of this invention may provide an effective
method of preventing such embryo implantation and thereby be
useful as birth control agents.
Finally, both RTKs and CTKs are currently suspected as
being involved in hyperimmune disorders.
A method for identifying a chemical compound that
modulates the catalytic activity of one or more of the above
discussed protein kinases is another aspect of this
invention. The method involves contacting cells expressing
the desired protein kinase with a compound of this invention
(or its salt or prodrug) and monitoring the cells for any
effect that the compound has on them. The effect may be any
observable, either to the naked eye or through the use of
instrumentation, change or absence of change in a cell
phenotype. The change or absence of change in the cell
phenotype monitored may be, for example, without limitation,
a change or absence of change in the catalytic activity of
the protein kinase in the cells or a change or absence of
change in the interaction of the protein kinase with a
natural binding partner.
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VEGF and Flk-1/KDR in Anaioaenesis and Colorectal Cancer
Tumor cells stimulate quiescent endothelial cells to
divide and form new blood vessels by releasing growth
factors, which bind to nearby endothelial cells (a paracrine
mode of action). Binding of vascular endothelial growth
factor ("VEGF") to one of its receptors begins the signaling
cascade that regulates cellular events involved in new blood
vessel formation.
A number of receptor tyrosine kinases are thought to be
directly or indirectly involved in angiogenesis. The search
for the receptor whose selective inhibition will prevent new
blood vessel growth to support growing tumors has been the
focus of basic research for the last ten years.. Although
there are multiple receptors whose expression is restricted
to endothelial cells (including Flk-1, Flt-1, Tie-1 and Tie-
2), it is believed that the Flk-1 receptor plays a critical
role in angiogenesis.
The temporal and spatial patterns of expression of VEGF
and its receptors support a role for these in normal
angiogenesis during embryonic development. VEGF, Flt-1 and
Flk-1 have also been implicated in pathological angiogenesis
to support the growth of many solid tumors, including
gliomas, breast cancer, bladder cancer, colon carcinoma and
other gastrointestinal tract cancers. A correlation has been
observed between VEGF expression and vessel density in breast
tumors, renal cell carcinoma and colon cancer. In highly
vascularized glioblastoma, transcripts for VEGF and its
receptors were identified by in itu hybridization;
transcripts were not detected in the less vascular, low grade
gliomas or in normal brain tissue. In this setting
(supporting a paracrine mode of action), Flk-1 receptors were
detected in the endothelial cells of the vessels while VEGF
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localized to the tumor cells. Expression of VEGF and its
receptors has been shown for hematopoietic tumor cell lines
including multiple myeloma.
VEGF is mitogenic for endothelial cells in vitro. In
such a system, neutralizing antibodies against Flk-1 inhibit
mitogenesis. Similarly, ribozymes that cleave flk-1 or flt-1
mRNAs reduce the growth of human microvasculature endothelial
cells, presumably by decreasing the number of receptors on
the cells.
A variety of in vivo techniques have been used to
investigate the role of VEGF signaling in tumor angiogenesis.
Flk-1 receptors which lack the intracellular kinase domain
block the activation of the endogenous Flk-1 receptor
activity in cultured cells, inhibiting the growth of tumors
1S implanted subcutaneously into nude mice. Any tumors that did
form in this animal model contained significantly reduced
vessel density. Also, reduction in VEGF expression with
antisense constructs inhibits the growth of C6 rat glioma
cells in nude mice with concurrent reduced blood vessel
density in these tumors and inhibits the growth of human
melanoma cells in nude/SCID mice. Likewise, reduction of
VEGF levels with neutralizing antibodies inhibits the growth
of human rhabdomyosarcoma, glioblastoma multiforme and
leiomyosarcoma in nude mice and fibrosarcoma in BALBc/nude
mice.
Taken together, these results provide strong evidence for
a critical role of VEGF signaling through Flk-1 in
angiogenesis in solid tumor growth. An inhibitor of Flk-1
may have therapeutic benefit in cancer patients.
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2. PHARMACOhOGY
Preclinical Studies with 3-I(2,4-dimethylpyrrol-5-
yl)methylidenvll-2-indolinone
In a cellular-based assay, 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone has been found to inhibit the
receptor phosphorylation that typically follows the
interaction of VEGF with its receptor. In vitro studies of
3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone have
demonstrated its ability to inhibit Flk-1 autophosphorylation
with ICso values of approximately 1 ~cM. In addition, 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone inhibits
in vitro proliferation of endothelial cells induced by VEGF
with ICso values of approximately 0.07 ACM. In this assay, 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone exerts a
time-dependent increase in potency, with detectable activity
first observed after a 5-minute exposure to drug. One-hour
exposure to 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone results in in vitro antiproliferative activity for
3 to 4 days thereafter. 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone has no direct inhibitory
effects on a variety of tumor cell lines at concentrations up
to 50 ~,M.
In vivo studies of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone, in which a variety of tumor
cell lines were subcutaneously implanted into
immunocompromised mice, 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone demonstrates a significant
suppression of tumor growth against a broad spectrum of tumor
types whose growth are driven by various growth factors such
as PDGF, EGF and Her2. Daily intraperitoneal dosing (ranging
from 12.5 - 25 mg/kg/day for 28 days) resulted in 30-80~
inhibition of tumor growth. In initial studies, 3-[(2,4-
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dimethylpyrrol-5-yl)methylidenyl]-2-indolinone administration
was started on Day 1 after tumor implantation. Later
studies, in which 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-
2-indolinone administration was delayed until tumors were
grown to a volume of approximately 50 mm3, demonstrated
equivalent efficacy in suppression of tumor growth.
Dose response studies with 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone (at doses between 6.25 - 25
mg/kg/day) were conducted with human melanoma cells implanted
subcutaneously in athymic mice. Daily administration of 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone at doses
as low as 1 mg/kg/day resulted in dose-dependent inhibition
of these cells. Additional studies with intraperitoneal
dosing of 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone in athymic mice using less frequent administration
(including twice weekly for four weeks) also resulted in
equivalent tumor growth inhibition when compared to daily
intraperitoneal administration (775 in twice weekly dosing
versus 68~ with daily dosing).
Daily administration of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone (25 mg/kg/day) was also shown
to significantly inhibit the growth of tumor cells surgically
implanted under the serosa of the colon. Treatment with 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone leads to
both decreased tumor size and decreased vascularization, as
evidenced by the pale appearance of tumors in 3-[(2,4-
dimethylpyrrol-5y1)methylidenyl]2-indolinone-treated animals.
Pharmacokinetics of 3-C(2,4-dimethyltwrrol-5-vl)methvl-
idenyll-2-indolinone.
Washout experiments in vitro have indicated a target
half-life of 96 hours, suggesting a very tight competitive
binding of 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
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indolinone to the ATP binding site of the receptor tyrosine
kinase. The in vivo intravenous pharmacokinetics of 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone was
characterized by rapid elimination of the parent compound
from the circulation in mice, rats and dogs. There was a
slightly longer elimination half-life determined for the rat
in comparison to mice and dogs. (Data not shown).
Pharmacokinetics of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone in rats are dose-dependent at
higher intravenous doses. At doses between 29.5 - 97.9
mg/mz, the elimination half-life of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone linearly increases as the dose
increased; the AUC increases 10-fold with only a 3-fold
increase in dose.
Subchronic toxicokinetic studies (28 day toxicity
studies) in rats and dogs indicated that the drug did not
accumulate in plasma upon repeated administration.
Whole body autoradiography using [1'C]-3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone demonstrated
widespread tissue distribution of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone followed by rapid elimination
following intravenous injection, with the highest levels
present in the small intestinal contents and urine (with
additional 3-((2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone observed in the liver, kidney, skin, testis, brown
fat, harderian gland and nasal turbinates). Total dose
recovered in 24 hours equaled 92~ of the total administered
dose, with 72~ excreted in feces and 16~ excreted in urine.
Biliary excretion is thought to be the major route of
elimination.
Studies with cold and [14C] -labeled 3- [ (2, 4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone demonstrate
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that the compound is rapidly metabolized following
intravenous administration in rats. Radiometabolite~profiling
indicated that greater than 90% of [1°C] -3- [ (2, 4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone was
metabolized within 3 hours following intravenous
administration. Data on metabolite identification suggest
that one metabolite has added a carboxyl group to one of the
methyl groups on the pyrrole ring, with a second metabolite
adding a methyl to the carboxyl group.
Preliminary pharmacokinetic data from a Phase 1 study in
patients with advanced malignancies in which patients were
treated at doses between 4.4 - 190 mg/mz indicates that the
drug has a half-life in humans of approximately 60 minutes.
The alpha half-life is rapid, with a mean 5.8 ~ 1.9 minutes.
The beta half-life or elimination phase has a mean value
43.4 ~ 21.9 minutes with a range from 10-111 minutes.
Clearance of 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-
indolinone from the systemic circulation is rapid, with a
mean value 1857 ~ 1016 liters of plasma cleared of 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone per day.
Clearance was independent of dose at these levels.
Individual clearance calculated based on BSA equaled 41.8 ~
22.1 L/hour/m2. After eight infusions of 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone, the rate of
clearance increases by 50 - 300% in all patients. The total
distributive volume of 3-[(2,4-dimethylpyrrol-5-
yl)methylidenyl]-2-indolinone, calculated by a one-
compartment model, is 53.6 ~ 11.3 liters, indicating that the
drug is distributed in the whole body fluid. At doses
tested in humans to date, AUC and C,,",~ increase linearly with
dose.
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The primary pathway for metabolism of 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone is through
sequential oxidation reactions of the 5-methyl group on the
pyrrol ring. Four metabolites are measurable in serum, all
of which involve serial oxidations of this methyl group on
the pyrrol ring. Data from in vitro metabolism studies shows
3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone is
metabolized via P-450 liver enzymes, most probably via CYP1A2
and CYP3A4, both of which are inducible enzymes. In
particular, CYP3A4 is induced by many xenobiotics, including
dexamethasone which is administered as a premedication prior
to all 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone
injections.
Fluorouracil and Fluorouracil/Leucovorin - General
The chemical structure of fluorouracil is 5-fluoro-2,4
(1H,3H)-pyrimidinedione. While the precise mode of action of
fluorouracil is not clear, the drug is thought to function as
an antimetabolite in at least three ways. In one aspect, as
its deoxyribonucleotide derivative, 5-fluoro-2'-deoxyuridine-
5'-phosphate (F-dUMP), the drug inhibits thymidylate synthetase
which results in inhibition of methylation of deoxyuridylic
acid to thymidylic acid. This, in turn, interferes with the
synthesis of DNA. In a second aspect, fluorouracil is found to
be incorporated into RNA to a an extent which, although small,
is sufficient to have a major effect on both the processing and
functions of the RNA. Finally, in a third aspect, fluorouracil
has been shown to block uracil phosphatase thus inhibiting
utilization of preformed uracil in RNA synthesis (Goodman and
Gilman's, '"The Pharmacological Basis of Therapeutics'", 1985,
pages 1268-1271).
Fluorouracil can be administered alone or in combination
with other drugs. The most common combination involves the use
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of leucovorin (folinic acid). Leucovorin potentiates the
cytotoxic effect of fluorouracil by, it is thought, increasing
the extracellular concentration of reduced folates which in
turn appears to stabilize the covalent ternary complex formed
by (F-dUMP), 5,10-methylenetetrahydrofolate and thymidine
synthetase. The stabilization of this complex enhances
inhibition of the synthetase, thereby increasing the efficacy
of fluorouracil.
Other chemotherapeutic combinations with fluorouracil for
the treatment of advanced stage colorectal cancer which have
been utilized include, without limitation, combination of
fluorouracil with: methotrexate, alone (Blijham, G., et al., J.
Clin Oncol., 1996, 14(8):2266-73) and in combination with
leucovorin (Romero, A. O., et al., Am. J. Clin. Onocol., 1998,
21(1):94-8); interferon alfa-2a (Greco, F. A., et al., J. Clin.
Oncol., 1996, 14(10):2674-81); interferon alpha 2b plus
leucovorin (Kohne, C. H., Oncology, 1997, 54(2):96-101):
platinum compounds, such as cisplatin and oxaliplatin, in
combination with leucovorin (Scheithauer, W., et al., Cancer,
1994, 73(6):1562-68); carboplatin plus methotrexate (prior to
fluorouracil administration) (Pronzato, P., J. Chemother.,
1998, 10(3):254-57);and Bleiberg, H, and Gramont, A., Semin.
Oncol., 1998, 25(2 Suppl. 5):32-39): lavamisole (Bandealy, M.
T., Clin. Cancer Res., 1998, 4(4):935-38); methyl lomustine
and leucovorin (Jones, Jr., D. V., Cancer, 1995, 76(IO):1709-
14); and, irinotecan, a topoisomerase-I inhibitor, (after
pretreatment with fluorouracil/leucovorin) (Rougier, P. et al.,
J. Clin. Oncol., 1997, 15(1):251-260).
While use of the above combinations is increasing, none
of them at present appear to provide a clear advantage over
fluorouracil alone or fluorouracil in combination with
leucovorin; that latter remains the standard initial
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treatment for patients with metastatic colorectal cancer.
As a single agent, it produces response rates of about 15%
with a median survival of six months. In combination with
leucovorin, the activity of the fluorouracil is increased
such that response rates of about 20% and median survival
times in advanced (Stage D) colorectal cancer of 11 - 13
months are observed (Wolmark, N., et al., J. Clin. Oncol.,
1993, 11:1879-1887).
Fluorouracil may be adminstered by either intravenous
bolus injection or continuous infusion. The volume of
distribution is slightly larger than the extracellular
space. Intravenous bolus doses of 370 to 720 mg/mz produce
an elimination half-life of 8 to l4 minutes with plasma
levels below 1 ~M within 2 hours, an approximate threshold
for cytotoxic effects. Less than 10% of the drug is
excreted in urine, while the balance is cleared through
metabolic pathways.
Frequently used administration schedules include short-
bolus injections over three to five days every 3-4 weeks,
continuous intravenous infusions of 96 - 120 hour duration
every 4 weeks, and weekly infusions for six weeks out of every
eight weeks. The incidence of serious clinical toxicity tends
to increase with higher systemic exposure (for example, with
higher steady-state plasma concentrations during constant
infusions and higher AUC with bolus administration).
Notably, each of the above schedules of treatment
includes substantial intervals during which no fluorouracil
is administered. This is due primarily to the inherent
toxicity of fluorouracil, which is exacerbated by the
addition of leucovorin. Unfortunately, this time interval
substantially reduces the efficacy of fluorouracil. That is,
initial treatment of a patient with fluorouracil or
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fluorouracil/leucovorin produces about a three log unit
(three orders of magnitude or 1000-fold) reduction in tumor
number and size. However, during the no-treatment "recovery"
period, tumor number and size rebound to the extent of about
two log units (100-fold). Thus, the overall effect of a
course of treatment with fluorouracil is only about one log
unit (an approximately 10-fold decrease in tumor number and
size) per administration of fluorouracil. Not only does
prolonged treatment with fluorouracil cause a problem with
regard to cost of treatment, patient quality of life, etc.,
it can result in secondary resistance to the drug. The
methods of this invention are directed to maintaining a more
substantial portion of the effect of each administration of
fluorouracil during the recovery period. Subsequent
administrations in the full course of treatment will thus be
confronted with tumors of reduced size and number, thus
improving the overall effectiveness of fluorouracil.
Clinical Trials with Fluorouracil and
Fluorouracil/Leucovorin in Advanced Colorectal Cancer
Frequently used continuous infusion schedules include
short-bolus injections over three to five days every 3-4
weeks, continuous intravenous infusions of 96 - 120 hours
every 4 weeks, and weekly infusions for six weeks out of every
eight weeks. The incidence of serious clinical toxicity tends
to increase with higher systemic exposure (for example, with
higher steady-state plasma concentrations during constant
infusions and higher AUC with bolus administration).
In a randomized clinical study conducted by the Mayo
Clinic and the North Central Cancer Treatment Group
(Mayo/NCCTG) in patients with advanced metastatic colorectal
cancer, three treatment regimens were compared: Leucovorin
(leucovorin) 200 mg/m2 and fluorouracil 370 mg/m2 versus
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leucovorin 20 mg/mz and fluorouracil 425 mg/m2 versus
fluorouracil 500 mg/m2. All drugs were administered by slow
intravenous infusion daily for 5 days repeated every 28-35
days. Response rates were 26~ (p = 0.04 versus fluorouracil
alone), 43~ (p = 0.001 versus fluorouracil alone) and 10~ for
the high dose leucovorin, low dose leucovorin and
fluorouracil alone groups respectively. Respective median
survival times were 12.2 months (p =0.037), 12 months (p =
0.050), and 7.7 months. The low dose leucovorin regimen gave
a statistically significant improvement in weight gain of
more than 5%, relief of symptoms, and improvement in
performance status. The high dose leucovorin regimen gave a
statistically significant improvement in performance status
and trended toward improvement in weight gain and in relief
of symptoms but these were not statistically significant.
In a second Mayo/NCCTG randomized clinical study the
fluorouracil alone arm was replaced by a regimen of
sequentially administered methotrexate (MTX), fluorouracil,
and leucovorin. Response rates with leucovorin 200 mg/m2 and
fluorouracil 370 mg/m2 versus leucovorin 20 mg/mZ and
fluorouracil 425 mg/m2 versus sequential MTX and fluorouracil
and leucovorin were respectively 31~ (p = <.O1), 42~ (p =
<.O1), and 14~. Respective median survival times were 12.7
months (p = <.04), 12.7 months (p = <.O1), and 8.4 months. No
statistically significant difference in weight gain of more
than 5~ or in improvement in performance status was seen
between the treatment arms.
In a third study comparing outcome and toxicities of low
(20 mg/m2) and high-dose (200 mg/m2) leucovorin, patients
received a 1-hour infusion of 400 mg/mz/day fluorouracil in
addition to leucovorin every 4 weeks. The two groups were
matched with no statistically significant differences in
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gender ratio, site of primary tumor, performance status, and
tumor extent. Toxicity in the two regimens was low and not
significantly different between the two groups. Overall
median survival was not significantly different between the
two groups: 346 days for those patients receiving low-dose
leucovorin and 323 days in those patients receiving high-dose
leucovorin. At 1 year, the test of equivalence was
significant (p < 0.01), demonstrating an absence of more than
20~ benefit in 1-year survival for the high-dose regimen. The
use of high-dose leucovorin combined with fluorouracil in the
5-day regimen does not significantly improve overall survival
for patients who have metastatic colorectal cancer.
Finally, in a fourth large randomized study, two of the
most common schedules of fluorouracil/leucovorin were
compared in the treatment of advanced colorectal cancer, as
each of these dosage administration schedules was
demonstrated to be superior to single-agent bolus
fluorouracil in previous controlled trials. Three hundred
seventy-two patients with metastatic colorectal cancer were
stratified according to performance status, and presence and
location of any measurable indicator lesions) and randomized
to receive chemotherapy with one of the two regimens: (1)
intensive-course fluorouracil plus low-dose leucovorin
(fluorouracil 425 mg/m2 plus leucovorin 20 mg/m2 intravenous
[IV] push daily for 5 days with courses repeated at 4- to 5-
week intervals); or (2) weekly fluorouracil plus high-dose
leucovorin (fluorouracil 600 mg/mz IV push plus leucovorin
500 mg/m2 as a 2-hour infusion weekly for 6 weeks with
courses repeated every 8 weeks). There were no significant
differences in therapeutic efficacy between the two
fluorouracil/leucovorin regimens tested with respect to the
following parameters: objective tumor response (35~ v 31~),
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survival (median, 9.3 v 10.7 months), and palliative effects
(as assessed by relief of symptoms, improved performance
status, and weight gain). There were significant (P < .05)
differences in toxicity, with more leukopenia and stomatitis
seen with the intensive-course regimen (Day 1-5), and more
diarrhea and increased requirement for hospitalization to
manage toxicity with the weekly regimen. Intensive-course
fluorouracil plus low-dose leucovorin appeared to have a
superior therapeutic index compared with weekly fluorouracil
plus high-dose leucovorin using the dosage administration
schedules applied in this study based on similar therapeutic
effectiveness, but with a decreased need for hospitalization
to manage chemotherapy toxicity.
3. PHARMACEUTICAL COMPOSITIONS AND USES
A compound of the present invention, a prodrug thereof
or a physiologically acceptable salt of either the compound
or its prodrug, can be administered as such to a human
patient or it can be administered in pharmaceutical
compositions in which the foregoing materials are mixed with
suitable carriers or excipient(s). Techniques for
formulation and administration of drugs may be found in
"Remington's Pharmacological Sciences," Mack Publishing Co.,
Easton, PA, latest edition.
Routes of Administration.
General
Suitable routes of administration may include, without
limitation, oral, rectal, transmucosal or intestinal
administration or intramuscular, subcutaneous,
intramedullary, intrathecal, direct intraventricular,
intravenous, intraperitoneal, intranasal, or intraocular
injections. The preferred routes of administration are oral
and parenteral.
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Alternatively, one may administer the compound in a
local rather than systemic manner, for example, via injection
of the compound directly into a solid tumor, often in a depot
or sustained release formulation.
Furthermore, one may administer the drug in a targeted
drug delivery system, for example, in a liposome coated with
tumor-specific antibody. The liposomes will be targeted to
and taken up selectively by the tumor.
Composition/Formulation
General
Pharmaceutical compositions of the present invention may
be manufactured by processes well known in the art; e.g., by
_ means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating,
entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with
the present invention may be formulated in conventional
manner using one or more physiologically acceptable carriers
comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which
can be used pharmaceutically. Proper formulation is
dependent upon the route of administration chosen.
For injection, the compounds of the invention may be
formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiological saline buffer. For
transmucosal administration, penetrants appropriate to the
barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
For oral administration, the compounds can be formulated
by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers
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enable the compounds of the invention to be formulated as
tablets, pills, lozenges, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral
ingestion by a patient. Pharmaceutical preparations for oral
use can be made using a solid excipient, optionally grinding
the resulting mixture, and processing the mixture of
granules, after adding other suitable auxiliaries if desired,
to obtain tablets or dragee cores. Useful excipients are, in
particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such
as, for example, maize starch, wheat starch, rice starch and
potato starch and other materials such as gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethylcellulose,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone
I5 (PVP). If desired, disintegrating agents may be added, such
as cross-linked polyvinylpyrrolidone, agar, or alginic acid.
A salt such as sodium alginate may also be used.
Dragee cores are provided with suitable coatings. For
this purpose, concentrated sugar solutions may be used which
may optionally contain gum arabic, talc, polyvinyl
pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be
added to the tablets or dragee coatings for identification or
to characterize different combinations of active compound
doses.
Pharmaceutical compositions which can be used orally
include push-fit capsules made of gelatin, as well as soft,
sealed capsules made of gelatin and a plasticizer, such as
glycerol or sorbitol. The push-fit capsules can contain the
active ingredients in admixture with a filler such as
lactose, a binder such as starch, and/or a lubricant such as
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talc or magnesium stearate and, optionally, stabilizers. In
soft capsules, the active compounds may be dissolved or
suspended in suitable liquids, such as fatty oils, liquid
paraffin, or liquid polyethylene glycols. Stabilizers may be
added in these formulations, also.
For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered
in the form of an aerosol spray using a pressurized pack or a
nebulizer and a suitable propellant, e.g., without
limitation, dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetra- fluoroethane or carbon dioxide. In the case
of a pressurized aerosol, the dosage unit may be controlled
by providing a valve to deliver a metered amount. Capsules
and cartridges of, for example, gelatin for use in an inhaler
or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
The compounds may also be formulated for parenteral
administration, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions may take such
forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulating materials such
as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral
administration include aqueous solutions of a water soluble
form, such as, without limitation, a salt, of the active
compound. Additionally, suspensions of the active compounds
may be prepared in a lipophilic vehicle. Suitable lipophilic
vehicles include fatty oils such as sesame oil, synthetic
fatty acid esters such as ethyl oleate and triglycerides, or
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materials such as liposomes. Aqueous injection suspensions
may contain substances which increase the viscosity of the
suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspension may also contain
suitable stabilizers and/or agents that increase the
solubility of the compounds to allow for the preparation of
highly concentrated solutions.
Alternatively, the active ingredient may be in powder
form for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water, before use.
The compounds may also be formulated in rectal
compositions such as suppositories or retention enemas,
using, e.g., conventional suppository bases such as cocoa
butter or other glycerides.
In addition to the fomulations described previously, the
compounds may also be formulated as depot preparations. Such
long acting formulations may be administered by implantation
(for example, subcutaneously or intramuscularly) or by
intramuscular injection. A compound of this invention may be
formulated for this route of administration with suitable
polymeric or hydrophobic materials (for instance, in an
emulsion with a pharamcologically acceptable oil), with ion
exchange resins, or as a sparingly soluble derivative such
as, without limitation, a sparingly soluble salt.
A non-limiting example of a pharmaceutical carrier for
the hydrophobic compounds of the invention is a cosolvent
system comprising benzyl alcohol, a nonpolar surfactant, a
water-miscible organic polymer and an aqueous phase such as
the VPD co-solvent system. VPD is a solution of 3~ w/v
benzyl alcohol, 8~s w/v of the nonpolar surfactant Polysorbate
80TM, and 65~ w/v polyethylene glycol 300, made up to volume
in absolute ethanol. The VPD co-solvent system (VPD:DSW)
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consists of VPD diluted 1:1 with a 5~ dextrose in water
solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon
systemic administration. The proportions of such a co-
y solvent system may be varied considerably without destroying
its solubility and toxicity characteristics. Furthermore,
the identity of the co-solvent components may be varied: for
example, other low-toxicity nonpolar surfactants may be used
instead of Polysorbate 80TM; the fraction size of
polyethylene glycol may be varied; other biocompatible
polymers may replace polyethylene glycol, e.g., polyvinyl
pyrrolidone; and other sugars or polysaccharides may
substitute for dextrose.
Alternatively, other delivery systems for hydrophobic
pharmaceutical compounds may be employed. Liposomes and
emulsions are well known examples of delivery vehicles or
carriers for hydrophobic drugs. In addition, certain organic
solvents such as dimethylsulfoxide also may be employed,
although often at the cost of greater toxicity.
Additionally, the compounds may be delivered using a
sustained-release system, such as semi-permeable matrices of
solid hydrophobic polymers containing the therapeutic agent.
Various sustained-release materials have been established
and are well known by those skilled in the art. Sustained-
release capsules may, depending on their chemical nature,
release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability
of the therapeutic reagent, additional strategies for protein
stabilization may be employed.
The pharmaceutical compositions herein also may comprise
suitable solid or gel phase carriers or excipients. Examples
of such carriers or excipients include, but are not limited
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to, calcium carbonate, calcium phosphate, various sugars,
starches, cellulose derivatives, gelatin, and polymers such
as polyethylene glycols.
3-I(2,4-dimethylpyrrol-5-yl)methylidenyll-2-indolinone
composition.
This compound may be formulated as any of the
compositions and formulations described above. A presently
preferred formulation, however, is comprised of 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone in sufficient
sterile parenteral solution to afford a 4.5 mg/ml final
concentration. Additional components of the formulation
include polyethylene glycol 400; polyoxyl 35 castor oil
(Cremophor°); benzyl alcohol and dehydrated alcohol. It
should be noted that this formulation, since it contains
Cremophor°, is not compatible with standard PVC-lined
syringes, intravenous bags and administration sets.
Fluorouracil/Leucovorin composition
Fluorouracil is commercially available in compositions
and formulations which are known to those skilled in the
chemotherapeutic art and may be administered in the methods
of this invention as those compositions/formulations.
Examples of such compositions/formulations are shown in the
Package Insert which accompanies commercial fluorouracil and
which is incorporated by reference as if fully set forth
herein. The use of any other or different
composition/formulation as such may be developed or become
available in the future is also within the scope of this
invention.
Likewise, leucovorin is also commercially available in
compositions/formulations known to those in the
chemotherapeutic art and may also be administered in the
methods of this invention as those compositions/formulations.
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Examples of such compositions/formulations are shown in the
Package Insert that accompanies commercial leucovorin and
which is incorporated as if fully set forth herein. As
above, any other or different composition/formulation as such
may be developed or become available in the future is also
within the scope of this invention.
DOSAGE
General
Pharmaceutical compositions suitable for use in the
present invention include compositions wherein the active
ingredients are contained in an amount sufficient to achieve
the intended purpose; i.e., the modulation of PK activity or
the treatment or prevention of a PK-related disorder.
More specifically, a therapeutically effective amount
means an amount of compound effective to prevent, alleviate
or ameliorate symptoms of disease or prolong the survival of
the subject being treated.
Determination of a therapeutically effective amount is
well within the capability of those skilled in the art,
especially in light of the detailed disclosure provided
herein.
For any compound used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from cell culture assays. Then, the dosage can be
formulated for use in animal models so as to achieve a
circulating concentration range that includes the ICso as
determined in cell culture (i.e., the concentration of the
test compound which achieves a half-maximal inhibition of the
PK activity). Such information can then be used to more
accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the compounds
described herein can be determined by standard pharmaceutical
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procedures in cell cultures or experimental animals, e.g., by
determining the ICSa and the LDso (both of which are discussed
elsewhere herein) for a subject compound. The data obtained
from these cell culture assays and animal studies can be used
in formulating a range of dosage for use in humans. The
dosage may vary depending upon the dosage form employed and
the route of administration utilized. The exact formulation,
route of administration and dosage can be chosen by the
individual physician in view of the patient's condition.
(See e.g., Fingl, et al., 1975, in "The Pharmacological Basis
of Therapeutics", Ch. 1 p.l).
Dosage amount and interval may be adjusted individually
to provide plasma levels of the active species, which are
sufficient to maintain the kinase modulating effects. These
plasma levels are referred to as minimal effective
concentrations (MECs). The MEC will vary for each compound
but can be estimated from in vitro data; e.g., the
concentration necessary to achieve 50-90~ inhibition of a
kinase may be ascertained using the assays described herein.
Dosages necessary to achieve the MEC will depend on
individual characteristics and route of administration. HPLC
assays or bioassays can be used to determine plasma
concentrations.
Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen that
maintains plasma levels above the MEC for 10-90~ of the time,
preferably between 30-90~ and most preferably between 50-905.
In cases of local administration or selective uptake,
the effective local concentration of the drug may not be
related to plasma concentration and other procedures known in
the art may be employed to determine the correct dosage
amount and interval.
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The amount of a composition administered will, of
course, be dependent on the subject being treated, the
severity of the affliction, the manner of administration, the
judgment of the prescribing physician, etc.
3-C(2,4-dimethylpyrrol-5-vl)methvlidenyll-2-indolinone
dosagre .
Based on the pharmacological data obtained regarding 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone(see
above), the compound may be administered in doses ranging
from about 4 mg/mZto about 195 mg/m2. In a presently
preferred embodiment, the dosage is between about 72.5 mg/m2
and about 145 mg/m2.
The dilution described in the above composition section
may be administered to a patient at a rate of from about 50
to about 350 cc/hour. Preferable, the rate is from about 150
to about 250 cc/hour. Most preferably, it is from about 175
to about 225 cc/hour.
By "about," wherever the term appears herein, is meant
~10%; i.e., about 175 cc/hour means from 157.5 cc/hour to
192.5 cc/hour, etc.
In a presently preferred embodiment, the 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone dose is
administered during rest periods when no fluorouracil or
fluorouracil/leucovorin is being administered to a patient.
As was made evident by the examples in the Pharmacology
section, above, fluorouracil or fluorouracil/leucovorin may
be administered in numerous treatment regimes, the choice of
which is within the knowledge and expertise of the treating
physician.
Fluorouracil and Fluorouracil/Leucovorin dosage
As can be seen in the clinical studies with fluorouracil
and fluorouracil/leucovorin described above, there are
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currently a variety of schedules used for administration of
fluorouracil or fluorouracil/leucovorin in advanced
colorectal cancer. However, there is a remarkable lack of
difference in the outcome using various administration doses
and schedules of fluorouracil and fluorouracil/leucovorin,
with most regimens producing leukopenia, diarrhea and
mucositis to a varying degree. Thus, while fluoruracil may be
administered in doses ranging from about 300 mg/m2 to about
800 mg/m2, schedules of fluorouracil which provide a dose
intensity of approximately 400 -500 mg/m2/week are presently
considered to be optimal therapy. When leucovorin is
included in the treatment, differences in clinical outcome
for low and high dose leucovorin are minimal which, given the
additional toxicity of the high dose regimen, the low dose
regimen presently appears most appropriate.
Thus, while the fluorouracil or fluorouracil/leucovorin
may, within the scope of this invention, be administered in
any presently approved manner or in any manner found in the
future to be efficacious, given the above data, a presently
preferred embodiment of this invention is to administer
fluorouracil at a dose of about 400 to 500 mg/m2 as a bolus
intravenous injection on day 1-5 of a 4 week cycle. The 4-
week cycle may be repeated as necessary or until adverse side
effects as recognized by the physician conducting the
treatment are encountered.
Leucovorin may be administered with the fluorouracil.
Leucovorin may be administered in doses of from about 20 to
about 500 mg/m2, preferably from about 20 to about 200 mg/mz
and in a presently preferred embodiment of this invention as
a low-dose administration of about 20 mg/m2, also as a bolus
injection, with each administration of fluorouracil.
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Fluorouracil or fluorouracil/leucovorin in combination
with 3- t (2,4-dimethylpvrrol-5-yl)met>~rlidenyll -2-
indolinone
It is an aspect of this invention that, when 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone is
administered in combination with fluorouracil or
fluorouracil/leucovorin, the compounds may be administered
simultaneously, sequentially, continuously, intermittantly,
etc. in accordance with a treatment regime calculated to take
maximum advantage of the characteristics of each of the
components. In a presently prefred embodiment 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone is
administered on days when no fluorouracil or
fluorouracil/leucovorin is administered. Thus, in one
embodiment of this invention, the above-described dose of 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone is
administered in any pattern desired; e.g., without
limitation, on each day, every other day, every third day,
etc. of a treatment regime selected for fluorouracil or
fluorouracil/leucovorin on which fluorouracil or
fluorouracil/leucovorin is not administered. The 3-[(2,4-
dimethylpyrrol-5-yl)methylidenyl]-2-indolinone may be
administered as a bolus intravenous injection or as a
continuous intravenous infusion. However, based upon in
vitro data, 3-[(2,4-dimethylpyrrol-5yl)methylidenyl]-2-
indolinone may be administered over a relatively short time
period (5 to 30 minutes) and exert antiproliferative activity
on the endothelial cells for 3 to 4 days thereafter.
Likewise, the in vivo data demonstrate that dosing with 3-
[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone at 3 to
4 day intervals was sufficient to inhibit tumor growth
without toxicity. Furthermore, no cumulative toxicity was
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observed in Phase I dose escalation studies in patients
treated with up to 52 weeks of treatment. Thus, in a
presently preferred embodiment of this invention, the
indicated dose of 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-
2-indolinone is administered twice weekly in weeks 2-4 of
each four week treatment regime.
Based on the disclosures herein, 3-[(2,4-Dimethylpyrrol-
5-yl) methylene]-2-indolinone might be expected to work in
combination with other chemotherapeutic agents as well. For
instance, the combination of 3-[(2,4-Dimethylpyrrol-5-yl)
methylene]-2-indolinone with other alkylating agents might
afford synergistic activity without concomitant increased
toxicity. Such alkylating agents could include, without
limitation, the alkyl sulfonates; e.g., busulfan (used for
treatment of chronic granulocytic leukemia), improsulfan and
piposulfan; the aziridines; e.g., benzodepa, carboquone,
meturedepa, and uredepa; the ethyleneimines and
methylmelamines; e.g., altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylolmelamine and the nitrogen mustards; e.g.,
chlorambucil (used in treatment of chronic lymphocytic
leukemia, primary macroglobulinemia and non-Hodgkin's
lymphoma), cyclophosphamide (used in treatment of Hodgkin's
disease, multiple myeloma, neuroblastoma, breast cancer,
ovarian cancer, lung cancer, Wilm's tumor and
rhabdomyosarcoma), estramustine, ifosfamide, novembrichin,
prednimustine and uracil mustard (for primary thrombocytosis,
non-Hodgkin's lymphoma, Hodgkin's disease and ovarian
cancer); and the triazines; e.g., dacarbazine (used for soft-
tissue sarcoma).
Likewise, 3-[(2,4-Dimethylpyrrol-5-yl) methylene]-2
indolinone could have a beneficial effect in combination with
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other antimetabolite chemotherapeutic agents such as, without
limitation, folic acid analogs (e.g., methotrexate (used in
treating acute lymphocytic leukemia, choriocarcinoma, mycosis
fungoides, breast, neck and head and lung cancer, osteogenic
sarcoma) and pteropterin) the purine analogs such as
mercaptopurine and thioguanine which find use in the
treatment of acute granulocytic, acute lymphocytic and
chronic granulocytic leukemias).
3-[(2,4-Dimethylpyrrol-5-yl) methylene]-2-indolinone
could also prove effective in combination with natural
product chemotherapeutic agents such as, without limitation,
the vinca alkaloids (vinblastine (used for breast and
testicular cancer), vincristine, vindesine), the
epipodophylotoxins (etoposide, teniposide (both used in the
treatment of testicular cancer and Kaposi~s sarcoma)), the
antibiotic chemotherapeutic agents (daunorubicin,
doxorubicin, bleomycin, mitomycin (used for stomach, cervix,
colon, breast, bladder and pancreatic cancer), dactinomycin,
plicamycin, bleomycin (used for skin, esophagus and
genitourinary tract cancer) and the enzymatic
chemotherapeutic agents such as L-Asparaginase.
Based on the disclosures of this invention, 3-[(2,4-
Dimethylpyrrol-5-yl) methylene]-2-indolinone might also
benefit the activity of chemotherapeutic agents such as
platinum coordination complexes (cisplatin, etc.),
substituted ureas (hyroxyurea), methylhydrazine derivatives
(procarbazine), adrenocortical suppressants (mitotane,
aminoglutethimide) as well as hormones and antagonists such
as adrenocorticosteroids (prednisone), progestins
(hydroxyprogesterone caproate), estrogens
(diethylstilbestrol), antiestrogens (tamoxifen) and androgens
(testosterone propionate).
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Finally, the combination of 3-[(2,4-Dimethylpyrrol-5-yl)
methylene]-2-indolinone with mitoxantrone or paclitaxel might
be expected to display especially beneficial results in the
treatment of solid tumors or leukemias such as, without
limitation, acute myelogenous (nonlymphocytic) leukemia.
It is to be understood that, while the above description
relates to the use of 3-[(2,4-dimethylpyrrol-5-yl)methylene]-
2-indolinone in combination with fluorouracil or
fluorouracil/leucovorin, other compounds of this invention,
in particular 3-[4-(2-carboxyethyl-3,5-dimethylpyrrol-2-
yl)methylidenyl]-2-indolinone, in combination with
fluorouracil or fluorouracil/leucovorin are also within the
scope and spirit of this invention.
PACKAGING
The compositions may, if desired, be presented in a pack
or dispenser device, such as an FDA approved kit, which may
contain one or more unit dosage forms containing the active
ingredient. The pack may for example comprise metal or
plastic foil, such as a blister pack. The pack or dispenser
device may be accompanied by instructions for administration.
The pack or dispenser may also be accompanied by a notice
associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use or sale
of pharmaceuticals, which notice is reflective of approval by
the agency of the form of the compositions or of human or
veterinary administration. Such notice, for example, may be
of the labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved
product insert. Compositions comprising a compound of the
invention formulated in a compatible pharmaceutical carrier
may also be prepared, placed in an appropriate container, and
labeled for treatment of an indicated condition. Suitable
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conditions indicated on the label may include treatment of a
tumor, inhibition of angiogenesis, treatment of fibrosis,
diabetes, and the like.
4. SYNTHESIS
The compounds of this invention, as well as the
precursor 2-oxindoles and aldehydes, may be readily
synthesized using techniques well known in the chemical arts.
It will be appreciated by those skilled in the art that other
synthetic pathways for forming the compounds of the invention
are available and that the following is offered by way of
example and not limitation.
I. 4-Methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemeth~rl~-
. _ .__ 1H-p~rrrole-2-carboxylic acid meth3rl ester
Phosphorus oxychloride (0.186 mL, 1.44 mmol) was added
dropwise to a solution of dimethyformamide (0.15 mL, 1.44
mmol) in dichloromethane (4mL) at 0 °C. The mixture was
warmed to room temperature and stirred for 30 minutes and then
cooled to 0° C. 4-Methyl-2-pyrrolecarboxylate methyl ester
(100 mg, 0.72 mmol) was added portion-wise and the mixture was
then stirred at 40-50° C for 4 hours. Sodium hydroxide (10%
aqueous solution, 2 ml) was added and the reaction mixture was
stirred for 30 minutes. The basic solution was then extracted
with ethyl acetate (3X) and the organic layer was washed with
brine to pH 6-7, dried over anhydrous sodium sulfate and
concentrated under vacuum to give 115.9 mg (96%) of 4-methyl-
5-formyl-2-pyrrolecarboxylate methyl ester as a yellow oil.
A mixture of oxindole (105 mg, 0.79 mmol), 4-methyl-5-
formyl-2-pyrrolecarboxylate methyl ester (110 mg, 0.67mmo1)
and piperidine (2 drops) in ethanol (2 mL) was stirred at 90
°C for 3 hours. The precipitate was collected by vacuum
filtration, washed with ethanol and dried under vacuum to
yield 153.2 mg (81%) of 4-methyl-5-(2-oxo-1,2-dihydroindol-3-
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ylidenomethyl)-1H-pyrrole-2-carboxylic acid methyl ester.
1HNMR (360 MHz, DMSO-d6) . 13.98(s, br, 1H, NH), 10.97
(s, br, 1H, NH), 7.82, (d, J=7.6Hz, 1H), 7.67 (s, 1H, H-
vinyl), 7.2 (dt, J= 1.2, 7.7HZ, 1H), 7.01 (dt, J=1.2, 7.7
Hz, 1H), 6.90 (d, J= 7.6 Hz, 1H), 6.77 (d, J= 2Hz, 1H).
MS (ES) 283 jM+1] (100$) .
2. 4-Methyl-5-(2-oxo-1,2-dihydroiadol-3-
ylidenemethvl)-1H-nvrrole-2-carbo ~lic acid
Phosphorus oxychloride (0.66 mL, 7.2 mmol) was added
dropwise to an ice-cold solution of dimethylforamide (0.6 mL,
7.2 mmol) in dichloromethane (30 mL). The mixture was
stirred at room temperature for 30 minutes and then cooled in
an ice-bath. 4-methyl-2-pyrrolecarboxylate ethyl ester (919
mg, 6 mmol) was added slowly to the reaction mixture. The
resulting reaction mixture was then stirred at room
temperature for 2.4 hours. The mixture was then cooled in an
ice-bath and 2N sodium hydroxide was added and the mixture
stirred for 30 minutes. The aqueous mixture was extracted
with ethyl acetate (2X), the organic layers combined and
washed with brine and then dried over anhydrous sodium
sulfate and concentrated under vacuum. The pink solid which
was obtained was dried under vacuum at room temperature for 3
days to yield 1.05g (96~) of 4-methyl-5-formyl-2-
pyrrolecarboxylate ethyl ester. The product was used without
further purification.
MS (APCI) [M-1]' 180 (80~) , [M-34] ' 146 (100%) .
A mixture of 4-methyl-5-formyl-2-pyrrolecarboxylate
ethyl ester (543.57 mg, 3 mmol) in 2N sodium hydroxide (1.2 g
in 15 mL of water) was refluxed for 1/2 hour. The reaction
mixture was cooled to room temperature and poured into ice
water. It was then acidified to pH -.3.5 with 2N hydrochloric
acid and extracted with ethyl acetate (2X). The organic
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layer was washed with brine, dried over anhydrous sodium
sulfate and concentrated under vacuum. The solid obtained
was dried under vacuum at 40 °C for 2 hours to yield 410 mg
(89%) of 4-methyl-5-formyl-2-pyrrolecarboxylic acid as a
white solid.
A mixture of oxindole (133.15 mg, 1 mmol), 4-methyl-5-
formyl-2-pyrrolecarboxylic acid (153.14 mg, 1 mmol), piperidine
(2 drops) in ethanol (2 mL) was refluxed for 3 hours. The
precipitate was collected by vacuum filtration, washed with
ethanol, neutralized with 2N hydrochloric acid, washed with
water and dried to yield 268.5 mg (1000 of 4-methyl-5-(2-oxo-
1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid
as an orange/red solid.
1NMR (360 MHz, DMSO-d6) . 13.84 (s, br, 1H, NH), 12.84
(s, br, 1H, COOH), 10.98 (s, br, 1H, NH), 7.82(d, J= 7.5Hz,
1H), 7.67 (s, 1H, H-vinyl), 7.18 (t, J= 7.5Hz, 1H), 7.01 (t,
J= 7.5Hz, 1H), 6.88(d, J= 7.5Hz, 1H), 6.71 (d, J= 2.2Hz, 1H),
2.32 (s, 3H, CH3) .
MS (negative mode) 266. 8 [M-1] '.
3. 3-(5-Hydroxymethyl-3-melthyl-1H-pvrrol-2-
ylmethylene)-1,3-dihydroindol-2-one and
4. 4-Methyl-5-(2-oxo-1,2-dihydro-indol-3;
ylidenomethvl)-1H-pyrrol-2-carboxaldehyde
To a suspension of 4-methyl-5-(2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-1H-pyrrole-2-carboxylic acid 4.02g, 15 mmol) in
tetrahydrofuran (50 mL) was slowly added oxalyl chloride
(3.80g, 30mmo1) at 0° C. After addition was complete, the
resultant suspension was stirred at room temperature for 2
hours. Sodium borohydride (1.148, 30mmol) was then added
portionwise to the mixture and the suspension was further
stirred at room temperature for 1 day. At that thme, a second
portion of 1.14 g of sodium borohydride was added followed by
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mL of dimethylformamide to dissolve the solids arid the
reaction mixture was stirred for another day at room
temperature. Ice water was added to the ice-cold reaction
mixture until no more gas evolved. The aqueous layer was
5 extracted with ethyl acetate. The precipitate which formed
between the organic and aqueous layer was filtered, washed with
water and ethyl acetate and dried to give 2.5g (60~) of a red
solid. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, concentrated and purified on a silica
10 gel column eluting with ethyl acetate-hexane to give 340 mg
(9~) of 3-(5-hydroxymethyl-3-methyl-1H- pyrrol-2-ylmethylone)-
1,3-dihydroindol-2-one as a yellow solid and 540 mg (14~ of 4-
methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-
carbaldehydeas an orange solid.
3-(5-Hydroxymethyl-3-methyl-1H-pyrrol-2-ylmethylene)-
I,3-dihydroindol-2-one: 1HNMR (360 MHz, DMSO-d6) . 13.39 (s,
br, 1H, NH), 10-69 (s, br, 1H, NH), 7.70(d, J= 7.6Hz, 1 H),
7.56(s, 1 H. H-vinyl), 7.09 (t, J= 7.6Hz, 1 H), 6.96 (t, J=
7.6Hz, 1H), 6.86 (d, J= 7.6Hz, 1 H), 6.06 (d, J=2.1 Hz, 1H),
5.33 (t, J=5,6Hz, 1H, OH), 4.51 (d, J=5,6Hz,2H, CH20H), 2.31
(s, 3H, CH3) .
MS 251 [M-1] i (1000
M.p. >350 °C
4-Methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrole-2-carbaldehyde: 1HNMR (360 MHz, DMSO-d6) 8: 13.87 (s,
br, 1H. NH), 11.05 (s, br, 1H, NH), 9.61(x, 1H CHO), 7.85 (d,
J=7.5Hz, 1H), 7.71(s, IH, H-vinyl), 7.23(t, J=7.5Hz, 1H), 7.03,
(t, J= 7.5Hz,lH), 6.97 (d, J=2.2Hz, 1H), 6.9(d, J=7.5Hz, 1H),
2.36 (s, 3H, CH3) .
MS 237.4 [M-OH]' 91000 .
M.p. 267.3-268.4 °C.
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5. BIOLOGICAL EVALUATION
It will be appreciated that, in any given series of
compounds, a spectrum of biological activities will be
obtained. In its preferred embodiments, this invention relates
to novel 3-heteroarylidenyl-2-indolinones demonstrating the
ability to modulate RTK, CTK, and STK activity. The following
assays are employed to select those compounds demonstrating the
optimal degree of the desired activity.
Assa~r Procedures .
IO The following in vitro assays may be used to determine
the level of activity and effect of the different compounds
of the present invention on one or more of the PKs. Similar
assays can be designed along the same lines for any PK using
techniques Well known in the art.
The cellular/catalytic assays described herein are
performed in an ELISA format. The general procedure is as
follows: a compound is introduced to cells expressing the
test kinase, either naturally or recombinantly, for a
selected period of time after which, if the test kinase is a
receptor, a ligand known to activate the receptor is added.
The cells are lysed and the lysate is transferred to the
wells of an ELISA plate previously coated with a specific
antibody recognizing the substrate of the enzymatic
phosphorylation reaction. Non-substrate components of the
cell lysate are washed away and the amount of phosphorylation
on the substrate is detected with an antibody specifically
recognizing phosphotyrosine compared with control cells that
were not contacted with a test compound. The
cellular/biologic assays described herein measure the amount
of DNA made in response to activation of a test kinase, which
is a general measure of a proliferative response. The
general procedure for this assay is as follows: a compound is
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introduced to cells expressing the test kinase, either
naturally or recombinantly, for a selected period of time
after which, if the test kinase is a receptor, a ligand known
to activate the receptor is added. After incubation at least
overnight, a DNA labeling reagent such as Bromodeoxyuridine
(BrdU) or 3H-thymidine is added. The amount of labeled DNA
is detected with either an anti-BrdU antibody or by measuring
radioactivity and is compared to control cells not contacted
with a test compound.
Cellular/Catalytic Assays
Enzyme linked immunosorbent assays (ELISA) may be
used to detect and measure the presence of PK activity. The
ELISA may be conducted according to known protocols which are
described in, for example, Voller, et al., 1980, "Enzyme-
Linked Immunosorbent Assay," In: Manual of Clinical
Immunology, 2d ed., edited by Rose and Friedman, pp 359-371
Am. Soc. Of Microbiology, Washington, D.C.
The disclosed protocol may be adapted for determining
activity with respect to a specific PK. That is, the
preferred protocols for conducting the ELISA experiments for
specific PKs is provided below. However, adaptation of these
protocols for determining a compound's activity for other
members of the RTK family, as well as for CTKs and STKs, is
well within the scope of knowledge of those skilled in the
art.
FLK-1 Assav
An ELISA assay is conducted to measure the kinase
activity of the FLK-1 receptor and more specifically, the
inhibition or activation of TK activity on the FLK-1
receptor. Specifically, the following assay can be conducted
to measure kinase activity of the FLK-1 receptor in cells
genetically engineered to express Flk-1.
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Materials and Reagents.
a. Corning 96-well ELISA plates (Corning Catalog No.
25805-96);
b. Cappel goat anti-rabbit IgG (catalog no. 55641);
c. PBS (Gibco Catalog No. 450-1300EB);
d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and
0.1% Tween-20);
e. Ethanolamine stock (10% ethanolamine (pH 7.0),
stored at 4°C);
IO f. HNTG buffer (20mM HEPES buffer (pH 7.5), 150mM
NaCl, 0.2% Triton X-100, and 10% glycerol);
g. EDTA (0.5 M (pH 7.0) as a 100X stock);
h. Sodium orthovanadate (0.5 M as a 100X stock);
i. Sodium pyrophosphate (0.2 M as a 100X stock);
j. NUNC 96 well V bottom polypropylene plates (Applied
Scientific Catalog No. AS-72092);
k. NIH3T3 C7#3 Cells (FLK-1 expressing cells);
1. DMEM with 1X high glucose L-Glutamine (catalog No.
11965-050);
m. FBS, Gibco (catalog no. 16000-028);
n. L-glutamine, Gibco (catalog no. 25030-016);
o. VEGF, PeproTech, Inc. (catalog no. 100-20)(kept as
1 ~g/100 ~1 stock in Milli-Q dH20 and stored at -20° C;
p. Affinity purified anti-FLK-1 antiserum;
q. UB40 monoclonal antibody specific for
phosphotyrosine (see, Fendley, et al., 1990, Cancer Research
50:1550-1558);
r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog
no. 172-1011);
s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic
acid (ABTS) solution (100mM citric acid (anhydrous}, 250 mM
Na2HP04 (pH 4.0), 0.5 mg/ml ABTS (Sigma catalog no. A-1888)),
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solution should be stored in dark at 4° C until ready for
use;
t. HzOz (30% solution)(Fisher catalog no. H325);
u. ABTS/ HzOz (15m1 ABTS solution, 2 ~tl HzOz) prepared
5 minutes before use and left at room temperature;
v. 0.2 M HC1 stock in H20;
w. dimethylsulfoxide (100%)(Sigma Catalog No. D-8418);
and
y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).
Protocol.
1. Coat Corning 96-well ELISA plates with 1.0 ~g per
well Cappel Anti-rabbit IgG antibody in O.1M NazC03 pH 9.6.
Bring final volume to 150 ~1 per well. Store plates
overnight at 4°C. Plates can be kept up to two weeks when
stored at 4°C.
2. Grow cells in Growth media (DMEM, supplemented with
2.0 mM L-Glutamine, 10% FBS) in suitable culture dishes until
confluent at 37°C, 5% CO2.
3. Harvest cells by trypsinization and seed in Corning
25850 polystyrene 96-well round bottom cell plates, 25,000
cells/well in 200 ~1 of growth media.
4. Grow cells at least one day at 37°C, 5% CO2.
5. Wash cells with D-PBS 1X.
6. Add 200 ~.1/well of starvation media (DMEM, 2.OmM 1-
Glutamine, 0.1% FBS). Incubate overnight at 37°C, 5% COZ.
7. Dilute Compounds 1:20 in polypropylene 96 well
plates using starvation media. Dilute dimethylsulfoxide 1:20
for use in control wells.
8. Remove starvation media from 96 well cell culture
plates and add 162 ~l of fresh starvation media to each well.
9. Add 18 ~tl of 1:20 diluted compound dilution (from
step 7) to each well plus the 1:20 dimethylsulfoxide dilution
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to the control wells (+/- VEGF), for a final dilution of
1:200 after cell stimulation. Final dimethylsulfoxide is
0.5~. Incubate the plate at 37°C, 5~ COz for two hours.
10. Remove unbound antibody from ELISA plates by
inverting plate to remove liquid. Wash 3 times with TBSW +
0.5~ ethanolamine, pH 7Ø Pat the plate on a paper towel to
remove excess liquid and bubbles.
11. Block plates with TBSW + 0.5~ ethanolamine, pH 7.0,
150 gel per well. Incubate plate thirty minutes while shaking
on a microtiter plate shaker.
12. Wash plate 3 times as described in step 10.
13. Add 0.5 ~g/well affinity purified anti-FLU-1
polyclonal-rabbit antiserum. Bring final volume to 150
~1/well with TBSW + 0.5~ ethanolamine pH 7Ø Incubate plate
for thirty minutes while shaking.
14. Add 180 ~,1 starvation medium to the cells and
stimulate cells with 20 ~1/well 10.0 mM sodium orthovanadate
and 500 ng/ml VEGF (resulting in a final concentration of 1.0
mM sodium orthovanadate and 50 ng/ml VEGF per well) for eight
minutes at 37°C, 5~ COz. Negative control wells receive only
starvation medium.
15. After eight minutes, media should be removed from
the cells and washed one time with 200 ~1/well PBS.
16. Lyse cells in 150 ~,1/well HNTG while shaking at
room temperature for five minutes. HNTG formulation includes
sodium ortho vanadate, sodium pyrophosphate and EDTA.
17. Wash ELISA plate three times as described in step 10.
18. Transfer cell lysates from the cell plate to ELISA
plate and incubate while shaking for two hours. To transfer
cell lysate pipette up and down while scrapping the wells.
19. Wash plate three times as described in step 10.
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20. Incubate ELISA plate with 0.02 ~g/well UB40 in TBSW
+ 05% ethanolamine. Bring final volume to 150 ~.1/well.
Incubate while shaking for 30 minutes.
21. Wash plate three times as described in step 10.
22. Incubate ELISA plate with 1:10,000 diluted EIA
grade goat anti-mouse IgG conjugated horseradish peroxidase
in TBSW plus 0.5% ethanolamine, pH 7Ø Bring final volume
to 150 ~l/well. Incubate while shaking for thirty minutes.
23. wash plate as described in step 10.
24. Add 100 ~1 of ABTS/HZOz solution to well. Incubate
ten minutes while shaking.
25. Add 100 ~.1 of 0.2 M HC1 for 0.1 M HC1 final
concentration to stop the color development reaction. Shake
1 minute at room temperature. Remove bubbles with slow
stream of air and read the ELISA plate in an ELISA plate
reader at 410 nm.
EGF Receptor-HER2 Chimeric Receptor Assay In Whole
Cells.
HER2 kinase activity in whole EGFR-NIH3T3 cells are
measured as described below:
Materials and Reagents.
a. EGF: stock concentration: 16.5 ILM; EGF 201,
TOYOBO, Co., Ltd. Japan.
b. 05-101 (UBI) (a monoclonal antibody
recognizing an EGFR extracellular domain).
c. Anti-phosphotyrosine antibody (anti-Ptyr)
(polyclonal)(see, Fendley, et al., supra).
d. Detection antibody: Goat anti-rabbit 1gG
horseradish peroxidase conjugate, TAGO, Inc., Burlingame, CA.
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e. TBST buffer:
Tris-HCl, pH 7.2 50 mM
NaCl 150 mM
Triton X-100 0.1
f. HNTG 5X stock:
HEPES 0.1 M
NaCl 0.75 M
Glycerol 50~
Triton X-100 1.0~
g. ABTS stock:
Citric Acid 100 mM
NazHP04 2 5 0 mM
HC1, conc. 0.5 mM
ABTS* 0.5mg/ml
* (2, 2' -azinobis (3-
ethylbenzthiazolinesulfonic acid)). Keep solution in dark at
4°C until use.
h. Stock reagents of:
EDTA 100 mM pH 7.0
Na3V04 0.5 M
Na4 ( P20, ) 0 . 2 M
Procedure.
Pre-coat ELISA Plate
1. Coat ELISA plates (Corning, 96 well, Cat.
#25805-96) with 05-101 antibody at 0.5 ~.g per well in PBS,
100 ~1 final volume/well, and store overnight at 4°C. Coated
plates are good for up to 10 days when stored at 4°C.
2. On day of use, remove coating buffer and
replace with 100 ~1 blocking buffer (5~ Carnation Instant
Non-Fat Dry Milk in PBS). Incubate the plate, shaking, at
room temperature (about 23°C to 25°C) for 30.minutes. Just
prior to use, remove blocking buffer and wash plate 4 times
with TBST buffer.
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Seeding Cells
1. An NIH3T3 cell line overexpressing a chimeric
receptor containing the EGFR extracellular domain and
intracellular HER2 kinase domain can be used for this assay.
2. Choose dishes having 80-90% confluence for the
experiment. Trypsinize cells and stop reaction by adding 10%
fetal bovine serum. Suspend cells in DMEM medium (10% CS DMEM
medium) and centrifuge once at 1500 rpm, at room temperature
for 5 minutes.
3. Resuspend cells in seeding medium (DMEM, 0.5%
bovine serum), and count the cells using trypan blue.
Viability above 90% is acceptable. Seed cells in DMEM medium
(0.5% bovine serum) at a density of 10,000 cells per well,
100 ~1 per well, in a 96 well microtiter plate. Incubate
seeded cells in 5% COz at 37°C for about 40 hours.
Assay Procedures
1. Check seeded cells for contamination using an
inverted microscope. Dilute drug stock (10 mg/ml in DMSO)
1:10 in DMEM medium, then transfer 5 ~1 to a TBST well for a
final drug dilution of 1:200 and a final DMSO concentration
of 1%. Control wells receive DMSO alone. Incubate in 5% COZ
at 37°C for two hours.
2. Prepare EGF ligand: dilute stock EGF in DMEM
so that upon transfer of 10 ~1 dilute EGF (1:12 dilution),
100 nM final concentration is attained.
3. Prepare fresh HNTG* sufficient for 100 ~1 per
well; and place on ice.
HNTG* ( 10 ml )
HNTG stock 2.0 ml
milli-Q H20 7.3 ml
EDTA, 100 mM, pH 7.0 0.5 ml
Na3V04 (0.5 M) 0.1 ml
Na4 ( PzO., ) ( 0 . 2 M ) 0 . 1 ml
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4. After 120 minutes incubation with drug, add
prepared SGF ligand to cells, 10 ~.1 per well, to a final
concentration of 100 nM. Control wells receive DMEM alone.
Incubate with shaking, at room temperature, for 5 minutes.
5. Remove drug, EGF, and DMEM. Wash cells twice
with PBS. Transfer HNTG* to cells, 100 ~,l per well. Place on
ice for 5 minutes. Meanwhile, remove blocking buffer from
other ELISA plate and wash with TBST as described above.
6. With a pipette tip securely fitted to a
l0 micropipettor, scrape cells from plate and homogenize cell
material by repeatedly aspirating and dispensing the HNTG*
lysis buffer. Transfer lysate to a coated, blocked, and
washed-ELISA-plate. Incubate shaking at room temperature for
one hour.
7. Remove lysate and wash 4 times with TBST.
Transfer freshly diluted anti-Ptyr antibody to ELISA plate at
100 ~1 per well. Incubate shaking at room temperature for 30
minutes in the presence of the anti-Ptyr antiserum (1:3000
dilution in TBST).
8. Remove the anti-Ptyr antibody and wash 4 times
with TBST. Transfer the freshly diluted TAGO anti-rabbit IgG
antibody to the ELISA plate at 100 ~1 per well. Incubate
shaking at room temperature for 30 minutes (anti-rabbit IgG
antibody: 1:3000 dilution in TBST).
9. Remove TAGO detection antibody and wash 4
times with TBST. Transfer freshly prepared ABTS/H202 solution
to ELISA plate, 100 ~1 per well. Incubate shaking at room
temperature for 20 minutes. (ABTS/H~02 solution: 1.0 ~.1 30%
H202 in 10 ml ARTS stock) .
10. Stop reaction by adding 50 ~1 5N H2S04
(optional), and determine O.D. at 410 nm.
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11. The maximal phosphotyrosine signal is
determined by subtracting the value of the negative controls
from the positive controls. The percent inhibition of
phosphotyrosine content for extract-containing wells is then
5. calculated, after subtraction of the negative controls.
PDGF-R Assav
All cell culture media, glutamine, and fetal bovine
serum can be purchased from Gibco Life Technologies (Grand
Island, NY) unless otherwise specified. All cells are grown
in a humid atmosphere of 90-95% air and 5-10% COz at 37°C.
All cell lines are routinely subcultured twice a week and are
negative for mycoplasma as determined by the Mycotect method
(Gibco) .
For ELISA assays, cells (U1242, obtained from Joseph
Schlessinger, NYU) are grown to 80-90% confluency in growth
medium (MEM with 10% FBS, NEAR, 1 mM NaPyr and 2 mM GLN) and
seeded in 96-well tissue culture plates in 0.5% serum at
25,000 to 30,000 cells per well. After overnight incubation
in 0.5% serum-containing medium, cells are changed to serum-
free medium and treated with test compound for 2 hr in a 5%
COz, 37°C incubator. Cells are then stimulated with ligand
for 5-10 minute followed by lysis with HNTG (20 mM Hepes, 150
mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na3V04, 0.2% Triton X-
100, and 2 mM NaPyr). Cell lysates (0.5 mg/well in PBS) are
transferred to ELISA plates previously coated with receptor-
specific antibody and which had been blocked with 5% milk in
TBST (50 mM Tris-HC1 pH 7.2, 150 mM NaCl and 0.1% Triton X-
100) at room temperature for 30 min. Lysates are incubated
with shaking for 1 hour at room temperature. The plates are
washed with TBST four times and then incubated with
polyclonal anti-phosphotyrosine antibody at room temperature
for 30 minutes. Excess anti-phosphotyrosine antibody is
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removed by rinsing the plate with TBST four times. Goat
anti-rabbit IgG antibody is added to the ELISA plate for 30
min at room temperature followed by rinsing with TBST four
more times. ABTS (100 mM citric acid, 250 mM Na2HP04 and 0.5
mg/mL 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid))
plus H202 ( 1. 2 mL 3 0 ~ HZOZ to 10 ml ABTS ) i s added to the
ELISA plates to start color development. Absorbance at 410
nm with a reference wavelength of 630 nm is recorded about 15
to 30 min after ABTS addition.
IGF-1 RECEPTOR Assay
The following protocol may be used to measure
phosphotyrosine level on IGF-1 receptor, which indicates IGF-
1 receptor tyrosine kinase activity.
Materials and Reagents.
a. The cell line used in this assay is 3T3/IGF-1R, a
cell line genetically engineered to overexpresses IGF-1
receptor.
b. NIH3T3/IGF-1R is grown in an incubator with 5~ COZ
at 37°C. The growth media is DMEM + 10~ FBS (heat
inactivated}+ 2mM L-glutamine.
c. Affinity purified anti-IGF-1R antibody 17-69.
d. D-PBS:
KHZPO4 0.20 g/1
KHZ PO4 2 . 16 g/ 1
KCl 0.20 g/1
NaCl 8.00 g/1 (pH 7.2)
e. Blocking Buffer: TBST plus 5% Milk (Carnation
Instant Non-Fat Dry Milk).
f. TBST buffer:
Tris-HC1 50 mM
NaCl 150mM (pH 7.2/HC1 lON)
Triton X-100 0.1~
Stock solution of TBS (10X) is prepared, and Triton
X-100 is added to the buffer during dilution.
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g. HNTG buffer:
HEPES 20 mM
NaCl 150 mM (pH 7.2/HC1 1N)
Glycerol 10~
Triton X-100 0.2~
Stock solution (5X) is prepared and kept at 4°C.
h. EDTA/HC1: 0.5 M pH 7.0 (NaOH) as 100X stock.
i. Na3V04: 0.5 M as 100X stock and aliquots are kept
at 80°C.
j . Na4Pz0,: 0.2 M as 100X stock.
k. Insulin-like growth factor-1 from Promega (Cat#
65111 ) .
1. Rabbit polyclonal anti-phosphotyrosine antiserum.
m. Goat anti-rabbit IgG, POD conjugate (detection
antibody), Tago (Cat. No. 4520, Lot No. 1802): Tago, Inc.,
Burlingame, CA.
n. ABTS (2,2'-azinobis(3-ethylbenzthiazolinesulfonic
acid)) solution:
Citric acid 100 mM
NazHPOq 250 mM (pH 4.0/1 N HC1)
ABTS 0.5 mg/ml
ABTS solution should be kept in dark and 4°C. The
solution should be discarded when it turns green.
o. Hydrogen Peroxide: 30~ solution is kept in the dark
and at 4°C.
Procedure.
All the following steps are conducted at room
temperature unless specifically indicated otherwise. All
ELISA plate washings are performed by rinsing the plate with
tap water three times, followed by one TBST rinse. Pat plate
dry with paper towels.
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Cell Seeding:
1. The cells, grown in tissue culture dish
(Corning 25020-100) to 80-90% confluence, are harvested with
Trypsin-EDTA (0.25%, 0.5 ml/D-100, GIBCO).
2. Resuspend the cells in fresh DMEM + 10% FBS +
2mM L-Glutamine, and transfer to 96-well tissue culture plate
(Corning, 25806-96) at 20,000 cells/well (100 ~1/well).
Incubate for 1 day then replace medium to serum-free medium
(90/1) and incubate in 5% COz and 37°C overnight.
ELISA Plate Coating and Blocking:
1. Coat the ELISA plate (Corning 25805-96) with
Anti-IGF-1R Antibody at 0.5 ~g/well in 100 ~1 PBS at least 2
hours.
2. Remove the coating solution, and replace with
100 ~1 Blocking Buffer, and shake for 30 minutes. Remove the
blocking buffer and wash the plate just before adding lysate.
Assay Procedures:
1. The drugs are tested under serum-free condition.
2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in
96-well poly-propylene plate, and transfer 10 ~,1/well of this
solution to the cells to achieve final drug dilution 1:100,
and final DMSO concentration of 1.0%. Incubate the cells in
5% CO2 at 37°C for 2 hours.
3. Prepare fresh cell lysis buffer (HNTG*)
HNTG 2 ml
EDTA 0.1 ml
Na3V04 0.1 ml
Na4 ( P20, ) 0 . 1 ml
HZO 7 . 3 ml
4. After drug incubation for two hours, transfer 10
~.1/well of 200nM IGF-1 Ligand in PBS to the cells (Final
Conc. is 20 nM), and incubate at 5% COz at 37°C for 10
minutes.
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5. Remove media and add 100 ~.1/well HNTG* and shake
for 10 minutes. Look at cells under microscope to see if
they are adequately lysed.
6. Use a 12-channel pipette to scrape the cells from
the plate, and homogenize the lysate by repeated aspiration
and dispensing. Transfer all the lysate to the antibody
coated ELISA plate, and shake for 1 hour.
7. Remove the lysate, wash the plate, transfer anti-
pTyr (1:3,000 with TBST) 100 ~1/well, and shake for 30
minutes.
8. Remove anti-pTyr, wash the plate, transfer TAGO
(1:3,000 with TBST) 100 ~l/well, and shake for 30 minutes.
9. Remove detection antibody, wash the plate, and
transfer fresh ABTS/H20z (1.2 ~1 HZOZ to 10 ml ABTS) 100
~tl/well to the plate to start color development.
Measure OD at 410 nm with a reference wavelength of 630
nm in Dynatec MR5000.
EGFR Assay
EGF Receptor kinase activity in cells genetically
engineered to express human EGF-R can be measured as
described below:
Materials and Reagents.
a. EGF Ligand: stock concentration = 16.5 ~M; EGF 201,
TOYOBO, Co., Ltd. Japan.
b. 05-101 (UBI) (a monoclonal antibody recognizing an
EGFR extracellular domain).
c. Anti-phosphotyosine antibody (anti-Ptyr)
(polyclonal}.
d. Detection antibody: Goat anti-rabbit 1gG horse
radish peroxidase conjugate, TAGO, Inc., Burlingame, CA.
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e. TBST buffer:
Tris-HC1, pH 7 50 mM
NaCl 150 mM
Triton X-100 0.1
f. HNTG 5X stock:
HEPES 0.1 M
NaCl 0.75 M
Glycerol 50
Triton X-100 1.0%
g. ABTS stock:
Citric Acid 100 mM
Na3VOq 250 mM
HCl, conc. 4.0 pH
ABTS* 0.5 mg/ml
Keep solution in dark at 4°C until used.
h. Stock reagents of:
EDTA 100 mM pH 7.0
Na3V04 0.5 M
Na4 ( PZ 0., ) 0 . 2 M
Procedure
Pre-coat ELISA Plate
1. Coat ELISA plates (Corning, 96 well, Cat. #25805-
96) with 05-101 antibody at 0.5 ~g per well in PBS, 150 ~,1
final volume/well, and store overnight at 4°C. Coated plates
are good for up to 10 days when stored at 4°C.
2. On day of use, remove coating buffer and replace
with blocking buffer (5~ Carnation Instant NonFat Dry Milk in
PBS). Incubate the plate, shaking, at room temperature (about
23°C to 25°C) for 30 minutes. Just prior to use, remove
blocking buffer and wash plate 4 times with TBST buffer.
Seeding Cells
1. NIH 3T3/C7 cell line (Honegger, et al., Cell
51:199-209, 1987) can be use for this assay.
2. Choose dishes having 80-905 confluence for the
experiment. Trypsinize cells and stop reaction by adding 10~
CS DMEM medium. Suspend cells in DMEM medium (10~ CS DMEM
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medium) and centrifuge once at 1000 rpm at room temperature
for 5 minutes. -
3. Resuspend cells in seeding medium (DMEM, 0.5%
bovine serum), and count the cells using trypan blue.
Viability above 90% is acceptable. Seed cells in DMEM medium
(0.5% bovine serum) at a density of 10,000 cells per well,
100 ~tl per well, in a 96 well microtiter plate. Incubate
seeded cells in 5% COZ at 37°C for about 40 hours.
Assay Procedures
1. Check seeded cells for contamination using an
inverted microscope. Dilute test compounds stock (10 mg/ml in
DMSO) 1:10 in DMEM medium, then transfer 5 ~l to a test well
for a test compounds drug dilution of 1:200 and a final DMSO
concentration of 1%. Control wells receive DMSO alone.
Incubate in 5% COz at 37°C for one hour.
2. Prepare EGF ligand: dilute stock EGF in DMEM so
that upon transfer of 10 ~1 dilute EGF (1:12 dilution), 25 nM
final concentration is attained.
3. Prepare fresh 10 ml HNTG* sufficient for 100 ~1 per
well wherein HNTG* comprises: HNTG stock (2.0 ml), milli-Q
H20 ( 7 . 3 ml ) , EDTA, 100 mM, pH 7 . 0 ( 0 . 5 ml ) , Na3V09 0 . 5 M ( 0 .
1
ml ) and Na4 ( P20, ) , 0 . 2 M ( 0 . 1 ml ) .
4. Place on ice.
5. After two hours incubation with drug, add prepared
EGF ligand to cells, 10 ~1 per well, to yield a final
concentration of 25 nM. Control wells receive DMEM alone.
Incubate, shaking, at room temperature, for 5 minutes.
6. Remove test compound, EGF, and DMEM. Wash cells
twice with PBS. Transfer HNTG* to cells, 100 ~,1 per well.
Place on ice for 5 minutes. Meanwhile, remove blocking
buffer from other ELISA plate and wash with TBST as described
above.
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7. With a pipette tip securely fitted to a
micropipettor, scrape cells from plate and homogenize cell
material by repeatedly aspirating and dispensing the HNTG*
lysis buffer. Transfer lysate to a coated, blocked, and
washed ELISA plate. Incubate shaking at room temperature for
one hour.
8. Remove lysate and wash 4 times with TBST. Transfer
freshly diluted anti-Ptyr antibody to ELISA plate at 100 ~.l
per well. Incubate shaking at room temperature for 30
minutes in the presence of the anti-Ptyr antiserum (1:3000
dilution in TBST).
9. Remove the anti-Ptyr antibody and wash 4 times with
TBST. Transfer the freshly diluted TAGO 30 anti-rabbit IgG
antibody to the ELISA plate at 100 ~1 per well. Incubate
shaking at room temperature for 30 minutes (anti-rabbit IgG
antibody: 1:3000 dilution in TBST).
10. Remove detection antibody and wash 4 times with
TBST. Transfer freshly prepared ABTS/H202 solution to ELISA
plate, 100 ~.1 per well. Incubate at room temperature for 20
minutes. ABTS/H202 solution: 1.2 ~1 30~ H202 in 10 ml ABTS
stock.
11. Stop reaction by adding 50 ~1 5N HZSOQ (optional),
and determine O.D. at 410 nm.
12. The maximal phosphotyrosine signal is determined by
subtracting the value of the negative controls from the
positive controls. The percent inhibition of phosphotyrosine
content for extract-containing wells is then calculated,
after subtraction of the negative controls.
Met Autophosphorylation Assav
This assay determines Met tyrosine kinase activity by
analyzing Met protein tyrosine kinase levels on the Met
receptor.
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Reagents
a. HNTG (5X stock solution): Dissolve 23.83 g HEPES
and 43.83 g NaCl in about 350 ml dHzO. Adjust pH to 7.2 with
HCl or NaOH, add 500 ml glycerol and 10 ml Triton X-100, mix,
add dHzO to 1 L total volume. To make 1 L of 1X working
solution add 200 ml 5X stock solution to 800 ml dH20, check
and adjust pH as necessary, store at 4°C.
b. PBS (Dulbecco's Phosphate-Buffered Saline), Gibco
Cat. # 450-1300EB (1X solution).
c. Blocking Buffer: in 500 ml dH20 place 100 g BSA,
12.1 g Tris-pH7.5, 58.44 g NaCl and 10 ml Tween-20, dilute to
1 L total volume.
d. Kinase Buffer: To 500 ml dH20 add 12.1 g TRIS (pH
7.2), 58.4 g NaCl, 40.7 g MgClz and 1.9 g EGTA; bring to 1 L
total volume with dH20.
e. PMSF (Phenylmethylsulfonyl fluoride), Sigma Cat. #
P-7626, to 435.5 mg, add 100 ethanol to 25 ml total volume,
vortex.
f. ATP (Bacterial Source), Sigma Cat. # A-7699, store
powder at -20°C; to make up solution for use, dissolve 3.31
mg in 1 ml dH20.
g. RC-20H HRPO Conjugated Anti-Phosphotyrosine,
Transduction Laboratories Cat. # E120H.
h. Pierce 1-Step '"" Turbo TMB-ELISA (3, 3' , 5, 5' -
tetramethylbenzidine, Pierce Cat. # 34022.
i . HZS04, add 1 ml conc . ( 18 N) to 35 ml dHzO.
j. TRIS HCL, Fischer Cat. # BP152-5; to 121.14 g of
material, add 600 ml MilliQ H20, adjust pH to 7.5 (or 7.2)
with HC1, bring volume to 1 L with MilliQ H20.
k. NaCl, Fischer Cat. # S271-10, make up 5M solution.
1. Tween-20, Fischer Cat. # 5337-500.
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m. Na3V04, Fischer Cat. # S454-50, to 1.8 g material
add 80 ml MilliQ HzO, adjust pH to 10.0 with HC1 or NaOH,
boil in microwave, cool, check pH, repeat procedure until pH
stable at 10.0, add MilliQ HZO to 100 ml total volume, make 1
ml aliquots and store at -80°C.
n. MgCl2, Fischer Cat. # M33-500, make up 1M solution.
o. HEPES, Fischer Cat. # BP310-500, to 200 ml MilliQ
HzO, add 59.6 g material, adjust pH to 7.5, bring volume to
250 ml total, sterile filter.
, p. Albumin, Bovine (BSA), Sigma Cat. # A-4503, to 30
grams material add sterile distilled water to make total
volume of 300 ml, store at 4°C.
q. TBST Buffer: to approx. 900 ml dH20 in a 1 L
graduated cylinder add 6.057 g TRIS and 8.766 g NaCl, when
dissolved, adjust pH to 7.2 with HC1, add 1.0 ml Triton X-100
and bring to 1 L total volume with dHzO.
r. Goat Affinity purified antibody Rabbit IgG (whole
molecule), Cappel Cat. # 55641.
s. Anti h-Met (C-28) rabbit polyclonal IgG antibody,
Santa Cruz Chemical Cat. # SC-161.
t. Transiently Transfected EGFR/Met chimeric cells
(EMR) (Komada, et al., Oncogene, 8:2381-2390 (1993).
u. Sodium Carbonate Buffer, (NaZC04, Fischer Cat. #
S495): to 10.6 g material add 800 ml MilliQ H20, when
dissolved adjust pH to 9.6 with NaOH, bring up to 1 L total
volume with MilliQ HzO, filter, store at 4°C.
Procedure
All of the following steps are conducted at room
temperature unless it is specifically indicated otherwise.
All ELISA plate washing is by rinsing 4X with TBST.
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EMR Lysis
This procedure can be performed the night before or
immediately prior to the start of receptor capture.
1. Quick thaw lysates in a 37° C waterbath with a
swirling motion until the last crystals disappear.
2. Lyse cell pellet with 1X HNTG containing 1 mM PMSF.
Use 3 ml of HNTG per 15 cm dish of cells. Add 1/2 the
calculated HNTG volume, vortex the tube for 1 min., add the
remaining amount of HNTG, vortex for another min.
3. Balance tubes, centrifuge at 10,000 x g for 10 min
at 4°C.
4. Pool supernatants, remove an aliquot for protein
determination.
5. Quick freeze pooled sample in dry ice/ethanol bath.
This step is performed regardless of whether lysate will be
stored overnight or used immediately following protein
determination.
6. Perform protein determination using standard
bicinchoninic acid (BCA) method (BCA Assay Reagent Kit from
Pierce Chemical Cat. # 23225).
ELISA Procedure
1. Coat Corning 96 well ELISA plates with 5 ~g per
well Goat anti-Rabbit antibody in Carbonate Buffer for a
total well volume of 50 ~.1. Store overnight at 4°C.
2. Remove unbound Goat anti-rabbit antibody by
inverting plate to remove liquid.
3. Add 150 ~1 of Blocking Buffer to each well.
Incubate for 30 min. with shaking.
4. Wash 4X with TBST. Pat plate on a paper towel to
remove excess liquid and bubbles.
5. Add l~,g per well of Rabbit anti-Met antibody
diluted in TBST for a total well volume of 100 ~.1.
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6. Dilute lysate in HNTG (90 ~g lysate/100~,1)
7. Add 100 ~1 of diluted lysate to each well. Shake
for 60 min.
8. Wash 4X with TBST. Pat on paper towel to remove
excess liquid and bubbles.
9. Add 50 ~.1 of 1X lysate buffer per well.
10. Dilute compounds/extracts 1:10 in 1X Kinase Buffer
in a polypropylene 96 well plate.
11. Transfer 5.5 ~l of diluted compound to ELISA plate
wells. Incubate at room temperature with shaking for 20 min.
12. Add 5.5 ~1 of 60 ~M ATP solution per well. Negative
controls do not receive any ATP. Incubate for 90 min., with
shaking.
13. Wash 4X with TBST. Pat plate on paper towel to
remove excess liquid and bubbles.
14. Add 100 ~tl per well of RC20 (1:3000 dilution in
Blocking Buffer). Incubate 30 min. with shaking.
15. Wash 4X with TBST. Pat plate on paper towel to
remove excess liquid and bubbles.
16. Add 100 ~1 per well of Turbo-TMB. Incubate with
shaking for 30-60 min.
17. Add 100 ~1 per well of 1M HZSO4 to stop reaction.
18. Read assay on Dynatech MR7000 ELISA reader. Test
Filter = 450 nm, reference filter = 410 nm.
Biochemical src assay
This assay is used to determine src protein kinase
activity measuring phosphorylation of a biotinylated peptide
as the readout.
Materials and Reagents:
a. Yeast transformed with src (Sugen, Inc., Redwood
City, California).
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b. Cell lysates: Yeast cells expressing src are
pelleted, washed once with water, re-pelleted and stored at -
80°C until use.
c. N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is
prepared by standard procedures well known to those skilled
in the art.
d. DMSO: Sigma, St. Louis, MO.
e. 96 Well ELISA Plate: Corning 96 Well Easy Wash,
Modified flat Bottom Plate, Corning Cat. #25805-96.
f. NUNC 96-well V-bottom polypropylene plates for
dilution of compounds: Applied Scientific Cat. # A-72092.
g. Vecastain ELITE ABC reagent: Vector, Burlingame,
CA.
h. Anti-src (327) mab: Schizosaccharomyces Pombe is
used to express recombinant Src (Superti-Furga, et al., EMBO
J., 12:2625-2634; Superti-Furga, et al., Nature Biochem.,
14:600-605). S. Pombe strain SP200 (h-s leu1.32 ura4 ade210)
is grown as described and transformations are pRSP expression
plasmids are done by the lithium acetate method (Superti-
Furga, su ra). Cells are grown in the presence of 1 ~tM
thiamine to repress expression from the nmtl promoter or in
the absence of thiamine to induce expression.
i. Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40
may be used instead).
j. Turbo TMB-ELISA peroxidase substrate: Pierce
Chemical.
Buffer Solutions
a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO
PBS, GIBCO Cat. # 450-1300EB.
b. Blocking Buffer: 5% Non-fat milk (Carnation) in
PBS.
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c. Carbonate Buffer: Na2C04 from Fischer, Cat. #
5495, make up 100 mM stock solution.
d. Kinase Buffer: 1.0 ml (from 1M stock solution)
MgClz; 0.2 ml (from a 1M stock solution) MnClz; 0.2 ml (from
a 1M stock solution) DTT; 5.0 ml (from a 1M stock solution)
HEPES; 0.1 ml TX-100; bring to 10 ml total volume with MilliQ
H20 .
e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.);
2.74 ml NaCl (from 5M stock solution); 10 ml glycerol; 1.0 mI
TX-100; 0.4 ml EDTA (from a 100 mM stock solution); 1.0 ml
PMSF (from a 100 mM stock solution); 0.1 ml Na3V04 (from a
0.1 M stock solution); bring to 100 ml total volume with
MilliQ H20.
f. ATP: Sigma Cat. # A-7699, make up 10 mM stock
solution (5.51 mg/ml).
g TRIS-HC1: Fischer Cat. # BP 152-5, to 600 ml
MilliQ H20 add 121.14 g material, adjust pH to 7.5 with HC1,
bring to 1 L total volume with MilliQ HzO.
h. NaCl: Fischer Cat. # S271-10, Make up 5M stock
solution with MilliQ H20.
i. Na3V04: Fischer Cat. # 5454-50; to 80 ml MilliQ
H20, add 1.8 g material; adjust pH to 10.0 with HC1 or NaOH;
boil in a microwave; cool; check pH, repeat pH adjustment
until pH remains stable after heating/cooling cycle; bring to
100 ml total volume with MilliQ HZO; make 1 ml aliquots and
store at -80°C.
j. MgCl2: Fischer Cat. # M33-500, make up 1M stock
solution with MilliQ HZO.
k. HEPES: Fischer Cat. # BP 310-500; to 200 ml MilliQ
H20, add 59.6 g material, adjust pH to 7.5, bring to 250 ml
total volume with MilliQ H20, sterile filter (1M stock
solution).
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1. TEST Buffer: TBST Buffer: To 900 ml dHzO add
6.057 g TRIS and 8.766 g NaCl; adjust pH to 7.2 with HC1, add
1.0 ml Triton-X100; bring to 1 L total volume with dH20.
m. MnClz: Fischer Cat. # M87-100, make up 1M stock
solution with MilliQ H20.
n. DTT: Fischer Cat. # BP172-5.
o. TBS (TRIS Buffered Saline): to 900 ml MilliQ Ha0
add 6.057 g TRIS and 8.777 g NaCl; bring to 1 L total volume
with MilliQ HZO.
p. Kinase Reaction Mixture: Amount per assay plate
(100 wells): 1.0 ml Kinase Buffer, 200 ~g GST-~ , bring to
final volume of 8.0 ml with MilliQ HzO.
q: Biotin labeled EEEYEEYEEEYEEEYEEEY: Make peptide
stock solution (lmM, 2.98 mg/ml) in water fresh just before
use.
r. Vectastain ELITE ABC reagent: To prepare 14 ml of
working reagent, add 1 drop of reagent A to 15 ml TBST and
invert tube several times to mix. Then add 1 drop of reagent
B. Put tube on orbital shaker at room temperature and mix
for 30 minutes.
Procedures
Preparation of src coated ELISA plate.
1. Coat ELISA plate with 0.5 ~g/well anti-src mab in
100 ~,1 of pH 9.6 sodium carbonate buffer; hold at 4°C
overnight.
2. Wash wells once with PBS.
3. Block plate with 0.15 ml 5~ milk in PBS for 30 min.
at room temperature.
4. Wash plate 5X with PBS.
5. Add 10 ~g/well of src transformed yeast lysates
diluted in Lysis Buffer (0.1 ml total volume per well).
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(Amount of lysate may vary between batches.) Shake plate for
20 minutes at room temperature.
Preparation of phosphotyrosine antibody-coated ELISA
plate.
1. 4610 plate: coat 0.5 ~g/well 4610 in 100 ~,1 PBS
overnight at 4°C and block with 150 ~tl of 5~ milk in PBS for
30 minutes at room temperature.
Kinase assay procedure.
1. Remove unbound proteins from plates and wash plates
5X with PBS.
2. Add 0.08 ml Kinase Reaction Mixture per well
(containing 10 ~tl of lOX Kinase Buffer and 10 ~M (final
concentration) biotin-EEEYEEYEEEYEEEYEEEY per well diluted in
water.
3. Add 10 ~.1 of compound diluted in water containing
10~ DMSO and pre-incubate for 15 minutes at room temperature.
4. Start kinase reaction by adding 10 ~1/well of 0.05
mM ATP in water (5 ~M ATP final).
5. Shake ELISA plate for 15 min. at room temperature.
6. Stop kinase reaction by adding 10 ~1 of 0.5 M EDTA
per well.
7. Transfer 90 ~,1 supernatant to a blocked 4610 coated
ELISA plate.
8. Incubate for 30 min. while shaking at room
temperature.
9. Wash plate 5X with THST.
10. Incubate with Vectastain ELITE ABC reagent (100
~.1/well) for 30 min. at room temperature.
11. Wash the wells 5X with TBST.
12. Develop with Turbo TMB.
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Biochemical lck Assay
This assay is used to determine lck protein kinase
activities measuring phosphorylation of GST-~ as the readout.
Materials and Reagents
a. Yeast transformed with lck. Schizosaccharomyces
Pombe is used to express recombinant Lck (Superti-Furga, et
al., EMBO J, 12:2625-2634; Superti-Furga, et al., Nature
Biotech., 14:600-605). S. Pombe strain SP200 (h-s leul.32
ura4 ade210) is grown as described and transformations with
pRSP expression plasmids are done by the lithium acetate
method (Superti-Furga, supra). Cells are grown in the
presence of 1 ~.M thiamine to induce expression.
b: Cell lysates: Yeast cells expressing lck are
pelleted, washed once in water, re-pelleted and stored frozen
at -80°C until use.
c. GST-~: DNA encoding for GST-~ fusion protein for
expression in bacteria obtained from Arthur Weiss of the
Howard Hughes Medical Institute at the University of
California, San Francisco. Transformed bacteria are grown
overnight while shaking at 25°C. GST-~ is purified by
glutathione affinity chromatography, Pharmacia, Alameda, CA.
d. DMSO: Sigma, St. Louis, MO.
e. 96-Well ELISA plate: Corning 96 Well Easy Wash,
Modified Flat Bottom Plate, Corning Cat. ##25805-96.
f. NUNC 96-well V-bottom polypropylene plates for
dilution of compounds: Applied Scientific Cat. # AS-72092.
g. Purified Rabbit anti-GST antiserum: Amrad
Corporation (Australia) Cat. #90001605.
h. Goat anti-Rabbit-IgG-HRP: Amersham Cat. # V010301.
i. Sheep ant-mouse IgG (H+L): Jackson Labs Cat. #
5215-005-003.
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j. Anti-Lck (3A5) mab: Santa Cruz Biotechnology Cat #
sc-433.
k. Monoclonal anti-phosphotyrosine UBI 05-321 (UB40
may be used instead).
Buffer solutions
a. PBS (Dulbecco's Phosphate-Buffered Saline) 1X
solution: GIBCO PBS, GIBCO Cat. # 450-1300EB.
b. Blocking Buffer: 100 g. BSA, 12.1 g. TRIS (pH7.5),
58.44 g NaCl, 10 ml Tween-20, bring up to 1 L total volume
with MilliQ HZO.
c. Carbonate Buffer: NaZC04 from Fischer, Cat. # S495;
make up 100 mM solution with MilliQ HZO.
d. Kinase Buffer: 1.0 ml (from 1M stock solution)
MgCIZ; 0.2 ml (from a 1M stock solution} MnCl2; 0.2 ml (from
a 1M stock solution) DTT; 5.0 ml (from a 1M stock solution)
HEPES; 0.1 ml TX-100; bring to 10 ml total volume with MilliQ
H20 .
e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.);
2.74 ml NaCl (from 5M stock solution); 10 ml glycerol; 1.0 ml
TX-100; 0.4 ml EDTA (from a 100 mM stock solution); 1.0 ml
PMSF (from a 100 mM stock solution); 0.1 ml Na3V04 (from a
0.1 M stock solution); bring to 100 ml total volume with
MilliQ H20.
f. ATP: Sigma Cat. # A-7699, make up 10 mM stock
solution (5.51 mg/ml}.
g TRIS-HC1: Fischer Cat. # BP 152-5, to 600 ml
MilliQ H20 add 121.14 g material, adjust pH to 7.5 with HC1,
bring to 1 L total volume with MilliQ HZO.
h. NaCl: Fischer Cat. # 5271-10, Make up 5M stock
solution with MilliQ HzO.
i Na1V04: Fischer Cat. # S454-50; to 80 ml MilliQ
H20, add 1.8 g material; adjust pH to 10.0 with HCl or NaOH;
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boil in a microwave; cool; check pH, repeat pH adjustment
until pH remains stable after heating/cooling cycle; bring to
100 ml total volume with MilliQ H20; make 1 ml aliquots and
store at -80°C.
j. MgCl2: Fischer Cat. # M33-500, make up 1M stock
solution with MilliQ H20.
k. HEPES: Fischer Cat. # BP 310-500; to 200 ml MilliQ
H20, add 59.6 g material, adjust pH to 7.5, bring to 250 ml
total volume with MilliQ HZO, sterile filter (1M stock
solution) .
1. Albumin, Bovine (BSA), Sigma Cat. # A4503; to 150
ml MilliQ H20 add 30 g material, bring 300 ml total volume
with.MilliQ H20, filter through 0.22 ~m filter, store at 4°C.
m. TEST Buffer: To 900 ml dHzO add 6.057 g TRIS and
8.766 g NaCl; adjust pH to 7.2 with HC1, add 1.0 ml Triton-
X100; bring to 1 L total volume with dH20.
n. MnCl2: Fischer Cat. # M87-100, make up 1M stock
solution with MilliQ H20.
o. DTT: Fischer Cat. # BP172-5.
p. TBS (TRIS Buffered Saline): to 900 ml MilliQ HZO
add 6.057 g TRIS and 8.777 g NaCl; bring to 1 L total volume
with MilliQ H20.
q Kinase Reaction Mixture: Amount per assay plate
(100 wells): 1.0 ml Kinase Buffer, 200 ~g GST-~, bring to
final volume of 8.0 ml with MilliQ H20.
Procedures
Preparation of Lck coated ELISA plate
1. Coat 2.0 ~g/well Sheep anti-mouse IgG in 100 ~1 of
pH 9.6 sodium carbonate buffer at 4°C overnight.
2. Wash well once with PBS.
3. Block plate with 0.15 ml of blocking Buffer for 30
min. at room temp.
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4. Wash plate 5X with PBS.
5. Add 0.5 ~g/well of anti-lck (mab 3A5) in 0.1 ml PBS
at room temperature for 1-2 hours.
6. Wash plate 5X with PBS.
7. Add 20 ~g/well of lck transformed yeast lysates
diluted in Lysis Buffer (0.1 ml total volume per well). Shake
plate at 4°C overnight to prevent loss of activity.
Preparation of phosphotyrosine antibody-coated ELISA
plate
1. UB40 plate: 1.0 ~g/well UB40 in 100 ~l of PBS
overnight at 4°C and block with 150 ~1 of Blocking Buffer for
at least 1 hour.
Kinase assay procedure
1. Remove unbound proteins from plates and wash plates
5X with PBS.
2. Add 0.08 ml Kinase Reaction Mixture per well
(containing 10 ~1 of lOX Kinase Buffer and 2 ~.g GST-~ per
well diluted with water).
3. Add 10 ~1 of compound diluted in water containing
10% DMSO and pre-incubate for 15 minutes at room temperature.
4. Start kinase reaction by adding l0~tl/well of 0.1 mM
ATP in water (10 ~M ATP final).
5. Shake ELISA plate for 60 min. at room temperature.
6. Stop kinase reaction by adding 10 ~,1 of 0.5 M EDTA
per well.
7. Transfer 90 ~tl supernatant to a blocked 4610 coated
ELISA plate from section B, above.
8. Incubate while shaking for 30 min. at room
temperature.
9. Wash plate 5X with TBST.
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10. Incubate with Rabbit anti-GST antibody at 1:5000
dilution in 100 ~l TBST for 30 min. at room temperature.
11. Wash the wells 5X with TBST.
12. Incubate with Goat anti-Rabbit-IgG-HRP at 1:20,000
dilution in 100 ~,1 of TBST for 30 min. at room temperature.
13. Wash the wells 5X with TBST.
14. Develop with Turbo TMB.
Assav measuring phosphorylatina function of RAF
The following assay reports the amount of RAF-catalyzed
phosphorylation of its target protein MEK as well as MEK's
target MAPK. The RAF gene sequence is described in Bonner et
al., 1985, Molec. Cell. Biol., 5:1400-1407, and is readily
accessible in multiple gene sequence data banks.
Construction of the nucleic acid vector and cell lines
utilized for this portion of the invention are fully
described in Morrison et al., 1988, Proc. Natl. Acad. Scit
USA, 85:8855-8859.
Materials and Reagents
1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL,
Gaithersburg, MD.
2. RIPA buffer: 20 mM Tris/HC1 pH 7.4, 137 mM NaCl,
10% glycerol, 1 mM PMSF, 5 mg/L Aprotenin, 0.5 % Triton X-
100;
3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK
expression and purification by affinity chromatography are
performed according to the manufacturer's procedures.
Catalog# K 350-O1 and R 350-40, Invitrogen Corp., San Diego,
CA
4. His-MAPK (ERK 2); His-tagged MAPK is expressed in
XL1 Blue cells transformed with pUCl8 vector encoding His-
MAPK. His-MAPK is purified by Ni-affinity chromatography.
Cat# 27-4949-O1, Pharmacia, Alameda, CA, as described herein.
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5. Sheep anti mouse IgG: Jackson laboratories, West
Grove, PA. Catalog, # 515-006-008, Lot# 28563
6. RAF-1 protein kinase specific antibody: URP2653
from UBI.
7. Coating buffer: PBS; phosphate buffered saline,
GIBCO-BRL, Gaithersburg, MD.
8. Wash buffer: TBST (50 mM Tris/HCL pH 7.2, 150 mM
NaCl, 0.1 % Triton X-100).
9. Block buffer: TBST, 0.1 % ethanolamine pH 7.4
10. DMSO, Sigma, St. Louis, MO
11. Kinase buffer (KB): 20 mM HEPES/HC1 pH 7.2, 150 mM
NaCl, 0.1 % Triton X-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 mM
sodium orthovanadate, 0.5 MM DTT.and 10 mM MgCl2.
12. ATP mix: 100 mM MgClz, 300 mM ATP, 10 mCi y33P ATP
(Dupont-NEN)/mL.
13 Stop solution: 1% phosphoric acid; Fisher,
Pittsburgh, PA.
14. Wallac Cellulose Phosphate Filter mats; Wallac,
Turku, Finnland.
15. Filter wash solution: 1% phosphoric acid, Fisher,
Pittsburgh, PA.
16. Tomtec plate harvester, Wallac, Turku, Finnland.
17. Wallac beta plate reader # 1205, Wallac, Turku,
Finnland.
18. NUNC 96-well V bottom polypropylene plates for
compounds Applied Scientific Catalog # AS-72092.
Procedure
All of the following steps are conducted at room
temperature unless specifically indicated otherwise.
1. ELISA plate coating: ELISA wells are coated with
100 ml of Sheep anti mouse affinity purified antiserum (1
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mg/100 mL coating buffer) over night at 4° C. ELISA plates
can be used for two weeks when stored at 4° C.
2. Invert the plate and remove liquid. Add 100 mL of
blocking solution and incubate for 30 min.
3. Remove blocking solution and wash four times with
wash buffer. Pat the plate on a paper towel to remove excess
liquid.
4. Add 1 mg of antibody specific for RAF-1 to each
well and incubate for 1 hour. Wash as described in step 3.
5. Thaw lysates from RAS/RAF infected Sf9 cells and
dilute with TBST to 10 mg/100 mL. Add 10 mg of diluted
lysate to the wells and incubate for 1 hour. Shake the plate
-. during incubation. Negative controls receive no lysate.
Lysates from RAS/RAF infected Sf9 insect cells are prepared
after cells are infected with recombinant baculoviruses at a
MOI of 5 for each virus, and harvested 48 hours later. The
cells are washed once with PBS and lysed in RIPA buffer.
Insoluble material is removed by centrifugation (5 min at
10,000 x g). Aliquots of lysates are frozen in dry
ice/ethanol and stored at -80 °C until use.
6. Remove non-bound material and wash as outlined
above (step 3).
7. Add 2 mg of T-MEK and 2 mg of His-MAEPK per well
and adjust the volume to 40 ml with kinase buffer. Methods
for purifying T-MEK and MAPK from cell extracts are provided
herein by example.
8. Pre-dilute compounds (stock solution 10 mg/ml DMSO}
or extracts 20 fold in TBST plus 1% DMSO. Add 5 ml of the
pre-diluted compounds/extracts to the wells described in step
6. Incubate for 20 min. Controls receive no drug.
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9. Start the kinase reaction by addition of 5 ml ATP
mix; Shake the plates on an ELISA plate shaker during
incubation.
10. Stop the kinase reaction after 60 min by addition
of 30 mL stop solution to each well.
11. Place the phosphocellulose mat and the ELISA plate
in the Tomtec plate harvester. Harvest and wash the filter
with the filter wash solution according to the manufacturer's
recommendation. Dry the filter mats. Seal the filter mats
and place them in the holder. Insert the holder into
radioactive detection apparatus and quantify the radioactive
phosphorous on the filter mats.
Alternatively, 40 mL aliquots from individual wells of
the assay plate can be transferred to the corresponding
positions on the phosphocellulose filter mat. After air
drying the filters, put the filters in a tray. Gently rock
the tray, changing the wash solution at 15 min intervals for
1 hour. Air-dry the filter mats. Seal the filter mats and
place them in a holder suitable for measuring the radioactive
phosphorous in the samples. Insert the holder into a
detection device and quantify the radioactive phosphorous on
the filter mats.
CDK2/Cyclin A - Inhibition Assay
This assay analyzes the protein kinase activity of CDK2
in exogenous substrate.
Reagents
A. Buffer A: (80 mM Tris ( pH 7.2), 40 mM MgCl2), 4.84
g. Tris (F. W. =121.1 g/mol), 4.07 g. MgClz (F. W.=203.31
g/mol) dissolved in 500 ml H20. Adjust pH to 7.2 with HC1.
B. Histone H1 solution (0.45 mg/ml Histone H1 and 20
mM HEPES pH 7.2: 5 mg Histone H1 (Boehinger Mannheim) in
11.111 ml 20 mM HEPES pH 7.2 (477 mg HEPES (F. W.= 238.3
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g/mol) dissolved in 100 ml ddHzO, stored in 1 ml aliquots at
-80° C.
C. ATP solution (60 ~M ATP, 300 ~g/ml BSA, 3 mM DTT):
120 ~1 10 mM ATP, 600 ~.l 10 mg/ml BSA to 20 ml, stored in 1
ml aliquots at -80° C.
D. CDK2 solution: cdk2/cyclin A in 10 mM HEPES pH 7.2,
25 mM NaCl, 0.5 mM DTT, 10~ glycerol, stored in 9 ~1 aliquots
at
-80° C.
Protocol
1. Prepare solutions of inhibitors at three times the
desired final assay concentration in ddH20/15~ DMSO by
volume.
2. Dispense 20 ~l of inhibitors to wells of
polypropylene 96-well plates (or 20 ~1 15~ DMSO for positive
and negative controls).
3. Thaw Histone Hi solution (1 ml/plate), ATP solution
(1 ml/plate plus 1 aliquot for negative control), and CDK2
solution (9 ~.1/plate). Keep CDK2 on ice until use. Aliquot
CDK2 solution appropriately to avoid repeated freeze-thaw
cycles.
4. Dilute 9 ~1 CDK2 solution into 2.1 ml Buffer A (per
plate). Mix. Dispense 20 ~.1 into each well.
5. Mix 1 ml Histone H1 solution with 1 ml ATP solution
(per plate) into a 10 ml screw cap tube. Add y33P ATP to a
concentration of 0.15 ~tCi/20~1 (0.15 ~Ci/well in assay). Mix
carefully to avoid BSA frothing. Add 20 ~l to appropriate
wells. Mix plates on plate shaker. For negative control, mix
ATP solution with an equal amount of 20 mM HEPES pH 7.2 and
add y3'P ATP to a concentration of 0.15 ~Ci/20~1 solution.
Add 20 ~,1 to appropriate wells.
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6. Let reactions proceed for 60 minutes.
7. Add 35 ~1 10% TCA to each well. Mix plates on
plate shaker.
8. Spot 40 ~1 of each sample onto P30 filter mat
squares. Allow mats to dry (approx. 10-20 minutes).
9 Wash filter mats 4 X 10 minutes with 250 ml 1%
phosphoric acid (10 ml phosphoric acid per liter ddH20).
10. Count filter mats with beta plate reader.
Cellular/Hiologic Assays
PDGF-Induced HrdU Incorporation Assay
Materials and Reagents
(1) PDGF: human PDGF B/B; 1276-956, Boehringer
Mannheim, Germany.
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(3} FixDenat: fixation solution (ready to use}, Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated
with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim,
Germany.
(5) TMB Substrate Solution: tetramethylbenzidine (TMB),
ready to use, Cat. No. 1 647 229, Boehringer Mannheim,
Germany.
(6) PBS Washing Solution . 1X PBS, pH 7.4 (Sugen, Inc.,
Redwood City, California).
(7) Albumin, Bovine (BSA): fraction V powder; A-8551,
Sigma Chemical Co., USA.
(8) 3T3 cell line genetically engineered to express
human PDGF-R.
Protocol
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(1) Cells are seeded at 8000 cells/well in DMEM,.10%
CS, 2mM Gln in a 96 well plate. Cells are incubated overnight
at 37°C in 5% COz.
(2) After 24 hours, the cells are washed with PBS, and
then are serum starved in serum free medium (0%CS DMEM with
0.1% BSA) for 24 hours.
(3) On day 3, ligand (PDGF, 3.8 nM, prepared in DMEM
with 0.1% BSA) and test compounds are added to the cells
simultaneously. The negative control wells receive serum
free DMEM with 0.1% BSA only; the positive control cells
receive the ligand (PDGF) but no test compound. Test
compounds are prepared in serum free DMEM with ligand in a 96
well plate, and serially diluted for 7 test concentrations.
(4) After 20 hours of ligand activation, diluted BrdU
labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the
cells are incubated with BrdU (final concentration=10 ~M) for
1.5 hours.
(5) After incubation with labeling reagent, the medium
is removed by decanting and tapping the inverted plate on a
paper towel. FixDenat solution is added (50 ~1/well) and the
plates are incubated at room temperature for 45 minutes on a
plate shaker.
(6) The FixDenat solution is thoroughly removed by
decanting and tapping the inverted plate on a paper towel.
Milk is added (5% dehydrated milk in PBS, 200 ~,1/well) as a
blocking solution and the plate is incubated for 30 minutes
at room temperature on a plate shaker.
(7) The blocking solution is removed by decanting and
the wells are washed once with PBS. Anti-BrdU-POD solution
(1:100 dilution in PBS, 1% BSA) is added (100 ~l/well) and
the plate is incubated for 90 minutes at room temperature on
a plate shaker.
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(8) The antibody conjugate is thoroughly removed by
decanting and rinsing the wells 5 times with PBS, and the
plate is dried by inverting and tapping on a paper towel.
(9) TMB substrate solution is added (100 ~,1/well) and
incubated for 20 minutes at room temperature on a plate
shaker until color development is sufficient for photometric
detection.
(10) The absorbance of the samples are measured at 410
nm (in "dual wavelength" mode with a filter reading at 490
nm, as a reference wavelength) on a Dynatech ELISA plate
reader.
EGF-Induced BrdU Incoruoration Assa
-.. Materials and Reagents
(1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan.
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated
with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim,
Germany.
(5) TMB Substrate Solution: tetramethylbenzidine (TMB),
ready to use, Cat. No. 1 647 229, Boehringer Mannheim,
Germany.
(6) PBS Washing Solution . 1X PBS, pH 7.4 (Sugen, Inc.,
Redwood City, California).
(7) Albumin, Bovine (BSA): fraction V powder; A-8551,
Sigma Chemical Co., USA.
(8) 3T3 cell line genetically engineered to express
human EGF-R.
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Protocol
(1) Cells are seeded at 8000 cells/well in 10~ CS, 2mM
Gln in DMEM, in a 96 well plate. Cells are incubated
overnight at 37°C in 5~ COZ.
(2) After 24 hours, the cells are washed with PHS, and
then are serum starved in serum free medium (0~ CS DMEM with
0.1~ BSA) for 24 hours.
(3) On day 3, ligand (EGF, 2 nM, prepared in DMEM with
0.1~ BSA) and test compounds are added to the cells
simultaneously. The negative control wells receive serum
free DMEM with 0.1~ BSA only; the positive control cells
receive the ligand (EGF) but no test compound. Test
compounds are prepared in serum free DMEM with li_gand in a 96
well plate, and serially diluted for 7 test concentrations.
(4) After 20 hours of ligand activation, diluted BrdU
labeling reagent (1:100 in DMEM, 0.1~ BSA) is added and the
cells are incubated with BrdU (final concentration = 10 ~M)
for 1.5 hours.
(5) After incubation with labeling reagent, the medium
is removed by decanting and tapping the inverted plate on a
paper towel. FixDenat solution is added (50 ~.1/well) and the
plates are incubated at room temperature for 45 minutes on a
plate shaker.
(6) The FixDenat solution is thoroughly removed by
decanting and tapping the inverted plate on a paper towel.
Milk is added (5~ dehydrated milk in PBS, 200 ~,1/well) as a
blocking solution and the plate is incubated for 30 minutes
at room temperature on a plate shaker.
(7) The blocking solution is removed by decanting and
the wells are washed once with PBS. Anti-BrdU-POD solution
(1:100 dilution in PBS, 1~ BSA) is added (100 ~,1/well) and
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the plate is incubated for 90 minutes at room temperature on
a plate shaker.
(8) The antibody conjugate is thoroughly removed by
decanting and rinsing the wells 5 times with PBS, and the
plate is dried by inverting and tapping on a paper towel.
(9) TMB substrate solution is added (100 ~tl/well) and
incubated for 20 minutes at room temperature on a plate
shaker until color development is sufficient for photometric
detection.
(10) The absorbance of the samples are measured at 410
nm (in "dual wavelength" mode with a filter reading at 490
nm, as a reference wavelength) on a Dynatech ELISA plate
reader.
EGF-Induced Her2-Driven BrdU Incort~oration
Materials and Reagents
(1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated
with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim,
Germany.
(5) TMB Substrate Solution: tetramethylbenzidine (TMB),
ready to use, Cat. No. 1 647 229, Boehringer Mannheim,
Germany.
(6) PBS Washing Solution: 1X PBS, pH 7.4, made in
house.
(7) Albumin, Bovine (BSA): fraction V powder; A-8551,
Sigma Chemical Co., USA.
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(8) 3T3 cell line engineered to express a chimeric
receptor having the extra-cellular domain of EGF-R and the
intra-cellular domain of Her2.
Protocol
(1) Cells are seeded at 8000 cells/well in DMEM, 10%
CS, 2mM Gln in a 96- well plate. Cells are incubated
overnight at 37° C in 5% CO2.
(2) After 24 hours, the cells are washed with PBS, and
then are serum starved in serum free medium (0% CS DMEM with
0.1% BSA} for 24 hours.
(3) On day 3, ligand (EGF = 2 nM, prepared in DMEM with
0.1% BSA) and test compounds are added to the cells
simultaneously. The negative control wells receive serum
free DMEM with 0.1% BSA only; the positive control cells
receive the ligand (EGF) but no test compound. Test compounds
are prepared in serum free DMEM with ligand in a 96 well
plate, and serially diluted for 7 test concentrations.
(4) After 20 hours of ligand activation, diluted BrdU
labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the
cells are incubated with BrdU (final concentration = ZO ~tM)
for 1.5 hours.
(5) After incubation with labeling reagent, the medium
is removed by decanting and tapping the inverted plate on a
paper towel. FixDenat solution is added (50 ~1/well) and the
plates are incubated at room temperature for 45 minutes on a
plate shaker.
(6) The FixDenat solution is thoroughly removed by
decanting and tapping the inverted plate on a paper towel.
Milk is added (5% dehydrated milk in PBS, 200 ~,l/well) as a
blocking solution and the plate is incubated for 30 minutes
at room temperature on a plate shaker.
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(7) The blocking solution is removed by decanting and
the wells are washed once with PBS. Anti-BrdU-POD solution
(1:100 dilution in PBS, 1~ BSA} is added (100 ~1/well) and
the plate is incubated for 90 minutes at room temperature on
a plate shaker.
(8) The antibody conjugate is thoroughly removed by
decanting and rinsing the wells 5 times with PBS, and the
plate is dried by inverting and tapping on a paper towel.
(9) TMB substrate solution is added (100 ~,1/well) and
incubated for 20 minutes at room temperature on a plate
shaker until color development is sufficient for photometric
detection.
(10) The absorbance of the samples are measured at 410
nm (in "dual wavelength" mode with a filter reading at 490
nm, as a reference wavelength) on a Dynatech ELISA plate
reader.
IGFl-Induced BrdU Incorporation Assay
Materials and Reagents
(1) IGF1 Ligand: human, recombinant; 6511, Promega Corp,
USA.
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat.
No. 1 647 229, Hoehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated
with peroxidase, Cat. No. 1 647'229, Boehringer Mannheim,
Germany.
(5) TMB Substrate Solution: tetramethylbenzidine (TMB),
ready to use, Cat. No. 1 647 229, Boehringer Mannheim,
Germany.
(6) PBS Washing Solution: 1X PBS, pH 7.4 (Sugen, Inc.,
Redwood City, California).
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(7} Albumin, Bovine (BSA): fraction V powder; A-8551,
Sigma Chemical Co., USA.
(8) 3T3 cell line genetically engineered to express
human IGF-1 receptor.
Protocol
(1) Cells are seeded at 8000 cells/well in DMEM, 10%
CS, 2mM Gln in a 96- well plate. Cells are incubated
overnight at 37°C in 5% COZ.
(2) After 24 hours, the cells are washed with PBS, and
then are serum starved in serum free medium (0%CS DMEM with
0.1% BSA} for 24 hours.
(3) On day 3, ligand (IGF1 = 3.3 nM, prepared in DMEM
with 0.1% BSA) and.test compounds axe added to the cells
simultaneously. The negative control wells receive serum
free DMEM with 0.1% BSA only; the positive control cells
receive the ligand (IGF1) but no test compound. Test
compounds are prepared in serum free DMEM with ligand in a 96
well plate, and serially diluted for 7 test concentrations.
(4) After 16 hours of ligand activation, diluted BrdU
labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the
cells are incubated with HrdU (final concentration=10 ~M) for
1.5 hours.
(5) After incubation with labeling reagent, the medium
is removed by decanting and tapping the inverted plate on a
paper towel. FixDenat solution is added (50 ~1/well) and the
plates are incubated at room temperature for 45 minutes on a
plate shaker.
(6) The FixDenat solution is thoroughly removed by
decanting and tapping the inverted plate on a paper towel.
Milk is added (5% dehydrated milk in PBS, 200 ~1/well) as a
blocking solution and the plate is incubated for 30 minutes
at room temperature on a plate shaker.
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(7) The blocking solution is removed by decanting and
the wells are washed once with PBS. Anti-BrdU-POD solution
(1:100 dilution in PBS, 1~ BSA) is added (100 ~l/well) and
the plate is incubated for 90 minutes at room temperature on
a plate shaker.
(8) The antibody conjugate is thoroughly removed by
decanting and rinsing the wells 5 times with PBS, and the
plate is dried by inverting and tapping on a paper towel.
(9} TMB substrate solution is added (100 ~1/well) and
incubated for 20 minutes at room temperature on a plate
shaker until color development is sufficient for photometric
detection.
(10) The absorbance of the samples are measured at 410
nm (in "dual wavelength" mode with a filter reading at 490
nm, as a reference wavelength) on a Dynatech ELISA plate
reader.
FGF-Induced HrdU incorporation Aesay
This assay measures FGF-induced DNA synthesis in
3Tc7/EGFr cells that express endogenous FGF receptors.
Materials and Reagents
1. FGF: human FGF2/bFGF (Gibco BRL, No. 13256-029).
2. BrdU Labeling reagent, (10 mM PBS (pH 7.4),
Boehringer Mannheim Cat No. 1 647 229).
3. Fixdenat fixation solution (Boehringer Mannheim Cat
No. 1 647 229).
4. Anti-BrdU-POD (mouse monoclonal antibody conjugated
with peroxidase, Boehringer Mannheim Cat. No. 1 647 229).
5. TMB (tetramethylbenzidine, Boehringer Mannheim Cat.
No. 1 647 229).
6. PBS washing solution, pH 7.4 (Sugen, Inc.).
7. Albumin, bovine (BSA), fraction V powder (Sigma
Chemical Co., Cat. No. A-8551)
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Procedure
1. 3T3 engineered cell line: 3T3c7/EGFr.
2. Cells are seeded at 8,000 cells/well in DMEM, 10%
CS and 2 mM Gln in a 96-well plate. Incubate 24 hours at 37°
C in 5% C02.
3. After 24 hours, wash cells with PBS then serum
starve in serum free medium (0% DMEM, 0.1% BSA} for 24 hours.
4. Add ligand (FGF2 (1.5 nM in DMEM with 0.1% BSA) and
test compound simultaneously. Negative control wells receive
serum free DMEM with 0.1% BSA only; positive control wells
receive FGF2 ligand but no test compound. Test compounds are
prepared in serum-free DMEM with ligand in a 96-well plate
and serially diluted to make seven {7) test concentrations.
5. After 20 hours, add diluted BrdU labeling reagent
(1:100 BrdU:DMEM, 0.1% BSA, final concentration is 10 ~.M) to
the cells and incubate for 1.5 hours.
6. Decant medium. Remove traces of material with
paper towel. Add FixDenat (50 ~1/well) and incubate at room
temperature for 45 minutes on a plate shaker.
7. Remove Fixdenat solution. Add blocking solution
(5% dehydrated milk in PBS (200 ~tl/well)) and incubate for 30
minutes at room temperature on a plate shaker.
8. Decant blocking solution; wash wells once with PBS.
Add anti-BrdU-POD solution (1:100 dilution in PBS, 0.1%
BSA); incubate for 90 minutes at room temperature on a plate
shaker.
9. Decant antibody conjugate; rinse wells 5 times with
PBS. Dry plate by inverting on paper towel and tapping.
10. Add TMB solution (100 ~1/well); incubate 20 minutes
at room temperature on a plate shaker until color development
is sufficient for photometric detection.
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11. Measure absorbance at 410 nM on a Dynatech ELISA
plate reader using "Dual wavelength" mode with a filter at
490 nM.
Biochemical EGFR Assav
This assay measures the in vitro kinase activity of
EGFR using ELISA.
Materials Aad Reagents
1. Corning 96-well Elisa plates (Corning Catalog No.
25805-96).
2. SUMO1 monoclonal anti-EGFR antibody (Biochemistry
Lab, SUGEN, Inc.).
3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco
Catalog No. 450-1300EB).
4. TBST Buffer
Reagent M.W. Working Amount
Concentration per L
Tris 121.14 50 mM 6.057 g
NaCl 58.44 150 mM 8.766 g
Triton X-100 NA 0.1~ 1.0 ml
5. Blocking Buffer:
Reagent M.W. Working Amount per
Concentration 100 ml
Carnation Instant 5~ 5.0 g
Non-Fat Milk
PBS NA NA 100 ml
6. A431 cell lysate (Screening Lab, SUGEN, Inc.)
7. THS Buffer:
Reagent M.W. Working Amount
Concentration per L
Tris 121.14 50 mM 6.057 g
NaCl 58.44 150 mM 8.766 g
8. TBS + 10~s DMSO
Reagent M.W. Working Amount
Concentration oer L
Tris 121.14 50 mM 1.514 g
NaCl 58.44 150 mM 2.192 g
DMSO NA 10~ 25 ml
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9. Adenosine-5'-triphosphate (ATP, from Equine muscle,
Sigma Cat. No. A-5394).
Prepare a 1.0 mM solution in dHzO. This reagent
should be made up immediately prior to use and kept on ice.
. MnCl2 .
Prepare a 1.0 M stock solution in dHzO.
11. ATP/MnCl2 phosphorylation mix
Reagent Stock Amount Working
10 solution per 10 ml Concentration
ATP 1 . 0 mM 3 0 0 ~,1 3 0 ~M
MnCl2 1.0 M 500 ~l 50 mM
dH20 9.2 ml
This reagent should be prepared immediately before
use and kept on ice
12. NUNC 96-well V bottom polypropylene plates (Applied
Scientific Cat. No. AS-72092).
13. Ethylenediaminetetraacetic acid (EDTA)
Prepare 200 mM working solution in dHzO. Adjust to
pH 8.0 with 10 N NaOH.
14. Rabbit polyclonal anti-phosphotyrosine serum
(Biochemistry Lab, SUGEN, Inc.)
15. Goat anti-rabbit IgG peroxidase conjugate
(Biosource Cat. No. ALI0404)
16. ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-
sulfonic acid), Sigma Cat. No. A-1888).
Reagent M.W. Working Amount
Concentration per L
Citric Acid 192.12 100 mM 19.21 g
Na2HP04 141.96 250 mM 35.49 g
ABTS NA 0.5 mg/ml 500 mg
Mix first two ingredients in about 900 ml dH20, adjust
pH to 4.0 with phosphoric acid. Add ABTS, cover, let sit
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about 0.5 hr., filter. The solution should be kept in the
dark at 4° C until ready to use.
17. Hydrogen peroxide 30~ solution (Fisher Cat. No.
H325)
18. ABTS/HZOz
Mix 15 ml ABTS solution and 2.0 ~1 H20z. Prepare 5
minutes before use.
19. 0.2 M HC1
Procedure
1. Coat Corning 96 well ELISA plates with 0.5 Pg SUMO1
in 100 ~1 PBS per well, store overnight at 4° C.
2. Remove unbound SUMO1 from wells by inverting plate
to remove liquid. Wash lx with dHzO. Pat the plate on a paper
towel to remove excess liquid.
3. Add 150 ~1 of Blocking Buffer to each well.
Incubate for 30 min. at room temperature with shaking.
4. Wash plate 3x with deionized water, then once with
TBST. Pat plate on a paper towel to remove excess liquid and
bubbles.
5. Dilute lysate in PBS (7 ~g lysate/100 ~1 PBS).
6. Add 100 ~1 of diluted lysate to each well. Shake at
room temperature for 60 min.
7. Wash plates as described in 4, above.
8. Add 120 ~l TBS to ELISA plate containing captured
EGFR.
9. Dilute test compound 1:10 in TBS in 96-well
polypropylene plates (ie. 10 ~l compound + 90 ~1 TBS).
10. Add 13.5 P1 diluted test compound to ELISA plate.
To control wells (wells which do not receive any test
compound), add 13.5 ~l TBS + 10~ DMSO.
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11. Incubate for 30 minutes while shaking at room
temperature.
12. Add 15 ~tl phosphorylation mix directly to all wells
except negative control well which does not receive ATP/MnCl2
(final well volume should be approximately 150 ~l with 3 ~,M
ATP/5 mM MnClz final concentration in each well.) Incubate 5
minutes while shaking.
13. After 5 minutes, stop reaction by adding 16.5 ~,l of
200 mM EDTA (pH 8.0) to each well, shaking continuously.
After the EDTA has been added, shake for 1 min.
14. Wash 4x with deionized water, twice with TBST.
15. Add 100 ~.1 anti-phosphotyrosine (1:3000 dilution in
TBST) per well. Incubate 30-45 min. at room temperature,
with shaking.
16. Wash as described in 4, above.
17. Add 100 ~.1 Biosource Goat anti-rabbit IgG
peroxidase conjugate (1:2000 dilution in TBST) to each well.
Incubate 30 min. at room temperature, with shaking.
18. Wash as described in 4, above.
19. Add 100 ~.1 of ABTS/HZOZ solution to each well.
20. Incubate 5 to 10 minutes with shaking. Remove any
bubbles.
21. If necessary stop reaction with the addition of 100
~1 0.2 M HC1 per well.
22. Read assay on Dynatech MR7000 ELISA reader. Test
Filter: 410 nM Reference Filter: 630 Nm.
Biochemical PDGFR Assav
This assay measures the in vitro kinase activity of
PDGFR using ELISA.
Materials and Reagents
Unless otherwise noted, the preparation of working
solution of the following reagents is the same as that for
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the Biochemical EGFR assay, above.
1. Corning 96-well Elisa plates (Corning Catalog No.
25805-96).
2. 28D4C10 monoclonal anti-PDGFR antibody
(Biochemistry Lab, SUGEN, Inc.).
3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco
Catalog No. 450-1300EB)
4. TBST Buffer.
5. Blocking Buffer.
6. PDGFR-(3 expressing NIH 3T3 cell lysate (Screening
Lab, SUGEN, Inc.).
7. TBS Buffer.
8. TBS + 10$ DMSO.
9. Adenosine-5'-triphosphate (ATP, from Equine muscle,
Sigma Cat. No. A-5394).
10 . MnClZ .
11. Kinase buffer phosphorylation mix.
Reagent Stock Amount Working
solution per 10 ml Concentration
Tris 1 M 250 ~1 25 mM
NaCl 5 M 200 ~1 100 mM
MnCl2 1 M 100 ~.1 10 mM
TX-100 100 mM 50 ~1 0.5 mM
12. NUNC 96-well V bottom polypropylene plates (Applied
Scientific Cat. No. AS-72092).
13. Ethylenediaminetetraacetic acid (EDTA).
14. Rabbit polyclonal anti-phosphotyrosine serum
(Biochemistry Lab, SUGEN, Inc.).
15. Goat anti-rabbit IgG peroxidase conjugate
(Biosource Cat. No. ALI0404).
16. 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS, Sigma Cat. No. A-1888).
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17. Hydrogen peroxide 30~ solution (Fisher Cat. No.
H325).
18 . ABTS /H202 .
19. 0.2 M HC1.
Procedure
1. Coat Corning 96 well ELISA plates with 0.5 ~.g
28D4C10 in 100 ~1 PBS per well, store overnight at 4° C.
2. Remove unbound 28D4C10 from wells by inverting
plate to remove liquid. Wash lx with dH20. Pat the plate on a
paper towel to remove excess liquid.
3. Add 150 ~,l of Blocking Buffer to each well.
Incubate for 30 min. at room temperature with shaking.
4. Wash plate 3x with deionized water,- then once with
TBST. Pat plate on a paper towel to remove excess liquid and
bubbles.
5. Dilute lysate in HNTG (10 ~g lysate/100 ~.l HNTG)
6. Add 100 ~1 of diluted lysate to each well. Shake at
room temperature for 60 min.
7. Wash plates as described in 4, above.
8. Add 80 ~1 working kinase buffer mix to ELISA plate
containing captured PDGFR.
9. Dilute test compound 1:10 in TBS in 96-well
polypropylene plates (i.e., 10 ~1 compound + 90 ~,1 TBS).
10. Add 10 ~.1 diluted test compound to ELISA plate. To
control wells (wells which do not receive any test compound),
add 10 ~1 TBS + 10$ DMSO.
11. Incubate for 30 minutes while shaking at room
temperature.
12. Add 10 ~1 ATP directly to all wells except negative
control well (final well volume should be approximately 100
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~.1 with 20 ~,M ATP in each well.) Incubate 30 minutes while
shaking.
13. After 30 minutes, stop reaction by adding 10 gel of
200 mM EDTA (pH 8.0) to each well.
14. Wash 4x with deionized water, twice with THST.
15. Add 100 ~1 anti-phosphotyrosine (1:3000 dilution in
TBST) per well. Incubate 30-45 min. at room temperature,
with shaking.
16. Wash as described in 4, above.
17. Add 100 ~tl Biosource Goat anti-rabbit IgG
peroxidase conjugate (1:2000 dilution in TBST) to each well.
Incubate 30 min. at room temperature, with shaking.
18. Wash as described in 4, above.
19. Add 100 ~tl of ABTS/H202 solution to each well.
20. Incubate 10 to 30 minutes with shaking. Remove any
bubbles.
21. If necessary stop reaction with the addition of 100
~,1 0.2 M HC1 per well.
22. Read assay on Dynatech MR7000 ELISA reader: test
filter: 410 nM, reference filter: 630 nM.
Biochemical FGFR Assav
This assay measures in vi ro kinase activity of the Myc-
GyrB-FGFR fusion protein using ELISA.
Materials And Reagents
~ 1. HNTG
Reagent M.W. 5x Stock Amount lx Working
Concentration per L Concentration
HEPES 238.3 100 mM 23.83 g 20 mM
NaCl 58.44 750 mM 43.83 g 150 mM
Glycerol NA 50% 500 ml 10%
Triton X-100. NA 5% 10 ml 1.0%
To make a liter of 5x stock solution, dissolve HEPES and
NaCl in about 350 ml dHzO, adjust pH to 7.2 with HC1 or NaOH
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(depending on the HEPES that is used), add glycerol; Triton
X-100 and then dHzO to volume.
2. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco
Catalog # 450-1300EB).
3. Blocking Buffer.
4. Kinase Buffer.
Reagent M.W, lOx Stock lx Working
Concentration Concentration
HEPES (pH 7.2) 238.3 500 mM 50 mM
MnClz 20 mM 2 mM
MgClZ 203.32 200 mM 10 mM
Triton-X-100 1 ~ 0.1~
DTT 380.35 5 mM 0.5 mM
5. Phenylmethylsulfonyl fluoride (PMSF, Sigma, Cat.
No. P-7626):
Working solution: 100 mM in ethanol.
6. ATP (Bacterial source, Sigma Cat. No. A-7699)
Use 3.31 mg per ml MilliQ Hz0 for a stock
concentration of 6 mM.
7. Biotin conjugated anti-phosphotyrosine mab (clone
4610, Upstate Biotechnology Inc. Cat. No. 16-103, Ser. No.
14495).
8. Vectastain Elite ABC reagent (Avidin peroxidase
conjugate, Vector Laboratories Cat. No. PK-6 100).
9. ABTS Solution.
10. Hydrogen peroxide 30~ solution ( Fisher Catalog #
H325) .
11. ABTS/HzOZ .
12. 0.2 M HC1.
13. TRIS HC1 (Fischer Cat. No. BP 152-5).
Prepare 1.0 mM solution in MilliQ HaO, adjust pH to
7.2 with HC1.
14. NaCl (Fisher Cat. No. 5271-10}.
Prepare 5 M solution in MilliQ H20.
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15. MgCl2 (Fisher Cat. No. M33-500).
Prepare 1 M solution in MilliQ HzO.
16. HEPES (Fisher Cat. No. BP310-500).
Prepare 1 M solution in MilI.iQ HzO, adjust pH to
7.5, sterile filter.
17. TBST Buffer.
18. Sodium Carbonate Buffer (Fisher Cat. No. S495).
Prepare 0.1 M solution in MilliQ H20, adjust pH to
9.6 with NaOH, filter.
19. Dithiothreitol (DTT, Fisher Cat. No. BP172-25).
Prepare 0.5 mM working solution in MilliQ H20 just
prior to use. Store at -20° C until used, discard any
leftover.
2 0 . MnCl2 .
21. Triton X-100.
22. Goat a-Rabbit IgG (Cappel).
23. Affinity purified Rabbit a GST GyrB (Biochemistry
Lab. SUGEN, Inc.).
Procedure
All of the following steps are conducted at room
temperature unless otherwise indicated.
1. Coat Corning 96-well ELISA plates with 2 ~g Goat a-
Rabbit antibody per well in Carbonate Buffer such that total
well volume is 100 ~tl. Store overnight at 4° C.
2. Remove unbound Goat a-Rabbit antibody by inverting
plate to remove liquid. Pat plate on a paper towel to remove
excess liquid and bubbles
3. Add 150 ~1 Blocking Buffer (5~ Low Fat Milk in PBS)
to each well. Incubate while shaking on a micro-titer plate
shaker for 30 min.
4. Wash 4x with TBST. Pat plate on a paper towel to
remove excess liquid and bubbles.
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5. Add 0.5 ~.g Rabbit a-GyrB antibody per well. Dilute
antibody in DPBS to a final volume of 100 ~1 per well.
Incubate with shaking on a micro-titer plate shaker at room
temperature for 1 hour.
6. Wash 4x with THST as described in step 4.
7. Add 2 ~,g COS/FGFR cell lysate (Myc-GyrB-FGFR
source) in HNTG to each well to give a final volume of 100 ~1
per well. Incubate with shaking on a micro-titer plate shaker
for 1 hour.
8. Wash 4X with TBST as described in step 4.
9. Add 80 ~1 of lx kinase buffer per well.
l0. Dilute test compound 1:10 in lx kinase buffer + 1~
DMSO in a polypropylene 96 well plate.
11. Transfer 10 ~1 of diluted test compound solution
and control wells from polypropylene plate wells to the
corresponding ELISA plate wells, incubate with shaking on a
micro-titer plate shaker for 20 minutes.
12. Add 10 ~.1 of 70 ~tM ATP diluted in kinase buffer to
positive control and test wells (Final ATP concentration is 7
~M/well). Add 10 ~1 lx kinase buffer to negative control
wells. Incubate with shaking on a micro-titer plate shaker
for 15 min.
13. Stop kinase reaction by adding 5 ~l 0.5 M EDTA to
all wells.
14. Wash 4x with TBST as described in step 4.
15. Add 100 ~.1 biotin conjugated a-phosphotyrosine mab
(b4G10) diluted in TBST to each well. Incubate with shaking
on a micro-titer plate shaker for 30 minutes.
16. Prepare Vectastain ABC reagent. Add 1 drop reagent
A to 15 ml TBST. Mix by inverting tube several times. Add 1
drop reagent B and mix again.
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17. Wash 4x with TBST as described in step 4.
18. Add 100 ~,1 ABC HRP reagent to each well. Incubate
with shaking on a micro-titer plate shaker for 30 minutes.
19. Wash 4x with TBST as described in step 4.
20. Add 100 ~1 of ABTS/Hz4z solution to each well.
22. Incubate 5 to 15 minutes with shaking. Remove any
bubbles.
23. If necessary stop reaction by adding 1 00 ~,1 of
0.2M HC1/well.
24. Read assay on Dynatech MR7000 ELISA Plate Reader;
test filter: 410 nM, reference filter: 630 nM.
Biochemical FLR-1 Assav
This assay evaluates flk-1 autophosphorylation activity
in vitro using ELISA.
Materials And Reagents
1. 15 cm tissue culture dishes
2. Flk-1/NIH cells: NIH fibroblast line over-
expressing human flk-1 clone 3 (SUGEN, Inc., obtained from
MPI, Martinsried, Germany).
3. Growth medium: DMEM plus heat inactivated 10% FBS
and 2 mM Glutamine (Gibco-BRL).
4. Starvation medium: DMEM plus 0.5% heat-inactivated
FBS, 2 mM Glutamine (Gibco-BRL).
5. Corning 96-well ELISA plates (Corning Cat. No.
25805-96).
6. L4 or E38 monoclonal antibody specific for flk-l;
Purified by Protein-A agarose affinity chromatography (SUGEN,
Inc . ) .
7. PBS (Dulbecco's Phosphate-Buffered Saline) Gibco
Cat. No. 450-1300EB).
8. HNTG (see BIOCHEMICAL FGFR for preparation).
9. Pierce BCA protein determination kit.
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10. Blocking buffer
11. TBST (pH 7.0)
12. Kinase Buffer
13. Kinase Stop Solution: 200 mM EDTA.
14. Biotinylated 4610, specific for phosphotyrosine
(UBI, Cat. No. No. 16-103).
15. AB kit (Vector Laboratories Cat. No. PK 4000).
16. DMSO
17. NUNC 96-well V bottom polypropylene plates (Applied
Scientific Cat. No. AS-72092).
18. Turbo-TMB (Pierce).
19. Turbo-TMB stop solution: 1 M HZSO4.
20. ATP (Sigma Cat. No. A-7699).
21. 20% DMSO in TBS (pH 7.0}.
Procedure
Cell Growth and Lysate Preparation
1. Seed cell into growth medium and grow for 2-3 days
to 90-100% confluency at 37° C and 5% C02. Do not exceed
passage #20.
2. Remove the medium and wash the cells twice with
PBS. Lyse with HNTG lysis buffer. Collect all lysates and
vortex mix them for 20-30 seconds.
3. Remove insoluble material by centrifugation (5-10
min at about 10,000 xg).
4. Determine the protein concentration using BCA kit.
5. Partition lysate into 1 mg aliquots, store at -80°
C.
Assay Procedure
1. Coat Corning 96-well ELISA plates with 2 ~.g/well
purified L4 (or E 38) in 100 ~1 of PBS. Store overnight at 4° C.
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2. Remove unbound proteins from wells by inverting the
plate to remove the liquid. Wash one time with dHzO, pat
plate on paper towel to remove excess liquid.
3. Block plates with 150 ~1 blocking buffer per well.
Incubate for 45-60 minutes with shaking at 4° C.
4. Remove the blocking buffer and wash the ELISA plate
three times with dH2o and one time with TBST. Pat plate on
paper towel to remove excess liquid.
5. Dilute lysate in PBS to give final concentration of
50 ~g/100 ~1. Add 100 ~1 of diluted lysate to each well.
Incubate with shaking at 4° C overnight.
6. Remove unbound proteins from wells by inverting the
plate. Wash as in step 4.
7. Add 80 ~1 of kinase buffer to wells (90 ~.1 to
negative control wells).
8. Dilute test compounds (normally 10-fold) into wells
of a polypropylene plate containing 20~ DMSO in TBS.
9. Add 10 ~tl of the diluted compounds to the ELISA
wells containing immobilized flk-1 and shake. Control wells
receive no compounds.
10. From stock 1 mM ATP, prepare 0.3 mM ATP solution in
dH20 (alternatively, kinase buffer may be used).
11. Add 10 ~1 of 0.3 mM ATP to all wells except the
negative controls. Incubate for 60 min. at room temperature
with shaking.
12. After 1 hr stop the kinase reaction by adding 11 ~tl
200 mM EDTA. Shake for 1-2 min.
13. Wash the ELISA plate 4 times with dHzO and twice
with TBST.
14. Add 100 ~.1 of 1:5000 biotinylated 4G10:TBST to all
wells. Incubate 45 min with shaking at room temperature.
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15. While the above is incubating, add 50 ~.1 of
solutions A & B from the ABC kit to 10 ml of TBST. These
solutions must be combined approximately 30 min prior to use.
16. Wash plates as in step 4.
17. Add 100 ~,1 of the preformed A & B complex to all
wells. Incubate 30 min with shaking at room temperature.
18. Wash plates as in step 4.I
19. Add 100 ~1 turbo-TMB. Shake at room temperature for
10- 15 min.
20. When the color in the positive control wells
reaches an absorbance of about 0.35 - 0.4, stop the reaction
with 100 ~1 of turbo-TMB stop solution.
21. Read plates on Dynatech MR7000 ELISA reader; test
filter: 450 nM, reference filter: 410 nM.
HW-EC-C Assav
The following protocol may also be used to measure a
compound's activity against PDGF-R, FGF-R, VEGF, aFGF or Flk-
1/KDR, all of which are naturally expressed by HW-EC cells.
DAY
1. Wash and trypsinize HW-EC-C cells (human umbilical
vein endothelial cells, (American Type Culture Collection;
catalogue no. 1730 CRL). Wash with Dulbecco's phosphate-
buffered saline (D-PBS; obtained from Gibco BRL; catalogue
no. 14190-029) 2 times at about 1 ml/10 cm2 of tissue culture
flask. Trypsinize with 0.05 trypsin-EDTA in non-enzymatic
cell dissociation solution (Sigma Chemical Company; catalogue
no. C-1544). The 0.05 trypsin is made by diluting 0.25
trypsin/1 mM EDTA (Gibco; catalogue no. 25200-049) in the
cell dissociation solution. Trypsinize with about 1 ml/25-30
cm2 of tissue culture flask for about 5 minutes at 37°C.
After cells have detached from the flask, add an equal volume
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of assay medium and transfer to a 50 ml sterile centrifuge
tube (Fisher Scientific; catalogue no. 05-539-6).
2. Wash the cells with about 35 ml assay medium in the
50 ml sterile centrifuge tube by adding the assay medium,
centrifuge for 10 minutes at approximately 200x g, aspirate
the supernatant, and resuspend with 35 ml D-PBS. Repeat the
wash two more times with D-PBS, resuspend the cells in about
1 ml assay medium/15 cm2 of tissue culture flask. Assay
medium consists of F12K medium (Gibco BRL; catalogue no.
21127-014) and 0.5% heat-inactivated fetal bovine serum.
Count the cells with a Coulter Counter~ (Coulter
Electronics, Inc.) and add assay medium to the cells to
obtain a concentration of 0.8-1.0 x 105 cells/ml.
3. Add cells to 96-well flat-bottom plates at 100
~C1/well or 0.8-1.0 x 10' cells/well; incubate ~24h at 37°C,
5 % C02 .
DAY 1
1. Make up two-fold test compound titrations in
separate 96-well plates, generally 50 ~M on down to 0 ~,M.
Use the same assay medium as mentioned in day 0, step 2
above. Titrations are made by adding 90 ~1/well of test
compound at 200 ~M (4X the final well concentration) to the
top well of a particular plate column. Since the stock test
compound is usually 20 mM in DMSO, the 200 ~,M drug
concentration contains 2% DMSO.
A diluent made up to 2% DMSO in assay medium (F12K +
0.5% fetal bovine serum) is used as diluent for the test
compound titrations in order to dilute the test compound but
keep the DMSO concentration constant. Add this diluent to
the remaining wells in the column at 60 ~l/well. Take 60 ~1
from the 120 ~tl of 200 ~.M test compound dilution in the top
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well of the column and mix with the 60 ~,1 in the second well
of the column. Take 60 ~l from this well and mix with the 60
~tl in the third well of the column, and so on until two-fold
titrations are completed. When the next-to-the-last well is
mixed, take 60 ~1 of the 120 ~.1 in this well and discard it.
Leave the last well with 60 ~1 of DMSO/media diluent as a
non-test compound-containing control. Make 9 columns of
titrated test compound, enough for triplicate wells each for:
(1) VEGF (obtained from Pepro Tech Inc., catalogue no. 100-
200; (2) endothelial cell growth factor (ECGF) (also known as
acidic fibroblast growth factor, or aFGF) (obtained from
Boehringer Mannheim Biochemica, catalogue no. 1439 600); or,
(3) human PDGF B/B (1276-956, Boehringer Mannheim, Germany)
and assay media control. ECGF comes as a preparation with
sodium heparin.
2. Transfer 50 ~1/well of the test compound dilutions
to the 96-well assay plates containing the 0.8-1.0x10'
cells/100 ~l/well of the HW-EC-C cells from day 0 and
incubate ~2 h at 37° C, 5% CO2.
3. In triplicate, add 50 ~1/well of 80 ~.g/ml VEGF, 20
ng/ml ECGF, or media control to each test compound condition.
As with the test compounds, the growth factor concentrations
are 4X the desired final concentration. Use the assay media
from day 0 step 2 to make the concentrations of growth
factors. Incubate approximately 24 hours at 37°C, 5% C02.
Each well will have 50 ~1 test compound dilution, 50 ~,1
growth factor or media, and 100 ~tl cells, which calculates to
200 P.1/well total. Thus the 4X concentrations of test
compound and growth factors become 1X once everything has
been added to the wells.
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DAY 2
1. Add 3H-thymidine (Amersham; catalogue no. TRK-686)
at 1 ~tCi/well (10 ~,1/well of 100 ~.Ci/ml solution made up in
RPMI media + 10% heat-inactivated fetal bovine serum) and
incubate ~24 h at 37°C, 5% COZ. RPMI is obtained from Gibco
BRL, catalogue no. 11875-051.
DAY 3
1. Freeze plates overnight at -20°C.
DAY 4
Thaw plates and harvest with a 96-well plate harvester
(Tomtec Harvester 96~) onto filter mats (Wallac; catalogue
no. 1205-401); read counts on a Wallac BetaplateT"" liquid
scintillation counter.
In Vivo Animal Models
Xenocrraft Animal Models
The ability of human tumors to grow as xenografts in
athymic mice (e. g., Balb/c, nu/nu) provides a useful in vivo
model for studying the biological response to therapies for
human tumors. Since the first successful xenotransplantation
of human tumors into athymic mice, (Rygaard and Povlsen, 1969,
Acta Pathol. Microbial. Scand. 77:758-760), many different
human tumor cell lines (e. g., mammary, lung, genitourinary,
gastro-intestinal, head and neck, glioblastoma, bone, and
malignant melanomas) have been transplanted and successfully
grown in nude mice. The following assays may be used to
determine the level of activity, specificity and effect of the
different compounds of the present invention. Three general
types of assays are useful for evaluating compounds:
cellular/catalytic, cellular/biological and in vivo. The
object of the cellular/catalytic assays is to determine the
effect of a compound on the ability of a TK to phosphorylate
tyrosines on a known substrate in a cell. The object of the
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cellular/biological assays is to determine the effect of a
compound on the biological response stimulated by a TK in a
cell. The object of the in vivo assays is to determine the
effect of a compound in an animal model of a particular
disorder such as cancer.
Suitable cell lines for subcutaneous xenograft
experiments include C6 cells (glioma, ATCC # CCL 107), A375
cells (melanoma, ATCC # CRL 1619), A431 cells (epidermoid
carcinoma, ATCC # CRL 1555), Calu 6 cells (lung, ATCC # HTB
56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cells
and NIH 3T3 fibroblasts genetically engineered to overexpress
EGFR, PDGFR, IGF-1R or any other test kinase. The following
protocol can be used to perform xenograft experiments:
Female athymic mice (BALB/c, nu/nu) are obtained from
Simonsen Laboratories (Gilroy, CA). All animals are
maintained under clean-room conditions in Micro-isolator
cages with Alpha-dri bedding. They receive sterile rodent
chow and water ad libitum.
Cell lines are grown in appropriate medium (for example,
MEM, DMEM, Ham's F10, or Ham's F12 plus 5% - 10% fetal bovine
serum (FBS) and 2 mM glutamine (GLN)). All cell culture
media, glutamine, and fetal bovine serum are purchased from
Gibco Life Technologies (Grand Island, NY) unless otherwise
specified. All cells are grown in a humid atmosphere of
90-95% air and 5-10% COZ at 37°C. All cell lines are
routinely subcultured twice a week and are negative for
mycoplasma as determined by the Mycotect method (Gibco).
Cells are harvested at or near confluency with 0.05%
Trypsin-EDTA and pelleted at 450 x g for 10 min. Pellets are
resuspended in sterile PBS or media~(without FBS) to a
particular concentration and the cells are implanted into the
hindflank of the mice (8 - 10 mice per group, 2 - 10 x 106
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cells/animal). Tumor growth is measured over 3 to 6 weeks
using venier calipers. Tumor volumes are calculated as a
product of length x width x height unless otherwise
indicated. P values are calculated using the Students t-test.
Test compounds in 50 - 100 ~L excipient (DMSO, or VPD:DSW)
can be delivered by IP injection at different concentrations
generally starting at day one after implantation.
Tumor Invasion Model
The following tumor invasion model has been developed
and may be used for the evaluation of therapeutic value and
efficacy of the compounds identified to selectively inhibit
KDR/FLK-1 receptor.
Procedure
8 week old nude mice (female) (Simonsen Inc.) are used
as experimental animals. Implantation of tumor cells can
be performed in a laminar flow hood. For anesthesia,
Xylazine/Ketamine Cocktail (100 mg/kg ketamine and 5 mg/kg
Xylazine) are administered intraperitoneally. A midline
incision is done to expose the abdominal cavity
(approximately 1.5 cm in length) to inject 10' tumor cells
in a volume of 100 ~1 medium. The cells are injected
either into the duodenal lobe of the pancreas or under the
serosa of the colon. The peritoneum and muscles are closed
with a 6-0 silk continuous suture and the skin is closed by
using wound clips. Animals are observed daily.
Analysis
After 2-6 weeks, depending on gross observations of
the animals, the mice are sacrificed, and the local tumor
metastases to various organs (lung, liver, brain, stomach,
spleen, heart, muscle) are excised and analyzed
(measurement of tumor size, grade of invasion,
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WO 00/38519 13 5 PCT/US99/31232
immunochemistry, in itu hybridization determination,
etc.).
Measurement Of Cell Toxicity
Therapeutic compounds should be more potent in inhibiting
receptor tyrosine kinase activity than in exerting a cytotoxic
effect. A measure of the effectiveness and cell toxicity of a
compound can be obtained by determining the therapeutic index;
i . a . , ICso/LDSO . ICso, the dose required to achieve 50~
inhibition, can be measured using standard techniques such as
those described herein. LDso,the dosage which results in 50~
toxicity, can also be measured by standard techniques as
well(Mossman, 1983, J. Immunol. Methods, 65:55-63), by
measuring the amount of LDH released (Korzeniewski and
Callewaert, 1983, J. Immunol. Methods, 64:313; Decker and
Lohmann-Matthes, 1988, J. Immunol. Methods, 115:61), or by
measuring the lethal dose in animal models. Compounds with a
large therapeutic index are preferred. The therapeutic index
should be greater than 2, preferably at least 10, more
preferably at least 50.
CONCLUSION
Thus, it will be appreciated that 3-heteroarylidenyl-2-
indolinones are expected to have a beneficial effect on the
chemotherapeutic efficacy of various chemotherapeutic agents,
in particular fluorinated pyrimidine compounds. Furthermore
3-[(2,4-Dimethylpyrrol-5-yl)methylene]-2-indolinone together
with fluorouracil or fluorouracil/leucovorin is expected to
be an effective chemotherapeutic combination for the
treatment of colorectal cancer.
It will also be appreciated that the compounds, methods
and pharmacological compositions of the present invention are
expected to modulate RTK and CTK activity and therefore to be
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WO 00/38519 13 6 PCT/US99/31232
effective as therapeutic agents against RTK- and CTK-related
disorders.
One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects
and obtain the ends and advantages mentioned, as well as
those inherent therein. The molecular complexes and the
methods, procedures, treatments, molecules, specific
compounds described herein are presently representative of
preferred embodiments and are exemplary and are not intended
as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art
which are encompassed within the spirit of the invention and
are defined by the scope of the claims.
It will be readily apparent to one skilled in the art
that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the
scope and spirit of the invention.
All patents and publications mentioned in the
specification are indicative of the levels of those skilled
in the art to which the invention pertains. All patents and
publications are herein incorporated by reference to the same
extent as if each individual publication was specifically and
individually indicated to be incorporated by reference.
The invention illustratively described herein suitably
may be practiced in the absence of any element or elements,
limitation or limitations which is not specifically disclosed
herein. Thus, for example, in each instance herein any of
the terms "comprising", "consisting essentially of" and
"consisting of" may be replaced with either of the other two
terms. The terms and expressions which have been employed
are used as terms of description and not of limitation, and
there is no intention that in the use of such terms and
CA 02357042 2001-06-27
WO 00/38519 13 ~ PCT/US99/31232
expressions indicates the exclusion of equivalents of the
features shown and described or portions thereof. It is
recognized that various modifications are possible within the
scope of the invention claimed. Thus, it should be
understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and
that such modifications and variations are considered to be
within the scope of this invention as defined by the appended
claims.
In addition, where features or aspects of the invention
are described in terms of Markush groups, those skilled in
the art will recognize that the invention is also thereby
described in terms of any individual member or subgroup of
members of the Markush group. For example, if X is described
as selected from the group consisting of bromine, chlorine,
and iodine, claims for X being bromine and claims for X being
bromine and chlorine are fully described.
Other embodiments are presented within the following
claims.
