U.S. patent application number 10/600868 was filed with the patent office on 2004-01-01 for methods of modulating c-kit tyrosine protein kinase function with indolinone compounds.
This patent application is currently assigned to Sugen, Inc.. Invention is credited to Lipson, Ken, McMahon, Gerald.
Application Number | 20040002534 10/600868 |
Document ID | / |
Family ID | 22624773 |
Filed Date | 2004-01-01 |
United States Patent
Application |
20040002534 |
Kind Code |
A1 |
Lipson, Ken ; et
al. |
January 1, 2004 |
Methods of modulating c-kit tyrosine protein kinase function with
indolinone compounds
Abstract
The present invention concerns compounds and their use to
inhibit the activity of a receptor tyrosine kinase. The invention
is preferably used to treat cell proliferative disorders such as
cancers characterized by over-activity or inappropriate activity
c-kit kinase.
Inventors: |
Lipson, Ken; (San Mateo,
CA) ; McMahon, Gerald; (Kenwood, CA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Sugen, Inc.
|
Family ID: |
22624773 |
Appl. No.: |
10/600868 |
Filed: |
June 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10600868 |
Jun 23, 2003 |
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09741842 |
Dec 22, 2000 |
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60171693 |
Dec 22, 1999 |
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Current U.S.
Class: |
514/414 ;
514/418 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 25/00 20180101; A61P 27/00 20180101; A61P 37/08 20180101; A61P
27/16 20180101; A61P 11/00 20180101; A61P 19/08 20180101; A61K
31/00 20130101; A61P 35/00 20180101; A61P 5/00 20180101; A61P 43/00
20180101; A61P 3/04 20180101; A61K 31/404 20130101; A61P 15/00
20180101; A61P 35/02 20180101; A61P 1/00 20180101; A61K 31/405
20130101; A61P 11/06 20180101 |
Class at
Publication: |
514/414 ;
514/418 |
International
Class: |
A61K 031/404 |
Claims
1. A method for treating or preventing an abnormal condition in an
organism, wherein said abnormal condition is associated with an
aberration in a signal transduction pathway mediated by a c-kit
kinase, wherein said method comprises the step of administering to
said organism a therapeutically effective amount of an indolinone
compound that modulates, in vitro, the catalytic activity of c-kit
kinase.
2. The method of claim 1 wherein said aberration in said signal
transduction pathway is mediated by an interaction between said
c-kit kinase and a natural binding partner, and said indolinone
compound modulates, in vitro, the interaction between said c-kit
kinase and said natural binding partner.
3. The method of claim 1 wherein said abnormal condition is a
disease related to inappropriate c-kit kinase signal
transduction.
4. The method of claim 1, wherein said abnormal condition is
selected from the group consisting of mastocytosis, the presence of
one or more mast cell tumors, asthma, and allergy associated
chronic rhinitis.
5. The method of claim 1, wherein said abnormal condition is
selected from the group consisting of small cell lung cancer,
non-small cell lung cancer, acute myelocytic leukemia, acute
lymphocytic leukemia, myelodysplastic syndrome, chronic myelogenous
leukemia, a colorectal carcinoma, a gastric carcinoma, a
gastrointestinal stromal tumor, a testicular cancer, a
glioblastoma, and an astrocytoma.
6. The method of claim 1 wherein said organism is a mammal.
7. The method of claim 1 wherein said organism is a human.
8. The method of any one of claims 1, 2 and 3 wherein said
indolinone compound is a compound of the structure set forth in
Formula I: 20wherein (a) Y is selected from the group consisting of
oxygen, sulfur and nitrogen substituted with a hydrogen; (b)
R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each independently
selected from the group consisting of hydrogen, alkyl, alkoxy,
aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R,
SO.sub.2 NRR', SO.sub.3R, SR, NO.sub.2, NRR', OH, CN, C(O)R,
OC(O)R, NHC(O)R, (CH.sub.2).sub.n CO.sub.2 R, and CONRR'; (c)
R.sub.5 is selected from the group consisting of hydrogen, alkyl,
alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl,
S(O)R, SO.sub.2 NRR', SO.sub.3R, SR, NO.sub.2, NRR', OH, CN, C(O)R,
OC(O)R, NHC(O)R, (CH.sub.2).sub.n CO.sub.2 R, CONRR', a
six-membered heteroaryl ring system containing 1 or 2 N, O, or S
atoms; and a six-membered aryl ring system; and (d) R.sub.6, and
R.sub.7 are each independently selected from the group consisting
of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy,
halogen, trihalomethyl, S(O)R, SO.sub.2 NRR', SO.sub.3R, SR,
NO.sub.2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH.sub.2).sub.n
CO.sub.2 R, and CONRR', 21
9. The method of any one of claims 1, 2 and 3 wherein said
indolinone compound is a compound of the structure set forth in
Formula II: 22wherein (a) Y is selected from the group consisting
of sulfur and nitrogen substituted with a hydrogen; (b) R.sub.1 is
independently selected from the group consisting of hydrogen and
methyl; (c) R.sub.2 is independently selected from the group
consisting of hydrogen, chlorine, bromine, --C(O)CH.sub.3,
--SO.sub.2NH.sub.2, and SO.sub.2N(CH.sub.3).sub.- 2; (d) R.sub.3 is
independently selected from the group consisting of hydrogen,
methyl, and --CH.sub.2CH.sub.2COOH; and (e) R.sub.4 and R.sub.5 are
independently selected from the group consisting of hydrogen,
methyl, --CH.sub.2CH.sub.2COOH, and substituents that when taken
together form a six-membered aliphatic or aromatic ring.
10. The method of any one of claims 1, 2 and 3 wherein said
indolinone compound is selected from the group consisting of
compounds 232425
Description
[0001] This application claims priority to U.S. Serial No.
60/171,693, filed Dec. 22, 1999, the entirety of which is hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods, compounds, and
compositions for inhibiting cell proliferative disorders. The
invention is particularly useful for inhibiting cell proliferative
disorders characterized by overactivity and/or inappropriate
activity of a c-kit kinase.
BACKGROUND OF THE INVENTION
[0003] The following description of the background of the invention
is provided to aid in understanding the invention, but is not
admitted to describe or constitute prior art to the invention.
[0004] Kit signaling is critical for fetal gonadal development, and
continues to play a role in adult fertility (Mauduit et al. (1999)
Human Reproduction Update 5:535-545). Spermatogenesis is inhibited
in the absence of SCF (Ohta et al. (2000) Development
127:2125-2131) or the ability of Kit to signal through the PI3
kinase pathway (Blume-Jensen et al. (2000) Nature Genetics
24:157-162; Kissel et al. (2000) EMBO Journal 19:1312-1326). Kit
expression has also been observed to be lower in sub-fertile testes
than in normal testicular tissue (Feng et al. (1999) Fertility
& Sterility 71:85-89). Kit signaling is also important for
oogenesis and folliculogenesis (Parrott & Skinner (1999)
Endocrinology 140:4262-4271; Driancourt et al. (2000) Reviews of
Reproduction 5:143-152). These observations suggest that Kit kinase
inhibitors would reduce both male and female fertility.
[0005] As a key mediator of mast cell function, Kit may play a role
in pathologies associated with mast cells. For example, mast cells
have been associated with interstitial fibrosis in chronic
rejection of human renal allografts (Pardo et al. (2000) Virchows
Archiv 437:167-172). Mast cells have also been implicated in liver
allograft rejection (Yamaguchi et al. (1999) Hepatology 29:133-139)
and in liver fibrosis, where hepatic stellate cells produce the SCF
that recruits the mast cells (Gaca et al. (1999) J. Hepatology
30:850-858). These observations suggest the Kit kinase inhibitors
may help prevent organ rejection and fibrosis.
[0006] Mast cells have also been implicated in the pathology of
multiple sclerosis (Secor et al. (2000) J. Experimental Medicine
191:813-822) and ischemia-reperfusion injury (Andoh et al. (1999)
Clinical & Experimental Immunology 116:90-93) in experimental
models using mice with mutant Kit receptors that are deficient in
mast cells. In both cases, the pathology of the disease was
significantly attenuated relative to mice with normal Kit and mast
cells populations. Thus, the role of mast cells in these diseases
suggests that Kit kinase inhibitors might be useful
therapeutics.
[0007] Cellular signal transduction is a fundamental mechanism
whereby extracellular stimuli are relayed to the interior of cells
and subsequently regulate diverse cellular processes. One of the
key biochemical mechanisms of signal transduction involves the
reversible phosphorylation of proteins. Phosphorylation of
polypeptides regulates the activity of mature proteins by altering
their structure and function. Phosphate most often resides on the
hydroxyl moiety (--OH) of serine, threonine, or tyrosine amino
acids in proteins.
[0008] Enzymes that mediate phosphorylation of cellular effectors
generally fall into two classes. The first class consists of
protein kinases which transfer a phosphate moiety from adenosine
triphosphate to protein substrates. The second class consists of
protein phosphatases which hydrolyze phosphate moieties from
phosphoryl protein substrates. The converse functions of protein
kinases and protein phosphatases balance and regulate the flow of
signals in signal transduction processes.
[0009] Protein kinases and protein phosphatases are generally
divided into two groups: receptor and non-receptor type proteins.
Most receptor-type protein tyrosine phosphatases contain two
conserved catalytic domains, each of which encompasses a segment of
240 amino acid residues. Saito, et al., 1991, Cell Growth and Diff.
2:59-65. Receptor protein tyrosine phosphatases can be
subclassified further based upon the amino acid sequence diversity
of their extracellular domains. Saito, et al., supra; Krueger, et
al., 1992, Proc. Natl. Acad. Sci. USA 89:7417-7421.
[0010] Protein kinases and protein phosphatases are also typically
divided into three classes based upon the amino acids they act
upon. Some catalyze the addition or hydrolysis of phosphate on
serine or threonine only, some catalyze the addition or hydrolysis
of phosphate on tyrosine only, and some catalyze the addition or
hydrolysis of phosphate on serine, threonine, and tyrosine.
[0011] Tyrosine kinases can regulate the catalytic activity of
other protein kinases involved in cell proliferation. Protein
kinases with inappropriate activity are also involved in some types
of cancer. Abnormally elevated levels of cell proliferation are
associated with receptor and non-receptor protein kinases with
unregulated activity.
[0012] In addition to their role in cellular proliferation, protein
kinases are thought to be involved in cellular differentiation
processes. Cell differentiation occurs in some cells upon nerve
growth factor (NGF) or epidermal growth factor (EGF) stimulation.
Cellular differentiation is characterized by rapid membrane
ruffling, cell flattening, and increases in cell adhesion. (Chao,
1992, Cell 68:995-997).
[0013] In an effort to discover novel treatments for cancer and
other diseases, biomedical researchers and chemists have designed,
synthesized, and tested molecules that inhibit the function of
protein kinases. Some small organic molecules form a class of
compounds that modulate the function of protein kinases. Examples
of molecules that have been reported to inhibit the function of
protein kinases are bis-monocyclic, bicyclic or heterocyclic aryl
compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO
94/14808), 1-cyclopropyl-4-pyridyl- -quinolones (U.S. Pat. No.
5,330,992), styryl compounds (by Levitzki, et al., U.S. Pat. No.
5,217,999, and entitled "Styryl Compounds which Inhibit EGF
Receptor Protein Tyrosine Kinase, Lyon & Lyon Docket No.
208/050), styryl-substituted pyridyl compounds (U.S. Pat. No.
5,302,606), certain quinazoline derivatives (EP Application No. 0
566 266 A1), seleoindoles and selenides (PCT WO 94/03427),
tricyclic polyhydroxylic compounds (PCT WO 92/21660), and
benzylphosphonic acid compounds (PCT WO 91/15495).
[0014] The compounds that can traverse cell membranes and are
resistant to acid hydrolysis are potentially advantageous
therapeutics as they can become highly bioavailable after being
administered orally to patients. However, many of these protein
kinase inhibitors only weakly inhibit the function of protein
kinases. In addition, many inhibit a variety of protein kinases and
will therefore cause multiple side-effects as therapeutics for
diseases.
[0015] Despite the significant progress that has been made in
developing compounds for the treatment of cancer, there remains a
need in the art to identify the particular structures and
substitution patterns that form the compounds capable of modulating
the function of particular protein kinases.
SUMMARY OF THE INVENTION
[0016] The present invention is directed in part towards indolinone
compounds and methods of modulating the function of protein kinases
with these compounds. In addition, the invention describes methods
of treating and preventing protein kinase-related abnormal
conditions in organisms with a compound identified by the methods
described herein. Furthermore, the invention pertains to
pharmaceutical compositions containing compounds identified by
methods of the invention.
[0017] The present invention features indolinone compounds that
potently inhibit receptor protein kinases of the c-kit family and
related products and methods. Other inhibitors and/or activators of
c-kit protein kinases can be obtained by adding chemical
substituents to an unsubstituted indolinone compound (See Formulas
I and II, below). The compounds of the invention provide
therapeutics and/or prophylactics for diseases associated with one
or more functional c-kit protein kinases. Certain types of cancer
fall into this class of diseases, along with certain immune
disorders associated with the over-production or over-stimulation
of mast cells. The compounds can be modified such that they are
specific to their target or targets and will subsequently cause few
side effects. These properties are significant improvements over
the currently utilized cancer therapeutics that cause multiple side
effects and deleteriously weaken patients.
[0018] The compounds, compositions, and methods of the invention
will minimize or obliterate certain types of solid tumors and
leukemias by inhibiting the activity of the c-kit receptor protein
kinases, or will at least modulate or inhibit tumor growth and/or
metastases. Certain types of cancer, such as Small Cell Lung Cancer
(SCLC), express both the c-kit receptor protein kinase and Stem
Cell Factor (SCF), a c-kit ligand.
[0019] While a precise understanding of the mechanism by which
compounds inhibit phosphotyrosine kinases (PTKs) (e.g., the c-kit
receptor kinase, a transmembrane tyrosine kinase growth factor
receptor) is not required in order to practice the present
invention, the compounds are believed to interact with the amino
acids of the PTKs' catalytic region. PTKs typically possess a
bi-lobate structure, and ATP appears to bind in the cleft between
the two lobes in a region where the amino acids are conserved among
PTKs; inhibitors of PTKs are believed to bind to the PTKs through
non-covalent interactions such as hydrogen bonding, Van der Waals
interactions, hydrophobic interactions, and ionic bonding, in the
same general region that ATP binds to the PTKs. More specifically,
it is thought that the oxindole component (See Formula III, below)
of the compounds of the present invention binds in the same general
space occupied by the adenine ring of ATP. Specificity of a PTK
inhibitor for a particular PTK may be conferred by interactions
between the constituents around the oxindole core with amino acid
domains specific to individual PKs. Thus, different substitutents
may contribute to preferential binding to particular PKs. The
ability to select those compounds active at different ATP binding
sites makes them useful in targeting any protein with such a site,
including not only protein tyrosine kinases, but also
serine/threonine kinases. Thus, such compounds have utility for in
vitro assays on such proteins and for in vivo therapeutic effect
through such proteins. For example, as mentioned above, certain
types of cancer express both the c-kit receptor protein kinase and
Stem Cell Factor (SCF) and this pairing could constitute an
autocrine loop stimulating the growth of these cancerous cells.
Therefore, inhibition of the c-kit protein kinase could disrupt
this autocrine loop and thereby retard tumor growth and/or
obliterate tumors via normal mechanisms of apoptosis.
[0020] Thus, in a first aspect, the invention provides a method for
treating or preventing an abnormal condition in an organism. The
abnormal condition is associated with an aberration in a signal
transduction pathway mediated by an interaction between a c-kit
kinase and a natural binding partner. The method involves
administering to the organism a therapeutically effective amount of
an indolinone compound. The indolinone compound modulates the
interaction between the c-kit kinase and a natural binding partner.
Therefore, promoting or disrupting (preferably disrupting) this
interaction is predicted to have therapeutic benefits to a given
population of patients in need of such treatment. In a preferred
embodiment, the amount of signaling through c-kit kinase is
abnormal, and the compound promotes or disrupts the signaling.
[0021] The term "treating" refers to having a therapeutic effect
and at least partially alleviating or abrogating an abnormal
condition in the organism. The term "treating" preferably refers to
ameliorating a symptom of the abnormal condition in a group of
patients to whom the indolinone is administered relative to a
control group that does not receive the indolinone. The effect of
the treatment can be monitored by measuring a change or an absence
of a change in cell phenotype, a change or an absence of a change
in cell proliferation, a change or an absence of a change in the
catalytic activity of this c-kit protein kinase, and a change or an
absence of a change in the interaction between this protein kinase
and a natural binding partner. The term "treating" or "treatment"
does not necessarily mean total cure. Any alleviation of any
undesired symptom of the disease to any extent or the slowing down
of the progress of the disease can be considered treatment.
Furthermore, treatment may include acts which may worsen the
patient's overall feeling of well being or appearance. For example,
the administration of chemotherapy in cancer patients which may
leave the patients feeling "sicker" is still considered
treatment.
[0022] The term "catalytic activity" used above, in the context of
the invention, defines the rate at which a protein kinase
phosphorylates a substrate. Catalytic activity can be measured, for
example, by determining the amount of a substrate converted to a
product as a function of time. Phosphorylation of a substrate
occurs at the active site of a protein kinase. The active site is
normally a cavity in which the substrate binds to the protein
kinase and is phosphorylated.
[0023] The term "substrate" as used above and herein refers to a
molecule phosphorylated by a protein kinase. The substrate is
preferably a peptide and more preferably a protein.
[0024] The term "preventing" refers to decreasing the probability
that an organism contracts or develops an abnormal condition. The
term "preventing" preferably refers to reducing the percentage of
individuals who develop the abnormal condition relative to a
control group that does not undergo administration of an
indolinone.
[0025] The term "abnormal condition" refers to a function in the
cells or tissues of an organism that deviates from its normal
functions in that organism. An abnormal condition can relate to
cell proliferation, cell differentiation, or cell survival.
Abnormal conditions include mastocytosis, the presence of one or
more mast cell tumors, asthma, allergy-associated chronic rhinitis,
small cell lung cancer, non-small cell lung cancer, acute
myelocytic leukemia, acute lymphocytic leukemia, myelodysplastic
syndrome, chronic myelogenous leukemia, colorectal carcinomas,
gastric carcinomas, gastrointestinal stromal tumors, testicular
cancers, glioblastomas, and astrocytomas. In a preferred
embodiment, these abnormal conditions, such as mast cell tumors and
mastocytosis, arise in non-human organisms and may thus be
prevented or treated during the practice of veterinary
medicine.
[0026] Abnormal cell survival conditions relate to conditions in
which programmed cell death (apoptosis) pathways are activated or
abrogated. A number of protein kinases are associated with the
apoptosis pathways. Aberrations in the function of any one of the
protein kinases could lead to cell immortality or premature cell
death.
[0027] The term "function" as used in relation to a protein kinase
above refers to the cellular role of a protein kinase, preferably a
c-kit kinase. The protein kinase family includes members that
regulate many steps in signaling cascades, including cascades
controlling cell growth, migration, differentiation, gene
expression, muscle contraction, glucose metabolism, cellular
protein synthesis, and regulation of the cell cycle. The "function"
of a membrane receptor kinase usually is to transduce a signal from
outside a cell's membrane to the interior of a cell. To accomplish
this it may perform one or all of these other functions: bind a
ligand, dimerize to another membrane receptor kinase, phosphorylate
other proteins within the cell, bind other proteins within the
cell, and cause the localization of proteins within the cell.
[0028] The term "organism" relates to any living entity comprised
of at least one cell. An organism can be as simple as one
eukaryotic cell or as complex as a mammal. The organism is
preferably a mammal, more preferably a human.
[0029] The term "mammal" refers preferably to such organisms as
mice, rats, rabbits, guinea pigs, sheep, and goats, more preferably
to cats, dogs, monkeys, and apes. In preferred embodiments, the
abnormal condition associated with mammals may include
mastocytosis, and the presence of one or more mast cell tumors.
[0030] The term "aberration," refers to a protein kinase, e.g., a
c-kit kinase that is over- or under-expressed in an organism,
mutated such that its catalytic activity is lower or higher than
wild-type protein kinase activity, mutated such that it can no
longer interact with a natural binding partner, no longer functions
in an autocrine loop within the cell, is no longer modified by
another protein kinase or protein phosphatase, or no longer
interacts with a natural binding partner. Preferably, the
aberration involves excessive or deficient signaling upon
interaction with a natural binding partner.
[0031] The term "signal transduction pathway" refers to the
molecules that propagate an extracellular signal through the cell
membrane to become an intracellular signal. This signal can then
stimulate a cellular response. The polypeptide molecules involved
in signal transduction processes include receptor and non-receptor
protein tyrosine kinases, receptor and non-receptor protein
phosphatases, proteins containing SRC homology 2 and 3 domains,
phosphotyrosine binding proteins (SRC homology 2 (SH2) and
phosphotyrosine binding (PTB and PH) domain containing proteins),
proline-rich binding proteins (SH3 domain containing proteins),
GTPases, phosphodiesterases, phospholipases, prolyl isomerases,
proteases, Ca.sup.2+ binding proteins, cAMP binding proteins,
guanyl cyclases, adenylyl cyclases, NO generating proteins,
nucleotide exchange factors, and transcription factors.
[0032] The term "mediated" refers to involvement in the control or
effect of the interaction between c-kit kinase and the natural
binding partners on the aberration in the signal transduction
pathway. Thus, the signal transduction pathway that has an
aberration and is associated with the abnormal condition, contains
a c-kit kinase in interaction with a natural binding partner.
[0033] The "interaction" of a c-kit kinase molecule is the binding
of that c-kit kinase molecule to a natural binding partner or
molecule within the cell or the phosphorylation by a c-kit kinase
molecule of another protein or molecule within the cell, or any
other association of c-kit kinase within a cell. These interactions
include non-covalent interactions such as hydrogen bonding, Van der
Waals interactions, hydrophobic interactions, and ionic
bonding.
[0034] The term "c-kit kinase" refers to a membrane receptor
protein tyrosine kinase which is preferably activated upon binding
Stem Cell Factor (SCF) to its extracellular domain (Yarden et al.,
1987; Qiu et al., 1988). The receptor tyrosine kinase c-kit kinase
contains 5 immunoglobulin-like motifs in the extracellular domain
and a cytoplasmic "split" kinase domain, FIG. 1. The full length
amino acid sequence of a c-kit kinase preferably is as set forth in
Yarden, et al., 1987, EMBO J. 11:3341-3351; and Qiu, et al., 1988,
EMBO J 7:1003-1011, which are incorporated by reference herein in
their entirety, including any drawings. Mutant versions of c-kit
kinase are encompassed by the term "c-kit kinase" and include those
that fall into two classes: (1) having a single amino acid
substitution at codon 816 of the human c-kit kinase, or its
equivalent position in other species (Ma et al., 1999, J. Invest
Dermatol 112:165-170), and (2) those which have mutations involving
the putative juxtamembrane z-helix of the protein (Ma, et al.,
1999, J Biol Chem 274:13399-13402). Both of these publications are
incorporated by reference herein in their entirety, including any
drawings.
[0035] The term "natural binding partner" refers to a polypeptide
or compound such as ATP that binds to a protein kinase in cells.
Natural binding partners can play a role in propagating a signal in
a protein kinase signal transduction process. A change in the
interaction between a protein kinase and a natural binding partner
can manifest itself as an increased or decreased probability that
the interaction forms, or an increased or decreased concentration
of the protein kinase/natural binding partner complex.
[0036] 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. A "therapeutically
effective amount," in reference to the treatment of a cancer refers
to an amount sufficient to bring about one or more of the following
results: reduce the size of the cancer, inhibit the metastasis of
the cancer, inhibit the growth of the cancer, stop the growth of
the cancer, relieve discomfort due to the cancer, or prolong the
life of a patient inflicted with the cancer. A "therapeutically
effective amount", in reference to the treatment of a cell
proliferative disorder other than a cancer refers to an amount
sufficient to bring about one or more of the following results:
inhibit the growth of cells causing the disorder, relieve
discomfort due to the disorder, or prolong the life of a patient
suffering from the disorder.
[0037] The term "indolinone" is used as that term is commonly
understood in the art and includes a large subclass of substituted
or unsubstituted compounds that are capable of being synthesized
from an aldehyde moiety and an oxindole moiety. In preferred
embodiments, the indolinones included in the present method have
the structures of Formulas I and II (see below), and more
preferably are selected from Compounds One through Thirteen (see
below).
[0038] Examples of representative indolinone compounds and the
synthesis thereof, are set forth in the following applications: (1)
PCT application number US99/06468, filed Mar. 26, 1999 by Fong, et
al. and entitled METHODS OF MODULATING TYROSINE PROTEIN KINASE
(Lyon & Lyon docket number 231/250 PCT), (2) U.S. Provisional
Application No. 60/131,192, filed Apr. 26, 1999 by Tang, et al. and
entitled DIARYL INDOLINONE COMPOUNDS AS KINASE INHIBITORS (Lyon
& Lyon docket number 239/205), (3) U.S. Provisional Application
No. 60/132,243, filed May 3, 1999 by Tang, et al. and entitled
SYNTHESIS OF 4-SUBSTITUTED OXINDOLE AND INDOLINONE COMPOUNDS AND
THEIR USE IN TREATMENT OF DISEASE (Lyon & Lyon docket number
231/251), (4) U.S. application Ser. No. 09/283,657, filed Apr. 1,
1999 by Tang, et al. and entitled METHODS OF MODULATING TYROSINE
PROTEIN KINASE FUNCTION WITH INDOLINONE COMPOUNDS (Lyon & Lyon
docket number 241/180), and (5) U.S. Pat. No. 5,792,783, issued
Aug. 11, 1998 by Tang et al., entitled 3-HETEROARYL-2-INDOLINONE
COMPOUNDS FOR THE TREATMENT OF DISEASE which are hereby
incorporated by reference in their entirety including any
drawings.
[0039] Preferably, the compounds used in the invention have a
structure set forth in Formula I, 1
[0040] wherein
[0041] (a) Y is selected from the group consisting of oxygen,
sulfur and nitrogen substituted with a hydrogen;
[0042] (b) R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each
independently selected from the group consisting of hydrogen,
alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen,
trihalomethyl, S(O)R, SO.sub.2 NRR', SO.sub.3 R, SR, NO.sub.2,
NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH.sub.2).sub.n CO.sub.2 R,
and CONRR';
[0043] (c) R.sub.5 is selected from the group consisting of
hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy,
halogen, trihalomethyl, S(O)R, SO.sub.2 NRR', SO.sub.3 R, SR,
NO.sub.2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH.sub.2).sub.n
CO.sub.2 R, CONRR', a six-membered heteroaryl ring system
containing 1 or 2 N, O, or S atoms; and a six-membered aryl ring
system; and
[0044] (c) R.sub.6, and R.sub.7 are each independently selected
from the group consisting of hydrogen, alkyl, alkoxy, aryl,
aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R,
SO.sub.2 NRR', SO.sub.3 R, SR, NO.sub.2, NRR', OH, CN, C(O)R,
OC(O)R, NHC(O)R, (CH.sub.2).sub.n CO.sub.2 R, and CONRR', where R
can be a wide variety of substituent groups.
[0045] More preferably, the compounds used in the invention have a
structure set forth in Formula II, 2
[0046] wherein
[0047] (a) Y is selected from the group consisting of sulfur and
nitrogen substituted with a hydrogen;
[0048] (b) R.sub.1 is selected from the group consisting of
hydrogen and methyl;
[0049] (c) R.sub.2 is selected from the group consisting of
[0050] (i) hydrogen;
[0051] (ii) chlorine;
[0052] (iii) bromine;
[0053] (iv) a ketone of the formula --CO--CH.sub.3,
[0054] (v) a sulfonamide of the formula --SO.sub.2NH.sub.2, or
--SO.sub.2NCH.sub.3CH.sub.3
[0055] (d) R.sub.3, R.sub.4, and R.sub.5 are each independently
selected from the group consisting of
[0056] (i) hydrogen;
[0057] (ii) methyl;
[0058] (iii) a carboxylic acid of formula --(CH.sub.2).sub.2--COOH;
and
[0059] (iv) R.sub.8 and R.sub.9 taken together form a six-membered
saturated carbon ring.
[0060] Most preferably, the compound is one of the following:
[0061] Compound One, below: 3
[0062] Compound Two, below: 4
[0063] Compound Three, below: 5
[0064] Compound Four, below: 6
[0065] Compound Five, below: 7
[0066] Compound Six, below: 8
[0067] Compound Seven, below: 9
[0068] Compound Eight, below: 10
[0069] Compound Nine, below: 11
[0070] Compound Ten, below 12
[0071] Compound Eleven, below: 13
[0072] Compound Twelve, below: 14
[0073] Compound Thirteen, below: 15
[0074] Compound Fourteen, below: 16
[0075] Compound Fifteen, below: 17
[0076] Compound Sixteen, below: 18
[0077] The term "oxindole" refers to an oxindole compound
substituted with chemical substituents. Oxindole compounds are of
the general structure shown in Formula III: 19
[0078] The term "substituted", in reference to the invention,
refers to an oxindole compound that is derivatized with any number
of chemical substituents.
[0079] The indolinone compounds of the invention preferably
modulate the activity of the protein tyrosine kinase in vitro.
These compounds preferably show positive results in one or more in
vitro assays for an activity corresponding to treatment of the
disease or disorder in question (such as the assays described in
the Examples below). The protein tyrosine kinase which is modulated
by the indolinone compounds of the invention is preferably the
c-kit kinase. Examples of the procedures for and the results of
such modulation are described in the Examples below.
[0080] The term "compound" means any identifiable molecule or a
pharmaceutically acceptable salt, ester, amide, prodrug, isomer, or
metabolite, thereof.
[0081] 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 then is
metabolically hydrolyzed to the carboxylic acid, the active entity,
once inside the cell where water solubility is beneficial.
[0082] A further example of a prodrug might be a short polypeptide,
for example, without limitation, a 2-10 amino acid polypeptide,
bonded through a terminal amino group to a carboxy group of a
compound of this invention wherein the polypeptide is hydrolyzed or
metabolized in vivo to release the active molecule.
[0083] The term "modulates" refers to altering the function of a
protein kinase by increasing or decreasing the probability that a
complex forms between a protein kinase and a natural binding
partner. A modulator preferably increases the probability that such
a complex forms between the protein kinase and the natural binding
partner, more preferably increases or decreases the probability
that a complex forms between the protein kinase and the natural
binding partner depending on the concentration of the compound
exposed to the protein kinase, and most preferably decreases the
probability that a complex forms between the protein kinase and the
natural binding partner. A modulator preferably activates the
catalytic activity of a protein kinase, more preferably activates
or inhibits the catalytic activity of a protein kinase depending on
the concentration of the compound exposed to the protein kinase, or
most preferably inhibits the catalytic activity of a protein
kinase.
[0084] The term "complex" refers to an assembly of at least two
molecules bound to one another. Signal transduction complexes often
contain at least two protein molecules bound to one another.
[0085] The term "activates" refers to increasing the function of a
protein kinase. The protein kinase function is preferably the
interaction with a natural binding partner and most preferably
catalytic activity.
[0086] The term "inhibit" refers to decreasing the function of a
protein kinase. The protein kinase function is preferably the
interaction with a natural binding partner and most preferably
catalytic activity.
[0087] A protein kinase's natural binding partner can bind to a
protein kinase's extracellular or intracellular region with high
affinity. High affinity represents an equilibrium binding constant
on the order of 10.sup.-6 M or less. In addition, a natural binding
partner can also transiently interact with a protein kinase's
extracellular or intracellular region and chemically modify it.
Protein kinase natural binding partners are chosen from a group
that includes, but is not limited to, SRC homology 2 (SH2) or 3
(SH3) domains, other phosphoryl tyrosine binding (PTB) domains,
guanine nucleotide exchange factors, protein phosphatases, other
protein kinases, and compounds such as ATP. Methods of determining
changes in interactions between protein kinases and their natural
binding partners are readily available in the art.
[0088] The term "related to" refers to a disease which has been
shown to be accompanied by inappropriate c-kit kinase expression
when compared to the same undiseased tissue isolated from an
organism. The inappropriate expression can be an elevation of
normal activities, a depression of normal activities, or the
presence of c-kit kinase activity where none is normally found.
[0089] The term "in vitro" refers to when the c-kit kinase enzyme
is tested outside of a living organism with a compound useful for
this invention whereby such compounds are screened for
efficaciousness. The term "in vitro" includes the use of tissue
culture cells.
[0090] The term "promotes or disrupts the abnormal interaction"
refers to a method that can be accomplished by administering a
compound of the invention to cells or tissues in an organism. A
compound can promote an interaction between a protein kinase and
natural binding partners by forming favorable interactions with
multiple atoms at the complex interface. Alternatively, a compound
can inhibit an interaction between a protein kinase and natural
binding partners by compromising favorable interactions formed
between atoms at the complex interface. In preferred embodiments,
the promotion or disruption of an abnormal interaction refers to
the compound of the invention promoting a conformational change in
one of the proteins.
[0091] In another aspect, the invention relates to synthesis of
indolinone compounds described herein, in particular indolinones of
Formula I, above, and especially Compounds One through Thirteen.
The general scheme for the synthesis of representative indolinone
compounds are set forth in the PCT publication US99/06468, filed
Mar. 26, 1999 by Fong et al. and entitled METHODS OF MODULATING
TYROSINE PROTEIN KINASE (Lyon & Lyon docket number 231/250 PCT)
and the U.S. Pat. No. 5,792,783, issued Aug. 11, 1998 by Tang et
al., entitled 3-HETEROARYL-2-INDOLINONE COMPOUNDS FOR THE TREATMENT
OF DISEASE which are hereby incorporated by reference in their
entirety including any drawings. Those skilled in the art know by
reviewing the above reference which oxindoles and which aldehydes
are to be reacted under which suitable conditions to form the
compounds of the present invention.
[0092] The invention also features a method of identifying
indolinone compounds, or compounds such as a pharmaceutically
acceptable salt, ester, amide, prodrug, isomer, or metabolite
thereof, that modulate the function of c-kit kinase, comprising the
following steps: (a) contacting cells expressing the c-kit kinase
with the compound; and (b) monitoring an effect upon the cells. The
effect upon the cells is preferably a change or an absence of a
change in cell phenotype, more preferably it is a change or an
absence of a change in cell proliferation, even more preferably it
is a change or absence of a change in the catalytic activity of the
c-kit kinase, and most preferably it is a change or absence of a
change in the interaction between the c-kit kinase with a natural
binding partner, as described herein.
[0093] The term "monitoring" refers to observing the effect of
adding the compound to the cells of the method. The "monitoring"
can be effected by comparing test cells with control cells.
[0094] The term "effect" describes a change or an absence of a
change in cell phenotype or cell proliferation. "Effect" can also
describe a change or an absence of a change in the catalytic
activity of the pro c-kit kinase. "Effect" can also describe a
change or an absence of a change in an interaction between the
c-kit kinase and a natural binding partner.
[0095] The term "cell phenotype" refers to the outward appearance
of a cell or tissue or the function of the cell or tissue. Examples
of cell phenotype are cell size (reduction or enlargement), cell
proliferation (increased or decreased numbers of cells), cell
differentiation (a change or absence of a change in cell shape,
cell function, or differences in protein expression), cell
survival, apoptosis (cell death), or the utilization of a metabolic
nutrient (e.g., glucose uptake). Changes or the absence of changes
in cell phenotype are readily measured by techniques known in the
art.
[0096] In a preferred embodiment, the invention features a method
for identifying the indolinones of the invention, comprising the
following steps: (a) lysing the cells to render a lysate comprising
c-kit kinase; (b) adsorbing the c-kit kinase to an antibody; (c)
incubating the adsorbed c-kit kinase with a substrate or
substrates; and (d) adsorbing a detecting antibody to the c-kit
kinase. The effect upon the kinases is then monitored and the step
of monitoring the effect on the kinases comprises measuring the
phosphate concentration incorporated into c-kit kinase.
[0097] The term "antibody" refers to an antibody (e.g., a
monoclonal or polyclonal antibody), or antibody fragment, having
specific binding affinity to c-kit kinase or its fragment or to
phosphotyrosine.
[0098] By "specific binding affinity" is meant that the antibody
binds to target (c-kit kinase) polypeptides with greater affinity
than it binds to other polypeptides under specified conditions.
Antibodies having specific binding affinity to a c-kit kinase may
be used in methods for detecting the presence and/or amount of a
c-kit kinase in a sample by contacting the sample with the antibody
under conditions such that an immunocomplex forms and detecting the
presence and/or amount of the antibody conjugated to the c-kit
kinase. Diagnostic kits for performing such methods may be
constructed to include a first container containing the antibody
and a second container having a conjugate of a binding partner of
the antibody and a label, such as, for example, a radioisotope. The
diagnostic kit may also include notification of an FDA approved use
and instructions therefor.
[0099] The term "polyclonal" refers to antibodies that are
heterogenous populations of antibody molecules derived from the
sera of animals immunized with an antigen or an antigenic
functional derivative thereof. For the production of polyclonal
antibodies, various host animals may be immunized by injection with
the antigen. Various adjuvants may be used to increase the
immunological response, depending on the host species.
[0100] "Monoclonal antibodies" are substantially homogenous
populations of antibodies to a particular antigen. They may be
obtained by any technique which provides for the production of
antibody molecules by continuous cell lines in culture. Monoclonal
antibodies may be obtained by methods known to those skilled in the
art. See, for example, Kohler, et al., Nature 256:495-497 (1975),
and U.S. Pat. No. 4,376,110.
[0101] The term "antibody fragment" refers to a portion of an
antibody, often the hypervariable region and portions of the
surrounding heavy and light chains, that displays specific binding
affinity for a particular molecule. A hypervariable region is a
portion of an antibody that physically binds to the polypeptide
target.
[0102] The summary of the invention described above is non-limiting
and other features and advantages of the invention will be apparent
from the following description of the preferred embodiments, and
from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0103] FIG. 1 is a schematic diagram showing the 5
immunoglobulin-like motifs in the extracellular domain and a
cytoplasmic "split" kinase domain of c-kit kinase. The half-loops
represent the immunoglobulin-like motifs, and the shaded boxes
represent the conserved kinase region of the receptors.
[0104] FIG. 2 shows the effects of indolinone derivatives on the
activity of c-kit kinase as measured by ELISA as described in
Example 1 in the experimental section.
DETAILED DESCRIPTION OF THE INVENTION
[0105] The present invention relates to methods, compounds and
compositions capable of regulating and/or modulating cellular
signal transduction and, in preferred embodiments, c-kit kinase
signal transduction.
[0106] Receptor kinase-mediated signal transduction is initiated by
extracellular interaction with a specific growth factor (ligand),
followed by receptor dimerization, transient stimulation of the
intrinsic protein kinase activity, and phosphorylation. Binding
sites are thereby created for intracellular signal transduction
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 to the
extracellular microenvironment). See, Schlessinger and Ullrich,
1992, Neuron 9:303-391.
[0107] Kinase signal transduction results in, among other
responses, cell proliferation, differentiation and metabolism.
Abnormal cell proliferation may result in a wide array of disorders
and diseases, including the development of neoplasia such as
carcinoma, sarcoma, leukemia, glioblastoma, hemangioma, psoriasis,
arteriosclerosis, arthritis and diabetic retinopathy (or other
disorders related to uncontrolled angiogenesis and/or
vasculogenesis).
[0108] This invention is therefore directed to methods, compounds,
and compositions which regulate, modulate and/or inhibit kinase
signal transduction by affecting the enzymatic activity of receptor
kinases and interfering with the signal transduced by such
proteins. More particularly, the present invention is directed to
methods, compounds and compositions which regulate, modulate and/or
inhibit the c-kit receptor tyrosine kinase and/or other kinase
mediated signal transduction pathways as a therapeutic approach to
cure many kinds of solid tumors and leukemias, including but not
limited to carcinoma, sarcoma, erythroblastoma, glioblastoma,
meningioma, astrocytoma, melanoma and myoblastoma. Indications may
include, but are not limited to lung cancers, including both small
cell lung cancers and non-small cell lung cancers, brain cancers,
bladder cancers, ovarian cancers, gastric cancers, pancreas
cancers, colon cancers, blood cancers, and bone cancers. The
present invention is also directed to the treatment and/or
prevention of those conditions characterized by the overexpression
of mast cells, or the inappropriate up-regulation of mast cells,
including, but not limited to, mastocytosis, and allergy-associated
chronic rhinitis, inflammation and asthma. These conditions are
described in greater detail below.
[0109] I. Target Diseases to be Treated by the Compounds of the
Invention.
[0110] The compounds described herein are useful for treating
disorders related to unregulated kinase signal transduction,
including cell proliferative disorders, fibrotic disorders and
metabolic disorders. Cell proliferative disorders which can be
treated or further studied by the present invention include
cancers, and mast cell proliferative disorders.
[0111] PTKs have been associated with such cell proliferative
disorders. For example, some members of the receptor tyrosine
kinase (RTK) family have been associated with the development of
cancer. Some of these receptors, like the EGFR (Tuzi, et al., 1991,
Br. J. Cancer 63:227-233; Torp, et al., 1992, APMIS 100:713-719)
HER2/neu (Slamon, et al., 1989, Science 244:707-712) and the PDGF-R
(Kumabe, et al., 1992, Oncogene 7:627-633) are overexpressed in
many tumors and/or persistently activated by autocrine loops. In
fact, in the most common and severe cancers these receptor
overexpressions (Akbasak and Suner-Akbasak, et al., 1992, J.
Neurol. Sci. 111:119-133; Dickson, et al., 1992, Cancer Treatment
Res. 61:249-273; Korc, et al., 1992, J. Clin. Invest. 90:1352-1360)
and autocrine loops (Lee and Donoghue, 1992, J. Cell. Biol.
118:1057-1070; Korc, et al., supra; Akbasak and Suner-Akbasak, et
al., supra) have been demonstrated. For example, the EGFR receptor
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. The PDGF-R has been associated with
glioblastoma, lung, ovarian, melanoma and prostate cancer.
[0112] The c-kit receptor kinase has been associated with such cell
proliferative disorders. For example, the c-kit kinase receptor has
been found to be aberrantly expressed in over half the SCLC cells
studied along with its ligand SCF (Hibi, et al., 1991, Oncogene
6:2291-2296). Potentially, inhibition of the c-kit kinase will
improve the long term survival of patients with SCLC.
[0113] The presence of c-kit RTK and/or SCF has also been
associated with other types of cancers, as described below. The
association between abnormalities in RTKs and disease are not
restricted to cancer, however. For example, the c-kit Receptor
Kinase has been associated with immune diseases such as
mastocytosis, asthmas and chronic rhinitis. Excessive activation of
c-kit might be associated with diseases resulting from an
over-abundance of mast cells. Mastocytosis is the term used to
describe a heterogeneous series of disorders characterized by
excessive mast cell proliferation (Metcalfe, 1991, J. Invest. Derm
93:2S-4S; Valent, 1996, Wein/Klin Wochenschr 108:385-397; and
Golkar, et al., 1997, Lancet 349:1379-1385). Elevated c-kit
expression was reported on mast cells from patients with aggressive
mastocytosis, but not on mast cells from patients with indolent
mastocytosis (Nagata, et al., 1998, Leukemia 12:175-181).
[0114] Additionally, mast cells and eosinophils represent key cells
involved in allergy, inflammation and asthma (Thomas, et al., 1996,
Gen. Pharmacol 27:593-597; Metcalfe, et al., 1997, Physiol Rev
77:1033-1079; Holgate, 1997, CIBA Found. Symp.; Naclerio, et al.,
1997, JAMA 278:1842-1848 and Costa, et al., 1997, JAMA
278:1815-1822). SCF, and hence c-kit, directly and indirectly
regulates activation of both mast cells and eosinophils, thereby
influencing the primary cells involved in allergy and asthma
through multiple mechanisms. Because of this mutual regulation of
mast cell and eosinophil function, and the role that SCF can play
in this regulation, inhibition of c-kit Kinase may provide a means
to treat allergy-associated chronic rhinitis, inflammation and
asthma.
[0115] II. c-kit Kinase
[0116] The c-kit kinase plays a critical role in the development of
melanocytes, mast, germ and hematopoietic cells. The protein
encoded by the S1 locus has been called kit ligand (KL), stem cell
factor (SCF) or mast cell growth factor (MGF), based on its
biological properties used to identify it (reviewed in Tsujimura,
1996, Pathol Int 46:933-938; Loveland, et al., 1997, J. Endocrinol
153:337-344; Vliagoftis, et al., 1997, Clin Immunol 100:435-440;
Broudy, 1997, Blood 90:1345-1364; Pignon, 1997, Hermatol Cell Ther
39:114-116; and Lyman, et al., 1998, Blood 91:1101-1134.). For
simplicity, we will use SCF to designate the ligand for the c-kit
RTK. SCF is synthesized as a transmembrane protein with a molecular
weight of 220 or 248 Dalton, depending on alternative splicing of
the mRNA to encode exon 6. The larger protein can be
proteolytically cleaved to form a soluble, glycosylated protein
which noncovalently dimerizes. Both the soluble and membrane-bound
forms of SCF can bind to and activate c-kit. For example, in the
skin, SCF is predominantly expressed by fibroblasts, keratinocytes,
and endothelial cells, which modulate the activity of melanocytes
and mast cells expressing c-kit. In bone, marrow stromal cells
express SCF and regulate hematopoiesis of c-kit expressing stem
cells. In the gastrointestinal tract, intestinal epithelial cells
express SCF and affect the interstitial cells of Cajal and
intraepithelial lymphocytes. In the testis, sertoli cells and
granulosa cells express SCF which regulates spermatogenesis by
interaction with c-kit on germ cells.
[0117] a. Target Malignancies of the Present Invention Involving
c-kit Kinase and/or SCF
[0118] Aberrant expression and/or activation of c-kit has been
implicated in a variety of tumors. Evidence for a contribution of
c-kit to neoplastic pathology includes its association with
leukemias and mast cell tumors, small cell lung cancer, testicular
cancer, and some cancers of the gastrointestinal tract and central
nervous system (see below). In addition, c-kit has been implicated
in playing a role in carcinogenesis of the female genital tract
(Inoue, et al., 1994, Cancer Res. 54(11):3049-3053), sarcomas of
neuroectodermal origin (Ricotti, et al., 1998, Blood 91:2397-2405),
and Schwann cell neoplasia associated with neurofibromatosis (Ryan,
et al., 1994, J. Neuro. Res. 37:415-432).
[0119] Leukemias: SCF binding to the c-kit RTK protects
hematopoietic stem and progenitor cells from apoptosis (Lee, et
al., 1997, J. Immunol. 159:3211-3219), thereby contributing to
colony formation and hematopoiesis. Expression of c-kit is
frequently observed in acute myelocytic leukemia (AML), but is less
common in acute lymphocytic leukemia (ALL) (for reviews, see
Sperling, et al., 1997, Haemat 82:617-621; Escribano, et al., 1998,
Leuk. Lymph. 30:459-466). Although c-kit is expressed in the
majority of AML cells, its expression does not appear to be
prognostic of disease progression (Sperling, et al, 1997, Haemat
82:617-621). However, SCF protected AML cells from apoptosis
induced by chemotherapeutic agents (Hassan, et al., 1996, Acta.
Hem. 95:257-262). Inhibition of c-kit by the present invention will
enhance the efficacy of these agents and may induce apoptosis of
AML cells.
[0120] The clonal growth of cells from patients with
myelodysplastic syndrome (Sawada, et al., 1996, Blood 88:319-327)
or chronic myelogenous leukemia (CML) (Sawai, et al., 1996, Exp.
Hem. 2:116-122) was found to be significantly enhanced by SCF in
combination with other cytokines. CML is characterized by expansion
of Philadelphia chromosome positive cells of the marrow
(Verfaillie, et al., 1998, Leuk. 12:136-138), which appears to
primarily result from inhibition of apoptotic death (Jones, 1997,
Curr. Opin. Onc. 9:3-7). The product of the Philadelphia
chromosome, p210.sup.BCR-ABL, has been reported to mediate
inhibition of apoptosis (Bedi, et al., 1995, Blood 86:1148-1158).
Since p210.sup.BCR-ABL and the c-kit RTK both inhibit apoptosis and
p62.sup.dok has been suggested as a substrate (Carpino, et al.,
1997, Cell 88:197-204), it is possible that clonal expansion
mediated by these kinases occurs through a common signaling
pathway. However, c-kit has also been reported to interact directly
with p210.sup.BCR-ABL (Hallek, et al., 1996, Brit. J Haem.
94:5-16), which suggests that c-kit may have a more causative role
in CML pathology. Therefore, inhibition of c-kit kinase will prove
useful in the treatment of the above disorders.
[0121] Gastrointestinal cancers: Normal colorectal mucosa does not
express c-kit (Bellone, et al., 1997, J. Cell Physiol. 172:1-11).
However, c-kit is frequently expressed in colorectal carcinoma
(Bellone, et al., 1997, J. Cell Physiol. 172: 1-11), and autocrine
loops of SCF and c-kit have been observed in several colon
carcinoma cell lines (Toyota, et al., 1993, Turn Biol 14:295-302;
Lahm, et al., 1995, Cell Growth &Differ 6:1111-1118; Bellone,
et al., 1997, J. Cell Physiol. 172:1-11). Furthermore, disruption
of the autocrine loop by the use of neutralizing antibodies (Lahm,
et al., 1995, Cell Growth & Differ. 6:1111-1118) and
downregulation of c-kit and/or SCF significantly inhibits cell
proliferation (Lahm, et al., 1995, Cell Growth & Differ
6:1111-1118; Bellone, et al., 1997, J. Cell Physiol. 172:1-11).
[0122] SCF/c-kit autocrine loops have been observed in gastric
carcinoma cell lines (Turner, et al., 1992, Blood 80:374-381;
Hassan, et al., 1998, Digest. Dis. Science 43:8-14), and
constitutive c-kit activation also appears to be important for
gastrointestinal stromal tumors (GISTs). GISTs are the most common
mesenchymal tumor of the digestive system. More than 90% of GISTs
express c-kit, which is consistent with the putative origin of
these tumor cells from interstitial cells of Cajal (ICCs) (Hirota,
et al., 1998, Science 279:577-580). ICCs are thought to regulate
contraction of the gastrointestinal tract, and patients lacking
c-kit in their ICCs exhibited a myopathic form of chronic
idiopathic intestinal pseudo-obstruction (Isozaki, et al., 1997,
Amer. J. of Gast. 9 332-334). The c-kit expressed in GISTs from
several different patients was observed to have mutations in the
intracellular juxtamembrane domain leading to constitutive
activation of this RTK (Hirota, et al., 1998, Science 279:577-580).
Hence, inhibition of c-kit kinase will be an efficacious means for
the treatment of these cancers.
[0123] Testicular cancers: Male germ cell tumors have been
histologically categorized into seminomas, which retain germ cell
characteristics, and nonseminomas which can display characteristics
of embryonal differentiation. Both seminomas and nonseminomas are
thought to initiate from a preinvasive stage designated carcinoma
in situ (CIS) (Murty, et al., 1998, Sem. Oncol. 25:133-144). Both
c-kit and SCF have been reported to be essential for normal gonadal
development during embryogenesis (Loveland, et al., 1997, J.6
Endocrinol 153:337-344). Loss of either the receptor or the ligand
resulted in animals devoid of germ cells. In postnatal testes,
c-kit has been found to be expressed in Leydig cells and
spermatogonia, while SCF was expressed in Sertoli cells (Loveland,
et al., 1997, J. Endocrinol 153:337-344). Testicular tumors develop
from Leydig cells with high frequency in transgenic mice expressing
human papilloma virus 16 (HPV16) E6 and E7 oncogenes (Kondoh, et
al., 1991, J. Virol. 65:3335-3339; Kondoh, et al., 1994, J. Urol.
152:2151-2154). These tumors express both c-kit and SCF, and an
autocrine loop may contribute to the tumorigenesis (Kondoh, et al.,
1995, Oncogene 10:341-347) associated with cellular loss of
functional p53 and the retinoblastoma gene product by association
with E6 and E7 (Dyson, et al., 1989, Science 243:934-937; Werness,
et al., 1990, Science 248:76-79; Scheffner, et al., 1990, Cell
63:1129-1136). Defective signaling mutants of SCF (Kondoh, et al.,
1995, Oncogene 10:341-347) or c-kit (Li, et al., 1996, Canc. Res.
56:4343-4346) inhibited formation of testicular tumors in mice
expressing HPV16 E6 and E7. The c-kit kinase activation is pivotal
to tumorigenesis in these animals and thus modulation of the c-kit
kinase pathway by the present invention will prevent or treat such
disorders.
[0124] Expression of c-kit on germ cell tumors shows that the
receptor is expressed by the majority of carcinomas in situ and
seminomas, but c-kit is expressed in only a minority of
nonseminomas (Strohmeyer, et al., 1991, Canc. Res. 51:1811-1816;
Rajpert-de Meyts, et al., 1994, Int. J. Androl. 17:85-92;
Izquierdo, et al., 1995, J. Pathol. 177:253-258; Strohmeyer, et
al., 1995, J. Urol. 153:511-515; Bokenmeyer, et al., 1996, J.
Cance. Res. Clin. Oncol. 122:301-306; Sandlow, et al., 1996, J.
Androl. 17:403-408). Therefore, inhibition of c-kit kinase will
provide a valuable new means for treating these disorders.
[0125] CNS cancers: SCF and c-kit are expressed throughout the CNS
of developing rodents, and the pattern of expression suggests a
role in growth, migration and differentiation of neuroectodermal
cells. Expression of both receptor and ligand have also been
reported in the adult brain (Hamel, et al., 1997, J. Neuro-Onc.
35:327-333). Expression of c-kit has also been observed in normal
human brain tissue (Tada, et al. 1994, J. Neuro 80:1063-1073).
Glioblastoma and astrocytoma, which define the majority of
intracranial tumors, arise from neoplastic transformation of
astrocytes (Levin, et al., 1997, Principles & Practice of
Oncology:2022-2082). Expression of c-kit has been observed in
glioblastoma cell lines and tissues (Berdel, et al., 1992, Canc.
Res. 52:3498-3502; Tada, et al. 1994, J. Neuro 80:1063-1073;
Stanulla, et al., 1995, Act Neuropath 89:158-165).
[0126] The association of c-kit with astrocytoma pathology is less
clear. Reports of expression of c-kit in normal astrocytes have
been made (Natali, et al., 1992, Int. J. Canc. 52:197-201), (Tada,
et al. 1994, J. Neuro 80:1063-1073), while others report it is not
expressed (Kristt, et al., 1993, Neuro. 33:106-115). In the latter
case, high levels of c-kit expression in high grade tumors were
observed (Kristt, et al., 1993, Neuro. 33:106-115), while the
former groups were unable to detect any expression in astrocytomas.
In addition, contradictory reports of c-kit and SCF expression in
neuroblastomas also exist. One study found that neuroblastoma cell
lines often express SCF, but rarely express c-kit. In primary
tumors, c-kit was detected in about 8% of neuroblastomas, while SCF
was found in 18% of tumors (Beck, et al., 1995, Blood
86:3132-3138). In contrast, other studies (Cohen, et al., 1994,
Blood 84:3465-3472) have reported that all 14 neuroblastoma cell
lines examined contained c-kit/SCF autocrine loops, and expression
of both the receptor and ligand were observed in 45% of tumor
samples examined. In two cell lines, anti-c-kit antibodies
inhibited cell proliferation, suggesting that the SCF/c-kit
autocrine loop contributed to growth (Cohen, et al., 1994, Blood
84:3465-3472). Hence, c-kit kinase inhibitors will prove
therapeutically useful as a means to treat these cancers.
[0127] b. Target Mast Cell Diseases Involving c-kit Kinase and/or
SCF to be Treated/Prevented by the Present Invention.
[0128] Mastocytosis: As mentioned above, SCF (also known as mast
cell growth factor) stimulation of c-kit has been reported to be
essential for the growth and development of mast cells (Hamel, et
al., 1997, J. Neuro-Onc. 35:327-333; Kitamura, et al., 1995, Int.
Arch. Aller. Immunol. 107:54-56). Mice with mutations of c-kit that
attenuate its signaling activity have exhibited significantly fewer
mast cells in their skin (Tsujimura, 1996, Pathol Int 46:933-938).
Excessive activation of c-kit might be associated with diseases
resulting from an over abundance of mast cells.
[0129] Mastocytosis is the term used to describe a heterogeneous
series of disorders characterized by excessive mast cell
proliferation (Metcalfe, 1991, J. Invest. Derm 93:2S-4S; Valent;
1996; Golkar, et al., 1997, Lancet 349:1379-1385). Mastocytosis is
limited to the skin in the majority of patients, but can involve
other organs in 15-20% of patients (Valent, 1996, Wein/Klin
Wochenschr 108:385-397; Golkar, et al., 1997, Lancet
349:1379-1385). Even among patients with systemic mastocytosis, the
disease can range from having a relatively benign prognosis to
aggressive mastocytosis and mast cell leukemia. (Valent, 1996,
Wein/Klin Wochenschr 108:385-397; Golkar, et al., 1997, Lancet
349:1379-1385). c-kit has been observed on malignant mast cells
from canine mast cell tumors (London, et al., 1996, J. Compar.
Pathol. 115:399-414), as well as on mast cells from patients with
aggressive systemic mastocytosis (Baghestanian, et al., 1996,
Leuk.:116-122; Castells, et al., 1996, J. Aller. Clin. Immunol.
98:831-840).
[0130] Elevated c-kit expression was reported on mast cells from
patients with aggressive mastocytosis, but not on mast cells from
patients with indolent mastocytosis (Nagata, et al., 1998,
Mastocytosis Leuk 12:175-181). SCF has been shown to be expressed
on stromal cells as a membrane-bound protein, and its expression
can be induced by fibrogenic growth factors such as PDGF (Hiragun,
et al. 1998). It has also been shown to be expressed on
keratinocytes as a membrane-bound protein in normal skin. However,
in the skin of patients with mastocytosis, an increased amount of
soluble SCF has been observed (Longley, et al., 1993, New Engl. J.
Med. 328:1302-1307).
[0131] Mast cell chymase has been reported to cleave
membrane-associated SCF to a soluble and biologically active form.
This mast cell-mediated process could serve to generate a feedback
loop to enhance mast cell proliferation and function (Longley, et
al., 1997, Proc. Natl. Acad. Sci. 94:9017-9021), and may be
important for the etiology of mastocytosis. Transgenic mice
overexpressing a form of SCF that could not be proteolytically
released from keratinocytes did not develop mastocytosis, while
similar animals expressing normal SCF in keratinocytes exhibited a
phenotype resembling human cutaneous mastocytosis (Kunisada, et
al., 1998, J. Exp. Med. 187:1565-1573). Formation of large amounts
of soluble SCF can contribute to the pathology associated with
mastocytosis in some patients and the present invention can treat
or prevent such disorders by modulating the interaction between SCF
and c-kit kinase. Several different mutations of the c-kit RTK that
resulted in constitutive kinase activity have been found in human
and rodent mast cell tumor cell lines (Furitsu, et al., 1993, J.
Clin. Invest. 92:1736-1744; Tsujimura, et al., 1994, Blood
9:2619-2626; Tsujimura, et al., 1995, Int. Arch. Aller. Immunol
106:377-385; Tsujimura, 1996, Pathol Int 46:933-938). In addition,
activating mutations of the c-kit gene have been observed in
peripheral mononuclear cells isolated from patients with
mastocytosis and associated hematologic disorders (Nagata, et al.,
1998, Mastocytosis Leuk 12:175-181), and in mast cells from a
patient with urticaria pigmentosa and aggressive mastocytosis
(Longley, et al., 1996, Nat. Gen. 12:312-314). Inhibition of c-kit
kinase will therefore prove to have an excellent therapeutic role
in the treatment of these disorders.
[0132] In some patients, activating mutations of the c-kit RTK may
be responsible for the pathogenesis of the disease and these
patients can be treated, or their diseases prevented, by modulation
of the SCF interaction with c-kit kinase. SCF activation of c-kit
as been shown to prevent mast cell apoptosis which may be critical
for maintaining cutaneous mast cell homeostasis (Iemura, et al.,
1994, Amer. J. Pathol 144:321-328; Yee, et al., 1994, J. Exp. Med.
179:1777-1787; Mekori, et al., 1994, J. Immunol 153:2194-2203;
Mekori, et al., 1995, Int. Arch. Allergy Immunol. 107:137-138).
Inhibition of mast cell apoptosis could lead to the mast cell
accumulation associated with mastocytosis. Thus, observation of
c-kit activation resulting from overexpression of the receptor,
excessive formation of soluble SCF, or mutations of the c-kit gene
that constitutively activate its kinase, provides a rationale that
inhibition of the kinase activity of c-kit will decrease the number
of mast cells and provide benefit for patients with
mastocytosis.
[0133] Asthma & Allergy: Mast cells and eosinophils represent
key cells in parasitic infection, allergy, inflammation, and asthma
(Thomas, et al., 1996, Gen. Pharmacol 27:593-597; Metcalfe, et al.,
1997, Physiol Rev 77:1033-1079; Holgate, 1997, CIBA Found. Symp.;
Naclerio, et al, 1997, JAMA 278:1842-1848; Costa, et al., 1997,
JAMA 778:1815-1822). SCF has been shown to be essential for mast
cell development, survival and growth (Kitamura, et al., 1995, Int.
Arch. Aller. Immunol. 107:54-56; Metcalfe, et al., 1997, Physiol
Rev 77:1033-1079). In addition, SCF cooperates with the
eosinophil-specific regulator, IL-5, to increase the development of
eosinophil progenitors (Metcalf, et al., 1998, Proc. Natl. Acad.
Sci., USA 95:6408-6412). SCF has also been reported to induce mast
cells to secrete factors (Okayama, et al., 1997, Int. Arch. Aller.
Immunol. 114:75-77; Okayama, et al., 1998, Eur. J. Immunol.
28:708-715) that promote the survival of eosinophils (Kay, et al.,
1997, Int. Arch. Aller. Immunol. 113:196-199), which may contribute
to chronic, eosinophil-mediated inflammation (Okayama, et al.,
1997, Int. Arch. Aller. Immunol. 114:75-77; Okayama, et al., 1998,
Eur. J. Immunol. 28:708-715). In this regard, SCF directly and
indirectly regulates activation of both mast cells and
eosinophils.
[0134] SCF induces mediator release from mast cells, as well as
priming these cells for IgE-induced degranulation (Columbo, et al.,
1992, J. Immunol 149:599-602) and sensitizing their responsiveness
to eosinophil-derived granule major basic protein (Furuta, et al.,
1998, Blood 92:1055-1061). Among the factors released by activated
mast cells are IL-5, GM-CSF and TNF-.alpha., which influence
eosinophil protein secretion (Okayama, et al., 1997, Int. Arch.
Aller. Immunol. 114:75-77; Okayama, et al., 1998, Eur. J. Immunol.
28:708-715). In addition to inducing histamine release from mast
cells (Luckacs, et al., 1996, J. Immunol. 156:3945-3951; Hogaboam,
et al., 1998, J. Immunol. 160:6166-6171), SCF promotes the mast
cell production of the eosinophil chemotactic factor, eotaxin
(Hogaboam, et al., 1998, J. Immunol. 160:6166-6171), and eosinophil
infiltration (Luckacs, et al., 1996, J. Immunol.
156:3945-3951).
[0135] SCF also directly influences the adhesion of both mast cells
(Dastych, et al., 1994, J. Immunol. 152:213-219; Kinashi, et al.,
1994, Blood 83:1033-1038) and eosinophils (Yuan, et al., 1997, J.
Exp. Med. 186:313-323), which in turn, regulates tissue
infiltration. Thus, SCF can influence the primary cells involved in
allergy and asthma through multiple mechanisms. Currently,
corticosteroids are the most effective treatment for chronic
rhinitis and inflammation associated with allergy (Naclerio, et
al., 1997, JAMA 278:1842-1848; Meltzer, 1997, Aller. 52:33-40).
These agents work through multiple mechanisms including reduction
of circulating and infiltrating mast cells and eosinophils, and
diminished survival of eosinophils associated with inhibition of
cytokine production (Meltzer, 1997, Aller. 52:33-40). Steroids have
also been reported to inhibit the expression of SCF by fibroblasts
and resident connective tissue cells, which leads to diminished
mast cell survival (Finotto, et al., 1997, J. Clin. Invest. 99
1721-1728). Because of the mutual regulation of mast cell and
eosinophil function, and the role that SCF can play in this
regulation, inhibition of c-kit kinase will provide a means to
treat allergy-associated chronic rhinitis, inflammation and
asthma.
[0136] c. Identification of Agonists and Antagonists to the c-kit
Receptor
[0137] In view of the deduced importance of RTKs in the control,
regulation and modulation of endothelial cell proliferation and
potentially carcinogenesis, many attempts have been made to
identify RTK "inhibitors" using a variety of approaches. These
include the use of mutant ligands (U.S. Pat. No. 4,966,849);
soluble receptors and antibodies (Application No. WO 94/10202;
Kendall and Thomas, 1994, Proc. Natl. Acad. Sci. USA
90:10705-10709; Kim, et al., 1993, Nature 362:841-844); and RNA
ligands (Jellinek, et al., 1994, Biochemistry 33:10450-10456).
[0138] Furthermore, kinase inhibitors (WO 94/03427; WO 92/21660; WO
91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al.,
1994, Proc. Am. Assoc. Cancer Res. 35:2268), and inhibitors acting
on receptor kinase signal transduction pathways, such as protein
kinase C inhibitors have been identified (Schuchter, et al., 1991,
Cancer Res. 51:682-687); Takano, et al., 1993, Mol. Bio. Cell
4:358A; Kinsella, et al., 1992, Exp. Cell Res. 199:56-62; Wright,
et al., 1992, J. Cellular Phys. 152:448-57).
[0139] More recently, attempts have been made to identify small
molecules which act as kinase inhibitors for use in the treatment
of cancer. Consequently, there is an unmet need for the
identification and generation of effective small compounds which
selectively inhibit the signal transduction of the c-kit RTK in
order to effectively and specifically suppress this autocrine
loop.
[0140] Some of the compounds of the present invention demonstrate
excellent activity in biological assays and thus these compounds
and related compounds are expected to be effective in treating
c-kit RTK-related disorders such as those described above.
Additionally, the assays and conditions described herein can be
utilized to identify further modulators of c-kit kinase
functions.
[0141] III. Biological Activity of the Compounds of the
Invention
[0142] The indolinone compounds of the present invention were
tested for their ability to inhibit most of protein kinase
activity. The biological assays and results of these inhibition
studies are reported herein. The methods used to measure indolinone
compound modulation of protein kinase function are similar to those
described in International Publication No. WO 98/07695, published
Mar. 26, 1998, by Tang et al., and entitled "INDOLINONE
COMBINATORIAL LIBRARIES AND RELATED PRODUCTS AND METHODS FOR THE
TREATMENT OF DISEASE," and U.S. Pat. No. 5,792,783, issued Aug. 11,
1998 by Tang et al., entitled 3-HETEROARYL-2-INDOLINONE COMPOUNDS
FOR THE TREATMENT OF DISEASE with respect to the high throughput
aspect of the method. The WO 98/07695 publication is incorporated
herein by reference in its entirety, including any drawings.
[0143] IV. Pharmaceutical Formulations and Routes of
Administration
[0144] The compounds described herein can be administered to a
human patient per se, or in pharmaceutical compositions where they
are mixed with other active ingredients, as in combination therapy,
or suitable carriers or excipient(s). Techniques for formulation
and administration of the compounds of the instant application may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition.
[0145] a) Routes of Administration
[0146] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intravenous, intramedullary injections, as well as intrathecal,
direct intraventricular, intraperitoneal, intranasal, or
intraocular injections.
[0147] Alternately, 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.
[0148] 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.
[0149] b) Composition/Formulation
[0150] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0151] Pharmaceutical compositions for use in accordance with the
present invention thus 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.
[0152] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks's 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.
[0153] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained by mixing
one or more solid excipients with one or more compounds of the
invention, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable 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, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0154] 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.
[0155] Pharmaceutical preparations 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 fillers such as lactose, binders such as starches,
and/or lubricants such as 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. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0156] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0157] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., 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.
[0158] The compounds may be formulated for parenteral
administration by injection, 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 formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0159] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or 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 or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0160] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0161] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0162] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0163] 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. The cosolvent system may be the VPD cosolvent
system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant Polysorbate 80, and 65% w/v polyethylene glycol
300, made up to volume in absolute ethanol. The VPD co-solvent
system (VPD:D5W) 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. Naturally, the proportions of a co-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 80; 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.
[0164] 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. Certain organic solvents such as
dimethylsulfoxide also may be employed, although usually at the
cost of greater toxicity. Additionally, the compounds may be
delivered using a sustained-release system, such as semipermeable
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.
[0165] Many of the PTK modulating compounds of the invention may be
provided as salts with pharmaceutically compatible counterions.
Pharmaceutically compatible salts may be formed with many acids,
including but not limited to hydrochloric, sulfuric, acetic,
lactic, tartaric, malic, succinic, etc. Salts tend to be more
soluble in aqueous or other protonic solvents than are the
corresponding free base forms.
[0166] c) Effective Dosage.
[0167] Pharmaceutical compositions suitable for use in the present
invention include compositions where the active ingredients are
contained in an amount effective to achieve its intended purpose.
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.
[0168] For any compound used in the methods of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. For example, a dose can be formulated in animal
models to achieve a circulating concentration range that includes
the IC.sub.50 as determined in cell culture (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of the PTK activity). Such information can be used to
more accurately determine useful doses in humans.
[0169] Toxicity and therapeutic efficacy of the compounds described
herein can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., for determining the
LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
between LD.sub.50 and ED.sub.50. Compounds which exhibit high
therapeutic indices are preferred. The data obtained from these
cell culture assays and animal studies can be used in formulating a
range of dosage for use in human. The dosage of such compounds lies
preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage may
vary within this range 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.1).
[0170] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain the kinase modulating effects, or minimal effective
concentration (MEC). 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 the kinase using the assays described
herein. Dosages necessary to achieve the MEC will depend on
individual characteristics and route of administration. However,
HPLC assays or bioassays can be used to determine plasma
concentrations.
[0171] Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen which maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%.
[0172] In cases of local administration or selective uptake, the
effective local concentration of the drug may not be related to
plasma concentration.
[0173] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
[0174] d) Packaging
[0175] The compositions may, if desired, be presented in a pack or
dispenser device 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 with
a notice associated with the container in 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 compound for human or veterinary
administration. Such notice, for example, may be the labeling
approved by the U.S. Food and Drug Administration for prescription
drugs, or the 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
conditions indicated on the label may include treatment of a tumor,
inhibition of angiogenesis, treatment of fibrosis, diabetes, and
the like.
[0176] Additional methods of preparing pharmaceutical formulations
of the compounds, methods of determining the amounts of compounds
to be administered to a patient, and modes of administering
compounds to an organism are disclosed in U.S. application Ser. No.
08/702,232 by Tang, et al., and entitled "Indolinone Combinatorial
Libraries and Related Products and Methods for the Treatment of
Disease," filed Aug. 23, 1996, and International patent publication
number WO 96/22976, by Buzzetti, et al., and entitled "Hydrosoluble
3-Arylidene-2-Oxindole Derivatives as Tyrosine Kinase Inhibitors,"
published Aug. 1, 1996, both of which are incorporated herein by
reference in their entirety, including any drawings. Those skilled
in the art will appreciate that such descriptions are applicable to
the present invention and can be easily adapted to it.
EXAMPLES
[0177] The examples below are non-limiting and are merely
representative of various aspects and features of the present
invention. The examples describe methods for synthesizing compounds
of the invention and methods for measuring an effect of a compound
on the function of protein kinases.
[0178] The cells used in the methods are available commercially or
from academic labs or were engineered from commercially available
cells. The nucleic acid vectors harbored by the cells are also
commercially available and the sequences of genes for the various
protein kinases are readily accessible in sequence data banks.
Thus, a person of ordinary skill in the art can readily recreate
the cell lines in a timely manner by combining the commercially
available cells, the commercially available nucleic acid vectors,
and the protein kinase genes using techniques readily available to
persons of ordinary skill in the art.
Assay Procedures
[0179] 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.
[0180] The cellular/catalytic assays described herein are performed
in an ELISA format. The general procedure is a follows: a compound
is introduced to cells expressing the test kinase, either naturally
or recombinantly, for some 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 assay could also be adapted to detection by Western
blotting.
[0181] 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 introduced to
cells expressing the test kinase, either naturally or
recombinantly, for some 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 bromodeoxy-uridine (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
[0182] 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.
[0183] The disclosed protocol may be adapted for determining
activity with respect to a specific PK, such as c-kit kinase. The
preferred protocols for conducting the ELISA experiments for the
specific PKs, c-kit kinase, is provided below. 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.
Example 1
The Activity of the Compounds of the Invention
[0184] The biochemical activity of some of the compounds of the
invention were tested using the assays described. The IC.sub.50
values were measured for several of the compounds of the invention.
The results are shown in FIG. 2.
[0185] A. Materials and Reagents
[0186] 1) HNTG: 5.times.stock concentration: 100 mM HEPES pH 7.2,
750 mM NaCl, 50% glycerol, 2.5% Triton X-100.
[0187] 2) PBS (Dulbecco's Phosphate-Buffered Saline): Gibco Catalog
# 450-1300EB
[0188] 3) 1.times. Blocking Buffer: 10 mM TRIS-pH 7.5, 1% BSA, 100
mM NaCl, 0.1% Triton X-100
[0189] 4) 1.times. Kinase Buffer: 25 mM HEPES, 100 mM NaCl, 10 mM
Mg Cl.sub.2, 6 mM Mn Cl.sub.2.
[0190] 5) PMSF Stock Solution=100 mM (Sigma Catalog # P-7626)
[0191] 6) 10 mM ATP (Bacterial source) Sigma A-7699, 5 g.
[0192] 7) UB40 anti-phosphotyrosine mAb.
[0193] 8) HRP conjugated sheep anti-Mouse IgG. (Amersham NA
931)
[0194] 9) ABTS (SPrime-3Prime 7-579844)
[0195] 10) TRIS HCL: Fisher BP 152-5
[0196] 11) NaCl: Fisher S271-10
[0197] 12) Triton X-100: Fisher BP151-100
[0198] 13) Na.sub.3VO.sub.4: Fisher S454-50
[0199] 14) MgCl.sub.2: Fisher M33-500
[0200] 15) MnCl.sub.2: Fisher M87-500
[0201] 16) HEPES: Fisher BP310-500
[0202] 17) Albumin, Bovine (BSA): Sigma A-8551
[0203] 18) TBST Buffer: 50 mM Tris pH 7.2, 150 mM NaCl, 0.1% Triton
X-100.
[0204] 19) Goat affinity purified antibody Rabbit IgG (whole
molecule): Cappel 55641.
[0205] 20) Anti Kit (C-20) rabbit polyclonal IgG antibody: Santa
Cruz sc-168
[0206] 21) Kit/CHO cells: CHO cells stably expressing GyrB/Kit,
which are grown in standard CHO medium, supplemented with 1 mg/ml
G418
[0207] 22) Indolinone Compounds: The indolinone compounds were
synthesized as set forth in the following application: PCT
application number US99/06468, filed Mar. 26, 1999 by Fong, et al.
and entitled METHODS OF MODULATING TYROSINE PROTEIN KINASE (Lyon
& Lyon docket number 231/250 PCT which is hereby incorporated
by reference in its entirety including any drawings.
[0208] B. Procedure
[0209] All of the following steps are conducted at room temperature
unless it is specifically indicated. All ELISA plate washing is by
rinsing 4.times.with TBST.
[0210] Kit Cell Lysis
[0211] This procedure is performed 1 hour prior to the start of
receptor capture.
[0212] 1) Wash a >95% confluent 15 cm dish with PBS and aspirate
as much as possible.
[0213] 2) Lyse the cells with 3 ml of 1.times.HNTG containing 1 mM
PMSF/15 cm dish. Scrape the cells from the plate and transfer to a
50 ml centrifuge tube.
[0214] 3) Pool supernatants, and allow to sit, on ice, for one hour
with occasional vortexing. Failure to do so with result in an
increased background (approximately 3-fold higher).
[0215] 4) Balance tubes and centrifuge at 10,000.times.g for 10 min
at 4.degree. C. Remove an aliquot for protein determination
[0216] 5) Perform protein determination as per the SOP for protein
determination using the bicinchoninic acid (BCA) method.
[0217] ELISA Procedure
[0218] 1) Coat Coming 96-well ELISA plates with 2 .mu.g per well
Goat anti-rabbit antibody in PBS for a total well volume of 100
.mu.l. Store overnight at 4.degree. C.
[0219] 2) Remove unbound Goat anti-rabbit antibody by inverting
plate to remove liquid.
[0220] 3) Add 100 .mu.l of Blocking Buffer to each well. Shake at
room temperature for 60 min.
[0221] 4) Wash 4.times. with TBST. Pat plate on a paper towel to
remove excess liquid and bubbles
[0222] 5) Add 0.2 .mu.g per well of Rabbit anti-Kit antibody
diluted in TBST for a total well volume of 100 .mu.l. Shake at room
temperature for 60 min.
[0223] 6) Dilute lysate in HNTG (180 .mu.g lysate/100 .mu.l)
[0224] 7) Add 100 .mu.l of diluted lysate to each well. Shake at
room temperature for 60 min.
[0225] 8) Wash 4.times. with TBST. Pat plate on a paper towel to
remove excess liquid and bubbles
[0226] 9) Dilute compounds/extracts (or as stated otherwise) in
1.times. kinase buffer, with 5 .mu.M ATP in a polypropylene 96 well
plate
[0227] 10) Transfer 100 .mu.l of diluted drug to ELISA plate wells.
Incubate at room temperature with shaking for 60 min.
[0228] 11) Stop reaction with the addition of 10 .mu.l of 0.5 M
EDTA. Plate is now stable for a reasonable period of time.
[0229] 12) Wash 4.times. with TBST. Pat plate on a paper towel to
remove excess liquid and bubbles
[0230] 13) Add 100 .mu.l per well of UB40 (1:2000 dilution in
TBST). Incubate 60 min at room temperature, with shaking.
[0231] 14) Wash 4.times. with TBST. Pat plate on a paper towel to
remove excess liquid and bubbles
[0232] 15) Add 100 .mu.l per well of sheep anti-mouse IgG--HRP
(1:5000 dilution in TBST). Incubate 60 min at room temperature,
with shaking.
[0233] 16) Wash 4.times. with TBST. Pat plate on a paper towel to
remove excess liquid and bubbles
[0234] 17) Add 100 .mu.l per well of ABTS. Incubate with shaking
for 15-30 min.
[0235] 18) Read assay on Dynatech MR7000 ELISA reader
1 Test Filter = 410 nm Reference Filter = 630 nm
Example 2
The Activity of the Compounds of the Invention
[0236] The biochemical activity of two of the compounds of the
invention were tested using the assays described below.
[0237] Methods:
[0238] Cell Lines
[0239] MO7E cells, a human myeloid leukemia cell line, were
maintained in RPMI-1640 medium supplemented with 10% fetal calf
serum and 10 ng/ml each IL-3 and GM-CSF.
[0240] Detection of c-kit Tyrosine Phosphorylation
[0241] MO7E cells were serum starved overnight in 0.1% serum. Cells
were pre-treated with Compound Eight for 2 hours, or with Compound
Six for 22 hours (concurrent with serum starvation), prior to
ligand stimulation. Cells were stimulated with 250 ng/ml rh-SCF for
15 minutes. Following stimulation, cells were lysed and
immunoprecipitated with an anti-c-kit antibody. Phosphotyrosine and
protein levels were determined by western blotting.
[0242] MTT Proliferation Assay
[0243] MO7E cells were serum starved and pre-treated with compound
as described for the phosphorylation experiments. Cells were plated
@ 4.times.10.sup.5 cells/well in a 96 well dish, in 100 .mu.l
RPMI+10% serum. rh-SCF (100 ng/mL) was added and the plate was
incubated for 48 hours. After 48 hours, 10 .mu.l of 5 mg/ml MTT
[3-(4,5-dimethythiazol-2-y- l)-2,5-diphenyl tetrazolium bromide)
was added and allowed to incubate for 4 hours. Acid isopropanol
(100 .mu.l of 0.04N HCl in isopropanol) was added and the optical
density was measured at a wavelength of 550 nm.
[0244] Apoptosis Assays
[0245] MO7E cells were incubated +/- SCF and +/- compound (Compound
Six or Compound Eight @ 5 and 25 .mu.M) in 10% FBS with
rh-GM-CSF(10 ng/mL) and rh-IL-3 (10 ng/mL). Samples were assayed at
24 and 48 hours. To measure activated caspase-3, samples were
washed with PBS and permeabilized with ice-cold 70% ethanol. The
cells were then stained with PE-conjugated polyclonal rabbit
anti-active caspase-3 and analyzed by FACS. To measure cleaved
PARP, samples were lysed and analyzed by western blotting with an
anti-PARP antibody.
[0246] Inhibition of Biological Functions of c-kit by Compound
Eight and Compound Six
[0247] Results:
[0248] Inhibition of Tyrosine Phosphorylation of c-kit
[0249] Compound Eight and Compound Six inhibit tyrosine
phosphorylation of c-kit in MO7E cells, a human myeloid leukemia
cell line, in response to ligand stimulation with stem cell factor
(SCF). In Compound Eight treated cells, no inhibition of
phosphorylation was observed at 0.01 .mu.M, partial inhibition was
observed at 0.1 .mu.M, and complete inhibition was observed at 1
and 10 .mu.M. In Compound Six treated cells, no inhibition of c-kit
tyrosine phosphorylation was observed at 0.01 .mu.M or 0.1 .mu.M,
partial inhibition was observed at 1 .mu.M, and complete inhibition
was observed at 10 .mu.M.
[0250] Inhibition of c-kit Mediated Proliferation
[0251] Compound Eight and Compound Six also inhibit c-kit mediated
signaling in MO7E cells in an MTT proliferation assay. The
IC.sub.50 value for Compound Eight inhibition of proliferation is
approximately 0.5-1.0 .mu.M, and the IC.sub.50 value for Compound
Six is approximately 5-7 .mu.M.
[0252] Induction of Apoptosis
[0253] Compound Eight and Compound Six also induce apoptosis in
MO7E cells, in a dose and time dependent fashion. Apoptosis was
assessed with two assays: a FACS analysis with an antibody that
recognizes activated caspase-3 in cells, which is induced during
apoptosis, and a western blotting assay that detects a cleaved
fragment of poly (ADP-ribose) polymerase, also induced during
apoptosis.
[0254] Using the caspase-3 assay, an approximately 50% increase in
the number of apoptotic cells was observed at 48 hours, upon SCF
stimulation and 25 .mu.M Compound Eight treatment, compared to
untreated SCF stimulated cells. A slight effect was observed at 48
hours with 25 .mu.M Compound Eight in the absence of SCF
stimulation. Treatment for 24 hours with 25 .mu.M Compound Eight
(+/- SCF stimulation), resulted in a measurable but smaller number
of apoptotic cells.
[0255] Treatment of cells with 5 .mu.M Compound Eight for 24 or 48
hours (+/-SCF stimulation) also resulted in a measurable but
smaller number of apoptotic cells.
[0256] Similar results were obtained for Compound Six, with the
exception of no effect observed with 5 .mu.M Compound Six at 24
hours, with or without SCF stimulation.
[0257] Using the PARP assay, treatment with 25 .mu.M Compound Eight
for 48 hours resulted in the greatest increase in the amount of
cleaved PARP. The effect was augmented slightly with SCF
stimulation. The 24 hour sample treated with 25 .mu.M Compound
Eight was similar to the 48 hour sample.
[0258] Treatment with 5 .mu.M Compound Eight, at both timepoints,
resulted in a very minimal increase in cleaved PARP.
[0259] Similar results were obtained for Compound Six.
CONCLUSION
[0260] 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 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 are
defined by the scope of the claims.
[0261] 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.
[0262] 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.
[0263] 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 expressions of excluding any equivalents of the features shown
and described or portions thereof, but 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.
[0264] 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.
[0265] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0266] Other embodiments are within the following claims.
* * * * *