Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-TUMOR COMPRISING BOROPROLINE COMPOUNDS
Field of the Invention
This invention relates to methods for the treatment of abnormal proliferative
disorders. The methods involve administering certain compounds to inhibit
proliferation and
angiogenesis in an abnormal proliferative cell mass.
Background of the Invention
Abnormal cell proliferation is usually characterized by an increased rate of
division
Io and_ in some cases uncontrolled growth. One example of a proliferative cell
disorder is a
tumor. In addition to posing a serious health risk in and of themselves,
primary malignant
tumors are particularly problematic given their tendency to invade surrounding
tissues and
metastasize to distant organs in the body. To date, the most frequently used
methods for
treating neoplasia, especially solid tumor forms of neoplasia, include
surgical procedures,
/5 radiation therapy, drug therapies, and combinations of the foregoing. These
methods involve
significant risk (e.g., of infection, death) to the patient. More importantly,
the probability of
eliminating all malignant cells is small particularly if the zone of malignant
growth is not
well defined or if the primary tumor has metastasized by the time of surgery.
Achieving
therapeutic doses effective for treating the cancer is often limited by the
toxic side effects of
2o the anti-cancer agent on normal, healthy tissue. An ideal anti-cancer agent
has tissue
specificity, thereby reducing side-effects on normal (dividing) cells.
Recently, a model of anti-cancer therapy has been proposed and validated that
targets
the vasculature of solid tumors rather than the malignant cells themselves.
Most, if not all,
solid tumors require a blood supply system for oxygenation, nutrient delivery
and waste
25 product removal. The need for neovascularization is particularly acute if
the tumor is to grow
beyond the confines of the normal blood supply system. As a result, tumors
which do attain a
certain size are able to elicit the growth of new blood vessels from the
surrounding
endothelial cells, through a process called angiogenesis, through the release
of angiogenic
factors.
3o In view of the foregoing, a need exists to identify agents for treating
cancer and
metastasis.
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Summary of the Invention
The invention solves these and other problems by providing methods and related
compositions for treating conditions characterized by abnormal cell
proliferation, including,
but not limited to, cancer and metastasis. The invention is based, in part, on
the observation
that compounds of Formula I are able to inhibit the enzymatic activity of
fibroblast activation
protein-alpha (FAP-a).
In one aspect, the invention provides a method for treating a subject having a
condition characterized by abnormal mammalian cell proliferation. The method
comprises
administering to a subject in need of such treatment, an agent in an amount
effective to
/o inhibit the proliferation, wherein the agent is a compound of Formula I:
Formula I
PR
wherein P is a targeting group which binds to the reactive site of FAP-a or
other post proline-
cleaving enzyme and can be a peptide or a peptidomimetic, and wherein R is a
reactive group
IS capable of reacting with a functional group in FAP-a or other post proline
cleaving enzyme,
preferably in the reactive site of FAP-a or other post proline cleaving
enzyme. The reactive
compound may be selected from the group consisting of organo boronates, organo
phosphonates, fluoroalkylketones, alphaketos, N-peptiolyl-O-
(acylhydroxylamines),
azapeptides, azetidines, fluoroolefins dipeptide isoesteres, peptidyl (alpha-
aminoalkyl)
2o phosphonate esters, aminoacyl pyrrolidine-2-nitriles and 4-
cyanothiazolidides.
One group of Formula I compounds useful in the invention can be further
defined by
Formula II
Formula II
H
/x,
Am A~ N C B-
\X
CHZ CH2 2
~H2C~
25 wherein m is an integer between 0 and 10, inclusive; A and A~ may be L- or
D-amino acid
residues such that each A in Am (i.e., where m> 1 ) may be a different amino
acid residue from
every other A in Am; the C bonded to B is in the L-configuration; the bond
between A~ and N
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and, in some embodiments, between A~ and Am, are peptide bonds; and each X~
and X2 is,
independently, a hydroxyl group or a group capable of being hydrolyzed to a
hydroxyl group
in aqueous solution at physiological pH. By "the C bonded to B is in the L-
configuration" is
meant that the absolute configuration of the C is like that of an L-amino
acid. Thus, the
group
~ X~
B'
X2
has the same relationship to the C as the --COON group of an L-amino acid has
to its a
carbon. In some embodiments, A and A~ are independently proline or alanine
residues. In
/o some embodiments, m is 0. In some embodiments, X~ and XZ are hydroxyl
groups. In some
embodiments, the inhibitor is L-Ala-L-boroPro. In still other embodiments,
inhibitor is L-
Pro-L-boroPro.
In addition to agents of Formula II, other agents useful in the invention
include those
in which the proline residue in Formula II is replaced with another amino acid
residue such
/5 as, for example, lysine, alanine or glycine. As well, derivatives of
Formula II in which the
boronate group is replaced with a reactive group as described above are also
useful in the
invention. In preferred embodiments, the agent is Val-boro-Pro.
In one aspect, the agent of Formula I is administered to a subject in need
thereof in an
amount effective to inhibit abnormal mammalian cell proliferation. The
subjects to be treated
2o are subjects having a condition characterized by abnormal mammalian cell
proliferation. In
certain embodiments, the subj ects preferably are otherwise free of symptoms
calling for
hemopoietic stimulation and, in particular, are free of symptoms calling for
treatment with a
compound for stimulating an immune response. In some embodiments, the subjects
to be
treated do not exhibit symptoms requiring hemopoietic stimulation and have
normal or
25 protective levels of hemopoietic cells. The subject to be treated may have
normal
hemopoietic activity. Included are subjects who are HIV positive but who have
normal
hemopoietic activity. In another embodiment, the subject is HIV negative. In
certain
embodiments, the subjects are not myeloid or lymphoid suppressed or are not
candidates for
treatment with an agent which causes such suppression at the time of treatment
with the
3o methods of the invention.
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The invention is further premised, in part, on the discovery that compounds of
Formula I (e.g., Val-boro-Pro) have anti-tumor activity in melanoma and
fibrosarcoma.
Thus, in another aspect, the subjects are treated with the agent of Formula I
in a
manner and in an amount so as to inhibit proliferation of a primary tumor, or
to inhibit
metastatic spread or growth while minimizing the potential for systemic
toxicity. In certain
embodiments, the abnormal mammalian cell proliferation is manifested as a
tumor. Some
conditions intended to be treated by the method of the invention include
benign (i.e., non-
cancerous), pre-malignant and malignant (i.e., cancerous) tumors. In some
embodiments, the
condition characterized by abnormal mammalian cell proliferation is further
characterized by
1o the presence of reactive stromal fibroblasts.
In other embodiments, the abnormal mammalian cell proliferation is selected
from the
group consisting of a carcinoma, a sarcoma, and a melanoma. In yet other
embodiments, the
condition is selected from the group consisting of breast cancer, colorectal
cancer, ovarian
cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer,
melanoma and
/5 fibrosarcoma. In still other embodiments, the condition is selected from
the group consisting
of bone and connective tissue sarcomas, examples of which include, but are not
limited to,
osteosarcoma and fibrosarcoma.
In still other embodiments, the abnormal mammalian cell proliferation is in
epithelial
cells, meaning that it is epithelial cells which are abnormally proliferating.
Some conditions
2o characterized by abnormal mammalian epithelial cell proliferation include
adenomas of
epithelial tissues such as the breast, colon and prostate, as well as
malignant tumors.
According to other embodiments of the invention, a method is provided for
treating a subject
having a metastasis of epithelial origin.
According to some embodiments of the invention, the agent is administered
locally.
25 In some embodiments, the agent is targeted to a tumor. This can be achieved
by the
particular mode of administration. For example, easily accessible tumors such
as breast or
prostate tumors may be targeted by direct needle injection to the site of the
lesion. Lung
tumors may be targeted by the use of inhalation as a route of administration.
In some embodiments, the agents may be administered in a systemic manner, via
3o administration routes such as, but not limited to, oral, intravenous,
intramuscular and
intraperitoneal administration. Systemic administration routes may be
preferred, for
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example, if the subject has metastatic lesions. In other embodiments, the
agent is
administered in a sustained release formulation.
In administering the compounds of the invention to subjects, dosing amounts,
dosing
schedules, routes of administration and the like may be selected so as to
affect the other
known activities of these compounds. For example, amounts, dosing schedules
and routes of
administration can be selected as described herein, whereby therapeutically
effective levels
for inhibiting proliferation are provided, yet therapeutically effective
levels for restoring
hemopoietic deficiency are not provided.
In addition, agents can be selected that are effective as anti-proliferative
agents or as
1o anti-angiogenic agents bui are relatively ineffective as hemopoietic cell
stimulatory or
activating agents. Thus, certain subjects who require both hemopoietic
stimulation and/or
activation and proliferation and/or angiogenic inhibition may be treated with
different agents
of the invention simultaneously, one each for the desired therapeutic effect,
or with a single
compound but in different dosages, schedules, and/or route to achieve both
hemopoietic
75 stimulation and proliferation inhibition at therapeutic levels.
In some embodiments of the invention, a method is provided in which the agent
is
administered in combination with an anti-proliferative compound, such as an
anti-cancer
compound. In another embodiment, the agent is administered in combination with
surgery to
remove an abnormal proliferative cell mass. In a related embodiment, the agent
is
2o administered to a patient who has had surgery to remove an abnormal
proliferative cell mass.
In another aspect, the invention provides a method for inhibiting angiogenesis
in a
subject having a condition characterized by abnormal mammalian cell
proliferation
comprising administering to a subject in need of such treatment, an agent in
an amount
effective to inhibit angiogenesis in an abnormal proliferative cell mass,
wherein the agent is a
25 compound of Formula I.
In some embodiments, the abnormal mammalian cell proliferation is manifested
as a
tumor. In another embodiment, the abnormal mammalian cell proliferation is
selected from
the group consisting of a carcinoma, a sarcoma, and a melanoma. In still
another
embodiment, the condition characterized by abnormal mammalian cell
proliferation is a
3o metastasis. In other embodiments, the condition is selected from the group
consisting of
breast cancer, colorectal cancer, ovarian cancer, prostate cancer, pancreatic
cancer, kidney
cancer, lung cancer, melanoma and fibrosarcoma. In another embodiment, the
abnormal
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mammalian cell proliferation is in epithelial cells, meaning that epithelial
cells are
abnormally proliferating.
In one embodiment, the agent is administered locally. In another embodiment,
the
agent is targeted to a tumor. In another embodiment, the agent is administered
in a sustained
release formulation. In yet another embodiment, the agent is administered
systemically.
In some embodiments, the agent is administered in combination with surgery to
remove an abnormal proliferative cell mass. In other embodiments, the agent is
administered
to a patient who has had surgery to remove an abnormal proliferative cell
mass.
In one embodiment, the agent is administered in combination with an anti-
proliferative compound such as an ant_~cancer compound. In other embodiments,
the agent is
administered in combination with an anti-angiogenic compound. In yet other
embodiments,
the agent is administered with an anti-cancer compound and an anti-angiogenic
compound.
In one embodiment, the subject has normal hemopoietic activity. In another
embodiment, the subject is HIV negative.
IS According to still another aspect of the invention, pharmaceutical
preparations are
provided that comprise an agent of Formula I, as described above, and a
pharmaceutically-
acceptable carrier. Formula I agents are present in the pharmaceutical
preparations in an
amount effective for inhibiting proliferation in primary or secondary (e.g.,
metastatic)
malignant lesions. These pharmaceutical preparations may contain the agent of
Formula I
2o alone or in combination with other compounds (e.g., anti-cancer compounds
and/or anti-
angiogenic compounds).
In one aspect, a pharmaceutical preparation is provided comprising an agent of
Formula I, at least one other anti-cancer compound (i.e., an anti-cancer
compound other than
an agent of Formula I), and a pharmaceutically acceptable carrier. In another
aspect, a
25 pharmaceutical preparation is provided which comprises an agent of Formula
I, at least one
other anti-angiogenic compound (i.e., an anti-angiogenic compound other than
an agent of
Formula I), and a pharmaceutically acceptable carrier.
In other embodiments, anti-cancer cocktails containing the agent of the
invention and
other anti-proliferative compounds and/or other anti-angiogenic compounds as
described
3o herein are also provided. In still other embodiments, the agents of Formula
I are used in the
preparation of a medicament for treating subjects having conditions
characterized by
abnormal mammalian cell proliferation.
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In still other embodiments, the agent may be targeted to a cell mass (e.g., a
tumor)
through the use of a targeting compound specific for a particular tissue or
tumor type. In
some embodiments, the agents of the invention may be targeted to primary or in
some
instances, secondary (i.e., metastatic) lesions through the use of targeting
compounds which
preferentially recognize a cell surface marker.
These and other aspects of the invention will be described in greater detail
below.
Throughout this disclosure, all technical and scientific terms have the same
meaning as
commonly understood by one of ordinary skill in the art to which this
invention pertains
unless defined otherwise.
Brief Description of the Figures
Figure 1 a is a comparison of response of Fischer D+ rat and BM stromal cells
to PT-
100 in vitro by measurement of interleukin-6 (IL-6) release.
Figure 1b is a comparison of response of Fischer D- rat and BM stromal cells
to PT-
/5 100 in vitro by measurement of interleukin-1 (IL-6) release.
Figure 2 is a profile of FAP-a and CD26 cell surface expression by human bone
marrow stromal cells.
Figure 3a is the PT-100 dose response of FAP-a+ CD26- primary human stromal
cells
by measurement of granulocyte-colony stimulating factor (G-CSF) release.
2o Figure 3b is the PT-100 dose response of FAP-a+ CD26- primary human stromal
cells
by measurement of inhibition of DPPIV-like activity.
Figure 4 is a bar graph showing the effect of PT-100 treatment on established
subcutaneous B 16-F 10 tumors.
Figure Sa is a bar graph showing the effect of PT-100 on WEHI subcutaneous
growth
25 (in terms of tumor volume) in vivo on day 20 post injection.
Figure Sb is a bar graph showing the effect of PT-100 on WEHI subcutaneous
growth
(in terms of tumor weight) in vivo on day 20 post injection.
Detailed Description of the Invention
3o The invention is directed to the inhibition of cellular proliferation and
angiogenesis in
conditions characterized by abnormal, unwanted mammalian cell proliferation.
The
invention is useful, inter alia, in the treatment of proliferative disorders
including benign, pre-
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_g_
malignant and malignant tumors. In certain embodiments, the methods are
directed towards
the treatment of subjects with carcinoma or metastatic lesions of epithelial
origin. The
invention is based, in part, on the finding that PT-100, the dipeptide valine-
proline-boronic
acid, i.e., (ValboroPro), is able to inhibit fibroblast activation protein
(FAP-a), and also
exhibits anti-tumor activity against cancers such as, for example, melanoma
and
fibrosarcoma.
According to one aspect of the invention, a method is provided for treating a
subject
having a condition characterized by an abnormal mammalian cell proliferation,
using
compounds of Formula I:
Formula I
PR
wherein P is a targeting group which binds to the reactive site of FAP-a or
other post proline
cleaving enzyme, and R is a reactive group capable of reacting with a
functional group in the
reactive site of FAP-a or other post proline-cleaving enzyme. Post proline-
cleaving enzymes
l5 are enzymes which have a specificity for removing Xaa-Pro (where Xaa
represents any
amino acid) dipeptides from the amino terminus of polypeptides. Examples of
post-proline
cleaving enzymes include, but are not limited to, CD26, dipeptidyl peptidase
IV (DP IV) and
dipeptidyl aminopeptidase IV.
The P targeting group can be composed of single or multiple residues of
peptide or
2o peptidomimetic nature, provided that such residues do not interfere
significantly, and most
preferably improve the site-specific recognition of FAP-a or other post
proline-cleaving
enzyme by the agent of Formula I. In certain embodiments, the portion of the P
targeting
group that is involved in binding to the reactive site of FAP-a or other post
proline-cleaving
enzyme is formed of amino acids and the remaining portion of P is formed of
non-amino acid
25 components. According to the particular embodiment, P can be composed
wholly of amino
acid residues, wholly of non-amino acid substituents, or a combination of
both.
In general, the targeting group (i.e., P) is covalently coupled to the
reactive group. In
some embodiments, the covalent coupling occurs via a carboxyl group at the
carboxyl
terminal amino acid in the P group. In certain embodiments, P may be 30, 20,
10 or less than
30 10 residues in length.
The development of phage display libraries and chemical combinatorial
libraries from
which synthetic compounds can be selected which mimic the substrate of a
protease permits
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the identification of further targeting groups to which an R group can be
covalently attached
to form a binding moiety which binds or associates with the reactive site of
the protease and
which forms a complex with a functional group in the protease reactive site.
Such libraries
can be screened to identify non-naturally occurring putative targeting groups
by assaying
protease cleavage activity in the presence and absence of the putative phage
display library
molecule or combinatorial library molecule and determining whether the
molecule inhibits
cleavage by the protease of a known substrate or of a substrate analog (e.g.,
a chromophoric
substrate analog which is easily detectable in a spectrophotometric assay).
Those phage
library and/or combinatorial library molecules which exhibit inhibition of,
for example, FAP-
IO a., then can be covalently coupled to the reactive groups disclosed herein
and again te5te~i to
determine whether these novel molecules selectively bind to, for example, FAP-
a. (e.g., by
repeating the above-noted screening assay). In this manner, a simple, high-
through-put
screening assay is provided for identifying non-naturally occurring targeting
groups of the
invention.
15 P targeting groups can be synthesized from peptides or other biomolecules
including
but not limited to saccharides, fatty acids, sterols, isoprenoids, purines,
pyrimidines,
derivatives or structural analogs of the above, or combinations thereof and
the like. Also
envisioned in the invention is the use of targeting groups made from peptoids,
random bio-
oligomers (U.S. Patent 5,650,489), benzodiazepines, diversomeres such as
dydantoins,
20 benzodiazepines and dipeptides, nonpeptidal peptidomimetics with a beta-D-
glucose
scaffolding, oligocarbamates or peptidyl phosphonates. Many, if not all, of
these compounds
can be synthesized using recombinant or chemical library approaches. A vast
array of
candidate targeting groups can be generated from libraries of synthetic or
natural
compounds. The methods of the invention utilize this library technology to
identify small
25 peptides which bind to protease reactive sites. One advantage of using
libraries for inhibitor
identification is the facile manipulation of millions of different putative
candidates of small
size in small reaction volumes (i.e., in synthesis and screening reactions).
Another advantage
of libraries is the ability to synthesize targeting groups which might not
otherwise be
attainable using naturally occurring sources, particularly in the case of non-
peptide moieties.
30 Examples of reactive groups useful in the invention include organo
boronates, organo
phosphonates, fluoroalkylketones, alphaketos, N-peptiolyl-O-
(acylhydroxylamines),
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azapeptides, azetidines, fluoroolefms dipeptide isoesteres, peptidyl (alpha-
aminoalkyl)
phosphonate esters, aminoacyl pyrrolidine-2-nitriles and 4-cyanothiazolidides.
It is to be understood that the terms 'agent' or 'agent of the invention' or
'agent of
Formula I' are used interchangeably to mean all of the derivatives of Formula
I described
herein.
Some representative agents of Formula I can be further defined by Formula II
as
follows:
Formula II
H
X1
Am A1 N C B'
CH2~ ~CHZ
H2C
wherein m is an integer between 0 and 10, inclusive; A and A, may be L- or D-
amino acid
residues (for glycine there is no such distinction) such that each A in Am may
be an amino
acid residue different from another or all other A in Am; the C bonded to B is
in the L-
configuration; the bond between A1 and N and, in some embodiments, the bond
between A
and A~ are peptide bonds; and each X~ and XZ is, independently, a hydroxyl
group or a group
capable of being hydrolyzed to a hydroxyl group in aqueous solution at
physiological pH. By
"the C bonded to B is in the L-configuration" is meant that the absolute
configuration of the
C is like that of an L-amino acid. Thus, the
X1
B'
\ X2
group has the same relationship to the C as the --COOH group of an L-amino
acid has to its a
carbon. In various embodiments, A and A~ are independently proline or alanine
residues; m
is 0; X1 and XZ are hydroxyl groups; the inhibitor is L-Ala-L-boroPro; and the
inhibitor is L-
Pro-L-boroPro.
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Other agents useful in the methods and compositions of the invention are
derivatives
of Formula II in which each and every A in A", may independently be a non-
amino acid
residue. Thus, the plurality of A (i.e., Am, wherein m>1) may be a peptide or
a
peptidomimetic which may include, in whole or in part, non-amino acid residues
such as
saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines,
derivatives or structural
analogs of the above, or combinations thereof and the like. The plurality of A
in Am may also
be comprised of a combination of amino acid and non-amino acid residues. It
also is possible
to substitute non-naturally occurring amino acids, such as 2-
azetidinecarboxylic acid or
pipecolic acid (which have 6-membered, and 4-membered ring structures
respectively) for the
to proline residue. Representati°:~e sr~ructures of transition-state
analog-base' inhibitors Xaa-
boroPro of Formula II, include Lys-BoroPro, Pro-BoroPro and Ala-BoroPro in
which
"boroPro" refers to the analog of proline in which the carboxylate group
(COOH) is replaced
with a boronyl group [B(OH)2]. Alternative compounds of the invention have an
analogous
structure in which the boronyl group is replaced by, for example, a
phosphonate or a
fluoroalkylketone, alphaketos, N-peptiolyl-O-(acylhydroxylamines),
azapeptides, azetidines,
fluoroolefins dipeptide isoesteres, peptidyl (alpha-aminoalkyl) phosphonate
esters, aminoacyl
pyrrolidine-2-nitriles and 4-cyanothiazolidides. It is to be understood that
each and every
reactive group described herein can be substituted for the reactive group of
Formula II (i.e.,
boronyl group).
2o All amino acids, with the exception of glycine, contain an asymmetric or
chiral
carbon and may contain more than one chiral carbon atom. The asymmetric a
carbon atom of
the amino acid is referred to as a chiral center and can occur in two
different isomeric forms.
These forms are identical in all chemical and physical properties with one
exception, the
direction in which they can cause the rotation of plane-polarized light. These
amino acids are
referred to as being "optically active," i.e., the amino acids can rotate the
plane-polarized
light in one direction or the other.
The four different substituent groups attached to the a carbon can occupy two
different arrangements in space. These arrangements are not superimposable
mirror images
of each other and are referred to as optical isomers, enantiomers, or stereo
isomers. A
3o solution of one stereo isomer of a given amino acid will rotate plane
polarized light to the left
and is called the levorotatory isomer [designated (-)J; the other stereo
isomer for the amino
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acid will rotate plane polarized light to the same extent but to the right and
is called
dextrorotatory isomer [designated (+)].
A more systematic method for classifying and naming stereo isomers is the
absolute
configuration of the four different substituents in the tetrahedron around the
asymmetric
carbon atom (e.g., the a carbon atom). To establish this system, a reference
compound was
selected (glyceraldehyde), which is the smallest sugar to have an asymmetric
carbon atom.
By convention in the art, the two stereo isomers of glyceraldehyde are
designated L and D.
Their absolute configurations have been established by x-ray analysis. The
designations, L
and D, also have been assigned to the amino acids by reference to the absolute
configuration
to of glyueraldehyde. Thus, the stereo isomers of chiral compounds having a
configuration
related to that of L-glyceraldehyde are designed L, and the stereo isomers
having a
configuration related to D-glyceraldehyde are designated D, regardless of the
direction in
which they rotate the plane-polarized light. Thus, the symbols, L and D, refer
to the absolute
configuration of the four substituents around the chiral carbon.
In general, naturally occurring compounds which contain a chiral center are
only in
one stereo isomeric form, either D or L. The naturally occurring amino acids
are the L stereo
isomers; however, the invention embraces amino acids which can be in the D
stereo isomer
configuration.
Most amino acids that are found in proteins can be unambiguously named using
the D
L system. However, compounds which have two or more chiral centers may be in
2° possible
stereo isomer configurations, where n is the number of chiral centers. These
stereo isomers
sometimes are designated using the RS system to more clearly specify the
configurations of
amino acids that contain two or more chiral centers. For example, compounds
such as
threonine isoleucine contain two asymmetric carbon atoms and therefore have
four stereo
isomer configurations. The isomers of compounds having two chiral centers are
known as
diastereomers. A complete discussion of the RS system of designating optical
isomers for
amino acids is provided in Principles in Biochemistry, editor A.L. Lehninger,
page 99-100,
supra. A brief summary of this system follows.
The RS system was invented to avoid ambiguities when a compound contains two
or
3o more chiral centers. In general, the system is designed to rank the four
different substituent
atoms around an asymmetric carbon atom in order of decreasing atomic number or
in order of
decreasing valance density when the smallest or lowest-rank group is pointing
directly away
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from the viewer. The different rankings are well known in the art and are
described on page
99 of Lehninger (supra). If the decreasing rank order is seen to be clock-
wise, the
configuration around the chiral center is referred to as R; if the decreasing
rank order is
counter-clockwise, the configuration is referred to as S. Each chiral center
is named
accordingly using this system. Applying this system to threonine, one skilled
in the art would
determine that the designation, L-threonine, refers to (2S, 3R)-threonine in
the RS system.
The more traditional designations of L-, D-, L-alto, and D-allo, for threonine
have been in
common use for some time and continue to be used by those of skill in this
art. However, the
R S system increasingly is used to designate the amino acids, particularly
those which contain
/o more than one chiral center.
Many of the agents of the invention and methods for their manufacture have
been
previously disclosed in U.S. Patent 4,935,493, the contents of which are
incorporated by
reference herein.
As mentioned earlier, the agents, including their individual targeting and
reactive
I5 groups, may be synthesized using recombinant or chemical library synthesis
approaches.
Libraries of interest in the invention include peptide libraries, synthetic
organic combinatorial
libraries, and the like. The artisan or ordinary skill is familiar with the
methodology for
library and combinatorial chemistry synthesis as well as the screening of such
compounds for
agents which are useful in the methods of the invention. The use of library
technology, such
20 as phage display, and combinatorial chemistry, such as compound array
methods, in the
synthesis and screening of protease inhibitors has been previously described
in U.S. Patent
Application entitled "Multivalent Compounds for Crosslinking Receptors and
Uses Thereof "
filed on April 12, 1999 and assigned U.S.S.N. 09/290,376, the contents of
which are
incorporated in their entirety by reference. Examples of parallel synthesis
mixtures and
25 parallel synthesis methods are provided in U.S.S.N. 08/177,497, filed
January 5, 1994 and its
corresponding PCT published patent application W095/18972, published July 13,
1995 and
U.S. Patent No. 5,712,171 granted January 27, 1998 and its corresponding PCT
published
patent application W096/22529, which are hereby incorporated by reference.
According to one aspect of the invention, a method for treating a subject
having a
3o condition characterized by an abnormal mammalian cell proliferation is
provided. As used
herein, subject means a mammal including humans, nonhuman primates, dogs,
cats, sheep,
goats, horses, cows, pigs and rodents. An abnormal mammalian cell
proliferation disorder or
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condition, as used herein, refers to a localized region of cells (e.g., a
tumor) which exhibit an
abnormal (e.g., increased) rate of division as compared to their normal tissue
counterparts.
Conditions characterized by an abnormal mammalian cell proliferation, as used
herein, include but are not limited to conditions involving solid tumor masses
of benign, pre-
y malignant or malignant character. Although not wishing to be bound by a
particular theory or
mechanism, some of these solid tumor masses arise from at least one genetic
mutation, some
may display an increased rate of cellular proliferation as compared to the
normal tissue
counterpart, and still others may display factor independent cellular
proliferation. Factor
independent cellular proliferation is an example of a manifestation of loss of
growth control
/o signals which some, if not all, tmrmis or cancers undergo.
Since the invention is premised, in part, on the finding that agents of
Formula I are
able to inhibit FAP-a, a cell surface marker of reactive stromal fibroblasts,
in one aspect, the
invention involves treating conditions involving a tumor mass which contains
or is dependent
upon the presence of reactive stromal fibroblasts at some point during its
development. As
/5 used herein, reactive fibroblasts are fibroblasts which have been activated
to express proteins
such as receptors and growth factors which, in some instances, have a positive
effect and, in
other instances, have a negative effect on cellular proliferation and growth
of the fibroblasts
themselves, and other cell types such as malignant cells of a carcinoma or
epithelial
metastasis.
2o In one aspect, the invention provides a method for treating subjects having
a condition
characterized by an abnormal epithelial cell proliferation. Epithelial cells
are cells occurring
in one or more layers which cover the entire surface of the body and which
line most of the
hollow structures of the body, excluding the blood vessels, lymph vessels, and
the heart
interior which are lined with endothelium, and the chest and abdominal
cavities which are
25 lined with mesothelium. Examples of epithelium include anterius corneae,
anterior
epithelium of cornea, Barrett's epithelium, capsular epithelium, ciliated
epithelium, columnar
epithelium, epithelium corneae, corneal epithelium, cubical epithelium,
cubical epithelium,
cuboidal epithelium, epithelium eductus semicircularis, enamel epithelium,
false epithelium,
germinal epithelium, gingival epithelium, glandular epithelium, glomerular
epithelium,
30 laminated epithelium, epithelium of lens, epithelium lends, mesenchymal
epithelium,
olfactory epithelium, pavement epithelium, pigmentary epithelium, pigmented
epithelium,
protective epithelium, pseudostratified epithelium, pyramidal epithelium,
respiratory
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epithelium, rod epithelium, seminiferous epithelium, sense epithelium, sensory
epithelium,
simple epithelium, squamous epithelium, stratified epithelium, subcapsular
epithelium,
sulcular epithelium, tessellated epithelium, transitional epithelium.
One category of conditions characterized by abnormal epithelial cell
proliferation is
proliferative dermatologic disorders. These include conditions such as
keloids, seborrheic
keratosis, papilloma virus infection (e.g. producing verruca vulbaris, verruca
plantaris,
verruca plana, condylomata, etc.) and eczema.
An epithelial precancerous lesion is a skin lesion which has a propensity to
develop
into a cancerous condition. Epithelial precancerous skin lesions also arise
from other
/0 proliferaiive skin disorders such as hemangiomas, keloids, eczema and
papilloma virus
infections producing verruca vulbaris, verruca plantaris and verruca planar.
The symptoms of
the epithelial precancerous lesions include skin-colored or red-brown macule
or papule with
dry adherent scales. Actinic keratosis is the most common epithelial
precancerous lesion
among fair skinned individuals. It is usually present as lesions on the skin
which may or may
not be visually detectable. The size and shape of the lesions varies. It is a
photosensitive
disorder and may be aggravated by exposure to sunlight. Bowenoid actinic
keratosis is
another form of an epithelial precancerous lesion. In some cases, the lesions
may develop
into an invasive form of squamous cell carcinoma and may pose a significant
threat of
metastasis. Other types of epithelial precancerous lesions include
hypertrophic actinic
2o keratosis, arsenical keratosis, hydrocarbon keratosis, thermal keratosis,
radiation keratosis,
viral keratosis, Bowen's disease, erythroplaquia of queyrat, oral
erythroplaquia, leukoplakia,
and intraepidermal epithelialoma.
Another category of conditions characterized by abnormal epithelial cell
proliferation
is tumors of epithelial origin. FAP-a has been observed in tumors of
epithelial origin Thus,
in one aspect, the invention provides a method for treating subjects having
epithelial tumors.
Epithelial tumors are known to those of ordinary skill in the art and include,
but are not
limited to, benign and premalignant epithelial tumors, such as breast
fibroadenoma and colon
adenoma, and malignant epithelial tumors. Malignant epithelial tumors include
primary
tumors, also referred to as carcinomas, and secondary tumors, also referred to
as metastases
of epithelial origin. Carcinomas intended for treatment with the methods of
the invention
include, but are not limited to, acinar carcinoma, acinous carcinoma, alveolar
adenocarcinoma (also called adenocystic carcinoma, adenomyoepithelioma,
cribriform
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carcinoma and cylindroma), carcinoma adenomatosum, adenocarcinoma, carcinoma
of
adrenal cortex, alveolar carcinoma, alveolar cell carcinoma (also called
bronchiolar
carcinoma, alveolar cell tumor and pulmonary adenomatosis), basal cell
carcinoma,
carcinoma basocellulare (also called basaloma, or basiloma, and hair matrix
carcinoma),
basaloid carcinoma, basosquamous cell carcinoma, breast carcinoma,
bronchioalveolar
carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform
carcinoma,
cholangiocellular carcinoma (also called cholangioma and cholangiocarcinoma),
chorionic
carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform
carcinoma,
carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical
cell
Io carcinoma, duct carcinoma, carcinoma durum, embryonal carcitfoix~a,
encephaloid carcinoma,
epibulbar carcinoma, epidermoid carcinoma, carcinoma epitheliale adenoides,
carcinoma
exulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma,
giant cell
carcinoma, gigantocellulare, glandular carcinoma, granulosa cell carcinoma,
hair-matrix
carcinoma, hematoid carcinoma, hepatocellular carcinoma (also called hepatoma,
malignant
hepatoma and hepatocarcinoma), Hurthle cell carcinoma, hyaline carcinoma,
hypernephroid
carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal
carcinoma,
intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma,
lenticular
carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial
carcinoma,
carcinoma mastitoides, carcinoma medullare, medullary carcinoma, carcinoma
melanodes,
melanotic carcinoma, mucinous carcinoma, carcinoma muciparum, carcinoma
mucocellulare,
mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma
myxomatodes, nasopharyngeal carcinoma, carcinoma nigrum, oat cell carcinoma,
carcinoma
ossificans, osteoid carcinoma, ovarian carcinoma, papillary carcinoma,
periportal carcinoma,
preinvasive carcinoma, prostate carcinoma, renal cell carcinoma of kidney
(also called
adenocarcinoma of kidney and hypernephoroid carcinoma), reserve cell
carcinoma,
carcinoma sarcomatodes, scheinderian carcinoma, scirrhous carcinoma, carcinoma
scrod,
signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid
carcinoma,
spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum,
squamous
carcinoma, squamous cell carcinoma, string carcinoma, carcinoma
telangiectaticum,
3o carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum,
tuberous
carcinoma, verrucous carcinoma, carcinoma vilosum. In preferred embodiments,
the
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methods of the invention are used to treat subjects having cancer of the
breast, cervix, ovary,
prostate, lung, colon and rectum, pancreas, stomach or kidney.
Other conditions characterized by an abnormal mammalian cell proliferation to
be
treated by the methods of the invention include sarcomas. Sarcomas are rare
mesenchymal
neoplasms that arise in bone and soft tissues. Different types of sarcomas are
recognized and
these include: liposarcomas (including myxoid liposarcomas and pleiomorphic
liposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheral nerve
sheath
tumors (also called malignant schwannomas, neurofibrosarcomas, or neurogenic
sarcomas),
Ewing's tumors (including Ewing's sarcoma of bone, extraskeletal [not bone]
Ewing's
1o sarcoma, and primitive neuroectoder~~~Qi tumor [PNET]), synovial sarcoma,
arsg~osarcomas,
hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma,
fibrosarcoma, desmoid tumor (also called aggressive fibromatosis),
dermatofibrosarcoma
protuberans (DFSP), malignant fibrous histiocytoma (MFH), hemangiopericytoma,
malignant
mesenchymoma, alveolar soft-part sarcoma, epithelioid sarcoma, clear cell
sarcoma,
IS desmoplastic small cell tumor, gastrointestinal stromal tumor (GIST) (also
known as GI
stromal sarcoma), osteosarcoma (also known as osteogenic sarcoma)-skeletal and
extraskeletal, and chondrosarcoma.
The methods of the invention are also directed towards the treatment of
subjects with
melanoma. Melanomas are tumors arising from the melanocytic system of the skin
and other
20 organs. Examples of melanoma include lentigo maligna melanoma, superficial
spreading
melanoma, nodular melanoma, and acral lentiginous melanoma.
Other conditions characterized by an abnormal mammalian cell proliferation are
cancers including, but not limited to, biliary tract cancer, endometrial
cancer, esophageal
cancer, gastric cancer, intraepithelial neoplasms, including Bowen's disease
and Paget's
25 disease, liver cancer, oral cancer, including squamous cell carcinoma,
sarcomas, including
fibrosarcoma and osteosarcoma, skin cancer, including melanoma, Kaposi's
sarcoma,
testicular cancer, including germinal tumors (seminoma, non-seminoma
(teratomas,
choriocarcinomas)), stromal tumors and germ cell tumors, thyroid cancer,
including thyroid
adenocarcinoma and medullar carcinoma, and renal cancer including
adenocarcinoma and
30 Wilms tumor.
According to other aspects of the invention, a method is provided for treating
a
subject having an abnormal proliferation originating in bone, muscle or
connective tissue.
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Exemplary conditions intended for treatment by the method of the invention
include primary
tumors (i.e., sarcomas) of bone and connective tissue.
The methods of the invention are also directed towards the treatment of
subjects with
metastatic tumors. In some embodiments, the metastatic tumors are of
epithelial origin.
s Carcinomas may metastasize to bone, as has been observed with breast cancer,
and liver, as is
sometimes the case with colon cancer. The methods of the invention are
intended to treat
metastatic tumors regardless of the site of the metastasis and/or the site of
the primary tumor.
In preferred embodiments, the metastases are of epithelial origin.
The method, in one embodiment, intends to treat subjects free of symptoms
calling for
hemop~iWic stimulation, by administering compounds of Formula I in an amount
effective to
inhibit proliferation. The ability to treat subjects having symptoms calling
for hemopoietic
stimulation with the some of compounds (e.g., ValboroPro) described herein has
been
previously disclosed in U. S. Patent Application entitled "Hematopoietic
Stimulation", Serial
No. 09/304,199, filed May 3, 1999, the contents of which are incorporated
herein in their
/5 entirety by reference. Thus, the instant invention intends, in certain
embodiments, to treat
subjects at a time when they are free of symptoms requiring hemopoietic
stimulating
treatment or to treat subjects who have such symptoms with amounts or dosages
or
administration schedules that differ from those used to protect or restore
normal or protective
levels of hemopoietic cells. A subject who has previously experienced a need
for
2o hemopoietic stimulation but has since recovered its hemopoietic cells to
normal or at least
protective levels may still be treated by the methods described herein.
As used herein, the terms hemopoietic and hematopoietic are used
interchangeably to
mean all blood cells including myeloid and lymphoid cells. Myeloid cells
include
erythrocytes (i.e., red blood cells), macrophages, monocytes, granulocytes
including
25 neutrophils, eosinophils and basophils, mast cells, megakaryoctyes,
platelets and dendritic
cells, and lymphoid cells include T and B lymphocytes, thymic dendritic cells
and natural
killer (NK) cells. Hemopoietic stimulation, as used herein, refers to the
increase in
hemopoietic cell numbers or activity to normal or protective levels.
An example of a symptom calling for hemopoietic stimulation is hemopoietic
cell
30 numbers below normal or protective levels. A "normal" level as used herein
may be a level
in a control population, which preferably includes subjects having similar
characteristics as
the treated individual, such as age and sex. The ''normal" level can also be a
range, for
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example, where a population is used to obtain a baseline range for a
particular group into
which the subject falls. Thus, the "normal" value can depend upon a particular
population
selected. Preferably, the normal levels are those of apparently healthy
subjects who have no
prior history of hematopoietic cell disorders. Such "normal" levels, then can
be established
as preselected values, taking into account the category in which an individual
falls.
Appropriate ranges and categories can be selected with no more than routine
experimentation
by those of ordinary skill in the art. Either the mean or another preselected
number within the
range can be established as the normal preselected value.
In general, the normal range for neutrophils is about 1800-7250 per p,1 (mean -
3650);
/o for basophils 0-150 per p,1 (mean -30); for eosinophils 0-700 per ~.i (mean
-150); for
macrophages and monocytes 200-950 per ~.1 (mean -430); for lymphocytes 1500-
4000 per g,1
(mean -2500); for erythrocytes 4.2 x 106 - 6.1 x 106 per ~,1; and for
platelets 133 x 103 - 333 x
103 per ~1. The foregoing ranges are at the 95% confidence level.
In connection with certain conditions, the medical community has established
certain
/5 preselected values. For example, mild neutropenia is characterized as
having a count of
between 1000 and 2000 per p.1, moderate neutropenia at between 500 and 1000
per q1 and
severe neutropenia at below 500 per g1. Likewise, in adults, a lymphocyte
count at less than
1500 is considered a medically undesirable condition. In children, the value
is less than
3000. Other preselected values will be readily known to those of ordinary
skill in the art.
2o A protective level of hematopoietic cells is the number of cells required
to confer
clinical benefit to the patient. The required level can be equal to or less
than the "normal
level". Such levels are well known to those of ordinary skill in the art. For
example, a
protective level of neutrophils is above 1000, preferably, at least 1500.
Thus the methods of the invention, according to some embodiments, are directed
25 towards subjects who possess normal or protective levels of hemopoietic
cells, as described
herein. Subjects with normal or protective levels of hemopoietic cells are
considered to have
normal hemopoietic activity. Likewise, in some embodiments, the invention is
directed for
use in subjects who are not immunocompromised. As used herein, the terms
immunocompromised and immunosuppressed are used interchangeably. An example of
an
3o immunocompromised subject is one infected with HIV and experiencing AIDS-
related
symptoms such as low CD4+ T lymphocyte levels. In still other embodiments, the
methods
may be used in subjects who are HIV-positive and who may be immunocompromised,
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provided that the agent is administered in an amount, a dosing regimen, and an
administration
schedule that have a therapeutic effect on abnormal proliferation, such as in
a Kaposi's
sarcoma tumor, but are not therapeutically effective in stimulating
hemopoiesis in the subject.
According to still other embodiments, subjects of the invention are those who
may
have previously received anti-cancer therapy or who will in the future receive
anti-cancer
therapy but who do not at the time of treatment need hemopoietic stimulation,
including a
blood transfusion or administration of a hemopoietic stimulant such as a
hemopoietic growth
factor.
Thus in certain embodiments, the subjects are not myeloid or lymphoid
suppressed or
IO are not candidates for treatment witl-~ ~ragent which causes such
suppression at the time of
treatment with the methods of the instant invention. Myeloid suppressing
conditions are
those which induce a reduction in myeloid cells such as erythrocytes,
neutrophils or platelets,
to below protective or normal levels. Exemplary myelosuppressed conditions are
hemopoietic malignancies, including leukemia and lymphoma and diseases such as
chronic
1s idiopathic neutropenia, cyclic neutropenia, anemia and thrombocytopenia.
Similarly,
lymphoid suppressing conditions are those which induce a reduction in lymphoid
cells such
as T lymphocytes. Suppression of lymphoid cells or some myeloid cells such as
granulocytes
is also referred to as immunosuppression since reduction in these cell types
makes an
individual susceptible to, inter alia, infection. Subjects may be exposed to
myeloid,
20 lymphoid or general immune suppressing conditions by the use of either
immunosuppressant
drugs such as cyclosporin or high dose chemotherapeutic compounds which affect
dividing
hemopoietic cells. Immuno-suppression may also arise as a result of treatment
modalities
such as total body irradiation or conditioning regimens prior to bone marrow
transplantation.
Viral infection, particularly as in the case of infection with human
immunodeficiency virus
25 (HIV), may also immunosuppress an individual. In some embodiments, subjects
are those
which have not been exposed and are not anticipated to be exposed to the above-
mentioned
conditions. In other embodiments, the instant invention aims to treat subjects
who may have
been myelosuppressed or immunosuppressed (e.g., by exposure to one or more of
the above
conditions), provided that at the time of treatment using the methods
described herein, the
3o subject has protective or normal levels of hemopoietic cells.
In still other embodiments, the invention aims to treat subjects who may
exhibit
symptoms calling for hemopoietic stimulation, provided that the agents are
administered in
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doses, routes and schedules that would not result in hemopoietic stimulation,
as explained
below. In certain embodiments, the methods of the invention are not intended
for use in the
treatment of malignancies in HIV infected (i.e., HIV positive or HIV+)
subjects who have
below normal or below protective levels of hemopoietic cells, unless the
agents are used
under conditions, such as administration routes, doses or dosing schedules,
that are
therapeutically effective in treating abnormal cell proliferation, as
described herein, and not
effective in stimulating hemopoiesis. For example, in some embodiments, the
agent may be
administered once a day, or twice a day, or three or more times a day, for
more than 7 days,
more than 10 days, more than 14 days or more than 20 days in order to achieve,
for example,
1o sustained desired systemic levels. In other embodiments, the agent may be
given at timed
intervals, such as, for example, every two days, every three days, every four
days, every week
or every two weeks. In still further embodiments, the agent may be delivered
intravenously
and continuously, for example, or by injection, such as, in single bolus
administrations.
According to another aspect of the invention, methods are provided for
inhibiting
angiogenesis in disorders having a pathology which requires angiogenesis.
Angiogenesis is
defined as the formation of new blood vessels. One subset of these disorders
is conditions
characterized by abnormal mammalian cell proliferation. Another subset is non-
cancer
conditions including diabetic retinopathy, neovascular glaucoma and psoriasis.
In preferred embodiments, the methods of the invention are aimed at inhibiting
tumor
angiogenesis. Tumor angiogenesis refers to the formation of new blood vessels
in the
vicinity or within a tumor mass. Solid tumor cancers require angiogenesis
particularly for
oxygen and nutrient supply. It has been previously shown that inhibition of
angiogenesis in
solid tumor can cause tumor regression in animal models. Thus in one aspect,
the invention
relates to a method for inhibiting angiogenesis by inhibiting the
proliferation, migration or
activation of endothelial cells and fibroblasts, provided this angiogenesis is
unrelated to
wound healing in response to injury, infection or inflammation.
Thus in certain embodiments, the methods of the invention are intended for the
treatment of diseases and processes that are mediated by angiogenesis
including, but not
limited to, hemangioma, solid tumors, tumor metastasis, benign tumors, for
example
3o hemangiomas, acoustic neuromas, neurofibromas and trachomas, Osler-Webber
Syndrome,
telangiectasia, myocardial angiogenesis, angiofibroma, plaque
neovascularization, coronary
collaterals, ischemic limb angiogenesis, corneal diseases, rubiosis,
neovascular glaucoma,
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diabetic retinopathy, retrolental fibroplasia, diabetic neovascularization,
macular
degeneration, keloids, ovulation, menstruation, and placentation.
The compositions and methods of the invention in certain instances may be
useful for
replacing existing surgical procedures or drug therapies, although in most
instances the
present invention is useful in improving the efficacy of existing therapies
for treating such
conditions. Accordingly combination therapy may be used to treat the subjects.
For
example, the agent may be administered to a subject in combination with
another anti-
proliferative (e.g., an anti-cancer) therapy. Suitable anti-cancer therapies
include surgical
procedures to remove the tumor mass, chemotherapy or localization radiation.
The other
1o anti-proliferative therapy may be administered before, concurrent with, or
after treatment
with the agent of the invention. There may also be a delay of several hours,
days and in some
instances weeks between the administration of the different treatments, such
that the agent
may be administered before or after the other treatment.
As an example, the agent may be administered in combination with surgery to
remove
75 an abnormal proliferative cell mass. As used herein, "in combination with
surgery" means
that the agent may be administered prior to, during or after the surgical
procedure. Surgical
methods for treating epithelial tumor conditions include intra-abdominal
surgeries such as
right or left hemicolectomy, sigmoid, subtotal or total colectomy and
gastrectomy, radical or
partial mastectomy, prostatectomy and hysterectomy. In these embodiments, the
agent may
2o be administered either by continuous infusion or in a single bolus.
Administration during or
immediately after surgery may include a lavage, soak or perfusion of the tumor
excision site
with a pharmaceutical preparation of the agent in a pharmaceutically
acceptable carrier. In
some embodiments, the agent is administered at the time of surgery as well as
following
surgery in order to inhibit the formation and development of metastatic
lesions. The
25 administration of the agent may continue for several hours, several days,
several weeks, or in
some instances, several months following a surgical procedure to remove a
tumor mass.
The subjects can also be administered the agent in combination with non-
surgical
anti-proliferative (e.g., anti-cancer) drug therapy. In one embodiment, the
agent may be
administered in combination with an anti-cancer compound such as a cytostatic
compound.
30 A cytostatic compound is a compound (e.g., a nucleic acid, a protein) that
suppresses cell
growth and/or proliferation. In some embodiments, the cytostatic compound is
directed
towards the malignant cells of a tumor. In yet other embodiments, the
cytostatic compound
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is one which inhibits the growth and/or proliferation of vascular smooth
muscle cells or
fibroblasts.
Suitable anti-proliferative drugs or cytostatic compounds to be used in
combination
with the agents of the invention include anti-cancer drugs. Anti-cancer drugs
are well known
and include: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;
Adozelesin;
Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;
Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine;
Azetepa;
Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride;
Bisnafide
Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine;
Busulfan;
to Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine;
Caruhicin
Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin;
Cladribine;
Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin;
Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine;
Dezaguanine
Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;
Droloxifene;
I5 Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate;
Eflornithine
Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin
Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine;
Estramustine Phosphate
Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole
Hydrochloride;
Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;
Flurocitabine;
2o Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride;
Hydroxyurea;
Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a;
Interferon Alfa-2b;
Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-
I b; Iproplatin;
Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate;
Liarozole
Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride;
Masoprocol;
25 Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol
Acetate;
Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;
Metoprine;
Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;
Mitomycin;
Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole;
Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;
Pentamustine;
3o Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone
Hydrochloride;
Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;
Procarbazine
Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine;
Rogletimide;
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Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;
Sparsomycin;
Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin;
Streptozocin;
Sulofenur; Talisomycin; Taxol; Taxotere; Tecogalan Sodium; Tegafur;
Teloxantrone
Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine;
Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride;
Toremifene
Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate
Glucuronate;
Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;
Verteporfin;
Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate;
Vinepidine Sulfate;
Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine
Sulfate;
to Vinzolidir~e 5~:ate; Vorozole; Zeniplatin; Zinostatin; Z,nrubicin
Hydrochloride.
Other anti-cancer drugs include: 20-epi-1,25 dihydroxyvitamin D3; 5-
ethynyluracil;
abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin;
ALL-TK
antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic
acid; amrubicin;
amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;
antagonist D;
antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic
carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;
aphidicolin glycinate;
apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-
PTBA; arginine
deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2;
axinastatin 3;
azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;
batimastat; BCR/ABL
2o antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives;
beta-alethine;
betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene;
bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate;
bropirimine; budotitane;
buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives;
canarypox IL-2;
capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3;
CARN 700;
cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine;
cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;
cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B;
combretastatin
A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol;
cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam;
cypemycin;
3o cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;
dehydrodidemnin B;
deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B;
didox;
diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin;
diphenyl
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spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;
duocarmycin
SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;
emitefur;
epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen
antagonists;
etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;
fenretinide; filgrastim;
finasteride; flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin
hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium
texaphyrin;
gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione
inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;
ibandronic acid;
idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones;
imiquimod;
1o immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor;
interferon
agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-
; irinotecan;
iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;
jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim;
lentinan sulfate;
leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha
interferon; leuprolide +
estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine
analogue;
lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide
7; lobaplatin;
lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;
lurtotecan; lutetium
texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;
marimastat; masoprocol;
maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril;
merbarone;
2o meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;
miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin
analogues; mitonafide; mitotoxin fibroblast growth factor-saporin;
mitoxantrone; mofarotene;
molgramostim; monoclonal antibody, human chorionic gonadotrophin;
monophosphoryl lipid
A + myobacterium cell wall sk; mopidamol; multiple drug resistance gene
inhibitor; multiple
tumor suppressor 1-based therapy; mustard anti cancer compound; mycaperoxide
B;
mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides;
nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin;
nartograstim; nedaplatin;
nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin;
nitric oxide
modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide;
okicenone;
oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine
inducer;
ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues;
paclitaxel derivatives;
palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;
parabactin;
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pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium;
pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate;
phosphatase
inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim;
placetin A; placetin
B; plasminogen activator inhibitor; platinum complex; platinum compounds;
platinum-
triamine complex; porfimer sodium; porfiromycin; propyl bis-acridone;
prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein kinase C
inhibitor;
protein kinase C inhibitors, microalgal; protein tyrosine phosphatase
inhibitors; purine
nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin
polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras
farnesyl protein
1n transferase inhibitors; ras inhibitors; ras-~A~ i~~i~ibitor; retelliptine
demethylated; rhenium
Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;
rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol
A;
sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense
oligonucleotides; signal transduction inhibitors; signal transduction
modulators; single chain
antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate;
solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D;
spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor;
stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive
vasoactive intestinal
peptide antagonist; suradista; suramin; swainsonine; synthetic
glycosaminoglycans;
2o tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan
sodium; tegafur;
tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline;
thrombopoietin;
thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist;
thymotrinan; thyroid
stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene
dichloride; topotecan;
topsentin; toremifene; totipotent stem cell factor; translation inhibitors;
tretinoin;
triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron;
turosteride; tyrosine kinase
inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived
growth inhibitory
factor; urokinase receptor antagonists; vapreotide; variolin B; vector system,
erythrocyte gene
therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;
vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; zinostatin stimalamer.
Anti-cancer supplementary potentiating compounds include: Tricyclic anti-
depressant
drugs (e.g., imipramine, desipramine, amitryptyline, clomipramine,
trimipramine, doxepin,
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nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic anti-
depressant drugs
(e.g., sertraline, trazodone and citalopram); Ca++ antagonists (e.g.,
verapamil, nifedipine,
nitrendipine and caroverine); Calmodulin inhibitors (e.g., prenylamine,
trifluoroperazine and
clomipramine); Amphotericin B; Triparanol analogues (e.g., tamoxifen);
antiarrhythmic
drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thiol
depleters (e.g.,
buthionine and sulfoximine) and multiple drug resistance reducing compounds
such as
Cremaphor EL.
Other compounds which are useful in combination therapy for the purpose of the
invention include the antiproliferation compound, Piritrexim Isethionate; the
antiprostatic
1o hypertrophy compound, Sitogluside; the benign prostatic lz;~perplasia
therapy compound,
Tamsulosin Hydrochloride; the prostate growth inhibitor, Pentomone;
radioactive compounds
such as Fibrinogen 1 125, Fludeoxyglucose F 18, Fluorodopa F 18, Insulin I
125, Insulin I
131, Iobenguane I 123, Iodipamide Sodium I 131, Iodoantipyrine I 131,
Iodocholesterol I
131, Iodohippurate Sodium I 123, Iodohippurate Sodium I 125, Iodohippurate
Sodium I 131,
Iodopyracet I 125, Iodopyracet I 131, Iofetamine Hydrochloride I 123, Iomethin
I 125,
Iomethin I 131, Iothalamate Sodium I 125, Iothalamate Sodium I 131, Iotyrosine
1 131,
Liothyronine I 125, Liothyronine I 131, Merisoprol Acetate Hg 197, Merisoprol
Acetate Hg
203, Merisoprol Hg 197, Selenomethionine Se 75, Technetium Tc 99m Antimony
Trisulfide
Colloid, Technetium Tc 99m Bicisate, Technetium Tc 99m Disofenin, Technetium
Tc 99m
2o Etidronate, Technetium Tc 99m Exametazime, Technetium Tc 99m Furifosmin,
Technetium
Tc 99m Gluceptate, Technetium Tc 99m Lidofenin, Technetium Tc 99m Mebrofenin,
Technetium Tc 99m Medronate, Technetium Tc 99m Medronate Disodium, Technetium
Tc
99m Mertiatide, Technetium Tc 99m Oxidronate, Technetium Tc 99m Pentetate,
Technetium
Tc 99m Pentetate Calcium Trisodium, Technetium Tc 99m Sestamibi, Technetium Tc
99m
Siboroxime, Technetium Tc 99m Succimer, Technetium Tc 99m Sulfur Colloid,
Technetium
Tc 99m Teboroxime, Technetium Tc 99m Tetrofosmin, Technetium Tc 99m Tiatide,
Thyroxine I 125, Thyroxine I 131, Tolpovidone I 131, Triolein I 125 and
Triolein I 131.
According to the methods of the invention, the agents of Formula I may be
administered prior to, concurrent with, or following the other anti-cancer
compounds. The
3o administration schedule may involve administering the different agents in
an alternating
fashion. In other embodiments, the agent may be delivered before and during,
or during and
after, or before and after treatment with other therapies. In some cases, the
agent is
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administered more than 24 hours before the administration of the other anti-
proliferative
treatment. In other embodiments, more than one anti-proliferative therapy may
be
administered to a subject. For example, the subject may receive the agents of
the invention, in
combination with both surgery and at least one other anti-proliferative
compound.
Alternatively, the agent may be administered in combination with more than one
anti-cancer
drug.
Other compounds useful in combination therapies with the inhibitor compounds
of the
invention include anti-angiogenic compounds such as angiostatin, endostatin,
fumagillin,
non-glucocorticoid steroids and heparin or heparin fragments and antibodies to
one or more
angiogenic peptides such as aFGF, (3FGF, VEGF, IL-8 and GM-CSF. These ianer
anti-
angiogenic compounds may be administered along with the inhibitor agents of
the invention
(i.e., the agents of Formula I) for the purpose of inhibiting proliferation or
inhibiting
angiogenesis in all of the aforementioned conditions as described herein. In
certain
embodiments, the agent may be administered in combination with an anti-
angiogenic
I5 compound and at least one of the anti-proliferative therapies described
above including
surgery or anti-proliferative drug therapy.
The above-described drug therapies are well known to those of ordinary skill
in the art
and are administered by modes known to those of skill in the art. The drug
therapies are
administered in amounts which are effective to achieve physiological goals
such as the
2o inhibition of proliferation or inhibition of angiogenesis, in combination
with the agents of the
invention. It is contemplated that the drug therapies may be administered in
amounts which,
when used alone, may not be capable of inhibiting proliferation or
angiogenesis but which,
when administered in combination with the agents of the invention, are capable
of achieving
the desired level of inhibition. Thus, in embodiments in which the agent of
Formula I is
25 administered with another therapeutic agent (e.g., an anti-proliferative
compound or an anti-
angiogenic compound), subtherapeutic doses of either or both agents may be
used. In still
other embodiments, anti-proliferative drug therapies may be administered in
conditions such
as doses or amounts which do not affect hemopoietic cell levels in the
subjects.
The agents of the invention are administered in therapeutically effective
amounts. An
3o effective amount is a dosage of the agent sufficient to provide a medically
desirable result.
The effective amount will vary with the particular condition being treated,
the age and
physical condition of the subject being treated, the severity of the
condition, the duration of
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the treatment, the nature of the concurrent or combination therapy (if any),
the specific route
of administration and like factors within the knowledge and expertise of the
health
practitioner. It is preferred generally that a maximum dose be used, that is,
the highest safe
dose according to sound medical judgment.
For example, in connection with methods directed towards treating subjects
having a
condition characterized by abnormal mammalian cell proliferation, an effective
amount to
inhibit proliferation would be an amount sufficient to reduce or halt
altogether the abnormal
mammalian cell proliferation so as to slow or halt the development of or the
progression of a
cell mass such as, for example, a tumor. As used in the embodiments, "inhibit"
embraces all
of the foregoing.
According to other aspects of the invention directed at inhibiting
angiogenesis in a
subject having a condition characterized by an abnormal mammalian cell
proliferation, an
effective amount to inhibit angiogenesis would be an amount sufficient to
lessen or inhibit
altogether smooth muscle cell proliferation so as to slow or halt the
development of or the
progression of tumor vascularization. As used in these embodiments, "inhibit"
embraces all
of the foregoing.
When used therapeutically, the agent is administered in therapeutically
effective
amounts. In general, a therapeutically effective amount means that amount
necessary to
delay the onset of, inhibit the progression of, or halt altogether the
particular condition being
treated. In some aspects of the invention, an therapeutically effective amount
will be that
amount necessary to inhibit mammalian cell proliferation. In other
embodiments, a
therapeutically effective amount will be an amount necessary to extend the
dormancy of
micrometastases or to stabilize any residual primary tumor cells following
surgical or drug
therapy.
In still other embodiments, the agent is delivered in an amount, a dose, and a
schedule
which is therapeutically effective in inhibiting proliferation yet which is
not therapeutically
effective in stimulating hemopoiesis in the subject. In administering the
agents of the
invention to subjects, dosing amounts, dosing schedules, routes of
administration and the like
can be selected so as to affect the other known activities of these compounds.
For example,
3o amounts, dosing schedules and routes of administration can be selected as
described below,
whereby therapeutically effective levels for inhibiting proliferation are
provided, yet
therapeutically effective levels for restoring hemopoietic deficiency are not
provided. As
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another example, local administration to tumors or protected body areas such
as the brain
may result in therapeutically effective levels for inhibiting proliferation,
but may be non-
therapeutically effective levels for hemopoietic cell stimulation.
In addition, agents of Formula I can be selected that are effective as anti-
proliferative
agents but are relatively ineffective as hemopoietic cell stimulatory or
activating agents.
Thus, certain subjects who require both hemopoietic stimulation and/or
activation and
proliferation inhibition may be treated with different agents of Formula I
simultaneously, one
each for the desired therapeutic effect, or with a single agent but in
different dosages,
schedules, and/or route to achieve both hemopoietic stimulation and
proliferation inhibition at
1o therapeutic levels.
Generally, a therapeutically effective amount will vary with the subject's
age,
condition, and sex, as well as the nature and extent of the disease in the
subject, all of which
can be determined by one of ordinary skill in the art. The dosage may be
adjusted by the
individual physician or veterinarian, particularly in the event of any
complication. A
/5 therapeutically effective amount typically varies from 0.01 mg/kg to about
1000 mg/kg,
preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from
about 0.2
mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or
more days.
In some embodiments, the agents are administered for more than 7 days, more
than 10 days,
more than 14 days and more than 20 days. In still other embodiments, the agent
is
2o administered over a period of weeks, or months. In still other embodiments,
the agent is
delivered on alternate days. For example, the agent is delivered every two
days, or every
three days, or every four days, or every five days, or every six days, or
every week, or every
month.
The agents of the invention can also be administered in prophylactically
effective
25 amounts, particularly in subjects diagnosed with benign or pre-malignant
tumors. In these
instances, the agents are administered in an amount effective to prevent the
development of
an abnormal mammalian cell proliferative mass or to prevent angiogenesis in
the solid tumor
mass, depending on the embodiment. The agents may also be administered in an
amount
effective to prevent metastasis of cells from a tumor to other tissues in the
body. In these
30 latter embodiments, the invention is directed to preventing the metastatic
spread of a primary
tumor.
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According to another aspect of the invention, a kit is provided. The kit is a
package
which houses a container which contains an agent of the invention and also
houses
instructions for administering the agent of the invention to a subject having
a condition
characterized by an abnormal mammalian cell proliferation. The kit may
optionally also
contain one or more other anti-proliferative compounds or one or more anti-
angiogenic
compounds for use in combination therapies as described herein.
In still another aspect of the invention, kits for administration of an agent
of the
invention to a subject is provided. The kits include a container containing a
composition
which includes at least one agent of the invention, and instructions for
administering the at
least one agent to a subject having a condition characterized by an abnormal
rr~ammalian cell
proliferation in an amount effective to inhibit proliferation. In certain
embodiments, the
container is a container for intravenous administration. In other embodiments
the agent is
provided in an inhaler. In still other embodiments, the agent is provided in a
polymeric
matrix or in the form of a liposome. In yet other embodiments, kits are
provided for the
75 administration of an agent of the invention to a subject having an abnormal
mammalian cell
mass for the purpose of inhibiting angiogenesis in the cell mass. In these
latter kits, the agent
is provided in an amount effective to inhibit angiogenesis along with
instructions for use in
subjects in need of such treatment.
The agent may be administered alone or in combination with the above-described
2o drug therapies by a variety of administration routes available. The
particular mode selected
will depend, of course, upon the agent selected, the condition being treated,
the severity of
the condition, whether the treatment is therapeutic or prophylactic, and the
dosage required
for efficacy. The methods of the invention, generally speaking, may be
practiced using any
mode of administration that is medically acceptable, meaning any mode that
produces
25 effective levels of the active compounds without causing clinically
unacceptable adverse
effects. The administration may, for example, be oral, intraperitoneal, intra-
cavity such as
rectal or vaginal, transdermal, topical, nasal, inhalation, mucosal,
interdermal, or parenteral
routes. The term "parenteral" includes subcutaneous, intravenous,
intramuscular, or infusion.
Intravenous or intramuscular routes may not particularly suitable for long
term therapy and
3o prophylaxis. In certain embodiments, however, it may be appropriate to
administer the agent
in a continuous infusion every several days, or once a week, or every several
weeks, or once a
month. Intravenous or intramuscular routes may be preferred in emergency
situations. Oral
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administration may be used for prophylactic treatment because of the
convenience to the
patient as well as the dosing schedule. Likewise, sustained release devices as
described
herein may be useful in certain embodiments for prophylactic or post surgery
treatment, for
example.
When using the agent of the invention in subjects in whom the primary site of
abnormal proliferation is well delineated and easily accessible, direct
administration to the
site may be preferred, provided the tumor has not already metastasized. For
example,
administration by inhalation for lung tumors or by suppositories in the
treatment of cervical,
ovarian or rectal tumors may be preferred. Likewise, melanoma, for example,
may be treated
7o with the agent via topical administration in and around the area of the
lesion. In still other
embodiments aimed at the treatment of subjects with breast or prostate cancer,
the agents
may be delivered by injection directly into the tissue with, for example, a
biopsy needle and
syringe.
Systemic administration may be preferred in some instances such as, for
example, if
15 the subject is known to have or is suspected of having metastases. In this
way, all tumor
sites, whether primary or secondary may receive the agent. Systemic delivery
may be
accomplished through for example, oral or parenteral administration.
Inhalation may be
used in either systemic or local delivery, as described below.
As discussed earlier, the agent may also be delivered to a tumor site during
or
2o immediately after a surgical procedure to remove the tumor by lavage into
the excision site or
by perfusion of the affected tissue with a physiologically acceptable solution
containing the
agent. Alternatively, the patient may be administered the agent prior to or
following the
surgical procedure by continuous infusion. In yet other embodiments, a
sustained release
device, as described below, such as a polymeric implant may be positioned
during surgery in
25 the vicinity of the excision site so as to provide a high local
concentration of the agent. These
latter embodiments may be appropriate to prevent regrowth of the tumor.
The agent of the invention may be administered alone or in combination with
the
above-described drug therapies as part of a pharmaceutical composition. Such a
pharmaceutical composition may include the agent in combination with any
standard
3o physiologically and/or pharmaceutically acceptable carriers which are known
in the art. The
compositions should be sterile and contain either a therapeutically or
prophylactically
effective amount of the agent in a unit of weight or volume suitable for
administration to a
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subject. The term "pharmaceutically-acceptable carrier" as used herein means
one or more
compatible solid or liquid filler, diluents or encapsulating substances which
are suitable for
administration into a subject of the invention. The term "carrier" denotes an
organic or
inorganic ingredient, natural or synthetic, with which the active ingredient
is combined to
facilitate the application. The components of the pharmaceutical compositions
also are
capable of being co-mingled with the molecules of the present invention, and
with each other,
in a manner such that there is no interaction which would substantially impair
the desired
pharmaceutical efficacy. Pharmaceutically-acceptable further means a non-toxic
material
that is compatible with a biological system such as a cell, cell culture,
tissue, or organism.
1o The characteristics of the carrier will depend on the route of
a~?r:inistration. Physiologically
and pharmaceutically-acceptable carriers include diluents, fillers, salts,
buffers, stabilizers,
solubilizers, and other materials which are well known in the art.
Compositions suitable for parenteral administration conveniently comprise a
sterile
aqueous preparation of the agent, which is preferably isotonic with the blood
of the recipient.
This aqueous preparation may be formulated according to known methods using
suitable
dispersing or wetting compounds and suspending compounds. The sterile
injectable
preparation also may be a sterile injectable solution or suspension in a non-
toxic parenterally-
acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
Among the
acceptable vehicles and solvents that may be employed are water, Ringer's
solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils are
conventionally employed
as a solvent or suspending medium. For this purpose, any bland fixed oil may
be employed
including synthetic mono- or di-glycerides. In addition, fatty acids such as
oleic acid may be
used in the preparation of injectables. Carrier formulations suitable for
oral, subcutaneous,
intravenous, intramuscular, etc. administrations can be found in Remington's
Pharmaceutical
Sciences, Mack Publishing Co., Easton, PA.
Preparations for parenteral administration include sterile aqueous or non-
aqueous
solutions. suspensions, and emulsions. Examples of non-aqueous solvents are
propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable
organic esters such
as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or
suspensions, including saline and buffered media. Parenteral vehicles include
sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's or fixed
oils. Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers
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(such as those based on Ringer's dextrose), and the like. Preservatives and
other additives
may also be present such as, for example, antimicrobials, anti-oxidants,
chelating compounds,
and inert gases and the like. The pharmaceutical compositions may conveniently
be
presented in unit dosage form and may be prepared by any of the methods well-
known in the
art of pharmacy.
Compositions suitable for oral administration may be presented as discrete
units, such
as capsules, tablets, lozenges, each containing a predetermined amount of the
agent. Other
compositions include suspensions in aqueous liquids or non-aqueous liquids
such as a syrup,
elixir or an emulsion.
1C In yet other embodiments. the preferred vehicle is a biocompatible
micropa_r«;,le or
implant that is suitable for implantation into the mammalian recipient.
Exemplary
bioerodible implants that are useful in accordance with this method are
described in PCT
International Application No. PCT/US/03307 (Publication No. WO 95/24929,
entitled
"Polymeric Gene Delivery System", claiming priority to U.S. patent application
serial no.
I5 213,668, filed March 15, 1994). PCT/LJS/0307 describes a biocompatible,
preferably
biodegradable polymeric matrix for containing a biological macromolecule. The
polymeric
matrix may be used to achieve sustained release of the agent in a subject. In
accordance with
one aspect of the instant invention, the agent described herein may be
encapsulated or
dispersed within the biocompatible, preferably biodegradable polymeric matrix
disclosed in
20 PCT/US/03307. The polymeric matrix preferably is in the form of a
microparticle such as a
microsphere (wherein the agent is dispersed throughout a solid polymeric
matrix) or a
microcapsule (wherein the agent is stored in the core of a polymeric shell).
Other forms of
the polymeric matrix for containing the agent include films, coatings, gels,
implants, and
stems. The size and composition of the polymeric matrix device is selected to
result in
25 favorable release kinetics in the tissue into which the matrix device is
implanted. The size of
the polymeric matrix devise further is selected according to the method of
delivery which is
to be used, typically injection into a tissue or administration of a
suspension by aerosol into
the nasal and/or pulmonary areas. The polymeric matrix composition can be
selected to have
both favorable degradation rates and also to be formed of a material which is
bioadhesive, to
3o further increase the effectiveness of transfer when the devise is
administered to a vascular or
pulmonary surface. The matrix composition also can be selected not to degrade,
but rather, to
release by diffusion over an extended period of time.
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Both non-biodegradable and biodegradable polymeric matrices can be used to
deliver
the agents of the invention to the subject. Biodegradable matrices are
preferred. Such
polymers may be natural or synthetic polymers. Synthetic polymers are
preferred. The
polymer is selected based on the period of time over which release is desired,
generally in the
order of a few hours to a year or longer. Typically, release over a period
ranging from
between a few hours and three to twelve months is most desirable. The polymer
optionally is
in the form of a hydrogel that can absorb up to about 90% of its weight in
water and further,
optionally is cross-linked with mufti-valent ions or other polymers.
In general, the agents of the invention may be delivered using the bioerodible
implant
by ~.~~ay of diffusion, or more preferably, by degradati~~3 of the polymeric
matrix. Exemplary
synthetic polymers which can be used to form the biodegradable delivery system
include:
polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene
oxides,
polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl
esters, poly-vinyl
halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes
and co-polymers
/5 thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers,
cellulose esters, nitro
celluloses, polymers of acrylic and methacrylic esters, methyl cellulose,
ethyl cellulose,
hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl
cellulose,
cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose
acetate phthalate,
carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt,
poly(methyl
2o methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),
poly(isobutyl
methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),
poly(lauryl
methacrylate), poly(phenyl methacrylate), poly(methyl acrylate),
poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, polyethylene
glycol), polyethylene oxide), polyethylene terephthalate), polyvinyl
alcohols), polyvinyl
25 acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.
Examples of non-biodegradable polymers include ethylene vinyl acetate,
poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
Examples of biodegradable polymers include synthetic polymers such as polymers
of
lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters,
polyurethanes, poly(butic
30 acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural
polymers such as
alginate and other polysaccharides including dextran and cellulose, collagen,
chemical
derivatives thereof (substitutions, additions of chemical groups, for example,
alkyl, alkylene,
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hydroxylations, oxidations, and other modifications routinely made by those
skilled in the
art), albumin and other hydrophilic proteins, zero and other prolamines and
hydrophobic
proteins, copolymers and mixtures thereof. In general, these materials degrade
either by
enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
Bioadhesive polymers of particular interest include bioerodible hydrogels
described
by H.S. Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-
587, the
teachings of which are incorporated herein, polyhyaluronic acids, casein,
gelatin, gluon,
polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl
methacrylates), poly(ethyl
methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate),
1o poly(hexylmethacrylate, poly(isodecyl methacrylate), poly(lauryl
mPthacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl
acrylate), and
poly(octadecyl acrylate). Thus, the invention provides a composition of the
above-described
agents of Formula I for use as a medicament, methods for preparing the
medicament and
methods for the sustained release of the medicament in vivo.
15 Other delivery systems can include timed release, delayed release or
sustained release
delivery systems. Such systems can avoid repeated administrations of the agent
of the
invention, increasing convenience to the subj ect and the physician. Many
types of release
delivery systems are available and known to those of ordinary skill in the
art. They include
the above-described polymeric systems, as well as polymer base systems such as
2o poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters,
polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing
polymers
containing drugs are described in, for example, U.S. Patent 5,075,109.
Delivery systems also
include non-polymer systems that are: lipids including sterols such as
cholesterol, cholesterol
esters and fatty acids or neutral fats such as mono- di- and tri-glycerides;
hydrogel release
25 systems; silastic systems; peptide based systems; wax coatings; compressed
tablets using
conventional binders and excipients; partially fused implants; and the like.
Specific examples
include, but are not limited to: (a) erosional systems in which the agent is
contained in a form
within a matrix such as those described in U.S. Patent Nos. 4,452,775,
4,675,189 and
5,736,152 and (b) diffusional systems in which an active component permeates
at a
3o controlled rate from a polymer such as described in U.S. Patent Nos.
3,854,480, 5,133,974
and 5,407,686. In addition, pump-based hardware delivery systems can be used,
some of
which are adapted for implantation.
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Use of a long-term sustained release implant may be particularly suitable for
treatment of chronic conditions, such as a the suspected presence of dormant
metastases.
Long-term release, are used herein, means that the implant is constructed and
arranged to
delivery therapeutic levels of the active ingredient for at least 30 days, at
least 60 days and
more preferably for several months. Long-term sustained release implants are
well-known to
those of ordinary skill in the art and include some of the release systems
described above.
In still other embodiments, the agent is targeted to a site of abnormal cell
proliferation, such as, a tumor, through the use of a targeting compound
specific for a
particular tissue or tumor type. The agents of the invention may be targeted
to primary or in
I~ some instances, secondary (i.e., metastatic) lesions through the use of
targeting corryounds
which preferentially recognize a cell surface marker. The targeting compound
may be
directly conjugated to the agents of the invention via a covalent linkage. The
agent may be
indirectly conjugated to a targeting compound via a linker. Alternatively, the
targeting
compound may be conjugated or associated with an intermediary compound such
as, for
75 example, a liposome within which the agent is encapsulated. Liposomes are
artificial
membrane vessels which are useful as a delivery vector in vivo or in vitro. It
has been shown
that large unilamellar vessels (LUV), which range in size from 0.2 - 4.0 g,m
can encapsulate
large macromolecules. Liposomes may be targeted to a particular tissue, such
as the vascular
cell wall, by coupling the liposome to a specific ligand such as a monoclonal
antibody, sugar,
20 glycolipid, or protein. Liposomes are commercially available from Gibco
BRL, for example,
as LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as
N-[1-
(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and
dimethyl
dioctadecylammonium bromide (DDAB). Methods for making liposomes are well
known in
the art and have been described in many publications. Liposomes also have been
reviewed
25 by Gregoriadis, G. in Trends in Biotechnology, V. 3, p. 235-241 (1985). In
still other
embodiments, the targeting compound may be loosely associated with the agents
of the
invention, such as within a microparticle comprising a polymer, the agent of
the invention
and the targeting compound.
Targeting compounds useful according to the methods of the invention are those
30 which direct the agent to a site of abnormal proliferation such as a tumor
site. The targeting
compound of choice will depend upon the nature of the tumor or the tissue
origin of the
metastasis. In some instances it may be desirable to target the agent to the
tissue in which the
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tumor is located. For example, agents can be delivered to breast epithelium by
using a
targeting compound specific for breast tissue. In preferred embodiments, the
target is
specific for malignant breast epithelium. Examples of compounds which may
localize to
malignant breast epithelium include, but are not limited to, estrogen and
progesterone,
epithelial growth factor (EGF) and HER-2/neu ligand, among others. The HER-
2/neu ligand
may also be used to target agents to ovarian cancers. Ovarian cancers are also
known to
express EGFR and c fms, and thus could be targeted through the use of ligands
for either
receptor. In the case of c fms which is also expressed by macrophages and
monocytes,
targeted delivery to an ovarian cancer may require a combination of local
administration such
Io as a vaginal suppository as well as a targeting compound. Frostate cancers
can be targeted
using compounds such as peptides (e.g., antibodies or antibody fragments)
which bind to
prostate specific antigen (PSA) or prostate specific membrane antigen (PSMA).
Other
markers which may be used for targeting of the agent to specific tissues
include, for example,
in liver: HGF, insulin-like growth factor I, II, insulin, OV-6, HEA-125,
hyaluronic acid,
collagen, N-terminal propeptide of collagen type III, mannose/N-
acetylglucosamine,
asialoglycoprotein, tissue plasminogen activator, low density lipoprotein,
carcinoembryonic
antigen; in kidney cells: angiotensin II, vasopressin, antibodies to CD44v6;
in keratinocytes
and skin fibroblasts: KGF, very low density lipoprotein, RGD-containing
peptides, collagen,
laminin; in melanocytes: kit ligand; in gut: cobalamin-intrinsic factor, heat
stable enterotoxin
of E. Coli; in breast epithelium: heregulin, prolactin, transferrin, cadherin-
11. Other markers
specific to particular tissues are available and would be known to one of
ordinary skill in the
art.
In still other embodiments, the agent of the invention may be targeted to
fibroblasts
specifically, via ligands or binding partners for fibroblast specific markers.
Examples of
these markers include, but are not limited to fibroblast growth factors (FGF)
and platelet
derived growth factor (PDGF). In some embodiments, it may be desirable to
target the agent
to FAP-a specifically through the use of binding peptides for FAP-a which do
not interfere
with inhibition by the agent of the invention. One such binding peptide is the
monoclonal
antibody F 19, used previously for immunodetection of reactive stromal
fibroblast expressing
3o FAP-a.
Agents useful in the invention can be identified using a screening assay
method for
determining whether a putative agent is able to inhibit the activity of FAP-a,
thereby
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inhibiting cell proliferation. The initial screening assay can be conducted in
an in vitro
system with a readout of FAP-a inhibition. In such screening assays, cells
expressing FAP-a
but not CD26 can be used as a source of FAP-a. Alternatively, recombinant or
purified FAP-
a can also be used in either a soluble or bound form. The choice of whether to
use FAP-a in
either a soluble or bound form may depend upon the source of the compounds to
be screened.
For example, if the compounds to be screened are present in phage libraries,
it may be
desirable to use soluble FAP-a. If, on the other hand, the compounds are
synthesized by
combinatorial chemistry techniques, then bound FAP-a may be more suitable. It
is possible
to immobilize FAP-a in 96 well plates through either direct binding to the
surface, or more
l0 preferably through the indirect binding via an anti-FAP-a antibody or
antibody fragment such
as that derived from F 19, a FAP-a specific antibody. Binding is achieved
through incubation
at room temperature for 2 hours, followed by washing with a phosphate buffered
saline
solution containing a suitable non-specific blocking agent such as albumin or
serum. After
significant washing, the substrate alanylprolyl-7-amido-4-trifluoromethyl-
coumarin (Ala-Pro-
-NH-F3-Mec, available from Bachem) is added to the plates and incubated for 1
hour at 37°C
in 100 mM Tris/HCI, pH7.8, 100 mM NaCI. At the end of the incubation, a
fluorometric
measurement is made for each well using an excitation wavelength of 390 nm and
an
emission wavelength of 538 nm. The substrate described above can also be used
in soluble
FAP-a enzyme inhibition assays are described in U.S. Patent Application
entitled
"Multivalent Compounds for Crosslinking Receptors and Uses Thereof " filed on
April 12,
1999 and assigned U.S.S.N. 09/290,376.
The above described procedure represents the control for the inhibition
screen. To
perform the inhibition screen, the agents of Formula I are incubated with FAP-
a for 5-10
minutes at 37°C prior to introduction of the substrate. The enzyme
reaction proceeds as
above, and a fluorescent measurement is used as a readout. A decrease in the
amount of
fluorescence is indicative of an inhibitor. Alternatively, the readout can be
a kinetic analysis
of the rate of fluorescence change, with a slower rate being indicative of an
inhibitory agent.
Once FAP-a inhibitors have been pre-screened in vitro, they can be tested in
proliferation assays in vitro or in vivo. In vitro proliferation assays could
analyze the effect
of Formula I compounds on the rate of proliferation of FAP-a expressing cells.
Proliferation
in these assays can be measured either by tritiated thymidine uptake or simply
by a cell count.
Carcinoma cell lines are not suitable for these types of in vitro assays since
these cells are
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generally FAP-a negative. Alternatively, inhibition of FAP-a protease activity
in vitro or in
vivo or inhibition of angiogenesis in vitro could also be used. Assay systems
for
angiogenesis inhibition are known in the art and are described in U.S. Patent
5,854,221 and
5,639,725, the entire contents of which are incorporated herein by reference.
In vivo assay
systems involve the initial induction of a suitable experimental tumor within
a mouse, usually
by the injection of a malignant cell line into a pre-defined location such as
the lungs or the
footpad. Following the implantation and growth of the tumor, the agent to be
tested is
administered to the mouse, again usually over a period of time, and at
differing doses. At the
end of the assay, the mouse is analyzed in terms of, among other things, tumor
growth and
1c tire presence of metastases. In assay systems aimed at studying the
prophylactic effic«;y of
an agent, the agent may be administered in close temporal proximity to the
tumor cell line
injection. In this way, one can determine whether the agent is able to prevent
tumor
formation altogether. These assay systems are described in more detail in the
following
Examples.
IS Identifying compounds and administration regimens which favor proliferation
inhibition over hemopoietic stimulation, including dosing amounts, dosing
schedules and
routes of administration, involves comparison of results of the above assays
to hemopoietic
stimulation assays described previously in U.S. Patent Application Serial No.
09/304,199,
filed May 3, 1999, entitled "Hematopoietic Stimulation", the contents of which
are
20 incorporated herein in their entirety by reference.
The invention will be more fully understood by reference to the following
examples.
These examples, however, are merely intended to illustrate the embodiments of
the invention
and are not to be construed to limit the scope of the invention.
All patents, patent applications, references and other documents identified
herein are
25 incorporated in their entirety herein by reference.
Examples
Example 1: This example illustrates the use of PT-100 (i.e., ValboroPro) to
inhibit FAP-a
protease activity and stromal cytokine secretion from FAP-a positive
CD26/DPPIV negative
3o rat and human stromal cells.
Materials and Methods:
Rat stromal cultures
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Cell Source. 8-12 week old female wild type CD26 positive (D+) and CD26
negative (D-)
Fischer rat (Charles River Labs, Japan) bone marrow.
Bone marrow preparation. Bone marrow was flushed from each femur, tibia and
fibula with
Dulbecco's phosphate buffered saline (D-PBS) using a 21-gauge needle and a 10
ml syringe.
After removal, the bone marrow was immediately aspirated through the needle to
make a
single cell suspension. The cells were washed twice with sterile PBS and
resuspended in
MyeloCult H5100 long-term culture (LTC) media, available from Stem Cell
Technologies,
and supplemented with freshly diluted 10-6 M hydrocortisone, available from
Sigma.
Establishment of adherent bone marrow stromal cell cultures. 1-2 x 107 cells
were seeded
/o into a T25 flask (Corning) containing 10 mls of LTC medium and incubated at
37°C in 100%
humidified 5% COZ in air. After 1 week half the medium was exchanged for fresh
medium
and incubated for approximately 1 week until a confluent cell layer formed.
PT 100 incubation. The adherent stromal cells were removed from the flask by
trypsin-
EDTA (Life Technologies, Inc.) digestion, washed once with sterile PBS and
resuspended in
long term culture medium at 1 x 105/m1. One ml of cells was seeded in each
well of a 12-
well tissue culture treated plate. PT-100 was diluted in medium and added
immediately to
the stromal cells at the appropriate concentrations ( 10-1 to 10-g M) and the
cultures were
incubated at 37°C in 100% humidified 5% C02 in air.
Supernatant collection. The supernatants were collected from the stromal
cultures after 1 or
2 days of incubation and either assayed immediately or stored at -20°C.
ELISA. The supernatants were assayed for IL-6 production using a commercially
available
ELISA kit (Biosource International).
DPPIV like activity assay, Following the removal of supernatant from the
stromal cultures,
the adherent cells were washed with D-PBS and assayed for DPPIV-like activity.
The
fluorogenic substrate Ala-Pro-AFC was diluted in DPPIV buffer (50 mM Hepes,
140 mM
NaCI) from the original stock of 10 mM (stored at -20°C as a solution
in dimethyl-
formamide) to 1 mM, directly before the assay. 30 ~.1 of the 1 mM stock was
added per ml of
buffer and the stromal cells were incubated for 10 minutes with 1 ml per well
of the diluted
substrate. The reaction was stopped by removing the substrate solution from
each well and
3o measured in the fluorescence spectrophotometer or stored -20°C.
Activity was measured
using an excitation wavelength of 400 nm and emission wavelength of 505 nm.
Human stromal cultures
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Cell source. Bone marrow was kindly donated by New England Medical Center,
Boston,
MA and Dr. Richard Benjamin from Brigham and Women's Hospital in Boston, MA.
Mononuclear cells were purified over Ficoll and used immediately or frozen in
liquid
nitrogen.
Establishment ofhuman stromal feeder loyer. 2 to 4 x 10' human bone marrow
mononuclear
cells were seeded into a T75 flask (Corning) containing 20 mls of LTC medium
and
incubated at 37°C in 100% humidified 5% COZ in air. After 1 week half
the medium was
exchanged for fresh medium and incubated for approximately 1 week when a
confluent cell
layer formed.
I0 MyeloCult H5100 long-term culture (LTC) media can be obtained from Stem
Cell
Technologies and contains 12.5% horse serum, 12.5% fetal bovine serum, 0.2 mM
I-inositol,
20 mM folic acid, 10~~ 2-(3-mercapto-ethanol, 2 mM L-glutamine ina-MEM. Prior
to using,
add freshly diluted 10-6 M hydrocortisone, available from Sigma.
ELISA of human cells. Cultured supernatants were assayed for a high
sensitivity ELISA
75 (R+D Systems).
Human cell surface staining. The phenotype of the established human bone
marrow stromal
cells as determined using immunofluorescent staining with monoclonal
antibodies specific
for CD26 and FAP alpha, followed by fluorescence activated cell sorting
(FACscan, Becton
Dickinson). FAP alpha was stained using a biotinylated monoclonal anti-human
antibody
20 (IgGl isotype) followed by Strepavidin-Fluorescein Isothiocyanin (SA-FITC).
The antibody
was purified over protein G, using cultured supernatants from the hybridoma F
19 (ATCC),
and biotinylated using biotinyl-p-nitrophenyl ester (Sigma). CD26 was stained
with Cy-
chrome or Phycoerythrin anti-human CD26 (clone MA261). Mouse IgGl (Pharmigen)
was
used for a negative control.
25 PT-100 incubation, supernatant collection and DPPIV-like activity assay for
human
and rat cells were carried out in an identical manner.
Results:
Table 1 shows three representative experiments which demonstrate normal DPPIV-
like activity on rat bone marrow stromal cells which lack CD26. The DPPIV-like
activity is
30 potently inhibited by PT-100. Bone marrow stromal cells were established
from the long
bones of Fischer D+ and D- rats and cultured for 2 days in the presence or
absence of PT-100.
Cells were assayed for DPPIV-like activity as described in the Materials and
Methods
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section. DPPIV-like activity is presented as velocity and the percent of
protease activity
inhibition with PT-100 is calculated relative to a 'no treatment' control.
Table 1
Comparison of DPPIV-Like Activity of Fischer D+ and D- Rat BM Stromal Cells
s
Experiment # Rat Phenotype DPPIV-Like Activity % Inhibition by
(Velocity = pMoles/min) 10-6 M PT-100
102098 D+ 23.4 76.9
D- 23.3 62.1
102198 D+ 22.4 35.5
D- 33.7 86.4
/s 102398 D- 35.8 73.8
Figure 1 shows the comparison of the response of Fischer D+ and D- rat and
bone
marrow stromal cells to PT-100 in in vitro cultures. Similar levels of IL-6
are secreted from
bone marrow stromal cells of D+ and D- rats. Moreover, IL-6 levels for both
strains were
2o enhanced by the addition of PT-100. Bone marrow stromal cells were
established from the
long bones of 3 Fischer D+ and D- rats as described in Materials and Methods.
The stromal
cells were incubated for 2 days in the presence or absence of the indicated
concentrations of
PT-100. IL-6 levels in the culture supernatant were determined by ELISA.
A fluorescent activated cell sorting (FACS) profile of human bone marrow
stromal
25 cells is shown in Figure 2. Human stromal cells can be established which
lack cell surface
CD26 but express the DPPIV-like protein FAP-a. Stromal cells were established
from the
bone marrow of a human volunteer as described in Materials and Methods.
Cultured cells
were removed by trypsin digestion and assayed for surface expression of CD26
and FAP-a
by immuno-fluorescence staining and cell sorting. The FAGS profile for FAP-a
(FL 1 ) and
3o CD26 (FL2) are represented as histograms. Negative controls used in this
experiment were
biotinylated mouse IgGI/SA-FITC and Cychrome-mouse IgGI (top panels). FAP
alpha was
stained with biotinylated F 19 followed by secondary staining with SA-FITC.
CD26 was
stained with PE-mouse anti-human CD26.
Figure 3 demonstrates that primary human stromal cells which express FAP, but
not
35 CD26, respond to PT-100. Human bone marrow cells that lack CD26 but express
FAP-a, as
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determined by FACS analysis, were potently stimulated by PT-100 to release G-
CSF. The
DPPIV-like activity associated with the intact cells was inhibited by PT-100.
Stromal cells
were established from the bone marrow of a human volunteer as described in the
materials
and methods. The stromal cells were incubated for 2 days in the presence or
absence of the
indicated concentrations of PT-100 and cultured supernatants were assayed for
IL-6 or
DPPIV-like activity as described in Materials and Methods.
Example 2: This example illustrates the anti-tumor activity of PT-100 (i.e.,
Val-boro-Pro)
in mouse models.
1o PT-100 administered orally significantly inhibits the growth of both the B
16
melanoma and the WEHI-164 fibrosarcoma in mice.
The highly metastic B16-FO tumor was implanted subcutaneously (s.c.) in
C57BL/6
mice. PT-100 administration was started on day 8 when palpable tumors were
apparent.
Tumor volumes were measured on days 8, 12, and 15. The data are shown in
Figure 4. In
mice treated with doses of 10 and 40 ~g of PT-100 administered orally (p.o.),
twice daily
(b.i.d.) from day 8 to 14, tumor growth was markedly inhibited compared with
the saline
treated control mice. A higher dose of 160 pg PT-100 administered for just two
days - day 8
and day 9 - was also effective in suppressing tumor growth. In the experiment
of Figure 4
each experimental group contained 10 animals.
Similar experiments with the WEHI-164 fibrosarcoma demonstrated that PT-100
could suppress the growth of an established s.c. tumor. In addition, when PT-
100
administration was started shortly after implantation of WEHI-164 on day 2, it
was found that
not only was tumor growth inhibited, but a proportion of the tumors also
became necrotic and
apparently regressed completely. Thus in the experiment of Figure 5, the large
reductions in
tumor volume recorded were in part due to the absence of a palpable tumor in
some of the
mice in each of the PT-100 treated groups (10 replicate animals per group).
For example, in
the mice treated with the 5 pg dose of PT-100 administered b.i.d., 6 of the
animals were
completely free of a detectable s.c. tumor. PT-100 treatment of the mice in
the experimental
groups was stopped on day 20, the day when the data of Figure 5 were recorded.
These mice
3o have now been monitored up to day 30 after WEHI-164 implantation, and to
date, none of the
mice recorded as tumor-free on day 20 has developed a detectable s.c. tumor.
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Example 3: The in vivo effects of the compounds of the invention can be
assayed through
various animal models known to those of ordinary skill in the art. Generally
such assays
involve the injection of a carcinoma cell line, of mouse or preferably, human,
origin, into a
cohort of mice. Following the passage of several days, as determined by the
proliferative rate
of the cell line, parameters such as tumor size, degree of metastasis and
cellular infiltration
into a region in the vicinity of the tumor site are evaluated. In most
experiments, two groups
of experimental mice are studied: a first, control, group which receives only
the cell line but
no agent of Formula I, and a second, test, group which receives at least one
agent of Formula
I such as PT-100. This second group is divided into several subgroups each of
which
/o receives a different dose of the ageni, preferably between 10 gg and 100 pg
per day for 10-20
days. On these days, the control group will be administered control doses
containing only
vehicle with no active agent. The evaluation of tumor growth and organ
specific metastasis
will vary according to the tumor type studied.
In yet other assay systems aimed at determining the effect of the agents on
metastatic
spread or development, the agents are administered at the time of innoculation
of the
malignant cells or short thereafter. Similarly, the agents may be administered
after the
innoculation, but before the full development of the tumor mass. In these
ways, the effects of
agents on different stages of malignant growth and metastasis can be tested.
The following are examples of different in vivo model systems for studied a
variety of
2o epithelial cancers.
Lung carcinoma: One such example for lung carcinoma involves the subcutaneous
flank
injection of the M109 mouse lung tumor cell line into syngeneic mice. On each
of days 4
through 8 after injection of the cell line, the mice receive single bolus
daily doses of the
compounds of the invention by tail vein injection. The compounds of the
invention are
prepared and administered in a carrier solution which is physiologically
compatible with both
the recipient environment and the stability of the compound. A preferable
carrier solution is
D-PBS with a carrier protein such as albumin. Mice are sacrificed at day 4
after cell line
injection and at two day intervals after the administration of inhibitory
compounds. The
tumors are excised and weighed. Measurement data can be standardized relative
to initial
body weight of the recipient mouse.
Alternatively, if the transplantable tumor line is able to grow to the extent
that it
causes a reproducible and significant effect on total mouse body weight, than
the recipients
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need not be sacrificed. In this readout system, starting at day 0 (i.e., prior
to the introduction
of the cell line), the mice are weighed daily to determine the tumor burden
and to evaluate the
effect of the injected compounds) on tumor burden. Tumor mass can be
calculated by the
difference in mouse body mass during the experiment and at day 0. Measurement
of control
mice which receive only carrier solution with carrier protein will be used to
standardize for
any unrelated weight gain.
Colon carcinoma: The effect of test agents on experimentally induced human
colorectal
tumors in mice can be deduced by transplanting into nude mice human colon
tumor cell lines
such as COLO 205, C-1H, 26M3.1, CT-26, LS174T, and HT29, in a manner similar
to that
to d~~-vribed above. In these models, pericecal tumor growth, angiogenesis,
ascites and
metastasis to the liver are suitable readouts to ascertain if the test
compounds are active.
Melanoma and metastatic melanoma: Melanoma cell lines (e.g., B 16 and SKMEL)
are
administered either intraperitoneally or intravenously or directly into the
footpad. Primary
tumor growth, survival time, resistance to tumor challenge, cellular
infiltrates characteristic
of melanoma tumors, and extent of tumor angiogenesis are all parameters of
interest which
can be evaluated. In certain models of melanoma, metastasis to the lung can be
readily
observed following surgical removal of the primary tumor.
Ovarian cancer: Human ovarian carcinoma cell lines such as JAM are
administered
subcutaneously to severe combined immunodeficiency (SCID) mice. After 21 days,
tumor
2o growth is generally established and the effects of the test agent after
this point can be
compared to vehicle-alone.
Breast cancer: Breast cancer cell lines such as MDA-MB-231 are injected
preferably into the
left cardiac ventricle of mice. Many breast cancers metastasize to bone. About
4 weeks after
inoculation, tumors and bone metastases can be evaluated as can the effect of
administration
of the test agent.
Squamous cell carcinoma: Human basaloid squamous cell carcinoma cells or
established
tumor lines such as HTB-1 are administered either subcutaneously or
submucosally into
mice. After allowing a sufficient time for primary tumor growth, the mice are
administered
test or control preparations, and the effects of the test agent on the
parameters described
3o above are determined.
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Other transplantable cell lines useful in these assays include, but are not
limited to,
human NCI-H522 lung tumor cell line (nude mice recipients), human SKOV3 cell
line, and
the M5076 cell line.
It should be understood that the preceding is merely a detailed description of
certain
preferred embodiments. It therefore should be apparent to those skilled in the
art that various
modifications and equivalents can be made without departing from the spirit
and scope of the
invention. It is intended to encompass all such modifications within the scope
of the
appended claims.
All references, patents and patent publications that are recited in this
application are
7o incorporated in their entirety herein by reference.
We claim:
