U.S. patent application number 13/309233 was filed with the patent office on 2012-04-12 for novel 1,4-benzodiazepine-2,5-diones with therapeutic properties.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to Gary D. Glick.
Application Number | 20120088757 13/309233 |
Document ID | / |
Family ID | 38006506 |
Filed Date | 2012-04-12 |
United States Patent
Application |
20120088757 |
Kind Code |
A1 |
Glick; Gary D. |
April 12, 2012 |
NOVEL 1,4-BENZODIAZEPINE-2,5-DIONES WITH THERAPEUTIC PROPERTIES
Abstract
The present invention relates to novel chemical compounds,
methods for their discovery, and their therapeutic use. In
particular, the present invention provides novel
1,4-benzodiazepine-2,5-dione compounds, and methods of using novel
1,4-benzodiazepine-2,5-dione compounds as therapeutic agents to
treat a number of conditions associated with the faulty regulation
of the processes of programmed cell death, autoimmunity,
inflammation, hyperproliferation, and the like.
Inventors: |
Glick; Gary D.; (Ann Arbor,
MI) |
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
MICHIGAN
Ann Arbor
MI
|
Family ID: |
38006506 |
Appl. No.: |
13/309233 |
Filed: |
December 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11591324 |
Nov 1, 2006 |
8088759 |
|
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13309233 |
|
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60732045 |
Nov 1, 2005 |
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Current U.S.
Class: |
514/221 ;
435/375; 540/506 |
Current CPC
Class: |
A61P 9/00 20180101; A61K
31/5513 20130101; A61P 35/00 20180101; C07D 243/14 20130101; A61P
37/06 20180101; A61P 43/00 20180101; A61P 29/00 20180101; A61P
37/00 20180101 |
Class at
Publication: |
514/221 ;
540/506; 435/375 |
International
Class: |
A61K 31/5513 20060101
A61K031/5513; A61P 35/00 20060101 A61P035/00; A61P 37/06 20060101
A61P037/06; C07D 243/24 20060101 C07D243/24; C12N 5/09 20100101
C12N005/09 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under AI
47450 awarded by the National Institutes of Health. The government
has certain rights in the invention.
Claims
1. A composition configured to induce apoptosis in a cell, said
composition comprising a compound described by a formula selected
from the group consisting of: ##STR00040## ##STR00041##
##STR00042## ##STR00043## substituted and unsubstituted, including
both R and S enantiomeric forms and racemic mixtures; wherein R1 is
an electron rich heterocycle; wherein R2 is selected from the group
consisting of H, alkyl, substituted alkyl, and R.sub.1; wherein
R.sub.3 is selected from the group consisting of H, alkyl, and
substituted alkyl; wherein R.sub.4 and R.sub.4' is independently
selected from the group consisting of CH.sub.3, halogen,
SO.sub.2R.sub.4'', SO.sub.2N(R.sub.4'').sub.2, OR.sub.4'',
N(R.sub.4'').sub.2, CON(R.sub.4'').sub.2, NHCOR.sub.4'',
NHSO.sub.2R.sub.4', alkyl, mono-substituted alkyl, di-substituted
alkyl, tri-substituted alkyl; wherein R.sub.4'' is selected from
the group consisting of halogen, H, alkyl, mono-substituted alkyl,
di-substituted alkyl, tri-substituted alkyl, aryl, mono-substituted
aryl, di-substituted aryl, tri-substituted aryl, cycloalipathic,
mono-substituted cycloalipathic, di-substituted cycloalipathic,
tri-substituted cycloalipathic; wherein R.sub.5 is selected from
the group consisting of H, alkyl, mono-substituted aryl,
di-substituted aryl, and tri-substituted aryl; and wherein R6 is
selected from the group consisting of C, N or S.
2. The composition of claim 1, wherein said electron rich
heterocycle contains 5 or more heterocyclic atoms.
3. The composition of claim 1, wherein R.sub.1 is selected from the
group consisting of ##STR00044## wherein R.sub.1' is selected from
the group consisting of cycloalipathic, aryl, substituted aryl,
heterocyclic, and substituted heterocyclic.
4. The composition of claim 1, wherein R1 is selected from the
group consisting of: In some embodiments, R1 is selected from the
group consisting of: ##STR00045## ##STR00046## ##STR00047##
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
and derivatives thereof.
5. The composition of claim 1, wherein said compound is described
by the following formula: ##STR00053##
6. The composition of claim 1, wherein said compound is selected
from the group consisting of: ##STR00054## ##STR00055##
7. The composition of claim 1, wherein said cells are selected from
the group consisting of B cells, T cells, granulocytes, and
proliferating cells.
8. A method for regulating cell death, comprising a. providing: i.
target cells; the position described in claim 1; and b. exposing
said target cells to said composition under conditions such that
said composition binds to said target cells so as to induce
cellular apoptosis.
9. The method of claim 8, wherein said target cells are in vitro
cells.
10. The method of claim 8, wherein said target cells are in vivo
cells.
11. The method of claim 8, wherein said target cells are ex vivo
cells.
12. The method of claim 8, wherein said target cells are cancer
cells.
13. The method of claim 8, wherein said target cells are selected
from the group consisting of B cells, T cells, and
granulocytes.
14. The method of claim 8, wherein said target cells are
proliferating cells.
15. A method of treating autoimmune disorders, comprising: a)
providing a subject and the composition described in claim 1,
wherein said composition is capable of inducing cellular apoptosis,
and wherein said composition demonstrates selective cytotoxicity
against T cells as compared to B cells; and b) administering said
composition to said subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of pending U.S.
patent application Ser. No. 11/591,324, filed Nov. 1, 2006, which
claims priority to expired U.S. Provisional Patent Application Ser.
No. 60/732,045, filed Nov. 1, 2005, the contents of which are
incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to novel chemical compounds,
methods for their discovery, and their therapeutic use. In
particular, the present invention provides novel
1,4-benzodiazepine-2,5-dione compounds, and methods of using novel
1,4-benzodiazepine-2,5-dione compounds as therapeutic agents to
treat a number of conditions associated with the faulty regulation
of the processes of programmed cell death, autoimmunity,
inflammation, hyperproliferation, vascular abnormalities, cancer,
anti-angiogenesis, and the like.
BACKGROUND OF THE INVENTION
[0004] Multicellular organisms exert precise control over cell
number. A balance between cell proliferation and cell death
achieves this homeostasis. Cell death occurs in nearly every type
of vertebrate cell via necrosis or through a suicidal form of cell
death, known as apoptosis. Apoptosis is triggered by a variety of
extracellular and intracellular signals that engage a common,
genetically programmed death mechanism.
[0005] Multicellular organisms use apoptosis to instruct damaged or
unnecessary cells to destroy themselves for the good of the
organism. Control of the apoptotic process therefore is very
important to normal development, for example, fetal development of
fingers and toes requires the controlled removal, by apoptosis, of
excess interconnecting tissues, as does the formation of neural
synapses within the brain. Similarly, controlled apoptosis is
responsible for the sloughing off of the inner lining of the uterus
(the endometrium) at the start of menstruation. While apoptosis
plays an important role in tissue sculpting and normal cellular
maintenance, it is also the primary defense against cells and
invaders (e.g., viruses) which threaten the well being of the
organism.
[0006] Not surprisingly many diseases are associated with
dysregulation of the process of cell death. Experimental models
have established a cause-effect relationship between aberrant
apoptotic regulation and the pathenogenicity of various neoplastic,
autoimmune and viral diseases. For instance, in the cell mediated
immune response, effector cells (e.g., cytotoxic T lymphocytes
"CTLs") destroy virus-infected cells by inducing the infected cells
to undergo apoptosis. The organism subsequently relies on the
apoptotic process to destroy the effector cells when they are no
longer needed. Autoimmunity is normally prevented by the CTLs
inducing apoptosis in each other and even in themselves. Defects in
this process are associated with a variety of autoimmune diseases
such as lupus erythematosus and rheumatoid arthritis.
[0007] Multicellular organisms also use apoptosis to instruct cells
with damaged nucleic acids (e.g., DNA) to destroy themselves prior
to becoming cancerous. Some cancer-causing viruses overcome this
safeguard by reprogramming infected (transformed) cells to abort
the normal apoptotic process. For example, several human papilloma
viruses (HPVs) have been implicated in causing cervical cancer by
suppressing the apoptotic removal of transformed cells by producing
a protein (E6) which inactivates the p53 apoptosis promoter.
Similarly, the Epstein-Barr virus (EBV), the causative agent of
mononucleosis and Burkitt's lymphoma, reprograms infected cells to
produce proteins that prevent normal apoptotic removal of the
aberrant cells thus allowing the cancerous cells to proliferate and
to spread throughout the organism.
[0008] Still other viruses destructively manipulate a cell's
apoptotic machinery without directly resulting in the development
of a cancer. For example, the destruction of the immune system in
individuals infected with the human immunodeficiency virus (HIV) is
thought to progress through infected CD4.sup.+ T cells (about 1 in
100,000) instructing uninfected sister cells to undergo
apoptosis.
[0009] Some cancers that arise by non-viral means have also
developed mechanisms to escape destruction by apoptosis. Melanoma
cells, for instance, avoid apoptosis by inhibiting the expression
of the gene encoding Apaf-1. Other cancer cells, especially lung
and colon cancer cells, secrete high levels of soluble decoy
molecules that inhibit the initiation of CTL mediated clearance of
aberrant cells. Faulty regulation of the apoptotic machinery has
also been implicated in various degenerative conditions and
vascular diseases.
[0010] It is apparent that the controlled regulation of the
apoptotic process and its cellular machinery is vital to the
survival of multicellular organisms. Typically, the biochemical
changes that occur in a cell instructed to undergo apoptosis occur
in an orderly procession. However, as shown above, flawed
regulation of apoptosis can cause serious deleterious effects in
the organism.
[0011] There have been various attempts to control and restore
regulation of the apoptotic machinery in aberrant cells (e.g.,
cancer cells). For example, much work has been done to develop
cytotoxic agents to destroy aberrant cells before they proliferate.
As such, cytotoxic agents have widespread utility in both human and
animal health and represent the first line of treatment for nearly
all forms of cancer and hyperproliferative autoimmune disorders
like lupus erythematosus and rheumatoid arthritis.
[0012] Many cytotoxic agents in clinical use exert their effect by
damaging DNA (e.g., cis-diaminodichroplatanim(II) cross-links DNA,
whereas bleomycin induces strand cleavage). The result of this
nuclear damage, if recognized by cellular factors like the p53
system, is to initiate an apoptotic cascade leading to the death of
the damaged cell.
[0013] However, existing cytotoxic chemotherapeutic agents have
serious drawbacks. For example, many known cytotoxic agents show
little discrimination between healthy and diseased cells. This lack
of specificity often results in severe side effects that can limit
efficacy and/or result in early mortality. Moreover, prolonged
administration of many existing cytotoxic agents results in the
expression of resistance genes (e.g., bcl-2 family or multi-drug
resistance (MDR) proteins) that render further dosing either less
effective or useless. Some cytotoxic agents induce mutations into
p53 and related proteins. Based on these considerations, ideal
cytotoxic drugs should only kill diseased cells and not be
susceptible to chemo-resistance.
[0014] Many autoimmune diseases and haematologic malignancies
result from the aberrant survival and expansion of B and T cells in
central and peripheral lymphoid organs. Current therapies for these
for these disorders generally employ cytotoxic drugs whose
mechanisms of action frequently involves DNA damage. Hence, the
selectivity of these drugs is limited and often relies on the
differential ability of diseased and healthy cells to tolerate and
repair drug-induced cellular damage.
[0015] What are needed are improved compositions and methods for
regulating the apoptotic processes in subjects afflicted with
diseases and conditions characterized by faulty regulation of these
processes (e.g., viral infections, hyperproliferative autoimmune
disorders, chronic inflammatory conditions, and cancers).
SUMMARY
[0016] The present invention relates to novel chemical compounds,
methods for their discovery, and their therapeutic use. In
particular, the present invention provides novel
1,4-benzodiazepine-2,5-dione compounds, and methods of using novel
1,4-benzodiazepine-2,5-dione compounds as therapeutic agents to
treat a number of conditions associated with the faulty regulation
of the processes of programmed cell death, cancer,
anti-angiogenesis, autoimmunity, inflammation, hyperproliferation,
vascular abnormalities, and the like. Such compounds and uses are
described throughout the present application and represent a
diverse collection of compositions and applications.
[0017] Certain preferred compositions and uses are described below.
The present invention is not limited to these particular
compositions and uses. The present invention provides a number of
useful compositions as described throughout the present
application.
[0018] In certain embodiments, the present invention provides a
composition comprising novel 1,4-benzodiazepine-2,5-dione
compounds. In certain embodiments, the present invention provide a
composition comprising a compound described by a formula selected
from the group consisting of:
##STR00001## ##STR00002## ##STR00003## ##STR00004##
substituted and unsubstituted, including both R and S enantiomeric
forms and racemic mixtures.
[0019] In some embodiments, R1 is an electron rich heterocycle. In
some embodiments, the electron rich heterocycle contains 5 or more
heterocyclic atoms.
[0020] In some embodiments, R.sub.1 is selected from the group
consisting of
##STR00005##
wherein R.sub.1' is selected from the group consisting of
cycloalipathic, aryl, substituted aryl, heterocyclic, and
substituted heterocyclic.
[0021] In some embodiments, R.sub.2 is selected from the group
consisting of H, alkyl, substituted alkyl, and R.sub.1.
[0022] In some embodiments, R.sub.3 is selected from the group
consisting of H, alkyl, and substituted alkyl.
[0023] In some embodiments, R3 is selected from the group
consisting of hydrogen; halogen; OH; a chemical moiety comprising
an aryl subgroup; a chemical moiety comprising a substituted aryl
subgroup; a chemical moiety comprising a cycloaliphatic subgroup; a
chemical moiety comprising a substituted cycloaliphatic subgroup; a
chemical moiety comprising a heterocyclic subgroup; a chemical
moiety comprising a substituted heterocyclic subgroup; a chemical
moiety comprising at least one ester subgroup; a chemical moiety
comprising at least one ether subgroup; a linear or branched,
saturated or unsaturated, substituted or non-substituted, aliphatic
chain having at least 2 carbons; a chemical moiety comprising
Sulfur; a chemical moiety comprising Nitrogen; --OR--, wherein R is
selected from the group consisting of a chemical moiety comprising
an aryl subgroup; a chemical moiety comprising a substituted aryl
subgroup; a chemical moiety comprising a cycloaliphatic subgroup; a
chemical moiety comprising a substituted cycloaliphatic subgroup; a
chemical moiety comprising a heterocyclic subgroup; a chemical
moiety comprising a substituted heterocyclic subgroup; a linear or
branched, saturated or unsaturated, substituted or non-substituted,
aliphatic chain having at least 2 carbons; a chemical moiety
comprising at least one ester subgroup; a chemical moiety
comprising at least one ether subgroup; a chemical moiety
comprising Sulfur; a chemical moiety comprising Nitrogen.
[0024] In some embodiments, R3 is selected from group consisting
of: napthalene; phenol; 1-Napthalenol; 2-Napthalenol;
##STR00006## ##STR00007##
quinolines, and all aromatic regioisomers.
[0025] In some embodiments, the R1 and R3 groups may be
interchanged (e.g., in some embodiments, the R1 group is positioned
at the first position of the benzodiazepine ring and the R3 group
is positioned at the third position of the benzodiazepine ring; in
some embodiments, the R1 group is positioned at the third position
of the benzodiazepine ring and the R3 group is positioned at the
first position of the benzodiazepine ring).
[0026] In some embodiments, R.sub.4 and R.sub.4' is independently
selected from the group consisting of CH.sub.3, halogen,
SO.sub.2R.sub.4'', SO.sub.2N(R.sub.4'').sub.2, OR.sub.4'',
N(R.sub.4'').sub.2, CON(R.sub.4'').sub.2, NHCOR.sub.4'',
NHSO.sub.2R.sub.4', alkyl, mono-substituted alkyl, di-substituted
alkyl, tri-substituted alkyl; wherein R.sub.4'' is selected from
the group consisting of halogen, H, alkyl, mono-substituted alkyl,
di-substituted alkyl, tri-substituted alkyl, aryl, mono-substituted
aryl, di-substituted aryl, tri-substituted aryl, cycloalipathic,
mono-substituted cycloalipathic, di-substituted cycloalipathic,
tri-substituted cycloalipathic.
[0027] In some embodiments, R.sub.5 is selected from the group
consisting of H, alkyl, mono-substituted aryl, di-substituted aryl,
and tri-substituted aryl.
[0028] In some embodiments, R6 is selected from the group
consisting of C, N or S.
[0029] In some embodiments, R1 is selected from the group
consisting of:
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015##
and substituted and unsubstituted, and derivatives thereof.
[0030] Certain 1,4-benzodiazepine-2,5-dione compounds of the
present invention include, but are not limited to,
##STR00016## ##STR00017##
[0031] In certain embodiments, the present invention provides a
method of treating cells, comprising a) providing i) target cells;
and ii) a composition comprising a 1,4-benzodiazepine-2,5-dione
compound having an electron rich heterocycle at the third carbon
position of the benzodiazepine structure; and b) exposing the
target cells to the composition under conditions such that said
composition interacts with the target cell so as to induce cellular
apoptosis. Such methods find use in research, drug screening, and
therapeutic applications.
[0032] In some embodiments, the target cells are in a subject
having, for example, an autoimmune disorder, a haematologic
malignancy, or a hyproliferative disorder. In some embodiments, the
target cells are selected from the group consisting of in vitro
cells, in vivo cells, and ex vivo cells. In other embodiments, the
target cells are cancer cells. In still other embodiments, the
target cells are selected from the group consisting of B cells, T
cells, and granulocytes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows the structure of Bz-423 and an exemplary
1,4-benzodiazepine-2,5-dione.
[0034] FIG. 2 shows exemplary compounds of the present invention
and their biological activities.
[0035] FIG. 3 shows ATP Synthesis and Hydrolysis Inhibition Graph
for 1,4-benzodiazepine-2,5-diones.
[0036] FIG. 4 shows exemplary compounds of the present invention
and their biological activities.
[0037] FIG. 5 presents additional selectivity data for additional
1,4-benzodiazepine-2,5-dione compounds of the present
invention.
[0038] FIG. 6 shows cellular ATP synthesis in the presence of the
compounds of the present invention.
DEFINITIONS
[0039] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below.
[0040] As used herein, the term "benzodiazepine" refers to a seven
membered non-aromatic heterocyclic ring fused to a phenyl ring
wherein the seven-membered ring has two nitrogen atoms, as part of
the heterocyclic ring. In some aspects, the two nitrogen atoms are
in the 1 and 4 positions or the 1 and 5 positions, as shown in the
general structures below:
##STR00018##
[0041] The term "larger than benzene" refers to any chemical group
containing 7 or more non-hydrogen atoms.
[0042] The term "chemical moiety" refers to any chemical compound
containing at least one carbon atom. Examples of chemical moieties
include, but are not limited to, aromatic chemical moieties,
chemical moieties comprising Sulfur, chemical moieties comprising
Nitrogen, hydrophilic chemical moieties, and hydrophobic chemical
moieties.
[0043] As used herein, the term "aliphatic" represents the groups
including, but not limited to, alkyl, alkenyl, alkynyl, and
acyclic.
[0044] As used herein, the term "aryl" represents a single aromatic
ring such as a phenyl ring, or two or more aromatic rings (e.g.,
biphenyl, naphthalene, anthracene), or an aromatic ring and one or
more non-aromatic rings. The aryl group can be optionally
substituted with a lower aliphatic group (e.g., alkyl, alkenyl,
alkynyl, or acyclic). Additionally, the aliphatic and aryl groups
can be further substituted by one or more functional groups
including, but not limited to, chemical moieties comprising N, S,
O, --NH.sub.2, --NHCOCH.sub.3, --OH, lower alkoxy
(C.sub.1-C.sub.4), and halo (--F, --Cl, --Br, or --I).
[0045] As used herein, the term "substituted aliphatic" refers to
an alkane, alkene, alkyne, or alcyclic moiety where at least one of
the aliphatic hydrogen atoms has been replaced by, for example, a
halogen, an amino, a hydroxy, an ether, a nitro, a thio, a ketone,
a sulfone, a sulfonamide, an aldehyde, an ester, an amide, a lower
aliphatic, a substituted lower aliphatic, or a ring (aryl,
substituted aryl, cycloaliphatic, or substituted cycloaliphatic,
etc.). Examples of such include, but are not limited to,
1-chloroethyl and the like.
[0046] As used herein, the term "substituted aryl" refers to an
aromatic ring or fused aromatic ring system consisting of at least
one aromatic ring, and where at least one of the hydrogen atoms on
a ring carbon has been replaced by, for example, a halogen, an
amino, a hydroxy, a nitro, a thio, a ketone, an aldehyde, an ether,
an ester, an amide, a sulfone, a sulfonamide, a lower aliphatic, a
substituted lower aliphatic, or a ring (aryl, substituted aryl,
cycloaliphatic, or substituted cycloaliphatic). Examples of such
include, but are not limited to, hydroxyphenyl and the like.
[0047] As used herein, the term "cycloaliphatic" refers to an
aliphatic structure containing a fused ring system. Examples of
such include, but are not limited to, decalin and the like.
[0048] As used herein, the term "substituted cycloaliphatic" refers
to a cycloaliphatic structure where at least one of the aliphatic
hydrogen atoms has been replaced by a halogen, a heteroatom, a
nitro, a thio, an amino, a hydroxy, a ketone, an aldehyde, an
ester, an amide, a lower aliphatic, a substituted lower aliphatic,
or a ring (aryl, substituted aryl, cycloaliphatic, or substituted
cycloaliphatic). Examples of such include, but are not limited to,
1-chlorodecalyl, bicyclo-heptanes, octanes, and nonanes (e.g.,
nonrbornyl) and the like.
[0049] As used herein, the term "heterocyclic" represents, for
example, an aromatic or nonaromatic ring containing one or more
heteroatoms. The heteroatoms can be the same or different from each
other. Examples of heteroatoms include, but are not limited to
nitrogen, oxygen and sulfur. Aromatic and nonaromatic heterocyclic
rings are well-known in the art. Some nonlimiting examples of
aromatic heterocyclic rings include pyridine, pyrimidine, indole,
purine, quinoline and isoquinoline. Nonlimiting examples of
nonaromatic heterocyclic compounds include piperidine, piperazine,
morpholine, pyrrolidine and pyrazolidine. Examples of oxygen
containing heterocyclic rings include, but not limited to furan,
oxirane, 2H-pyran, 4H-pyran, 2H-chromene, and benzofuran. Examples
of sulfur-containing heterocyclic rings include, but are not
limited to, thiophene, benzothiophene, and parathiazine. Examples
of nitrogen containing rings include, but not limited to, pyrrole,
pyrrolidine, pyrazole, pyrazolidine, imidazole, imidazoline,
imidazolidine, pyridine, piperidine, pyrazine, piperazine,
pyrimidine, indole, purine, benzimidazole, quinoline, isoquinoline,
triazole, and triazine. Nonlimiting examples of heterocyclic rings
containing two different heteroatoms include, but are not limited
to, phenothiazine, morpholine, parathiazine, oxazine, oxazole,
thiazine, and thiazole. The heterocyclic ring is optionally further
substituted with one or more groups selected from aliphatic, nitro,
acetyl (i.e., --C(.dbd.O)--CH.sub.3), or aryl groups.
[0050] As used herein, the term "substituted heterocyclic" refers
to a heterocylic structure where at least one of the ring hydrogen
atoms is replaced by oxygen, nitrogen or sulfur, and where at least
one of the aliphatic hydrogen atoms has been replaced by a halogen,
hydroxy, a thio, nitro, an amino, an ether, a sulfone, a
sulphonamide, a ketone, an aldehyde, an ester, an amide, a lower
aliphatic, a substituted lower aliphatic, or a ring (aryl,
substituted aryl, cycloaliphatic, or substituted cycloaliphatic).
Examples of such include, but are not limited to
2-chloropyranyl.
[0051] As used herein, the term "electron-rich heterocycle," means
cyclic compounds in which one or more ring atoms is a heteroatom
(e.g., oxygen, nitrogen or sulfur), and the heteroatom has unpaired
electrons which contribute to a 6-.pi. electronic system. Exemplary
electron-rich heterocycles include, but are not limited to,
pyrrole, indole, furan, benzofuran, thiophene, benzothiophene and
other similar structures.
[0052] As used herein, the term "linker" refers to a chain
containing up to and including eight contiguous atoms connecting
two different structural moieties where such atoms are, for
example, carbon, nitrogen, oxygen, or sulfur. Ethylene glycol is
one non-limiting example.
[0053] As used herein, the term "lower-alkyl-substituted-amino"
refers to any alkyl unit containing up to and including eight
carbon atoms where one of the aliphatic hydrogen atoms is replaced
by an amino group. Examples of such include, but are not limited
to, ethylamino and the like.
[0054] As used herein, the term "lower-alkyl-substituted-halogen"
refers to any alkyl chain containing up to and including eight
carbon atoms where one of the aliphatic hydrogen atoms is replaced
by a halogen. Examples of such include, but are not limited to,
chlorethyl and the like.
[0055] As used herein, the term "acetylamino" shall mean any
primary or secondary amino that is acetylated. Examples of such
include, but are not limited to, acetamide and the like.
[0056] As used herein, the term "a moiety that participates in
hydrogen bonding" as used herein represents a group that can accept
or donate a proton to form a hydrogen bond thereby. Some specific
non-limiting examples of moieties that participate in hydrogen
bonding include a fluoro, oxygen-containing and nitrogen-containing
groups that are well-known in the art. Some examples of
oxygen-containing groups that participate in hydrogen bonding
include: hydroxy, lower alkoxy, lower carbonyl, lower carboxyl,
lower ethers and phenolic groups. The qualifier "lower" as used
herein refers to lower aliphatic groups (C.sub.1-C.sub.4) to which
the respective oxygen-containing functional group is attached.
Thus, for example, the term "lower carbonyl" refers to inter alia,
formaldehyde, acetaldehyde. Some nonlimiting examples of
nitrogen-containing groups that participate in hydrogen bond
formation include amino and amido groups. Additionally, groups
containing both an oxygen and a nitrogen atom can also participate
in hydrogen bond formation. Examples of such groups include nitro,
N-hydroxy and nitrous groups. It is also possible that the
hydrogen-bond acceptor in the present invention can be the .pi.
electrons of an aromatic ring.
[0057] The term "derivative" of a compound, as used herein, refers
to a chemically modified compound wherein the chemical modification
takes place either at a functional group of the compound (e.g.,
aromatic ring) or benzodiazepine backbone. Such derivatives
include, but are not limited to, esters of alcohol-containing
compounds, esters of carboxy-containing compounds, amides of
amine-containing compounds, amides of carboxy-containing compounds,
imines of amino-containing compounds, acetals of
aldehyde-containing compounds, ketals of carbonyl-containing
compounds, and the like.
[0058] As used herein, the term "subject" refers to organisms to be
treated by the methods of the present invention. Such organisms
preferably include, but are not limited to, mammals (e.g., murines,
simians, equines, bovines, porcines, canines, felines, and the
like), and most preferably includes humans. In the context of the
invention, the term "subject" generally refers to an individual who
will receive or who has received treatment (e.g., administration of
a compound of the present invention and optionally one or more
other agents) for a condition characterized by the dysregulation of
apoptotic processes.
[0059] The term "diagnosed," as used herein, refers to the to
recognition of a disease by its signs and symptoms (e.g.,
resistance to conventional therapies), or genetic analysis,
pathological analysis, histological analysis, and the like.
[0060] As used herein, the terms "anticancer agent," or
"conventional anticancer agent" refer to any chemotherapeutic
compounds, radiation therapies, or surgical interventions, used in
the treatment of cancer.
[0061] As used herein the term, "in vitro" refers to an artificial
environment and to processes or reactions that occur within an
artificial environment. In vitro environments include, but are not
limited to, test tubes and cell cultures. The term "in vivo" refers
to the natural environment (e.g., an animal or a cell) and to
processes or reaction that occur within a natural environment.
[0062] As used herein, the term "host cell" refers to any
eukaryotic or prokaryotic cell (e.g., mammalian cells, avian cells,
amphibian cells, plant cells, fish cells, and insect cells),
whether located in vitro or in vivo.
[0063] As used herein, the term "cell culture" refers to any in
vitro culture of cells. Included within this term are continuous
cell lines (e.g., with an immortal phenotype), primary cell
cultures, finite cell lines (e.g., non-transformed cells), and any
other cell population maintained in vitro, including oocytes and
embryos.
[0064] In preferred embodiments, the "target cells" of the
compositions and methods of the present invention include, refer
to, but are not limited to, lymphoid cells or cancer cells.
Lymphoid cells include B cells, T cells, granulocytes, dendritic
cells, and antigen presenting cells. Granulocycles include
eosinophils and macrophages. In some embodiments, target cells are
continuously cultured cells or uncultured cells obtained from
patient biopsies.
[0065] Cancer cells include tumor cells, neoplastic cells,
malignant cells, metastatic cells, and hyperplastic cells.
Neoplastic cells can be benign or malignant. Neoplastic cells are
benign if they do not invade or metastasize. A malignant cell is
one that is able to invade and/or metastasize. Hyperplasia is a
pathologic accumulation of cells in a tissue or organ, without
significant alteration in structure or function.
[0066] In one specific embodiment, the target cells exhibit
pathological growth or proliferation. As used herein, the term
"pathologically proliferating or growing cells" refers to a
localized population of proliferating cells in an animal that is
not governed by the usual limitations of normal growth.
[0067] As used herein, the term "un-activated target cell" refers
to a cell that is either in the G.sub.o phase or one in which a
stimulus has not been applied.
[0068] As used herein, the term "activated target lymphoid cell"
refers to a lymphoid cell that has been primed with an appropriate
stimulus to cause a signal transduction cascade, or alternatively,
a lymphoid cell that is not in G.sub.o phase. Activated lymphoid
cells may proliferate, undergo activation induced cell death, or
produce one or more of cytotoxins, cytokines, and other related
membrane-associated proteins characteristic of the cell type (e.g.,
CD8.sup.+ or CD4.sup.+). They are also capable of recognizing and
binding any target cell that displays a particular antigen on its
surface, and subsequently releasing its effector molecules.
[0069] As used herein, the term "activated cancer cell" refers to a
cancer cell that has been primed with an appropriate stimulus to
cause a signal transduction. An activated cancer cell may or may
not be in the G.sub.0 phase.
[0070] An activating agent is a stimulus that upon interaction with
a target cell results in a signal transduction cascade. Examples of
activating stimuli include, but are not limited to, small
molecules, radiant energy, and molecules that bind to cell
activation cell surface receptors. Responses induced by activation
stimuli can be characterized by changes in, among others,
intracellular Ca.sup.2+, hydroxyl radical levels; the activity of
enzymes like kinases or phosphatases; or the energy state of the
cell. For cancer cells, activating agents also include transforming
oncogenes.
[0071] As used herein, the term "effective amount" refers to the
amount of a compound (e.g., a compound of the present invention)
sufficient to effect beneficial or desired results. An effective
amount can be administered in one or more administrations,
applications or dosages and is not limited intended to be limited
to a particular formulation or administration route.
[0072] As used herein, the term "dysregulation of the process of
cell death" refers to any aberration in the ability of (e.g.,
predisposition) a cell to undergo cell death via either necrosis or
apoptosis. Dysregulation of cell death is associated with or
induced by a variety of conditions, including for example,
autoimmune disorders (e.g., systemic lupus erythematosus,
rheumatoid arthritis, myasthenia gravis, Sjogren's syndrome, etc.),
chronic inflammatory conditions (e.g., graft-versus-host disease,
psoriasis, asthma and Crohn's disease), hyperproliferative
disorders (e.g., tumors, B cell lymphomas, T cell lymphomas, etc.),
viral infections (e.g., herpes, papilloma, HIV), and other
conditions such as osteoarthritis and atherosclerosis.
[0073] It should be noted that when the dysregulation is induced by
or associated with a viral infection, the viral infection may or
may not be detectable at the time dysregulation occurs or is
observed. That is, viral-induced dysregulation can occur even after
the disappearance of symptoms of viral infection.
[0074] A "hyperproliferative disorder," as used herein refers to
any condition in which a localized population of proliferating
cells in an animal is not governed by the usual limitations of
normal growth. Examples of hyperproliferative disorders include
tumors, neoplasms, lymphomas and the like. A neoplasm is said to be
benign if it does not undergo, invasion or metastasis and malignant
if it does either of these. A metastatic cell or tissue means that
the cell can invade and destroy neighboring body structures.
Hyperplasia is a form of cell proliferation involving an increase
in cell number in a tissue or organ, without significant alteration
in structure or function. Metaplasia is a form of controlled cell
growth in which one type of fully differentiated cell substitutes
for another type of differentiated cell. Metaplasia can occur in
epithelial or connective tissue cells. A typical metaplasia
involves a somewhat disorderly metaplastic epithelium.
[0075] The pathological growth of activated lymphoid cells often
results in an autoimmune disorder or a chronic inflammatory
condition. As used herein, the term "autoimmune disorder" refers to
any condition in which an organism produces antibodies or immune
cells which recognize the organism's own molecules, cells or
tissues. Non-limiting examples of autoimmune disorders include
autoimmune hemolytic anemia, autoimmune hepatitis, Berger's disease
or IgA nephropathy, Celiac Sprue, chronic fatigue syndrome, Crohn's
disease, dermatomyositis, fibromyalgia, graft versus host disease,
Grave's disease, Hashimoto's thyroiditis, idiopathic
thrombocytopenia purpura, lichen planus, multiple sclerosis,
myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,
scleroderma, Sjorgren syndrome, systemic lupus erythematosus, type
1 diabetes, ulcerative colitis, vitiligo, tuberculosis, and the
like.
[0076] As used herein, the term "chronic inflammatory condition"
refers to a condition wherein the organism's immune cells are
activated. Such a condition is characterized by a persistent
inflammatory response with pathologic sequelae. This state is
characterized by infiltration of mononuclear cells, proliferation
of fibroblasts and small blood vessels, increased connective
tissue, and tissue destruction. Examples of chronic inflammatory
diseases include, but are not limited to, Crohn's disease,
psoriasis, chronic obstructive pulmonary disease, inflammatory
bowel disease, multiple sclerosis, and asthma. Autoimmune diseases
such as rheumatoid arthritis and systemic lupus erythematosus can
also result in a chronic inflammatory state.
[0077] As used herein, the term "co-administration" refers to the
administration of at least two agent(s) (e.g., a compound of the
present invention) or therapies to a subject. In some embodiments,
the co-administration of two or more agents/therapies is
concurrent. In other embodiments, a first agent/therapy is
administered prior to a second agent/therapy. Those of skill in the
art understand that the formulations and/or routes of
administration of the various agents/therapies used may vary. The
appropriate dosage for co-administration can be readily determined
by one skilled in the art. In some embodiments, when
agents/therapies are co-administered, the respective
agents/therapies are administered at lower dosages than appropriate
for their administration alone. Thus, co-administration is
especially desirable in embodiments where the co-administration of
the agents/therapies lowers the requisite dosage of a known
potentially harmful (e.g., toxic) agent(s).
[0078] As used herein, the term "toxic" refers to any detrimental
or harmful effects on a cell or tissue as compared to the same cell
or tissue prior to the administration of the toxicant.
[0079] As used herein, the term "pharmaceutical composition" refers
to the combination of an active agent with a carrier, inert or
active, making the composition especially suitable for diagnostic
or therapeutic use in vivo, in vivo or ex vivo.
[0080] As used herein, the term "pharmaceutically acceptable
carrier" refers to any of the standard pharmaceutical carriers,
such as a phosphate buffered saline solution, water, emulsions
(e.g., such as an oil/water or water/oil emulsions), and various
types of wetting agents. The compositions also can include
stabilizers and preservatives. For examples of carriers,
stabilizers and adjuvants. (See e.g., Martin, Remington's
Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa.
[1975]).
[0081] As used herein, the term "pharmaceutically acceptable salt"
refers to any pharmaceutically acceptable salt (e.g., acid or base)
of a compound of the present invention which, upon administration
to a subject, is capable of providing a compound of this invention
or an active metabolite or residue thereof. As is known to those of
skill in the art, "salts" of the compounds of the present invention
may be derived from inorganic or organic acids and bases. Examples
of acids include, but are not limited to, hydrochloric,
hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic,
phosphoric, glycolic, lactic, salicylic, succinic,
toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,
ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,
benzenesulfonic acid, and the like. Other acids, such as oxalic,
while not in themselves pharmaceutically acceptable, may be
employed in the preparation of salts useful as intermediates in
obtaining the compounds of the invention and their pharmaceutically
acceptable acid addition salts.
[0082] Examples of bases include, but are not limited to, alkali
metals (e.g., sodium) hydroxides, alkaline earth metals (e.g.,
magnesium), hydroxides, ammonia, and compounds of formula
NW.sub.4.sup.+, wherein W is C.sub.1-4 alkyl, and the like.
[0083] Examples of salts include, but are not limited to: acetate,
adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
palmoate, pectinate, persulfate, phenylpropionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate,
undecanoate, and the like. Other examples of salts include anions
of the compounds of the present invention compounded with a
suitable cation such as Na.sup.+, NH.sub.4.sup.+, and
NW.sub.4.sup.+ (wherein W is a C.sub.1-4 alkyl group), and the
like.
[0084] For therapeutic use, salts of the compounds of the present
invention are contemplated as being pharmaceutically acceptable.
However, salts of acids and bases that are non-pharmaceutically
acceptable may also find use, for example, in the preparation or
purification of a pharmaceutically acceptable compound.
[0085] As used herein, the term "pathogen" refers a biological
agent that causes a disease state (e.g., infection, cancer, etc.)
in a host. "Pathogens" include, but are not limited to, viruses,
bacteria, archaea, fungi, protozoans, mycoplasma, prions, and
parasitic organisms.
[0086] The terms "bacteria" and "bacterium" refer to all
prokaryotic organisms, including those within all of the phyla in
the Kingdom Procaryotae. It is intended that the term encompass all
microorganisms considered to be bacteria including Mycoplasma,
Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of
bacteria are included within this definition including cocci,
bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included
within this term are prokaryotic organisms which are gram negative
or gram positive. "Gram negative" and "gram positive" refer to
staining patterns with the Gram-staining process which is well
known in the art. (See e.g., Finegold and Martin, Diagnostic
Microbiology, 6th Ed., CV Mosby St. Louis, pp. 13-15 [1982]). "Gram
positive bacteria" are bacteria which retain the primary dye used
in the Gram stain, causing the stained cells to appear dark blue to
purple under the microscope. "Gram negative bacteria" do not retain
the primary dye used in the Gram stain, but are stained by the
counterstain. Thus, gram negative bacteria appear red.
[0087] As used herein, the term "microorganism" refers to any
species or type of microorganism, including but not limited to,
bacteria, archaea, fungi, protozoans, mycoplasma, and parasitic
organisms. The present invention contemplates that a number of
microorganisms encompassed therein will also be pathogenic to a
subject.
[0088] As used herein, the term "fungi" is used in reference to
eukaryotic organisms such as the molds and yeasts, including
dimorphic fungi.
[0089] As used herein, the term "virus" refers to minute infectious
agents, which with certain exceptions, are not observable by light
microscopy, lack independent metabolism, and are able to replicate
only within a living host cell. The individual particles (i.e.,
virions) typically consist of nucleic acid and a protein shell or
coat; some virions also have a lipid containing membrane. The term
"virus" encompasses all types of viruses, including animal, plant,
phage, and other viruses.
[0090] The term "sample" as used herein is used in its broadest
sense. A sample suspected of indicating a condition characterized
by the dysregulation of apoptotic function may comprise a cell,
tissue, or fluids, chromosomes isolated from a cell (e.g., a spread
of metaphase chromosomes), genomic DNA (in solution or bound to a
solid support such as for Southern blot analysis), RNA (in solution
or bound to a solid support such as for Northern blot analysis),
cDNA (in solution or bound to a solid support) and the like. A
sample suspected of containing a protein may comprise a cell, a
portion of a tissue, an extract containing one or more proteins and
the like.
[0091] As used herein, the terms "purified" or "to purify" refer,
to the removal of undesired components from a sample. As used
herein, the term "substantially purified" refers to molecules that
are at least 60% free, preferably 75% free, and most preferably
90%, or more, free from other components with which they usually
associated.
[0092] As used herein, the term "antigen binding protein" refers to
proteins which bind to a specific antigen. "Antigen binding
proteins" include, but are not limited to, immunoglobulins,
including polyclonal, monoclonal, chimeric, single chain, and
humanized antibodies, Fab fragments, F(ab')2 fragments, and Fab
expression libraries. Various procedures known in the art are used
for the production of polyclonal antibodies. For the production of
antibody, various host animals can be immunized by injection with
the peptide corresponding to the desired epitope including but not
limited to rabbits, mice, rats, sheep, goats, etc. In a preferred
embodiment, the peptide is conjugated to an immunogenic carrier
(e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole
limpet hemocyanin [KLH]). Various adjuvants are used to increase
the immunological response, depending on the host species,
including but not limited to Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum.
[0093] For preparation of monoclonal antibodies, any technique that
provides for the production of antibody molecules by continuous
cell lines in culture may be used (See e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.). These include, but are not
limited to, the hybridoma technique originally developed by Kohler
and Milstein (Kohler and Milstein, Nature, 256:495-497 [1975]), as
well as the trioma technique, the human B-cell hybridoma technique
(See e.g., Kozbor et al., Immunol. Today, 4:72 [1983]), and the
EBV-hybridoma technique to produce human monoclonal antibodies
(Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96 [1985]).
[0094] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778;
herein incorporated by reference) can be adapted to produce
specific single chain antibodies as desired. An additional
embodiment of the invention utilizes the techniques known in the
art for the construction of Fab expression libraries (Huse et al.,
Science, 246:1275-1281 [1989]) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity.
[0095] Antibody fragments that contain the idiotype (antigen
binding region) of the antibody molecule can be generated by known
techniques. For example, such fragments include but are not limited
to: the F(ab')2 fragment that can be produced by pepsin digestion
of an antibody molecule; the Fab' fragments that can be generated
by reducing the disulfide bridges of an F(ab')2 fragment, and the
Fab fragments that can be generated by treating an antibody
molecule with papain and a reducing agent.
[0096] Genes encoding antigen binding proteins can be isolated by
methods known in the art. In the production of antibodies,
screening for the desired antibody can be accomplished by
techniques known in the art (e.g., radioimmunoassay, ELISA
(enzyme-linked immunosorbant assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitin reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), Western Blots,
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays, etc.), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.) etc.
[0097] As used herein, the term "immunoglobulin" or "antibody"
refer to proteins that bind a specific antigen. Immunoglobulins
include, but are not limited to, polyclonal, monoclonal, chimeric,
and humanized antibodies, Fab fragments, F(ab').sub.2 fragments,
and includes immunoglobulins of the following classes: IgG, IgA,
IgM, IgD, IbE, and secreted immunoglobulins (sIg). Immunoglobulins
generally comprise two identical heavy chains and two light chains.
However, the terms "antibody" and "immunoglobulin" also encompass
single chain antibodies and two chain antibodies.
[0098] The term "epitope" as used herein refers to that portion of
an antigen that makes contact with a particular immunoglobulin.
When a protein or fragment of a protein is used to immunize a host
animal, numerous regions of the protein may induce the production
of antibodies which bind specifically to a given region or
three-dimensional structure on the protein; these regions or
structures are referred to as "antigenic determinants". An
antigenic determinant may compete with the intact antigen (i.e.,
the "immunogen" used to elicit the immune response) for binding to
an antibody.
[0099] The terms "specific binding" or "specifically binding" when
used in reference to the interaction of an antibody and a protein
or peptide means that the interaction is dependent upon the
presence of a particular structure (i.e., the antigenic determinant
or epitope) on the protein; in other words the antibody is
recognizing and binding to a specific protein structure rather than
to proteins in general. For example, if an antibody is specific for
epitope "A," the presence of a protein containing epitope A (or
free, unlabelled A) in a reaction containing labeled "A" and the
antibody will reduce the amount of labeled A bound to the
antibody.
[0100] As used herein, the terms "non-specific binding" and
"background binding" when used in reference to the interaction of
an antibody and a protein or peptide refer to an interaction that
is not dependent on the presence of a particular structure (i.e.,
the antibody is binding to proteins in general rather that a
particular structure such as an epitope).
[0101] As used herein, the term "modulate" refers to the activity
of a compound (e.g., a compound of the present invention) to affect
(e.g., to promote or retard) an aspect of cellular function,
including, but not limited to, cell growth, proliferation,
apoptosis, and the like.
[0102] The term "test compound" refers to any chemical entity,
pharmaceutical, drug, and the like, that can be used to treat or
prevent a disease, illness, sickness, or disorder of bodily
function, or otherwise alter the physiological or cellular status
of a sample (e.g., the level of dysregulation of apoptosis in a
cell or tissue). Test compounds comprise both known and potential
therapeutic compounds. A test compound can be determined to be
therapeutic by using the screening methods of the present
invention. A "known therapeutic compound" refers to a therapeutic
compound that has been shown (e.g., through animal trials or prior
experience with administration to humans) to be effective in such
treatment or prevention. In preferred embodiments, "test compounds"
are agents that modulate apoptosis in cells.
GENERAL DESCRIPTION OF THE INVENTION
[0103] As a class of drugs, benzodiazepine compounds have been
widely studied and reported to be effective medicaments for
treating a number of disease. For example, U.S. Pat. Nos.
4,076,823, 4,110,337, 4,495,101, 4,751,223 and 5,776,946, each
incorporated herein by reference in its entirety, report that
certain benzodiazepine compounds are effective as analgesic and
anti-inflammatory agents. Similarly, U.S. Pat. No. 5,324,726 and
U.S. Pat. No. 5,597,915, each incorporated by reference in its
entirety, report that certain benzodiazepine compounds are
antagonists of cholecystokinin and gastrin and thus might be useful
to treat certain gastrointestinal disorders.
[0104] Other benzodiazepine compounds have been studied as
inhibitors of human neutrophil elastase in the treating of human
neutrophil elastase-mediated conditions such as myocardial
ischemia, septic shock syndrome, among others (See e.g., U.S. Pat.
No. 5,861,380 incorporated herein by reference in its entirety).
U.S. Pat. No. 5,041,438, incorporated herein by reference in its
entirety, reports that certain benzodiazepine compounds are useful
as anti-retroviral agents.
[0105] Despite the attention benzodiazepine compounds have drawn,
it will become apparent from the description below, that the
present invention provides novel compounds (e.g.,
1,4-benzodiazepine-2,5-dione compounds) and related compounds and
methods of using the novel compounds, as well as known compounds,
for treating a variety of diseases.
[0106] Benzodiazepine compounds are known to bind to benzodiazepine
receptors in the central nervous system (CNS) and thus have been
used to treat various CNS disorders including anxiety and epilepsy.
Peripheral benzodiazepine receptors have also been identified,
which receptors may incidentally also be present in the CNS. The
present invention demonstrates that 1,4-benzodiazepine-2,5-dione
compounds with, for example, an electron rich heterocycle moiety at
the C3 position of the benzodiazepine ring have pro-apoptotic
properties consistent with a mechanism that does not result from
interaction with the mitochondrial F.sub.1F.sub.0-ATPase. The
present invention also provides 1,4-benzodiazepine-2,5-dione
compounds that demonstrate selective cytotoxicity against T cells
as compared to B cells.
DETAILED DESCRIPTION OF THE INVENTION
[0107] The present invention provides novel chemical compounds,
methods for their discovery, and their therapeutic, research, and
diagnostic use. In particular, the present invention provides
1,4-benzodiazepine-2,5-dione compounds, and methods of using
1,4-benzodiazepine-2,5-dione compounds as therapeutic agents to
treat a number of conditions associated with the faulty regulation
of the processes of programmed cell death, autoimmunity,
inflammation, and hyperproliferation, and the like.
[0108] Exemplary compositions and methods of the present invention
are described in more detail in the following sections: I.
Modulators of Cell Death; II. Exemplary Compounds; III.
Pharmaceutical compositions, formulations, and exemplary
administration routes and dosing considerations; IV. Drug screens;
and V. Therapeutic Applications.
[0109] The present invention herein incorporates by reference known
uses of benzodiazepine compounds, including, but not limited to the
uses described in Otto, M. W., et al., (2005) J. Clin. Psychiatry
66 Suppl. 2:34-38; Yoshii, M., et al., (2005) Nippon Yakurigaku
Zasshi 125(1):33-36; Yasuda, K. (2004) Nippon Rinsho. 62 Suppl.
12:360-363; Decaudin, D. (2004) 15(8):737-745; Bonnot, O., et al.
(2003) Encephale. 29(6):553-559; Sugiyama, T. (2003) Ryoikibetsu
Shokogun Shirizu. 40:489-492; Lacapere, J. J., et al., (2003)
Steroids. 68(7-8):569-585; Galiegue, S., et al., (2003) Curr. Med.
Chem. 10(16):1563-1572; Papadopoulo, V. (2003) Ann. Pharm. Fr.
61(1):30-50; Goethals, I., et al., (2002) Eur. J. Nucl. Med. Mol.
Imaging. 30(2):325-328; Castedo, M., et al., (2002) J. Exp. Med.
196(9):1121-1125; Buffett-Jerrott, S. E., et al., (2002) Curr.
Pham. Des. 8(1):45-58; Beurdeley-Thomas, A., et al., (2000) J.
Nuerooncol. 46(1):45-56; Smyth, W. F., et al., (1998)
Electrophoresis 19(16-17):2870-2882; Yoshii, M., et al., (1998)
Nihon Shinkei Seishin Yakurigaku Zasshi. 18(2):49-54; Trimble, M.
and Hindmarch, I. (2000) Benzodiazepines, published by Wrighton
Biomedical Publishing; and Salamone, S. J. (2001) Benzodiazepines
and GHB--Detection and Pharmacology, published by Humana Press.
[0110] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of organic chemistry,
pharmacology, molecular biology (including recombinant techniques),
cell biology, biochemistry, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature, such as, "Molecular cloning: a laboratory manual"
Second Edition (Sambrook et al., 1989); "Oligonucleotide synthesis"
(M. J. Gait, ed., 1984); "Animal cell culture" (R. I. Freshney,
ed., 1987); the series "Methods in enzymology" (Academic Press,
Inc.); "Handbook of experimental immunology" (D. M. Weir & C.
C. Blackwell, eds.); "Gene transfer vectors for mammalian cells"
(J. M. Miller & M. P. Calos, eds., 1987); "Current protocols in
molecular biology" (F. M. Ausubel et al., eds., 1987, and periodic
updates); "PCR: the polymerase chain reaction" (Mullis et al.,
eds., 1994); and "Current protocols in immunology" (J. E. Coligan
et al., eds., 1991), each of which is herein incorporated by
reference in its entirety.
I. Modulators of Cell Death
[0111] In preferred embodiments, the present invention regulates
apoptosis through the exposure of cells to the compounds of the
present invention (e.g., 1,4-benzodiazepine-2,5-diones). In
particular, the present invention demonstrates that
1,4-benzodiazepine-2,5-diones with, for example, certain
heterocycles at the C3 position of the benzodiazepine ring have
pro-apoptotic properties consistent with a mechanism that does not
result from interaction with the mitochondrial
F.sub.1F.sub.0-ATPase. The present invention also demonstrates that
1,4-benzodiazepine-2,5-diones with an electron rich heterocycle
moiety at the C3 position of the benzodiazepine ring can have
pro-apoptotic selectivity for T cells over B cells.
[0112] The effect of compounds can be measured by detecting any
number of cellular changes. Cell death may be assayed as described
herein and in the art. In preferred embodiments, cell lines are
maintained under appropriate cell culturing conditions (e.g., gas
(CO.sub.2), temperature and media) for an appropriate period of
time to attain exponential proliferation without density dependent
constraints. Cell number and or viability are measured using
standard techniques, such as trypan blue exclusion/hemo-cytometry,
or MTT dye conversion assay. Alternatively, the cell may be
analyzed for the expression of genes or gene products associated
with aberrations in apoptosis or necrosis.
II. Exemplary Compounds
[0113] Exemplary compounds of the present invention are provided
below. Certain 1,4-benzodiazepine-2,5-dione derivatives have been
described (see, e.g., U.S. patent application Ser. No. 09/700,101;
U.S. Pat. No. 6,506,744; Kamal, et al., 2004 Synlett 14:2533-2535;
Hulme, et al., 1998 J. Org. Chem. 63:8021-8022; Raboisson et al.,
2005 Bioorg. Med. Chem. Lett. 15:1857-1861; Raboisson et al., 2005
Bioorg. Med. Chem. Lett. 15:765-770; Rabiosson et al., 2005 J. Med.
Chem. 48:909-912; each herein incorporated by reference in their
entireties). The present invention provides novel
1,4-benzodiazepine-2,5-dione compounds, and uses for
1,4-benzodiazepine-2,5-dione compounds.
[0114] Certain embodiments provide a composition comprising a
compound described by a formula selected from the group consisting
of:
[0115] In certain embodiments, the present invention provides a
composition comprising novel 1,4-benzodiazepine-2,5-dione
compounds. In certain embodiments, the present invention provide a
composition comprising a compound described by a formula selected
from the group consisting of:
##STR00019## ##STR00020## ##STR00021## ##STR00022##
substituted and unsubstituted, including both R and S enantiomeric
forms and racemic mixtures.
[0116] In some embodiments, R1 is an electron rich heterocycle. In
some embodiments, the electron rich heterocycle contains 5 or more
heterocyclic atoms.
[0117] In some embodiments, R.sub.1 is selected from the group
consisting of
##STR00023##
wherein R.sub.1' is selected from the group consisting of
cycloalipathic, aryl, substituted aryl, heterocyclic, and
substituted heterocyclic.
[0118] In some embodiments, R.sub.2 is selected from the group
consisting of H, alkyl, substituted alkyl, and R.sub.1.
[0119] In some embodiments, R.sub.3 is selected from the group
consisting of H, alkyl, and substituted alkyl.
[0120] In some embodiments, R3 is selected from the group
consisting of hydrogen; halogen; OH; a chemical moiety comprising
an aryl subgroup; a chemical moiety comprising a substituted aryl
subgroup; a chemical moiety comprising a cycloaliphatic subgroup; a
chemical moiety comprising a substituted cycloaliphatic subgroup; a
chemical moiety comprising a heterocyclic subgroup; a chemical
moiety comprising a substituted heterocyclic subgroup; a chemical
moiety comprising at least one ester subgroup; a chemical moiety
comprising at least one ether subgroup; a linear or branched,
saturated or unsaturated, substituted or non-substituted, aliphatic
chain having at least 2 carbons; a chemical moiety comprising
Sulfur; a chemical moiety comprising Nitrogen; --OR--, wherein R is
selected from the group consisting of a chemical moiety comprising
an aryl subgroup; a chemical moiety comprising a substituted aryl
subgroup; a chemical moiety comprising a cycloaliphatic subgroup; a
chemical moiety comprising a substituted cycloaliphatic subgroup; a
chemical moiety comprising a heterocyclic subgroup; a chemical
moiety comprising a substituted heterocyclic subgroup; a linear or
branched, saturated or unsaturated, substituted or non-substituted,
aliphatic chain having at least 2 carbons; a chemical moiety
comprising at least one ester subgroup; a chemical moiety
comprising at least one ether subgroup; a chemical moiety
comprising Sulfur; a chemical moiety comprising Nitrogen.
[0121] In some embodiments, R3 is selected from group consisting
of: napthalene; phenol; 1-Napthalenol; 2-Napthalenol;
##STR00024## ##STR00025##
quinolines, and all aromatic regioisomers.
[0122] In some embodiments, the R1 and R3 groups may be
interchanged (e.g., in some embodiments, the R1 group is positioned
at the first position of the benzodiazepine ring and the R3 group
is positioned at the third position of the benzodiazepine ring; in
some embodiments, the R1 group is positioned at the third position
of the benzodiazepine ring and the R3 group is positioned at the
first position of the benzodiazepine ring).
[0123] In some embodiments, R.sub.4 and R.sub.4' is independently
selected from the group consisting of CH.sub.3, halogen,
SO.sub.2R.sub.4'', SO.sub.2N(R.sub.4'').sub.2, OR.sub.4'',
N(R.sub.4'').sub.2, CON(R.sub.4'').sub.2, NHCOR.sub.4'',
NHSO.sub.2R.sub.4', alkyl, mono-substituted alkyl, di-substituted
alkyl, tri-substituted alkyl; wherein R.sub.4'' is selected from
the group consisting of halogen, H, alkyl, mono-substituted alkyl,
di-substituted alkyl, tri-substituted alkyl, aryl, mono-substituted
aryl, di-substituted aryl, tri-substituted aryl, cycloalipathic,
mono-substituted cycloalipathic, di-substituted cycloalipathic,
tri-substituted cycloalipathic.
[0124] In some embodiments, R.sub.5 is selected from the group
consisting of H, alkyl, mono-substituted aryl, di-substituted aryl,
and tri-substituted aryl.
[0125] In some embodiments, R6 is selected from the group
consisting of C, N or S.
[0126] In some embodiments, R1 is selected from the group
consisting of:
##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##
##STR00031## ##STR00032## ##STR00033##
substituted and unsubstituted, and derivatives thereof.
[0127] Certain 1,4-benzodiazepine-2,5-dione compounds of the
present invention include, but are not limited to,
##STR00034## ##STR00035##
[0128] From the above description, it is apparent that many
specific examples are represented by the generic formulas presented
above. A wide variety of sub combinations arising from selecting a
particular group at each substituent position are possible and all
such combinations are within the scope of this invention. The
experimental examples, provided below, describe biological
activities of these compounds and provide assays for assessing
activities of derivatives or other related compounds.
[0129] In summary, a large number of compounds are presented
herein. Any one or more of these compounds can be used to treat a
variety of dysregulatory disorders related to cellular death as
described elsewhere herein. Additionally, any one or more of these
compounds can be used in combination with at least one other
therapeutic agent (e.g., potassium channel openers, calcium channel
blockers, sodium hydrogen exchanger inhibitors, antiarrhythmic
agents, antiatherosclerotic agents, anticoagulants, antithrombotic
agents, prothrombolytic agents, fibrinogen antagonists, diuretics,
antihypertensive agents, ATPase inhibitors, mineralocorticoid
receptor antagonists, phosphodiesterase inhibitors, antidiabetic
agents, anti-inflammatory agents, antioxidants, angiogenesis
modulators, antiosteoporosis agents, hormone replacement therapies,
hormone receptor modulators, oral contraceptives, antiobesity
agents, antidepressants, antianxiety agents, antipsychotic agents,
antiproliferative agents, antitumor agents, antiulcer and
gastroesophageal reflux disease agents, growth hormone agents
and/or growth hormone secretagogues, thyroid mimetics,
anti-infective agents, antiviral agents, antibacterial agents,
antifungal agents, cholesterol/lipid lowering agents and lipid
profile therapies, and agents that mimic ischemic preconditioning
and/or myocardial stunning, antiatherosclerotic agents,
anticoagulants, antithrombotic agents, antihypertensive agents,
antidiabetic agents, and antihypertensive agents selected from ACE
inhibitors, AT-1 receptor antagonists, ET receptor antagonists,
dual ET/AII receptor antagonists, and vasopepsidase inhibitors, or
an antiplatelet agent selected from GPIIb/IIIa blockers, P2Y.sub.1
and P2Y.sub.12 antagonists, thromboxane receptor antagonists, and
aspirin) cellular activating agents in along with a
pharmaceutically-acceptable carrier or diluent in a pharmaceutical
composition. The above-described compounds can also be used in drug
screening assays and other diagnostic and research methods.
III. Pharmaceutical Compositions, Formulations, and Exemplary
Administration Routes and Dosing Considerations
[0130] Exemplary embodiments of various contemplated medicaments
and pharmaceutical compositions are provided below.
[0131] A. Preparing Medicaments
[0132] The compounds of the present invention are useful in the
preparation of medicaments to treat a variety of conditions
associated with dysregulation of cell death, aberrant cell growth
and hyperproliferation.
[0133] In addition, the compounds are also useful for preparing
medicaments for treating other disorders wherein the effectiveness
of the compounds are known or predicted. Such disorders include,
but are not limited to, autoimmune disorders disorders. The methods
and techniques for preparing medicaments of a compound of the
present invention are well-known in the art. Exemplary
pharmaceutical formulations and routes of delivery are described
below.
[0134] One of skill in the art will appreciate that any one or more
of the compounds described herein, including the many specific
embodiments, are prepared by applying standard pharmaceutical
manufacturing procedures. Such medicaments can be delivered to the
subject by using delivery methods that are well-known in the
pharmaceutical arts.
[0135] B. Exemplary Pharmaceutical Compositions and Formulation
[0136] In some embodiments of the present invention, the
compositions are administered alone, while in some other
embodiments, the compositions are preferably present in a
pharmaceutical formulation comprising at least one active
ingredient/agent, as defined above, together with a solid support
or alternatively, together with one or more pharmaceutically
acceptable carriers and optionally other therapeutic agents (e.g.,
a benzodiazepine compound as described in 60/812,270, 60/802,394,
60/607,599, and 60/641,040, and U.S. patent application Ser. Nos.
11/445,010, 11/324,419, 11/176,719, 11/110,228, 10/935,333,
10/886,450, 10/795,535, 10/634,114, 10/427,211, 10/427,212,
10/217,878, 09/767,283, 09/700,101, and related applications; each
herein incorporated by reference in their entireties). Each carrier
should be "acceptable" in the sense that it is compatible with the
other ingredients of the formulation and not injurious to the
subject.
[0137] Contemplated formulations include those suitable oral,
rectal, nasal, topical (including transdermal, buccal and
sublingual), vaginal, parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) and pulmonary
administration. In some embodiments, formulations are conveniently
presented in unit dosage form and are prepared by any method known
in the art of pharmacy. Such methods include the step of bringing
into association the active ingredient with the carrier which
constitutes one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association (e.g., mixing) the active ingredient with liquid
carriers or finely divided solid carriers or both, and then if
necessary shaping the product.
[0138] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets, wherein each preferably contains a
predetermined amount of the active ingredient; as a powder or
granules; as a solution or suspension in an aqueous or non-aqueous
liquid; or as an oil-in-water liquid emulsion or a water-in-oil
liquid emulsion. In other embodiments, the active ingredient is
presented as a bolus, electuary, or paste, etc.
[0139] In some embodiments, tablets comprise at least one active
ingredient and optionally one or more accessory agents/carriers are
made by compressing or molding the respective agents. In preferred
embodiments, compressed tablets are prepared by compressing in a
suitable machine the active ingredient in a free-flowing form such
as a powder or granules, optionally mixed with a binder (e.g.,
povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert
diluent, preservative, disintegrant (e.g., sodium starch glycolate,
cross-linked povidone, cross-linked sodium carboxymethyl cellulose)
surface-active or dispersing agent. Molded tablets are made by
molding in a suitable machine a mixture of the powdered compound
(e.g., active ingredient) moistened with an inert liquid diluent.
Tablets may optionally be coated or scored and may be formulated so
as to provide slow or controlled release of the active ingredient
therein using, for example, hydroxypropylmethyl cellulose in
varying proportions to provide the desired release profile. Tablets
may optionally be provided with an enteric coating, to provide
release in parts of the gut other than the stomach.
[0140] Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavored basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0141] Pharmaceutical compositions for topical administration
according to the present invention are optionally formulated as
ointments, creams, suspensions, lotions, powders, solutions,
pastes, gels, sprays, aerosols or oils. In alternative embodiments,
topical formulations comprise patches or dressings such as a
bandage or adhesive plasters impregnated with active ingredient(s),
and optionally one or more excipients or diluents. In preferred
embodiments, the topical formulations include a compound(s) that
enhances absorption or penetration of the active agent(s) through
the skin or other affected areas. Examples of such dermal
penetration enhancers include dimethylsulfoxide (DMSO) and related
analogues.
[0142] If desired, the aqueous phase of a cream base includes, for
example, at least about 30% w/w of a polyhydric alcohol, i.e., an
alcohol having two or more hydroxyl groups such as propylene
glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and
polyethylene glycol and mixtures thereof.
[0143] In some embodiments, oily phase emulsions of this invention
are constituted from known ingredients in a known manner. This
phase typically comprises a lone emulsifier (otherwise known as an
emulgent), it is also desirable in some embodiments for this phase
to further comprises a mixture of at least one emulsifier with a
fat or an oil or with both a fat and an oil.
[0144] Preferably, a hydrophilic emulsifier is included together
with a lipophilic emulsifier so as to act as a stabilizer. In some
embodiments it is also preferable to include both an oil and a fat.
Together, the emulsifier(s) with or without stabilizer(s) make up
the so-called emulsifying wax, and the wax together with the oil
and/or fat make up the so-called emulsifying ointment base which
forms the oily dispersed phase of the cream formulations.
[0145] Emulgents and emulsion stabilizers suitable for use in the
formulation of the present invention include Tween 60, Span 80,
cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and
sodium lauryl sulfate.
[0146] The choice of suitable oils or fats for the formulation is
based on achieving the desired properties (e.g., cosmetic
properties), since the solubility of the active compound/agent in
most oils likely to be used in pharmaceutical emulsion formulations
is very low. Thus creams should preferably be a non-greasy,
non-staining and washable products with suitable consistency to
avoid leakage from tubes or other containers. Straight or branched
chain, mono- or dibasic alkyl esters such as di-isoadipate,
isocetyl stearate, propylene glycol diester of coconut fatty acids,
isopropyl myristate, decyl oleate, isopropyl palmitate, butyl
stearate, 2-ethylhexyl palmitate or a blend of branched chain
esters known as Crodamol CAP may be used, the last three being
preferred esters. These may be used alone or in combination
depending on the properties required. Alternatively, high melting
point lipids such as white soft paraffin and/or liquid paraffin or
other mineral oils can be used.
[0147] Formulations suitable for topical administration to the eye
also include eye drops wherein the active ingredient is dissolved
or suspended in a suitable carrier, especially an aqueous solvent
for the agent.
[0148] Formulations for rectal administration may be presented as a
suppository with suitable base comprising, for example, cocoa
butter or a salicylate.
[0149] Formulations suitable for vaginal administration may be
presented as pessaries, creams, gels, pastes, foams or spray
formulations containing in addition to the agent, such carriers as
are known in the art to be appropriate.
[0150] Formulations suitable for nasal administration, wherein the
carrier is a solid, include coarse powders having a particle size,
for example, in the range of about 20 to about 500 microns which
are administered in the manner in which snuff is taken, i.e., by
rapid inhalation (e.g., forced) through the nasal passage from a
container of the powder held close up to the nose. Other suitable
formulations wherein the carrier is a liquid for administration
include, but are not limited to, nasal sprays, drops, or aerosols
by nebulizer, an include aqueous or oily solutions of the
agents.
[0151] Formulations suitable for parenteral administration include
aqueous and non-aqueous isotonic sterile injection solutions which
may contain antioxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents, and liposomes
or other microparticulate systems which are designed to target the
compound to blood components or one or more organs. In some
embodiments, the formulations are presented/formulated in unit-dose
or multi-dose sealed containers, for example, ampoules and vials,
and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for
example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules and tablets of the kind previously
described.
[0152] Preferred unit dosage formulations are those containing a
daily dose or unit, daily subdose, as herein above-recited, or an
appropriate fraction thereof, of an agent.
[0153] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention
may include other agents conventional in the art having regard to
the type of formulation in question, for example, those suitable
for oral administration may include such further agents as
sweeteners, thickeners and flavoring agents. It also is intended
that the agents, compositions and methods of this invention be
combined with other suitable compositions and therapies. Still
other formulations optionally include food additives (suitable
sweeteners, flavorings, colorings, etc.), phytonutrients (e.g.,
flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, and
other acceptable compositions (e.g., conjugated linoelic acid),
extenders, and stabilizers, etc.
[0154] C. Exemplary Administration Routes and Dosing
Considerations
[0155] Various delivery systems are known and can be used to
administer therapeutic agents (e.g., exemplary compounds as
described in Section II above) of the present invention, e.g.,
encapsulation in liposomes, microparticles, microcapsules,
receptor-mediated endocytosis, and the like. Methods of delivery
include, but are not limited to, intra-arterial, intra-muscular,
intravenous, intranasal, and oral routes. In specific embodiments,
it may be desirable to administer the pharmaceutical compositions
of the invention locally to the area in need of treatment; this may
be achieved by, for example, and not by way of limitation, local
infusion during surgery, injection, or by means of a catheter.
[0156] The agents identified can be administered to subjects or
individuals susceptible to or at risk of developing pathological
growth of target cells and correlated conditions. When the agent is
administered to a subject such as a mouse, a rat or a human
patient, the agent can be added to a pharmaceutically acceptable
carrier and systemically or topically administered to the subject.
To identify patients that can be beneficially treated, a tissue
sample is removed from the patient and the cells are assayed for
sensitivity to the agent. Therapeutic amounts are empirically
determined and vary with the pathology being treated, the subject
being treated and the efficacy and toxicity of the agent.
[0157] In some embodiments, in vivo administration is effected in
one dose, continuously or intermittently throughout the course of
treatment. Methods of determining the most effective means and
dosage of administration are well known to those of skill in the
art and vary with the composition used for therapy, the purpose of
the therapy, the target cell being treated, and the subject being
treated. Single or multiple administrations are carried out with
the dose level and pattern being selected by the treating
physician.
[0158] Suitable dosage formulations and methods of administering
the agents are readily determined by those of skill in the art.
Preferably, the compounds are administered at about 0.01 mg/kg to
about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100
mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg.
When the compounds described herein are co-administered with
another agent (e.g., as sensitizing agents), the effective amount
may be less than when the agent is used alone.
[0159] The pharmaceutical compositions can be administered orally,
intranasally, parenterally or by inhalation therapy, and may take
the form of tablets, lozenges, granules, capsules, pills, ampoules,
suppositories or aerosol form. They may also take the form of
suspensions, solutions and emulsions of the active ingredient in
aqueous or nonaqueous diluents, syrups, granulates or powders. In
addition to an agent of the present invention, the pharmaceutical
compositions can also contain other pharmaceutically active
compounds or a plurality of compounds of the invention.
[0160] More particularly, an agent of the present invention also
referred to herein as the active ingredient, may be administered
for therapy by any suitable route including, but not limited to,
oral, rectal, nasal, topical (including, but not limited to,
transdermal, aerosol, buccal and sublingual), vaginal, parental
(including, but not limited to, subcutaneous, intramuscular,
intravenous and intradermal) and pulmonary. It is also appreciated
that the preferred route varies with the condition and age of the
recipient, and the disease being treated.
[0161] Ideally, the agent should be administered to achieve peak
concentrations of the active compound at sites of disease. This may
be achieved, for example, by the intravenous injection of the
agent, optionally in saline, or orally administered, for example,
as a tablet, capsule or syrup containing the active ingredient.
[0162] Desirable blood levels of the agent may be maintained by a
continuous infusion to provide a therapeutic amount of the active
ingredient within disease tissue. The use of operative combinations
is contemplated to provide therapeutic combinations requiring a
lower total dosage of each component antiviral agent than may be
required when each individual therapeutic compound or drug is used
alone, thereby reducing adverse effects.
[0163] D. Exemplary Co-Administration Routes and Dosing
Considerations
[0164] The present invention also includes methods involving
co-administration of the compounds described herein with one or
more additional active agents. Indeed, it is a further aspect of
this invention to provide methods for enhancing prior art therapies
and/or pharmaceutical compositions by co-administering a compound
of this invention. In co-administration procedures, the agents may
be administered concurrently or sequentially. In one embodiment,
the compounds described herein are administered prior to the other
active agent(s). The pharmaceutical formulations and modes of
administration may be any of those described above. In addition,
the two or more co-administered chemical agents, biological agents
or radiation may each be administered using different modes or
different formulations.
[0165] The agent or agents to be co-administered depends on the
type of condition being treated. For example, when the condition
being treated is cancer, the additional agent can be a
chemotherapeutic agent or radiation. When the condition being
treated is an autoimmune disorder, the additional agent can be an
immunosuppressant or an anti-inflammatory agent. When the condition
being treated is chronic inflammation, the additional agent can be
an anti-inflammatory agent. The additional agents to be
co-administered, such as anticancer, immunosuppressant,
anti-inflammatory, and can be any of the well-known agents in the
art, including, but not limited to, those that are currently in
clinical use. The determination of appropriate type and dosage of
radiation treatment is also within the skill in the art or can be
determined with relative ease.
[0166] Treatment of the various conditions associated with abnormal
apoptosis is generally limited by the following two major factors:
(1) the development of drug resistance and (2) the toxicity of
known therapeutic agents. In certain cancers, for example,
resistance to chemicals and radiation therapy has been shown to be
associated with inhibition of apoptosis. Some therapeutic agents
have deleterious side effects, including non-specific
lymphotoxicity, renal and bone marrow toxicity.
[0167] The methods described herein address both these problems.
Drug resistance, where increasing dosages are required to achieve
therapeutic benefit, is overcome by co-administering the compounds
described herein with the known agent. The compounds described
herein sensitize target cells to known agents (and vice versa) and,
accordingly, less of these agents are needed to achieve a
therapeutic benefit.
[0168] The sensitizing function of the claimed compounds also
addresses the problems associated with toxic effects of known
therapeutics. In instances where the known agent is toxic, it is
desirable to limit the dosages administered in all cases, and
particularly in those cases were drug resistance has increased the
requisite dosage. When the claimed compounds are co-administered
with the known agent, they reduce the dosage required which, in
turn, reduces the deleterious effects. Further, because the claimed
compounds are themselves both effective and non-toxic in large
doses, co-administration of proportionally more of these compounds
than known toxic therapeutics will achieve the desired effects
while minimizing toxic effects.
IV. Drug Screens
[0169] In some embodiments of the present invention, the compounds
of the present invention, and other potentially useful compounds,
are screened for pro-apoptotic properties consistent with a
mechanism that does not entail interaction with the mitochondrial
F.sub.1F.sub.0-ATPase. In preferred embodiments of the present
invention, the compounds of the present invention, and other
potentially useful compounds, are screened for pro-apoptotic
selectivity for T cells over B cells.
[0170] A number of suitable screens for measuring the binding
affinity of drugs and other small molecules to receptors are known
in the art. In some embodiments, binding affinity screens are
conducted in in vitro systems. In other embodiments, these screens
are conducted in in vivo or ex vivo systems.
V. Therapeutic Application
[0171] In particularly preferred embodiments, the compositions of
the present invention are contemplated to provide therapeutic
benefits to patients suffering from any one or more of a number of
conditions (e.g., diseases characterized by dysregulation of
necrosis and/or apoptosis processes in a cell or tissue, disease
characterized by aberrant cell growth and/or hyperproliferation,
autoimmune diseases, haematologic malignancies resulting from
aberrant survival and expansion of B and T cells in central and
peripheral lymphoid organs, etc.) by modulating (e.g., inhibiting
or promoting) apoptosis in affected cells or tissues. In further
preferred embodiments, the compositions of the present invention
are used to treat autoimmune/chronic inflammatory conditions.
[0172] In particularly preferred embodiments, the compositions of
the present invention regulate apoptosis through the exposure of
cells to the compounds of the present invention (e.g.,
1,4-benzodiazepine-2,5-diones). In particular, the present
invention demonstrates that 1,4-benzodiazepine-2,5-diones with, for
example, an electron rich heterocycle moiety at the C3 position of
the benzodiazepine ring have pro-apoptotic properties consistent
with a mechanism that does not result from interaction with the
mitochondrial F.sub.1F.sub.0-ATPase. The present invention also
demonstrates that 1,4-benzodiazepine-2,5-diones with an electron
rich heterocycle moiety at the C3 position of the benzodiazepine
ring have pro-apoptotic selectivity for T cells over B cells.
[0173] Accordingly, preferred methods embodied in the present
invention provide therapeutic benefits to patients by providing
compounds of the present invention that modulate (e.g., inhibiting
or promoting) cellular apoptosis in affected cells or tissues
without interacting with the mitochondrial
F.sub.1F.sub.0-ATPase.
[0174] In some embodiments, compounds potentially useful in methods
of the present invention are screened against the National Cancer
Institute's (NCI-60) cancer cell lines for efficacy. (See e.g., A.
Monks et al., J. Natl. Cancer Inst., 83:757-766 [1991]; and K. D.
Paull et al., J. Natl. Cancer Inst., 81:1088-1092 [1989]).
Additional screens suitable screens (e.g., autoimmunity disease
models, etc.) are within the skill in the art.
[0175] In one aspect, derivatives (e.g., pharmaceutically
acceptable salts, analogs, stereoisomers, and the like) of the
exemplary compounds or other suitable compounds are also
contemplated as being useful in the methods of the present
invention.
[0176] Those skilled in the art of preparing pharmaceutical
compounds and formulations will appreciate that when selecting
optional compounds for use in the methods disclosed herein, that
suitability considerations include, but are not limited to, the
toxicity, safety, efficacy, availability, and cost of the
particular compounds.
EXAMPLES
[0177] The following examples are provided to demonstrate and
further illustrate certain preferred embodiments of the present
invention and are not to be construed as limiting the scope
thereof.
Example 1
[0178] This example describes the formulation of exemplary
1,4-benzodiazepine-2,5-diones. As shown in FIG. 1, Bz-423 is a
pro-apoptotic 1,4 benzodiazepine with potent activity in animal
models of lupus (see, e.g., Blatt, N. B., et al., J. Clin. Invest.
2002, 10, 1123; Bednarski, J. J., et al., Arthritis Rheum. 2003,
48, 757; each herein incorporated by reference in their
entiretiese). Bz-423 binds to the oligomycin sensitivity conferring
protein (OSCP) component of the mitochondrial F.sub.1F.sub.0-ATPase
(see, e.g., Johnson, K. M., et al., Chemistry and Biology. 2005,
12, 485; herein incorporated by reference in its entirety). Bz-423
inhibits the enzyme, which produces a state 3 to state 4 transition
within the mitochondrial respiratory chain (MRC), ultimately
resulting in the production of O.sub.2.sup.- from MRC complex III.
This reactive oxygen species functions as a signal-initiating a
tightly-regulated apoptotic process (see, e.g., Johnson, K. M., et
al., Chemistry and Biology. 2005, 12, 485; herein incorporated by
reference in its entirety).
[0179] Previous studies revealed that a hydrophobic aromatic
substituent at C3 along with the phenolic hydroxyl group is
required for the cytotoxic activity of Bz-423. As part of efforts
to further define the elements of Bz-423 required for inhibiting
the F.sub.1F.sub.0-ATPase, a series of
1,4-benzodiazepine-2,5-diones were synthesized as intermediates for
other chemistry. Most of these compounds were cytotoxic and unlike
Bz-423, many were more selective for T cells compared to B cells
(FIG. 2). Replacing the napthyl moiety with other hydrophobic
groups of comparable size (2, 3), but which occupy different space,
had relatively small effects on activity compared with 1, and in
some cases altered the selectivity. By contrast, reducing the size
of the C3 group (4, 5) was not tolerated. Given the similarity
between the structures in FIG. 2 and the pro-apoptotic
1,4-benzodiazepines previously reported (see, e.g., Johnson, K. M.,
et al., Chemistry and Biology. 2005, 12, 485; herein incorporated
by reference in its entirety), experiments were conducted to
determine if the 1,4-benzodiazepine-2,5-dione compounds inhibit the
F.sub.0F.sub.0-ATPase and generate O.sub.2.sup.- in the same manner
as Bz-423. Compounds listed in FIG. 2 neither blocked the
F1F0-ATPase nor inhibited by agents that specifically scavenge
superoxide. These results indicated that the
1,4-benzodiazepine-2,5-dione compounds shown in FIG. 2 have a
different molecular target and apoptotic mechanism than Bz-423. By
contrast, the m-biphenyl analog inhibited ATP hydrolysis while not
blocking synthesis, similar to previously reported
1,4-benzodiazepine (see, e.g., Hamann, L. G., et al., Bio. Med.
Chem. Lett. 2004, 14, 1031; herein incorporated by reference in its
entirety).
Example 2
[0180] This example describes the optimization of the novel
1,4-benzodiazepine-2,5-dione compounds of the present invention.
The data in FIG. 2 show that the size of the C3 is important for
the activity of the 1,4-benzodiazepine-2,5-dione compounds.
Moreover, these data suggest that it is possible to optimize
potency and selectivity based on the biphenyl or 2-napthylene C3
side chains. A range of substituted biphenyls can be prepared
readily by Suzuki couplings of aryl halides with commercially
available boronic acids (see, e.g., Suzuki, A. Acc. Chem. Res.
1982, 15, 178; herein incorporated by reference in its entirety).
Therefore, the relationship between the stereoelectronics of the C3
side chain and cytotoxicity of the 1,4-benzodiazepine-2,5-dione
compounds, was further evaluated by synthesizing substituted
analogs of 10 (FIG. 3).
[0181] In the first group of derivatives a methyl group or chlorine
atom was substituted at each of the 2', 3', or 4'-positions.
Analysis of these compounds revealed that substitution had little
effect of killing T cells but improved the potency against B cells.
Since substitution at either the 3' or 4' position led to the
greatest improvement in potency, substitutions at those positions
was further explored. The addition of electron rich substituents to
the meta and para positions (26, 27) increased potency, whereas the
carboxylic acid 28 had the reverse effect. In addition, electron
rich, heterocyclic aromatic rings (30-33) provided selectively
potent compounds, namely (30, 31). Collectively, this data indicate
that electron rich aromatic heterocycles at R.sub.1 of FIG. 4
provide optimal activity and selectivity, although the present
invention is not limited to such compounds.
[0182] FIG. 5 presents additional selectivity data for additional
1,4-benzodiazepine-2,5-dione compounds of the present invention.
Ramos EC50 refers to the concentration of drug required to 50% of
Ramos B cells and Jurkat EC50 refers to the concentration of drug
required to 50% of Jurkat T cells. Selectivity was calculated by
dividing the B cell EC50 data by the that for the T cells. All
measurements were conducted as described previously (see, e.g., T.
Francis, et al., Bioorg. Med. Chem. Lett. 2006 16, 2423-2427;
herein incorporated by reference in its entirety).
Example 3
[0183] This example demonstrates that 1,4-benzodiazepine-2,5-dione
compounds of the present invention do not inhibit ATP synthesis.
The following four compounds were exposed to cells, and ATP
synthesis measured:
##STR00036## ##STR00037##
As shown in FIG. 6, the following compounds,
##STR00038##
failed to inhibit ATP synthesis, while
##STR00039##
did inhibit ATP synthesis.
[0184] All publications and patents mentioned in the above
specification are herein incorporated by reference. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention that are obvious to those skilled in the relevant fields
are intended to be within the scope of the following claims.
* * * * *