Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS RELATING TO NOVEL
BENZODIAZEPINE COMPOUNDS AND TARGETS THEREOF
This application is a Continuation in Part of U.S. Patent Application Serial
No.:
09/767,283, filed January 22, 2001, which is a continuation of U.S. Patent
Application
Serial No.: 09/700,101, filed November 8, 2000, which is the National entry of
PCTUS00/11599 filed April 27, 2000, which claims priority to U.S. Provisional
Application
Serial No.: 60/131,761, filed April 30, 1999, to U.S. Provisional Application
Serial No.:
60/165,511, filed November 15, 1999, and to U.S. Provisional Application
Serial No.:
60/191,855, filed March 24, 2000. This application also claims priority to
U.S. Provisional
Application Serial No.: 60/312,560, filed August 15, 2001, to U.S. Provisional
Application
Serial No.: 60/313,689, filed August 20, 2001, and to U.S. Provisional
Application Express
Mail No.: EV092300423, filed July 18, 2002. Each aforementioned application is
specifically incorporated herein by reference in it entirety.
This invention was supported in part with NIH grants GM46831 and AI47450. The
United States government may have rights in this invention.
FIELD OF THE INVENTION
The present invention relates to novel chemical compounds, methods for their
discovery, and their therapeutic use. In particular, the present invention
provides
benzodiazepine derivatives and methods of using benzodiazepine derivatives 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
life.
BACKGROUND OF THE INVENTION
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,
l~nown as
apoptosis. Apoptosis is triggered by a variety of extracellular and
intracellular signals that
engage a common, genetically programmed death mechanism.
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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.
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.
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
Burlcitt'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.
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
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immune system in individuals infected with the human immunodeficiency virus
(HIV) is
thought to progress through infected CD4+ T cells (about 1 in 100,000)
instructing
uninfected sister cells to undergo apoptosis.
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.
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 tmdergo apoptosis occur in an
orderly procession.
However, as shown above, flawed regulation of apoptosis can cause serious
deleterious
effects in the organism.
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 aII forms of cancer aild hyperproliferative
autoimmune
disorders like lupus erythematosus and rheumatoid arthritis.
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
iutiate an apoptotic cascade leading to the death of the damaged cell.
However, existing cytotoxic chemotherapeutic agents have serious drawbaclcs.
For
example, many l~nown 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 ofresistance genes (e.g.,
bcl-2 family or
mufti-drug resistance (MDR) proteins) that render further dosing either less
effective or
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useless. Some cytotoxic agents induce mutations intop53 and related proteins.
Based on
v
these considerations, ideal cytotoxic drugs should only kill diseased cells
and not be
susceptible to chemo-resistance.
One strategy to selectively lcill diseased cells is to develop drugs that
selectively
recogiuze molecules expressed in diseased cells. Thus, effective cytotoxic
chemotherapeutic agents, would recognize disease indicative molecules and
induce (e.g.,
either directly or indirectly) the death of the diseased cell. Although
markers on some types
of cancer cells have been identified and targeted with therapeutic antibodies
and small
molecules, unique traits for diagnostic and therapeutic exploitation are not
lmown for most
cancers. Moreover, for diseases life lupus, specific molecular targets for
drug development
have not been identified.
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
autoimrnune
disorders, chronic inflammatory conditions, and cancers).
SUMMARY OF THE INVNETION
The present invention relates to novel chemical. compounds, methods for their
discovery, and their therapeutic use. In particular, the present invention
provides
benzodiazepine derivatives and methods of using benzodiazepine derivatives 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.
For example, the present invention provides methods for regulating cell death
comprising the step of providing: target cells having mitochondria; an agent
that binds to
oligomycin sensitivity confernng protein; and exposing the cells to the agent
under
conditions such that the agent binds to the oligomycin sensitivity conferring
protein so as to
increase superoxide levels or alter ATP levels in the cells. In some of
embodiments, the
target cells are in vitro cells. In other embodiments, the target cells are in
vivo cells. In still
further embodiments, the target cells are ex vivo cells.
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The present invention contemplates a number of target cells are suitable for
with the
methods and compositions of the present invention, for example, target cells
include, but are
not limited to, cancer cells, B cells, T cells, and granulocytes. Preferred
agents for use
in the methods of the present invention are the~benzodiazepine and benzodione
derivatives
disclosed herein. The present invention is not intended to be limited,
however, to the
benzodiazepine and benzodione derivatives disclosed herein. Indeed, in some
additional
embodiments, any agent that binds to the oligomycin~sensitivity conferring
protein (OSCP)
portion of the mitochondria) ATP synthase complex and that is useful for
treating any one
or more of a number of conditions including, but not limited to,
hyperproliferative,
autoirnlmne, inflammatory disease, graft-versus-host disease, transplant
rejection, cancer,
and the life, is used in the methods of the present invention. The present
invention is not
limited to agents that bind the OSCP portion of mitochondria) ATP synthase. As
described
herein, a number of non-limiting mechanisms and therapeutic agents useful in
the methods
of the present invention are provided.
In particularly preferred embodiments, the methods of the present invention
provide
an effective amount (e.g., therapeutically useful) ,of BZ-432 to patient. The
slcilled artisan
will appreciate the numerous formulations thatynay'be employed (e.g., tablets,
powders,
salves, creams, oral suspensions, injectable formulations, etc.). The slcilled
artisan is also
familiar with dosing considerations and with the regulatory and administrative
steps that
must be tal~en when administering pharmaceutical compositions to a patient. In
preferred
embodiments, contemplated patients include but are not limited to mammals. In
particularly preferred embodiments the methods and compositions of the present
invention
are directed to, and optimized for, administration to. humans.
The present invention fiuther provides compositions (e.g., benzodiazepine or a
benzodione derivative) and methods (e.g., administration) directed to
increasing cell death
in target cells.
The present invention also provides compositions (e.g., benzodiazepine or a
benzodione derivative) and methods (e.g., administration) for inhibiting
proliferation in
cells comprising the steps of providing: proliferating target cells having
mitochondria; an
agent that binds to mitochondria) ATP synthase complex; and exposing the cells
to the
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agent under conditions such that the agent binds to the mitochondria) ATP
synthase
complex so as to increase superoxide levels or alter ATP levels in the cells.
In some embodiments of the present invention, the contemplated agents of the
present invention binds to the oligomycin sensitivity c'onfernng protein such
that superoxide
levels in the treated cells/tissues increase. In some of these embodiments,
the target cells
are proliferating cells. In particularly preferred embodiments, therapeutic
levels of Bz-423
are administered to patient.
In still further embodiments, the present invention provides pharmaceutical
compositions comprising: a sufficient dose of an;agent that binds to
oligomycin sensitivity
conferring protein so as to increase superoxide or alter ATP levels in cells
of a subject
exposed to the agent; and instructions for using the agent for treating a
condition (e.g.,
cancer, proliferative diseases, autoimmune diseases'~graft-versus-host
disease, transplant
rej ection, and the life).
The present invention provides, in still further 'embodiment, pharmaceutical
compositions comprising: a sufficient dose of an agent (e.g., benzodiazepine
or a
benzodione derivative) that binds to mitochondria) ATP synthase complex so as
to increase
superoxide or alter ATP levels in cells of a subject exposed to the agent; and
instructions for
using the agent for treating an autoimmune disease, a proliferative disease,
or cancer. In
some of these embodiments, a preferred agent comprises Bz-432.
Additional embodiments of the present invention provide methods for
identifying
agents useful for treating proliferative disease, autoimmune diseases, or
cancer comprising:
providing: mitochondria) ATP synthase complex; benzodiazepine or a benzodione
derivative; a candidate agent; exposing the mitochondria) ATP synthase complex
to the
benzodiazepine or a benzodione derivative and the candidate agent; and
comparing the
binding of the benzodiazepine or a benzodione derivative and the candidate
agent to the
;.
mitochondria) ATP synthase complex. The methods of the present invention are
limited by
the measure being observed. For instance, in some embodiments, the comparing
comprises
observing cell death, growth rate, or cell number in cells containing the
mitochondria) ATP
synthase complex. In other embodiments, the comparing comprises measuring
superoxide
levels in cells containing the mitochondria) ATP synthase complex. In still
further
embodiments, the comparing comprises measuring binding affinities of the
benzodiazepine
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or a benzodione derivative and the candidate agent to the mitochondrial ATP
synthase
complex. Alternatively, other embodiments contemplate a comparing step
comprising
detecting the binding of the candidate agent to oligomycin sensitivity
conferring protein.
Also provided are methods for identifying pharmaceutical agents, comprising:
providing an agent that binds to mitochondrial ATP synthase complex so as to
generate
superoxide free radicals, alter ATP levels, initiate cell death, or alter
cellular proliferation;
chemically modifying the agent to generate a library''of candidate
pharmaceutical agents;
aald selecting one or more individual members of the library of candidate
agents based on
their increased ability to generate superoxide free radicals, initiate cell
death, or alter
cellular proliferation compared to the agent. Additionally, some embodiments
further
comprise the step of testing the one or more individual members of the library
for toxicity in
a tissue or animal.
The therapeutic methods disclosed herein may; additionally comprise the step
of
submitting the one or more individual members of the library to a regulatory
agency for
approval as a commercial product (e.g., The U.S. Food and Drug
Administration).
Yet other embodiments of the present invention provide methods for screening
for
agents that selectively induce cell death or inhibit the; growth or
proliferation of activated
cells, comprising: providing: a first cell sample comprising at least one
unactivated cell; a
second cell sample comprising at least one unactivated cell; a third cell
sample comprising
at least one unactivated cell; an effective amount of an. activating agent;
and an effective
amount of a candidate agent; an effective ratio and amount of the activating
agent and the
candidate agent; and contacting the first cell sample with the effective
amount of the
activating agent; contacting the second cell sample with the effective amount
of the
candidate agent; contacting the third cell sample with the effective ratio and
amount of the
activating agent and the candidate agent; comparing the level of cell death or
cell number in
the third cell sample to the level of cell death or cell number in the first
cell sample and the
second cell sample; and comparing the amount of cell, death or growth
inhibition in the third
cell sample to the level of cell death or growth inhibition in the first cell
sample and the
second cell sample. Optionally some of these embodiments fiuther comprise the
step of
selecting a candidate agent contacted to the third sample if the level of cell
death or growth
inlubition in the third cell sample is greater than the cell death in the
first cell sample and
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the second cell sample. Suitable samples for use in these embodiments comprise
B cells, T
cells, granulocytes, cancer cells, and the like.
Activating agents suitable for use in the methods of the present invention
include,
but are not limited to, T cell ligand, BAFF ligand, TNF, Fas ligand (FasL),
Toll ligand,
APRIL, CD40 ligand, cytol~ines, chemokines, hormones, steroids, a B cell
ligand, gamma
irradiation, W irradiation, an agent or condition that enhances cell stress,
and antibodies
that specifically recognize and bind cell surface receptors (e.g., anti-CD4,
anti-CDB, anti-
CD20, anti-BAFF, anti-TNF, anti-CD40, anti-CD3, anti-CD28, anti-B220, anti-
Toll
receptor, anti-APRIL receptor, anti-B cell receptor, anti-T cell receptor, and
the like).
Additional embodiments of the present invention also provide methods for
inhibiting
induced cell death in am activated target cell (e.g., in vitro or i~ vivo
activated target cells)
by contacting the activated target cell with an effective amount of an agent
(e.g., tacrolimus
or the like) that inhibits the formation of superoxide in said activated
target cell prior to
mitochondrial permeability transition.
Some other embodiments of the present invention provide methods for screening
for
agents that selectively induce cell death or inhibit the growth or
proliferation of activated
cells, comprising: providing: a first cell sample comprising at least one
unactivated cell;
second cell sample comprising at least one unactivated cell; a candidate
agent; and
contacting the first cell sample with the candidate agent; and comparing the
intracellular
concentration of superoxide in first and second cells. Some of these methods
further
comprise the additional step of selecting the candidate agent contacted to the
first cell
sample if the intracellular concentration of superoxide; is greater in the
first cell sample than
in the second cell sample. In still further embodiments, the present invention
further
provides in these methods the additional step of providing: an agent lrnown to
increase
superoxide levels in treated unactivated cells; a third cell sample comprising
at least one
unactivated cell; a fourth cell sample comprising at least one miactivated
cell; treating the
third cell sample with the agent known to increase superoxide levels; treating
the fourth cell
sample with said with said agent known to increase superoxide levels and said
selected
candidate agent; and identifying whether or not the candidate agent
synergistically increases
superoxide levels with the agent known to increase superoxide levels by
determining
whether superoxide levels are higher in the treated fourth cell sample as
compared to the
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treated third cell sample. In yet another embodiment, the present invention
provides a
pharmaceutical cocktail comprising the agent known to, increase superoxide
levels and the
identified candidate agent.
DESCRIPTION OF THE FIGURES
Figure 1 shows cell death of (NZB x NZW)F, ("NZB/W") splenocytes measured by
permeability to propidium iodide ("PI") after in vivo treatment (24 h)
comparing Bz-423 (10
p,M) to other benzodiazepine receptor ligands at the same concentration.
Control = media,
Bz = Bz-423, Naph =1-naphthol, Cz = clonazepam, Dz = diazepam, Cl-Dz = 4'-
chlorodiazepam, PK = PK 11195.
Figures 2A through 2H show that Bz-423 reduces autoimmune nephritis and
splenic
hypeiplasia in NZB/W mice. Renal histopathology (400X) in control (Figures 2A
and 2C)
and treated animals (Figures 2B an 2D) after 12 wks :o.f dosing identified by
hemotoxilin
and eosin ("H&E") (Figures 2A and 2B) or immunofluoresent staining of IgG
deposition
(Figures 2C and 2D). Spleen sections from a control (Figure 2E) and treated
mouse (Figure
2F) stained with anti-B220. Germinal centers ("GC") from control (Figure 2G)
and treated
(Figure 2H) spleen sections (frozen) stained for simultaneous detection of
B220
(background) and fragmented DNA (white spots).
Figures 3A and 3B show the effect of activation and co-stimulation on Bz-423-
induced apoptosis. Data are expressed as percent of cells PI-positive at 24 h.
Figure 3A
shows dose-response of Ramos cells to Bz-423 with (soluble anti-IgM) or
without
stimulation. Figure 3B shows the specific killing, of primary B cells in the
presence of
indicated stimuli and 4 ~M Bz-423. White bays = anti-IgM, black bas°s =
Bz-423, and gray
bass = anti-IgM plus Bz-423.
Figures 4A through 4C show that Bz-423 increases superoxide which functions as
an
apoptotic signal in B cell receptor ("BCR")-activated cells. Ramos cells were
treated with
anti-IgM alone (control, --) or with Bz-423 at the,indicated concentrations (-
). Inserts show
effect of vitamin E. (Figure 4A) Superoxide levels 1 hr after treatment with
Bz-423.
(Figure 4B) PI staining after 24 h demonstrates hypodiploid DNA content
consistent with
apoptosis. (Figure 4C) Interference contrast microscopy (400X) demonstrates
similar
apoptotic appearance of BCR-activated and un-activated cells treated with Bz-
423.
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Figures SA through SD show properties of Bz-423. (Figure SA) Structure of Bz-
423
and inactive congeners. (Figure SB) Effect of Bz-423, 4'-chlorodiazepam (4-
CIDz),
PKl 1195, SNAP, and OOH on Ramos cell viability at 24 h determined by
permeability to
PI. (Figure SC) Morphology of cells treated for 24 h with vehicle, Bz-423 (10
~M), or Bz-
423 (10 ~.M) plus z-VAD (100 ~.M) determined by interference contrast
microscopy
(400X). (Figure SD) After treatment as in (Figure SC), cells were analyzed by
flow
cytometry to determine DNA content. Panels A-D are representative of >5
separate
determinations.
Figures 6A and 6B show mediators of Bz-423-induced apoptosis. (Figure 6A)
Ramos cells were incubated with vehicle or Bz-423 for 1 h. Fluorescence
intensity
increased above control (shaded histogram) with 5 and 10 ~M Bz-423 (green and
red
histograms, respectively). (Figure 6B) Time-course~'of changes detected in
Ramos cells
upon treatment with Bz-423 (10 ~,M). Data are presented as the percentage of
cells with
indicated response relative to time-matched vehicle controls. No changes in
cells treated
with vehicle during this time frame were observed. Inset- Cytochrome c
release. Lane 1-
purified cytochrome c (20 ng); Lane 2 - cytosolic fraction isolated 5 h after
incubation with
vehicle; Lanes 3 and 4 - cytosolic fractions isolated 1 and 5 h after
incubation with Bz-423,
respectively. Panels A and B represent data from >5 'separate determinations.
Figures 7A through 7E show Bz-423 generates ROS in isolated mitochondria. Rat
liver mitochondria were incubated with DCFH-DA and the desired agent, and the
fluorescence intensity was monitored. These measurement were conducted in
duplicate and
repeated with four mitochondrial preparations. (Figure 7A) Data obtained in
state 3
respiration. The slopes of each curve after the induction phase (1200-1500 s)
are 2.1, 1.5,
1.1 (x10-3 ~ 15%) for, antimycin A (0.5 ~,M), Bz-423 (10 ~,M), and vehicle,
respectively. A
greater slope corresponds to a higher level of ROS production. (Figure 7B)
Swelling of
mitochondria in state 3 buffer. Only CaZ+ (400 ~.M) triggers the MPT pore,
which results in
swelling. (Figure 7C) Data obtained in state 3 respiration using the S15
fraction. (Figure
7D) Data obtained with mitochondria in state 4 respiration. (Figure 7E)
Representative
micrographs (630X) of mitochondria stained with DHE. (red) or with DIOC3(6)
(green) in
states 3 and 4.
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Figures 8A and 8B show regulating ROS preserves mitochondria) function and
blocks Bz-423-induced killing. (Figure 8A) After pre-incubating Ramos cells
for 30 min
with either FK506 (1 ~M, blue), vitamin E (100 ~,M; red), MnTBAP (100 ~,M,
green), or no
inhibitor (black), Bz-423 (10 ~,M) was added to the cultures. White bars
indicate control
samples treated with vehicle alone. Using flow cytometry, DHE fluorescence (Oz
) was
measured at 1 h, caspase activation at 5 h, ~'Ifn, (DiOCb(3)) at 5 h, and PI
permeability and
hypodiploid DNA at 24 h. (Figure 8B) Effect on, cell-viability of adding FK506
at times
relative to the addition of Bz-423. The results in panels A and B represent >5
separate
determinations.
Figure 9 depicts disease progression analysis for MRL-lpr mice treated
according to
the methods described herein (solid line) as compared to controls (dotted
line). The
percentage of disease-free animals (y-axis) is plotted over time (x-axis).
Figures l0A-lOC depict footpad swelling in:MRL-lpr mice treated according to
the
methods described herein (Figure )0A) as compared to controls (Figure )0B).
Figure l OC
is a graphical analysis.
Figure 11 is a bar graph depicting the effrcacy of using benzodiazepine to
kill D2
neuroblastoma cells i~ vita~.
Figl~re 12 is a graph that shows that ovarian cancer cells are killed by
application of
benzodiazepine ira vit~~o.
,. .
DEFINITIONS
To facilitate an understanding of the present invention, a number of terms and
phrases are defined below.
As used herein, the teen "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 1 and
4 positions, as shown in the general structure below.,
N
g 1 2
g 3
7 4
6 S N
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The benzodiazepine can be substituted with one lceto group (typically at the 2-
position), or with two keto groups, one each at the 2- and 5- positions. When
the
benzodiazepine has two keto groups, one each at the 2- and 5- positions, it is
referred to as
benzodiazepine-2,5-dione. Most generally, the benzodiazepine is further
substituted either
on the six-membered phenyl ring or on the seven-membered heterocyclic ring or
on both
rings by a variety of substituents. These substituents are described more
fully herein.
As used herein, the term "substituted aliphatic" refers to an all~ane
possessing less
than 10 carbons where at least one of the aliphatic hydrogen atoms has been
replaced by a
halogen, an amino, a hydroxy, a vitro, a thio, 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, etc.). Examples of such include, but are not
limited to, 1
chloroethyl and the like.
As used herein, the term "substituted aryl" refers to an aromatic ring or
fused
aromatic ring system consisting of no more than three, fused rings at least
one of which is
aromatic, and where at least one of the hydrogen atoms on a ring carbon has
been replaced
by a halogen, an amino, a hydroxy, a vitro, a tluo, 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, hydroxyphenyl and the like.
As used herein, the term "cycloaliphatic" refers to a cycloalkane possessing
less than
8 carbons or a fused ring system consisting of no more than three fused
cycloaliphatic rings.
Examples of such include, but are not limited to, decalin and the lilce.
As used herein, the term "substituted cycloaliphatic" refers to a cycloallcane
possessing less than 8 carbons or a fused ring system consisting of no more
than three fused
rings, and where at least one of the aliphatic hydrogen atoms has been
replaced by a
halogen, a vitro, a thio, an amino, a hydroxy, a lcetone, 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 and the like.
As used herein, the term "heterocyclic" refers to a cycloall~ane and/or an
aryl ring
system, possessing less than 8 carbons, or a fused ring system consisting of
no more than
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three fused rings, where at least one of the ring carbon atoms is replaced by
oxygen,
nitrogen or sulfur. Examples of such include, but are not limited to,
morpholino and the
like.
As used herein, the term "substituted heterocyclic" refers to a cycloalkane
and/or an
aryl ring system, possessing less than 8 carbons, or a fused ring system
consisting of no
more than three fused rings, where at least one of the ring carbon 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, a lcetone, 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. , ,
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.
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.
i
As used herein, the term "lower-alkyl-substituted-halogen" refers to any alkyl
chain
containng 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. , ,
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 lilce.
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 or on the aromatic ring. Non-limiting examples of 1,4-
benzodiazepine derivatives of the present invention may include N-acetyl, N-
methyl, N-
hydroxy groups at any of the available nitrogens in the compound. Additional
derivatives
may include those having a trifluoromethyl group on the phenyl ring.
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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 benzodiazepine compound(s), and optionally one or more other
agents)
for a condition characterized by the dysregulation of apoptotic processes.
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 azlalysis, and the like.
As used herein, the terms "anticancer agent," or "conventional anticancer
agent"
refer to any chemotherapeutic compounds, radiation tfierapies, or surgical
interventions,
used in the treatment of cancer.
As used herein the term, "iya vitro" refers to an artificial environment and
to
processes or reactions that occur within an artificial environment. IyZ vit~~o
environments can
consist of, 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.
As used herein, the term "host cell" refers to any eulcaryotic or prokaryotic
cell
(e.g.; marrunalian cells, avian cells, amphibian cells, plant cells, fish
cells, and insect cells),
whether located in vitro or ifz vivo.
As used herein, the term "cell culture" refersto' 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 ire vitro, including oocytes and embryos.
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, and granulocytes. Granulocyctes
include
eosinopluls and macrophages. In some embodiments, target cells are
continuously cultured
cells or uncultered cells obtained from patient biopsies.
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Cancer cells include tumor cells, neoplastic cells, malignmt 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.
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.
As used herein, the term "un-activated target cell" refers to a cell that is
either in the Go
phase or one in which a stimulus has not been applied.
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 Go phase. Activated lymphoid
cells may
proliferate, undergo activation induced cell death, or produce one or more of
cytotoxins,
cytol~ines, and other related membrane-associated proteins characteristic of
the cell type
(e.g., CD8+ or CD4''~). 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.
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 Go phase.
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 Ca2+, superoxide, or hydroxyl radical levels; the
activity of
enzymes lilce l~inases or phosphatases; or the energy state of the cell. For
cancer cells,
activating agents also include transforming oncogenes.
In one aspect, the activating agent is any agent that binds to a cell surface
activation
receptor. These can be selected from the group consisting of a T cell receptor
ligand, a B cell
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activatW g factor ("BAFF"), a TNF, a Fas ligand (FasL), a CD40 ligand, a
proliferation
iizducing ligand ("APRIL"), a cytokine, a chemokine, a hormone, an amino acid
(e.g.,
glutamate), a steroid, a B cell receptor ligand, gamma irradiation, UV
irradiation, an agent or
condition that enhances cell stress, or an antibody that specifically
recognizes and binds a cell
surface activation receptor (e.g., anti-CD4, anti-CDB, anti-CD20, anti-TACI,
anti-BCMA,
anti-TNF receptor, anti-CD40, anti-CD3, anti-CD28, anti-B220, anti-CD38, and-
CD19, and
anti-CD21). BCMA is B cell maturation antigen receptor and TACI is
transmembrane
activator and CAML interactor. (J.A. Gross et al., Nature, 404:995-999 [2000];
X. Laabi et
al., EMBO J., 11:3897-3904 [1992]). Antibodies. include monoclonal or
polyclonal or a
mixture thereof.
Examples of a T cell ligand include, but axe not limited to, a peptide that
binds to an
MHC molecule, a peptide MHC complex, or an mtibody that recognizes components
of the T
cell receptor.
Examples of a B cell ligand include, but are not limited to, a molecule or
antibody that
binds to or recognizes components of the B cell receptor.
Examples of reagents that bind to a cell surface activation receptor include,
but are not
limited to, the natural ligands of these receptors or antibodies raised
against them (e.g., anti-
CD20). RITZJXIN (Genentech, Inc., San Francisco, CA) is a commercially
available anti-CD
chimeric monoclonal antibody.
20 Examples of agents or conditions that enhance cell stress include heat,
radiation,
oxidative stress, or growth factor withdrawal and the life. Examples of growth
factors
include, but are not limited to serum, IL-2, platelet derived growth factor
("PDGF"), and the
life.
As used herein, the term "effective amount" refers to the amount of a compound
(e.g., benzodiazepine) 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.
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
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erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia
gravis, Sjogren's
syndrome, etc.), chronic inflammatory conditions (e.g., 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.
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 dysregul'ation can occur even
after the
disappearance of symptoms of viral infection.
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 lilce. A neoplasm is said to be benign if it does
not undergo,
7.
invasion or metastasis and malignaxzt 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 epitheliuril.
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 my 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 rheumatoid arthritis, Sjogren's syndrome, graft
versus host
disease, myasthenia gravis, systemic lupus erythematosus ("SLE"), and the
like.
As used herein, the term "chronic inflammatory,condition" refers to a
condition
wherein the organism's immune cells are activated. Such 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. Ea~amples of chronic
inflammatory
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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.
As used herein, the term "co-administration" refers to the administration of
at least
two agents) (e.g., benzodiazepines) 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 shill
in the art
understand that the formulations and/or routes of administration of the
various
agents/therapies used may va3.y. 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-achninistration 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) ageut(s).
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.
As used herein, the teen "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 ih vivo, ih vivo or ex vivo.
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]).
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 lcnown to those of
skill in the art,
"salts" of the compounds of the present invention may be derived from
inorganic or organic
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acids and bases. Examples of acids include, but are not limited to,
hydrochloric,
hydrobromic, sulfuric, nitric, perchloric, fumaric, malefic, phosphoric,
glycolic, lactic,
salicylic, succinic, toluene-p-sulfonic, tartaric, acetic;ecitric,
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.
Examples of bases include, but are not limited to, alkali metals (e.g.,
sodium)
hydroxides, allcaline earth metals (e.g., magnesium), hydroxides, ammonia, and
compounds
of formula NW4+, wherein W is C,_4 alkyl, and the like,
Examples of salts include, but axe 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*, NH4+, and
NW4+ (wherein
W is a Cl_d alkyl group), and the like.
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.
As used herein, the terms "solid phase supports" or "solid supports," are used
in their
broadest sense to refer to a number of supports that are available and known
to those of
ordinary skill in the art. Solid phase supports include, but are not limited
to, silica gels,
resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina
gels, and the
like. As used herein, "solid supports" also include synthetic antigen-
presenting matrices,
cells, liposomes, and the like. A suitable solid phase support may be selected
on the basis of
desired end use and suitability for various protocols. For example, for
peptide synthesis,
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solid phase supports may refer to resins such as polystyrene (e.g., PAM-resin
obtained from
Bachem, Inc., Peninsula Laboratories, etc.), POI,YHIPE) resin (obtained from
Aminotech,
Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene
resin grafted
with polyethylene glycol (TENTAGEL, Rapp Polymere, Tubingen, Germany) or
polydimethylacrylamide resin (obtained from Milligen/Biosearch, California).
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.
The terms "bacteria" and "bacterium" refer to all prolearyotic 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 teen are prokaryotic organisms which are gram
negative or gram
positive. "Gram negative" and "gram positive" refer t'o 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 [19820. "Gram
positive
bacteria" are bacteria which retain the primary dye used in the Gram stain,
causing the
stained cells to appear darlc 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.
As used herein, the term "microorgasusm" refers to any species or type of
microorganism, including but not limited to, bacteria,,4archaea, fungi,
protozoans,
mycoplasma, and parasitic organisms. The present invention contemplates that a
number of
microorganisms encompassed therein will also be pathogenic to a subject.
As used herein, the term "fungi" is used in reference to eukaryotic organisms
such
as the molds and yeasts, including dimorphic fungi.
As used herein, the term "virus" refers to minute infectious .gents, 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
CA 02457405 2004-02-11
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containing membrane. The term 'virus" encompasses all types of viruses,
including animal,
plant, phage, and other viruses.
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 Southeni 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 lilce. 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. ,
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.
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 lceyhole
limpet hemocyanin
[I~I,H]). 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.
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.,
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Harlow azid Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press,
Cold Spring Harbor, NY). These include, but are not limited to, the hybridoma
technique
originally developed by Kohler and Milstein (Kohler arid Milstein, Nature,
256:495-497
[1975]), as well as the trioma technique, the human B-cell hybridoma technique
(See e.g.,
Kozbor et al., Tmmunol. 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]).
According to the invention, techniques described for the production of single
chain
antibodies (U.S. 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 lmown 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.
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. . .
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,~irr situ immunoassays
(using
colloidal gold, enzyme or radioisotope labels, for example), Western Blots,
precipitation
reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination assays,
ete.), complement fixation assays, immunofluorescence assays, protein A
assays, and
immunoelectrophoresis assays, etc.) etc.
As used herein, the term "immunoglobulin" or "antibody" refer to proteins that
bind
a specific antigen. Tmmunoglobulins include, but are not limited to,
polyclonal,
monoclonal, chimeric, and humanized antibodies, Fab fragments, F(ab')Z
fragments, and
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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.
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.
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
a
to the antibody.
r
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).
As used herein, the term "modulate" refers to the activity of a compound
(e.g.,
benzodiazepine compound) 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.
As used herein, the term "competes for binding" is used in reference to a
first
molecule (e.g., a first benzodiazepine derivative) with an activity that binds
to the same
substrate (e.g., the oligomycin sensitivity confernng protein in mitochondria)
ATP synthase)
as does a second molecule (e.g., a second benzodiazepine derivative or other
molecule that
binds to the oligomycin sensitivity conferring protein in mitochondria) ATP
synthase, etc.).
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The efficiency (e.g., kinetics or thermodynamics) of binding by the first
molecule may be
the same as, or greater than, or less than, the efficiency of the substrate
binding to the
second molecule. For example, the equilibrium binding constant (KD) for
binding to the
substrate may be different for the two molecules.
As used herein, the term "instructions for administering said
benzodiazepine compound to a subject," and grammatical equivalents thereof,
includes
instructions for using the compositions contained in a kit for the treatment
of conditions
characterized by the dysregulation of apoptotic processes in a cell or tissue.
The term also
specifically refers to instructions for using the compositions contained in
the kit to treat
autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid
arthritis, graft-
versus-host disease, myasthenia gravis, Sjogren's.syndrome, etc.), chronic
inflammatory
conditions (e.g., psoriasis, asthma and Crohn's disease), hyperproliferative
disorders (e.g.,
tumors, B cell lymphomas, T cell lymphomas, etc.), viral infections (e.g.,
herpes virus,
papilloma virus, HIV), and other conditions such as osteoarthritis and
atherosclerosis, and
the like.
In some embodiments, the instructions further~comprise a statement of the
recommended or usual dosages of the compositions contained within the kit
pursuant to 21
CFR ~201 et seq. Additional information concerning labeling and instruction
requirements
applicable to the methods and compositions of the present are available at the
Internet web
page of the U.S. Food and Drug Administration (FDA).
In some embodiments, the instructions further comprise the statement of
intended
use required by the FDA in labeling in vitro diagnostic products. The FDA
classifies ih
vitro diagnostics as medical devices and required.that they be approved
through the 510(k)
procedure. Information required in an application under 510(k) includes: 1)
The isz vitro
diagnostic product name, including the trade or proprietary name, the common
or usual
name, and the classification name of the device; 2) The intended use of the
product; 3) The
establishment registration number, if applicable, of the owner or operator
submitting the
510(1c) submission; the class in which the in vitro diagnostic product was
placed under
section 513 of the FD&C Act, if lmown, its appropriate panel, or, if the owner
or operator
determines that the device has not been classified under such section, a
statement of that
24
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WO 03/015703 PCT/US02/26171
determination and the basis for the determination that 'the ih vitro
diagnostic product is not
so classified; 4) Proposed labels, labeling and advertisements sufficient to
describe the ih
vitro diagnostic product, its intended use, and directions for use, including
photographs or
engineering drawings, where applicable; 5) A statement indicating that the
device is similar
to and/or different from other in vitro diagnostic products of comparable type
in commercial
distribution in the U.S., accompanied by data to support the statement; 6) A
510(k)
smnmary of the safety and effectiveness data upon which the substantial
equivalence
determination is based; or a statement that the 510(k) safety and
effectiveness information
supporting the FDA fording of substantial equivalence will be made available
to any person
within 30 days of a written request; 7) A statement that the submitter
believes, to the best of
their lmowledge, that all data and information submitted in the premarket
notification are
truthful and accurate and that no material fact has been omitted; and ~) Any
additional
information regarding the ih. vitro diagnostic product requested that is
necessary for the
FDA to make a substantial equivalency determination. Additional information is
available
at the Internet web page of the U.S. FDA.
The term "test compound" refers to any chemical entity, pharmaceutical, drug,
and
the like, that can be used to treat or prevent a disease, illiless, sickness,
or disorder of bodily
function, or otherwise alter the physiological or cellular status of a sample
(e.g., the level of
dysregulatiomof apoptosis in a cell or tissue). Test,compounds comprise both
lcnown 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.
As used herein, the term "third party" refers to any entity engaged in
selling,
warehousing, distributing, or offering for sale a test compound contemplated
for
administered with a benzodiazepine compound. for treating conditions
characterized by the
dysregulation of apoptotic processes.
CA 02457405 2004-02-11
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GENERAL DESCRIPTION OF THE INVENTION
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.
4,076823, 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. 5,324,726 and U.S.
5,59'7,915, each
incorporated by reference in its entirety, report that certain benzodiazepine
compounds are
antagonists of cholecystol~inin and gastrin and thus might be useful to treat
certain
gastrointestinal disorders.
Other benzodiazepine compounds have. been studied as inlubitors 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.
5,861,380
incorporated herein by reference in its entirety). U.S. 5,041,438,
incorporated herein by
reference in its entirety, reports that certain benzodiazepine compounds are
useful as anti-
retroviral agents.
Despite the attention benzodiazepine compounds have drawn, it will become
apparent from the description below, that the present.invention provides novel
benzodiazepine compounds and methods of using these compounds that are useful
in
treating a variety of disease characterized by the dysregulation of processes
associated with
cell death.
Experimental models have established a cause=effect relationship between
derangement in the mechanism regulating apoptosis or necrosis and the
pathenogenicity of
various neoplastic, autoimmune, and viral diseases. (C.B. Thompson, Science,
267:1456-
1462 [1995]). A well-defined example is the effect of aberrant, high-level
expression of
bcl-2 on lymphoma development. The bcl-2 oncogene was originally identified as
the
genetic element located at the t(14:18) chromosomal~translocation brealepoint
present in
many B-cell follicular lymphomas. (S.J. Korsmeyer, Blood, 359:554-556 [1992]).
Since
that discovery, it has been reported that the bcl-2 gene product inhibits
apoptosis induced by
a variety of stimuli and that its oncogenic potential stems from its ability
to derail apoptosis.
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(C.L. Sentman et al., Cell, 67:878-888 [1994]; and T.J. McDonnell S.J. and
Korsmeyer,
Nature, 349:254-256 [1991]).
Failed or reduced apoptosis has been reported'to be associated with the
development
of human autoimmune lynphoproliferative syndrome as well as mouse models of
this
disease. MRL-lp~ or gld mice develop lymphadenopathy, splenomegaly, nephritis
and
arthritis, as well as producing large quantities of autoantibodies. (P.L.
Cohen, and R.A.
Eisenberg, Amml. Rev. Immunol., 9:243-269 [1991]).
MRL-lp~ mice carry loss of function mutations in the genes encoding FAS and
Fas
ligand, respectively. (M. Adachi et al., Proc. Natl. Acad. Sci. USA, 90:1756-
1760 [1993]);
T. Talcal~ashi et al., Cell, 76:969-976 [1994]). FAS; a ubiquitously expressed
cell surface
receptor, normally generates an apoptotic response upon binding with Fas
ligand. (N. Itoh,
et al., Cell, 66:233-243 [1991]). In mice carrying these loss of function
mutations, the
disruption of FAS signaling renders T cells resistant to.peripheral deletion
by apoptosis. (H.
Russell et al., Proc. Natl. Acad. Sci., USA 90:4409-4413). The inappropriate
survival of
these cells results in a pathologic accumulation of T and B cells evidenced by
the
neoplastic-life growth of lymphoid tissues and high,-level autoantibody
production. In
humans, autoimmune lymphoproliferative syndromes shares similarities with the
mouse
phenotype including lymphadenopathy, splenomegaly, autoantibodies and
autoimmune
manifestations. Patients with this disease lilcewise have been reported to
carry mutations in
the FAS gene. (See, S. Nagata, J. Hum. Genet., 43:2; 8 [1998]).
Benzodiazepine compounds are lalown 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.
Benzodiazepines
and related structures have pro-apoptotic and cytotoxic properties useful in
the treatment of
transformed cells grown in tissue culture. There is therapeutic potential for
this class of
..
agents against cancer and other neoplastic diseases. Two specific examples
shown are
neuroblastoma and ovarian cancer.
Neuroblastoma is the most common extracranial solid tumor found in children.
Modern treatments, which include chemotherapy, radiation therapy and surgery,
have not
sig~lificantly reduced the mortality of metastatic neuroblastoma. Novel
therapies are needed
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to improve survival of children with this disease. Some embodiments of the
present
invention provide compositions and methods that slow the growth of these
tumors.
Likewise, ovarian cancer is difficult to treat due to chemoresistance shown by
the
patient to standard chemotherapy drugs. Treatment failures are usually
attributed to the
emergence of chemotherapy resistant cells. Some embodiments of the present
invention
provide benzodiazepine compounds that lcill chemoresistant cancer cells (e.g.,
ovarian
cancer cells).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel chemical compounds, methods for their
discovery, and their therapeutic use. In particular, the present invention
provides
;~4.
benzodiazepine derivatives and methods of using benzodiazepine derivatives 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
lilce. Additionally, the present invention provides compositions and methods
to regulate the
processes of programmed cell death, autoimmunity,~ inflammation, and
hyperproliferation,
and the like, under pathological conditions.
Exemplary compositions and methods of the present invention are described in
more
detail in the following sections: I. Benzodiazepine derivative modulators of
cell death; II.
Benzodiazepine derivative modulators of cell growth and proliferation; III.
Synthesis of
exemplary benzodiazepine derivatives; IV. Pharmaceutical compositions,
formulations, and
exemplary administration routes and dosing considerations; V. Mitochondrial
ATP synthase
(mitochondrial FoF, ATPase) activity modulators; and VI. Drug screens.
The practice of the present invention employsa 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 8z C.C. Blackwell, eds.); "Gene transfer vectors for
mammalian
28
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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).
I. Benzodiazepine derivative modulators of cell death
In some embodiments of the present invention, the benzodiazepine compounds
have
the structure:
or
R2
or its enantiomer, wherein, R, is aliphatic or aryl; Rz is aliphatic, aryl, -
NH2, -HC(=O)-R5, or
a moiety that participates in hydrogen bond formation, wherein RS is aryl,
heterocyclic, -R~
NH-C(=O)-R~ or -R~ C(=O)-NH-R~, wherein R6 is an aliphatic linl~er of 1-6
carbons and R,
is aliphatic, aryl, or heterocyclic; and each of R3 and R4 is independently
hydrogen, hydroxy,
all~oxy, halo, amino, lower-allcyl-substituted-amino, acylamino, hydroxyamino,
an aliphatic
group having 1-8 caxbons and 1-20 hydrogens, aryl, or heteroaryl; or a
pharmaceutically
acceptable salt, prodrug or derivative thereof.
The cell death can be induced by necrosis, apoptosis or regulation of the FAS
pathway. The conditions associated with the dysregulation of a process of cell
death include
but are not limited to: autoimmune diseases such as systemic lupus
erythematosus,
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rheumatoid arthritis, Sjogren's syndrome, graft-versus-host-disease, and
myasthenia gravis;
chronic inflammatory conditions such as psoriasis, asthma, and Crohn's
disease;
hyperproliferative disorders or neoplasms such as a B-cell or a T-cell
lymphomas; and other
conditions such as osteoarthritis and atherosclerosis. Methods are also
provided for using
the benzodiazepine compounds to treat the conditions associated with the
dysregulation of
cell death, wherein the condition is induced by a viral infection. hi
addition, in some
aspects, methods are provided to treat a viral infection by using the
benzodiazepines of the
present invention.
Methods are also provided to co-administer one or more additional agents with
the
benzodiazepines of the present invention, wherein such additional agents may
include
antineoplastic agents, immunosuppressants, anti-inflammatory agents, antiviral
agents, or
radiation.
The cell death to be achieved by the methods and compositions of this
invention
involve the cell or cells present in a tissue that are: autoimmunogenic or
affected by an
autoimmune disorder; inflammatory or affected by inflammation;
hyperproliferative; viral-
infected; atherosclerosed or osteoarthritic.
Assay and diagnostic methods are also provided to identify agents useful to
treat a
condition associated with dysregulation of the process, of cell death in a
subj ect wherein the
ability of a potential candidate agent to induce cell death is assayed by
contacting the
dysregulated cell with a benzodiazepine compound. The assay includes
maintaining the
suitable cell or tissue preferably in a low serum.
Methods are also presented to prepare medicaments to treat a condition
associated
with dysregulation of the process of cell death in a subject, wherein the
conditions, the
affected cells or tissue and the benzodiazepine compounds axe described as
above. The
invention also provides novel 1,4-benzodiazepine compounds having the
structure:
CA 02457405 2004-02-11
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or
R~
R2
or its enantiomer, wherein, R, is aliphatic or aryl; RZ is -NHC(=O)-R5,
wherein RS is aryl,
heterocyclic, -R~ NH-C(=O)-R~ or R~ C(=O)-NH-R~, wherein R~ is an aliphatic
linker of 1-
6 carbons and R, is aliphatic, aryl, or heterocyclic; and each of R3 and R4 is
independently
hydrogen, hydroxy, allcoxy, halo, amino, lower-alkyl-substituted-amino,
acylamino,
hydroxyamino, an aliphatic group having 1-8 carbons and 1-20 hydrogens, aryl,
or
heterocyclic; or a pharmaceutically acceptable salt, prodrug or derivative
thereof.
Exemplary target diseases and methods for identifying disease targets are
presented
below.
A. Target Diseases and Conditions
The present invention provides methods of treating conditions that are, in
some
embodiments, related in that they arise as the result of dysregulation of the
normal processes
of cell death (e.g., necrosis and/or apoptosis) in the cells or tissues of a
subject. For the
purpose of illustration only, such conditions include, but are not limited to,
autoirnmune
disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, graft-
versus-host
disease, Sjogren's syndrome and myasthenia gravis); .hyperproliferative
disorders (e.g., B or
T cell lymphoma, neuroblastoma, glioblastoma, chronic lymphocytic leukemia,
breast
cancer, prostate cancer, lung cancer, skin cancer, pancreatic cancer, colon
cancer,
melanoma, ovarian cancer, brain cancer, head and neck cancer, liver cancer,
bladder cancer,
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non-small lung cancer, and cervical carcinoma); chronic inflammatory
conditions (e.g.,
psoriasis, asthma, or Crohn's disease); other conditions such as
osteoarthritis and
atherosclerosis; and other conditions induced by DNA and/or RNA viral
infections, wherein
the viruses include, but are not limited to, herpes virus, papilloma virus and
human
innnunodeficiency virus (HIV).
These disorders are treated by administering an effective amount of the
benzodiazepine compounds described herein. The various benzodiazepine
compounds are
described more fully below. In preferred embodiments, these compounds are
therapeutically effective on their own, and have few or no toxic effects when
administered
in large doses. Furthermore in some additional embodiments, as described in
detail below,
co-administration of these compounds with other agents provides an unexpected
synergistic
therapeutic benefit. In methods of co-administration;,the claimed compounds
axe also
useful in reducing deleterious side-effects of known therapeutic agents by
decreasing the
amount which must be administered to the subject.
The conditions which benefit from treatment with the compounds described
herein
appear to share the common etiology of dysregulation of the process of cell
death. Normal
apoptosis occurs via several pathways, with each pathway having multiple
steps. The
compositions and methods described herein are useful_in treating dysregulated
apoptosis
regardless of the pathway or the step in the pathway where the dysfunction is
occurring. In
some embodiments, the conditions are caused by dysregulation of the FAS
apoptotic
pathway.
Similarly, the compounds are also useful in treating dysregulated necrosis
regardless
of the pathway or the step in the pathway where the dysfunction is occurring.
Dysregulation of the process of cell death is associated with many conditions.
In
neoplasms, for example, normal cell death is inhibited, allowing
hyperproliferative growth
of cells. Aberrant functioning of this process can also result in serious
pathologies
including autoimmune disorders, viral infections, conditions induced by viral
infections,
neurodegenerative disease, and the like. The present invention provides
methods of treating
these and other conditions. While the present invention is not limited to any
particular
mechanism, nor to any understanding of the action of the agents being
administered, it
seems that the compounds described herein induce or promote cell death when
this process
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is malfunctioning. The compounds of the present invention, however, are also
useful for
treating conditions not caused by defects in the apoptotic processes. For
example, in certain
viral infections, while there may not be any apoptotic defect, cell death may
be promoted by
inducing necrosis.
The condition to be treated is generally determined by noting the presence of
symptoms in the subj ect or by noting phenotypic or genotypic changes in the
cells of the
subject, in particular, the inability of the cell to undergo apoptosis or
necrosis.
Phenotypic changes associated with the neoplastic state of a cell (e.g., a set
of in vitro
characteristics associated with a tumorigenic ability ih vivo) include more
rounded cell
morphology, looser substratum attachment, loss of contact inhibition, loss of
anchorage
dependence, release of proteases, increased sugar transport, decreased serum
requirement,
expression of fetal antigens, etc. (See e.g., Luria et al., GENERAL VIROLOGY,
3rd
edition, pp. 436-446 [1978] John Wiley ~ Sons, New Yorlc).
In some embodiments, treating cells or tissues refers to inducing cell death
(wherein
the cell death is either apoptotic or necrotic) in the cells or tissue which
are causative
(primary or distal) of the disorder being treated. For, example, in
hyperproliferative
disorders, the method will treat the disorder by inducing apoptosis of the
hyperproliferative
cells, such as neoplastic cells. In this embodiment, reduction in tumor size
or tumor burden
is one means to identify that the object of the method has been met. In other
aspects,
treating encompasses restoration of immune function or regulation of immune
dysfunction,
as in autoimmune disorders and chronic inflammatory conditions. In further
embodiments,
treating encompasses ameliorating the symptoms associated with a particular
disease (e.g.,
cachexia in cancer or HIV infection or inflammation in arthritis). In still
further
embodiments, prophylactic as well as therapeutic uses of the compounds and
methods of
this invention are intended.
In some embodiments, where the condition being treated is an autoimrnune
disease,
the use of the methods disclosed herein reduce autoantibody production and
lead to a
decrease in inflammation and tissue destruction. Thus, a cell that is being
treated may be
the cell that itself is autoimmunogenic or is affected distally by an
autoimmune reaction,
wherein it is desirable to induce cell death in such a cell or in tissues
containing such cells.
Similarly, in the case of treating inflammatory conditions, the cell that is
being treated may
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be the inflammatory cell itself or it may be distally affected by inflammation
wherein it is
desirable to induce cell death in such cells or tissues containing such cells
and thus reduce
inflammation.
In further embodiments, the cell being treated is a virally infected cell or a
cell or
tissue that previously has been infected. In some embodiments, successful
therapies induce
cell death and therefore a reduction in viral titer. This result is easily
determined by
assaying viral titer or by noting a reduction in cell number. It should be
noted that in some
instances it is desirable to induce cell death even among cells that do not
have any viral
remnants or other signs of viral infections at the time,of treatment because a
viral infection
that occurred much earlier in time can cause disruption of cell death at a
much later time.
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
(C02), 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.
In other embodiments of the present invention, the compounds of the present
invention have antiviral activity independent of their efficacy to induce cell
death. One
aspect or method for inhibiting viral replication andlor propagation comprises
contacting the
virus with an effective amount of one or more compounds and/or compositions of
the
'r
present invention. The contacting is conducted under~suitable conditions to
inhibit viral
replication and/or propagation. In further embodiments, the methods comprises
preventing
viral infection andlor propagation in a cell or tissue by contacting the cell
or tissue with an
effective amount of the compounds and/or compositions as defined above. The
contacting
is conducted under suitable conditions to such that viral infection and/or
propagation is
inhibited.
It is contemplated that by inhibiting and reducing viral replication and
proliferation,
viral infectivity is also inhibited and reduced and he host cells are suitably
treated for viral
,..
~ infection with the additional benefit that associated pathologies also are
treated.
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The viruses that are contemplated under the present methods include, but are
not
limited to, RNA and/or DNA viruses. By way of example only, such viruses are
of herpes,
non-herpes and retroviral origins. Major examples of human pathogens of the
herpes virus
family include herpes simplex viruses (HSV) 1, 2, aiid cercopithecine herpes
virus 1 (B-
virus); varicella-zoster; Epstein-Barr virus (EBV); Lymphocryptovirus; human
herpes
viruses 6-8 (HHV6-S); kaposi-associated herpes virus (KI3V); herpes virus
simiae, and
human cytomegalovirus (HCMV). (,See e.g., J.E. Gallant et al., J. Infect.
Dis., 166:1223-
1227 [1992]).
Animal pathogens of herpes viral origin include infectious bovine
rhinotracheitis
virus, bovine mammillitis virus, bovine leukemia virus; (BLV) and
cercopithecine herpes
virus (B-virus), among others.
The human viruses of non-herpes origin include, but are not limited to,
influenza
viruses A, B and C; parainfluenza viruses -1,2, 3~and 4; adenovirus; reovirus;
respiratory
syncytial virus; rhinovirus; coxsaclcle virus; echo. virus; -rubeola virus;
hepatitis viruses of
the types Band C (HBV and HCV); and papovavirus.
The animal viruses of non-herpes origin include, but are not limited to,
pseudorabies
virus (PRV, of swine), equine rhinopneumonitis, coital ~exanthema viruses
(varicella
viruses); lymphocryptovirus; Marek's disease virus, Bovine Herpes virus-1 (BHV-
1), herpes
virus Pseudorabies virus (PRV).
The viruses of retroviral origin that are contemplated to be treatable by the
.;
compounds and compositions of this invention. include; but are not limited to,
human
immunodeficienoy viruses (HIV) of the types 1 and 2 and human lymphotropic 1
and 2
viruses (HTLV-I and II).
B. Methods of identifying potential therapeutic agents
Also provided herein are assays to identify potential agents to treat
conditions
associated with the dysregulation of the apoptotic or necrotic pathway. In
some
embodiments, the methods comprise contacting the dysregulated cell, i.e., a
cell affected by
the disorder (e.g., a tumor cell when the condition is hyperproliferative) or
an immune cell
(a neutrophil, basophil, eosinophil, monocyte, or lymphocyte) when the
condition is a
chronic inflammatory condition or an autoimmune disorder) with the potential
therapeutic
CA 02457405 2004-02-11
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agent. In further aspects of the invention, control cells are further assayed
with or without a
benzodiazepine compound. The benzodiazepine compound may be a 1,4-
benzodizepine
compound as described herein. Cell death as compared to the control cells is
also noted and
compared. To identify potential therapeutic agents, appropriate assay
conditions (e.g.,
incubation time, temperature, culture maintenance medium, etc.) are used and
can be readily
determined by one of slcill in the art. Serum may be obtained from any
commercial source,
for example, fetal bovine serum from Gibco BRL (Gaithersburg, NM). The cells
are
cultured with the test agent for a sufficient amount of time for the test
agent to affect
apoptotic processes and/or necrosis. Following the appropriate incubation
period, cell death
is assayed by any means lcnown, for example by 1VITT dye trypan exclusion.
Novel
cytotoxic agents are identified by their ability to induce the death of
dysregulated cells
versus cell death in control cells.
The present inventors have discovered that when the cells are maintained in
low
serum conditions, cytotoxicity is greatly exacerbated and the incubation time
is reduced to
about 2 hours or less. This is quite an unexpected result since under standard
incubation
conditions, which employ higher serum levels, the required incubation time is
often several
hours, approaching in some cases 24 hours or more. The term "low serum," as
used herein,
refers to culture media containing less than about 10% per volume down to or
equal to less
than about 0.1 % (v/v). It should be understood that within this range the
concentration is
flexible, and the applicants contemplate any possible 'subrange in increments
of about 0.1
witlun this range, for example, less than or equal,to about any of 0.1, 0.2,
0.3, 0.4, 0.5, 0.6,
0.7, 0.8, . . . 1.0, . . . 1.5, . . . 2.0, . . . 2.5, . . . 3.0, . .~~: ~3.5,
. . . 5%, etc., to about 9.9, and to
about 10% of serum (%v/v).
Thus, in some embodiments, the benzodiazepines of this invention induce
apoptosis
in low serum as defined above.
In further embodiments, the compositions of the invention are further
characterized
and identified by their inability to bind either to a central benzodiazepine
receptor or to bind
with low affinity to a peripheral benzodiazepine receptor. However, in
particularly
preferred embodiments, the compositions and methods of the present invention
do not target
(e.g., .selectively bind) either the central or peripheral benzodiazepine
receptors. These
compounds can be identified by using methods well-known in the art.
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For example, the binding affinity of a benzodiazepine compound for a
peripheral
benzodiazepine receptor can be determined according to well-established
methodology as
described in H. Schoemalcer et al., J. Phann. Exp' Ther., 225:61-69 (1983);
and A. Doble et
al., Brain Res. Bull., 18:49 1987).
Briefly, the method comprises comparing the potency of a benzodiazepine
compound with that of a well-known high affinity binding agent such as 1-(2-
chlorophenyl)-N-methyl-N-(-1, methylpropyl)-3-isoqu'inolinecarboxamide
(PI~11195),
wherein the ability of the benzodiazepine compound to displace PK511195 from
the
peripheral benzodiazepine receptors in a competitive binding assay.
In any of the above assay methods, the benzodiazepine compound can be
detectably
labeled. Suitable detectable labels include, but are not limited, isotopes,
chromophores,
fluorophores, magnetic particles, high affinity binding partners (e.g.,
strepavidin/biotin), and
antibodies, etc. Examples of isotope labeling include stable or radioactive
isotopes of one
or more atoms on the benzodiazepine molecule.
Methods for introducing detectable labels and for detecting the labels are
well-
l~nown in the art. For example, the radioisotope label~can be detected using
special
instnunentation, including electron spin resonance spectrometers. Stable
isotopes can be
detected using mass spectrometers, or magnetic resonance spectrometers.
Fluorescent
labels can be detected using fluorescent spectrometers: These instruments are
commercially
available and their operation is within the ordinary shill, in the art.
The benzodiazepine compounds that can.be used in the assay and diagnostic
methods are described in greater detail below. It should be understood that
all the
compounds described therein, including the many general and specific
embodiments, can be
used in the assay and diagnostic methods.
, ,.
II. Benzodiazepine derivative modulators of cell.growth and proliferation
The selectivity of many cytotoxic agents is limited and generally relies on
the
differential ability of diseased and healthy cells to tolerate and repair drug-
induced cellular
damage. However, developing cytotoxic therapies that exploit disease-specific
targets
remains challenging. For many diseases, suitable targets have not been
identified, and in
cases where targets exist (P. Huang and A. Oliff, Trends Cell. Biol., 11:343-
248 [2001]),
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relatively few have been validated to the extent that it is known that
bloclcing their function
controls disease (D.W. Nicholson, Nature, 407:810=816 [2000]).
Diversity-oriented synthesis and phenotype screening (sometimes referred to as
"chemical genetics") have been advanced as robust methods for identifying
bioactive
compounds (S.L. Schreiber, Science, 287:1964-1969 [2000]). In these methods,
arrays of
molecules are synthesized and screened to identify lead compounds based
directly on
function rather than affinity for a target. Preferred embodiments, of the
present invention
provides compositions and methods that therapeutically target cytotoxic agents
to diseased
cells.
In one embodiment, the present invention provides selective compositions and
methods for the therapeutic treatment of systemic lupus, erythematosus (SLE)
in a subject.
SLE is characterized by a spectrum of antibodies that recognize self antigens.
Autoantibodies are the products of B cells that escape peripheral tolerance.
Autoantibody-
antigen immune complexes deposit in the tissues of the heart, brain, lungs,
and kidney,
incite tissue destruction and impair organ function by;activating complement
and recruiting
inflammatory cells. The kidney is an important target; at least 40% of all SLE
patients have
renal involvement, and lupus is associated with a mortality rate of 28% over
10 years.
Effective drugs to treat lupus include immunosuppressive lymphotoxic agents.
Although effective, immunosuppressive drugs often induce severe side effects
that account
for a significant portion of lupus-related deaths. Agents with greater
specificity toward
disease-causing lymphocytes would clearly advance the treatment of SLE and
related
disorders. However, it is not yet possible to precisely identify the
population of autoreactive
B cells causing disease. Therefore, a specific therapy that only targets
disease-causing cells
does not exist. Despite its drawbacks, lymphocyte toxicity offers a reasonable
basis by
which to select new therapeutics.
The present invention provides methods for screening for candidate agents that
selectively kill activated cells or inhibit cell growth, or proliferation of
an activated target cell
by first contacting unactivated counterpart target cells (e.g., cells that
have not been exposed to
an activation stimulus) with varying amounts of the candidate agent and a
separate sample of
cells with equal varying amounts of buffer or an equivalent thereof, and
selecting those
candidate agents that increase the intracellular concentration of superoxide
prior to the
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mitochondrial permeability transition ("MPT") in the W activated cell. As used
herein, the
term "an increase in intracellular superoxide prior to the MPT" is any
statistically significant
(p < 0.05) increase induced by a candidate agent compared to cells treated
with buffer alone.
Several methods to measure intracellular superoxide in cells are known in the
art. One method
employs hyclioethidimn as described by L. Benov et al., Free Radic. Biol.
Med., 25:826-831
(1998). Several methods to measure the MPT in cells are lrnown in the art. One
method
employs the dyes DIOC~(3) and JC-1 as described by Zanzami et al. (1995).
Agents that produce increased superoxide concentration in the unactivated
target cell
prior to the MPT, are contacted with cells in the presence of micromolar
amounts of FK506
or MnTBAP (or their equivalents) and the cells are assayed for levels of
superoxide after
treatment. These "inhibitors" can be added to the cells prior to or at the
same time as the
candidate compound. The FK506 screen identifies agents that, at their 50%
effective
concentrations (ECS°), have the change in superoxide concentration
inhibited (to 75%
inhibition) whereby FK506 is not acting solely by inhibiting the calcineurin
pathway. (R.
Zini et al., Life Sciences, 63(5):357-368 [19980. TheFMnTBAP screen identifies
agents
that have the change in superoxide concentration,inhibited (to >50%
inhibition) with
micromolar amounts of manganese (III) meso-tetrakis (4-benzoic acid) porphyrin
("MnTBAP"). In preferred embodiments, only after a candidate agent: 1)
increases
superoxide concentration in an unactivated cell prior to MPT; and 2) the
increase in
superoxide concentration in the unactivated cell is inhibited or reduced by
FK506 or
MnTBAP (or an equivalent) is the candidate agent selected for the third step
of the screen.
Both the second and third steps of the screen distinguishe the compounds
having the
preferred properties from those known in the axt to also increase the
intracellular
concentration of superoxide prior to MPT. (See e.g., D.A Fennell et al., Br.
J. of Cancer,
84(10):1397-1404 [2001]).
These agents are then further screened for theix ability to inhibit the growth
or
proliferation (e.g., affect cell number), or induce death of an activated
counterpart target cell
to a greater extent than the combined effects of the activating agent alone
and the candidate
agent on unactivated cells.
In a separate embodiment, the screen is performed by first selecting an
unactivated
target, which can be a cultured cell or one obtained from a tissue biopsy.
Serial dilutions
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from 0.01 nM through 100 mM of the compound or, agent are made for testing.
Serial
dilutions are contacted with the cells by mixing with culture medium.
Superoxide levels
and the MPT in the unactivated target cells are measured for each dilution,
and an ECSO for
each endpoint is determined. In an alternative aspect, the test cells receive
serial dilutions
(as above) of an inlubitor prior to contacting with the candidate agent or
compound, and
superoxide levels are then measured. The inhibition assay screens for
compounds whose
production of superoxide is inhibited by FK506 or MnTBAP or their equivalents
at the
agent's ECSO. Candidate agents that are inhibited by FK506 and MnTBAP (or
their
equivalents) are then assayed against counterpart activated target cells by
malting serial
dilutions of the candidate agent cell (from 0.01 nM through 100 mM) and an
activating
agent (of a suitable amount) for the target which are then separately
contacted with the
unactivated target cells. An agent that inhibits growth or proliferation, or
kills activated
cells to a greater extent than the combined effects of the activating agent
alone and the
candidate compound on unactivated cells is a compound of this invention.
These candidate agents have the ability to selectively inhibit the growth or
proliferation, or kill activated target cells and therefore, are useful for
therapies to treat
conditions or diseases associ~.ted with the pathological growth of the
relevant target cell
type in a subject. The agents also are useful to ameliorate the symptoms
associated with the
presence of pathologically growing activated target cells in a subject.
Agents or compounds identified by this screen have the general structure:
R~
O
nnnR2
R3
or
R~
R~
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or their enantiomers, wherein Rl is selected from the group consisting of an
aliphatic, an
aryl, a substituted aliphatic, and a substituted aryl and wherein RZ is
selected from the group
consisting of a substituted aliphatic, a substituted aryl, a cycloaliphatic, a
substituted
cycloaliphatic, a heterocyclic, and a substituted heterocyclic NHZ, NHC(=O)-RS
and any
substituent that participates in hydrogen bond formation. RS is selected from
the group
consisting of an aryl, a heterocycle, and -R~ NH-C(=O)-NH-R~, wherein R~ is an
aliphatic
linker of 1 to 6 carbons and R, is selected from the group consisting of an
aliphatic, an aryl,
and a heterocycle. Each of R3 and R4 may be the same or different and is
selected from the
group consisting of hydrogen, hydroxy, alkoxy, halo, amino, thio, nitro, lower-
alkyl-
substituted-amino, lower-alkyl-substituted-halo, acetylamino, hydroxyamino, an
aliphatic
group having 1 to 8 carbons, aryl, substituted aryl, cycloaliphatic,
substituted cycloaliphatic
or heterocyclic, a leetone, an aldehyde, an ester and an amide. In one aspect:
R, is H, R4 is a
halogen, R3 is hydroxyl or halogen, and RZ is an aromatic or heterocycle.
Unless
specifically recited, all substituents are substituted or unsubstituted.
Pharmaceutically
acceptable salts of such compounds are further provided by this invention.
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More specific examples of compounds identified by the screen are compounds
having the structures:
O O
H
H3
N~ N
Rz R2
CI N CI N
HO HU
Hs
O
O
N~ H
N_
R~
R~
CI
CI
O O\
\CH3 \CH
3
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wherein RZ is selected from the group consisting of
i% j n
N
a"a
/ N~ / /
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and dimethylphenyl (all isomers) and ditrifluoromethyl (all isomers), and
HO
HO
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This invention also provides the compound Bz-423.
CH3
CI
OH
Bz-423 differs from benzodiazepines in clinical use by the presence of a
hydrophobic substituent at C-3. This substitution renders binding to the
peripheral
benzodiazepine receptor ("PBR") wealc (Kd ca. 1 pM) and prevents binding to
the central
benzodiazepine receptor so that Bz-423 is not a sedative.
The agents or compounds that possess the above noted three properties are
examples
of agents of this invention. The compounds of this invention were identified
by this screen
and confirmed in an animal model. Further provided by this invention are the
agents
identified by this screen.
Although Applicants utilized this screen for small molecules that possess the
desired
properties, this invention encompasses other therapeutic modalities that are
identified using
this screen, e.g., polynucleotides or polypeptides, and thus are intended
within the term
"agents."
The invention also provides methods for selectively killing and/or inhibiting
cell growth
or proliferation of activated target cells by contacting a target cell with an
effective amount of
an agent of this invention. Further provided are methods for selectively
inducing cell death of
a target cell in either its activated or un-activated state by contacting the
target cell with an
effective amount of an activating agent and an effective amount of an agent of
this invention.
Also provided by the invention are methods for inhibiting cell death of a
target cell by
contacting the target cell with an effective amount of an agent that inhibits
the formation of
CA 02457405 2004-02-11
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superoxide iii the target cell prior to the cells' mitochondrial permeability
transition, e.g.,
FK506 or its equivalent. These methods ameliorate the symptoms of
neurodegenerative
diseases such as Alzheimers and ischemia reprofusion injury, e.g., neuromotor
problems and
the like, and strobe, respectively.
The above methods of this invention can be practiced ih vivo or ih vivo. When
practiced
ih vivo, the method provides a convenient animal model to confirm biological
efficacy of
agents identified by the screen of this invention.
Further provided is a method for selectively inhibiting the pathology of
activated target
cells in a subj ect in need of such therapy by administering to the subj ect
an effective amount
of an agent of this invention. Administration of an effective amount of the
agents of this
invention also serves to ameliorate the symptoms associated with the presence
of
...~ a
pathologically growing activated target cells or to treat diseases associated
with their presence
in a subject. Suitable subjects include, but are not limited to a non-
Hodgkin's lymphoma
patient, a chronic lymphocytic leukemia patient, a cutaneous T cell leukemia
patient, a patient
with an autoiminune disorder, or a cancer patient (solid tumors, lymphomas,
leubemias).
Therapeutic compounds for use in these methods include the compounds described
herein as
well as those provided by PCT/US00/0057~.
A method for selectively inhibiting the pathological growth of unactivated
target
cells in a subject in need of such therapy is further provided by this
invention. An effective
amount of an agent that selectively activates the target cells and an
effective amount of an
agent of this invention are administered to the subject. Administration of
agents of the
present invention can be simultaneous or sequential.
In one embodiment of the present invention, Bz-423 was selected from a 1,4-
benzodiazepine library based on its ability to induce lymphoid cell death in
vivo.
Comparison of Bz-423 with other benzodiazepines and ligands of the peripheral
berizodiazepine receptor reveals that Bz-423 has unique cytotoxicity. (Figure
1). The
activity of Bz-423 against NZB/W lymphocytes is concentrated on B cells such
that
treatment lulls twice as many B cells as T cells. In some embodiments, the
potential ih vivo
lymphotoxicity of Bz-423 was tested by administering (60 mg/lcg/d for 7 d) of
Bz-423 to
autoimmune NZB/W and normal BALB/c mice. After treatment, sphenic lymphocytes
were
analyzed for evidence of cell death. In the NZB/W mice, lymphocyte viability
was
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decreased and B cell apoptosis increased (Table 1). Table 1 shows splenocyte
viability (PI)
and lineage specific apoptosis (TUNEL) after 7 days. By contrast, Bz-423
neither decreased
viability nor increased apoptosis of BALB/c splenic lymphocytes. Thus, Bz-423
specifically effects B cells from the autoimmune mice.
Table 1
Cell Apoptotic Apoptotic
Viability B cells T cells
Control 85 ~ 1 25 ~ 2 12 ~ 1
Bz-423 811 322 , 123
p=0.006 p=0.037 p=0.27
Given the selective action on autoimmune B1 cells, further studies were made
to
determine if Bz-423 affects the progression of autoimmune nephritis. For
example, female
NZB/W mice were given Bz-423 (60 mg/lcg), or vehicle every other day from 6.5
to 9.5
months of age. Control mice developed diffuse, proliferative
glomerulonephritis with
expansion of all cellular elements and occasional wire-loop formation
resulting in an
average histopathologic score of 3+. (Figure 2A). In contrast, mice dosed with
Bz-423 had
milder nephritic changes with an average score of 1+ (p = 0.002) (Figure 2B).
Bz-423
treated mice also had less glomerular IgG (Figures 2C and 2D) (IgG, p = 0.002;
C3, p =
0.034). At the time of sacrifice, 83%of control mice had abnormally lugh BLTN
levels (>
30 mg/dL), compared to 27% of the treatment group (chi-square: p = 0.001). A
strong
correlation was identified between the histological score for nephritis and
proteinuria (>100
mg/dL; p = 0.001) or elevated BUN (p = 0.039). In aggregate, these
measurements indicate
less disease in treated animals compared to controls in some embodiments.
In still further embodiments, NZB/W mice with disease-related lymphoid
hyperplasia characterized by the pathologic expansion of GC B cells were used
in further
studies. Flow cytometric measurements revealed that Bz-423 reduced the
fraction of B cells
in the spleen (49% ~ 3 vs. 58% ~ 3 in controls; p = 0.05) with no statistical
change in the
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fractional representation of T cells. Irnmunohistochemical staining
demonstrated that this
decrease resulted from a specific effect on GC B cells (Figures 2E and 2F).
Mice treated
with Bz-423 had fewer GCs relative to control mice (17 ~ 5 vs. 10 ~ 7 per 10
mm2, p =
0.01) and the GCs present in the drug group were almost 50% smaller than those
present in
the control animals (p = 0.01). Furthermore, Bz-423-treated animals had
increased TUNEL-
positive B cells (ca. 3+ vs. 1+, p = 0.038) that were predominantly
concentrated in GCs
(Figures 2G and 2H). This observation indicates that the decrease in GC size
and number
results from apoptosis within this compartment in some embodiments.
GC B cells require B cell receptor (BCR) stimulation for development and
survival,
a property that distinguishes them from other mature cells. To account for the
selective
reduction of GC in treated animals whether activation via BCR stimulation
facilitates killing
by Bz-423 was investigated. To test this hypothesis, two models of BCR
stimulation were
used that differ with respect to the degree of receptor cross-linking. In the
first, Ramos cells
were treated with soluble anti-IgM Fabz to provide a modest BCR signal that is
itself
insufficient to induce apoptosis. Ramos cells were utilized here because they
display
surface marlcers characteristic of GC cells, demonstrate a response to BCR
ligation
characteristic of mature B cells, and survive in culture with little
spontaneous death.
Soluble anti-IgM Fab2, that alone fails to induce death, sensitizes to Bz-423
(Figure 3A). In
fact, treatment of activated Ramos cells with Bz-423 results in a synergistic,
supra-additive
death response. Stimulatory antibody directed against CD40, a signal that
abrogates anti-
IgM activation-induced cell death (AICD), offers no protection against Bz-423
alone, or
anti-IgM sensitization to Bz-423 (Figure 3B).
In a second activation model of BCR stimulation, primary NZB/W B cells were
incubated with immobilized, whole anti-IgM which extensively cross-links BCRs
and Fc
receptors and provokes AICD in normal immune B cells. Consistent with previous
reports,
NZB/W B cells are resistant to apoptosis induced by receptor cross-linl~ing
compared to
normal immune B cells. Nevertheless, stimulation with anti-IgM in the presence
of Bz-423
lcills NZB/W cells. While co-stimulation of CD40 completely blocks AICD in
BALB/c
cells, it provides little protection against Bz-423 alone, or death in the
synergistic
conditions. These results show that irrespective of the degree of receptor
cross-linking,
activated cells are more sensitive to Bz-423 in some embodiments.
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The signals and markers associated with apoptosis in BCR simulated and un-
activated Ramos cells were compared. With respect to morphology, treatment of
both
activated and unactivated cells results in cytoplasmic vaculization, nuclear
condensation,
and plasma membrane blebbing (Figure 4C). . '
The dependence of sensitized death on pro-apoptotic signals that function in B
cells
was also tested. Chelating extracellular calcium with.l,2-bis(2-
aminophenoxy)ethane-
N,N,N',N'-tetraaceticacid (BAPTA) minimally reduced non-sensitized death while
substantially protecting sensitized cells (7% vs. 71% inhibition,
respectively). The effect of
BAPTA is unrelated to its ability to blunt the rapid BCR-induced calcium flux
because it is
protective even when added 1 hour after stimulation. Inhibition of caspase
activity with z-
VAD (un-activated: 31 % vs. activated: 86%), the mitochondrial permeability
transition
(MPT) with cyclosporin (CsA) (un-activated: 9% vs. activated: 54%) and protein
synthesis
with cyclohexamide (un-activated: 19% vs. activated: 64%) only protected
sensitized cells.
Inhibiting calcineurin (with FK506 l OnM) was not protective in either
condition. The
antioxidants vitamin E and superoxide (OZ )-specific MnTBAP each protected
activated and
unactivated cells to a similar extent (~80%) suggesting that Oz is an
essential event in Bz-
423-induced signaling. These experiments show that high concentrations of Bz-
423
generates superoxide which kills cells independently of these other mediators
of apoptosis.
OZ in activated and un-activated cells using hydroethidium which is a
selective
indicator of OZ , was also measured. 0a increases in both un-activated and
activated cells
within 1 hour of exposure to Bz-423 (Figure 4A) which is prior to the MPT.
MnTBAP and
vitamin E reduced OZ levels, but z-VAD, BAPTA and CsA did not. The amount of
O~
increases with increasing concentration of Bz-423, and in the absence of BCR
stimulation,
correlates with cell death measured at 24 h. However, for a given
concentration of Bz-423,
BCR stimulation does not increase OZ relative to un-activated cells (Figures
4A-4C).
Hence, the role of superoxide in cell death differs ~in activated cells. BCR
activated cells are
billed by lower concentration of Bz-423 through a mechanism in which BCR cross-
linl~ing
sensitizes cells to OZ , gene expression, caspase, and mitochondria-dependent
processes to
occur.
These results demonstrate that in some preferred embodiments, the elements) of
the
BCR response render B cells vulnerable to killing by low concentrations of Bz-
423.
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Accordingly, in preferred embodiments, the selective toxicity of low
concentrations of Bz-
423 to BCR activated B cells provides a mechanism for this compound to target
the
expanded GC population based on their unique dependence on BCR stimulation.
Although
an understanding of the mechaiusm is not necessary to practice the present
invention and the
present invention is not so limited, it is contemplated that Bz-423
compensates for either of
the two pathogenic mechanisms proposed to support persistent GCs and
autoreactive B
cells, by acting in concert with either modest or robust BCR cross-linking
resulting in a
supra-additive death response. Thus, the same stimulus that supports
autoimmune
pathogenesis provides a selective therapeutic target for some diseased cells.
In human SLE,
moreover, hyperactivated B cells are also implicated as, critical determinants
of disease and
are similarly dependent on BCR-generated responses.-
In preferred embodiments, benzodiazepines are selected because they are
amenable
to combinatorial synthesis (See, B.A. Bunin et al., Proc. Natl. Acad. Sci.
U.S.A., 91:4708-
4712 [1994]), and under certain conditions; some benzodiazepines influence
cell survival
(A. Beurdeley-Thomas et al., J. Neurooncol., 46:45-56 [2000]). Also, because
benzodiazepines do not damage DNA or interfere with.nucleotide metabolism,
cytotoxic
benzodiazepines would likely possess unique modes of action.
As described above, some preferred embodiments of the present invention
identified
Bz-423 (Figure 5A) as a potent lead compound. Unlike benzodiazepines with
anxiolytic
properties, Bz-423 does not bind to the central benzodiazepine receptor.
Certain
embodiments of the present invention show that incubation of transformed Ramos
B cells
with Bz-423 rapidly generates Oi and this reactive oxygen species (ROS)
functions as an
upstream signal to commence an apoptotic death process. Although an
understanding of the
mechanism is not necessary to practice the present invention and the present
invention is not
so limited, it is contemplated that the Oz response results from the
interaction between Bz-
423 and a target within mitochondria, possibly in the mitochondrial
respiratory chain
(MRC). Because Ramos cells model aspects of germinal center (GC) B cell
physiology
(C.T. Gregory et al., J. Immunol., 139:313-318 [1987]), the present invention
contemplates
that Bz-423 has activity against GCs in vivo. In some embodiments, the
activity of Bz-423
is shoran using the (NZB x NZV~F1 (NZB/V~ model of lupus where aberrant
survival and
expansion of GC B cells drives disease (See, J.P. Portanova et al., Mol.
Immunol., 32:117-
CA 02457405 2004-02-11
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135 [1987]; and M.J. Shlomchik et al., Nat. Rev. hmnunol., 1:147-153 [2001]).
In
preferred embodiments, Bz-423 specifically controls GC hyperplasia and the
subsequent
development of glomerulonephritis in the NZB x NZW)F1 (NZBIW) mice model.
Accordingly, the present invention contemplates a novel role for Oz in B cell
apoptosis and
identifies Bz-423 as a novel lead compound for the development of selective
cytotoxic
molecules to manage SLE and related disorders.
High levels of ROS that accompany the late stages of apoptosis damage
macromolecules, whereas physiologic concentrations regulate a range of
intracellular
signaling pathways (T. Finlcel, J. Leulcoc. Biol., 65:337-340 [1999]). Some
apoptotic
stimuli (e.g., ceramide [See, C. Garcia-Ruiz et al., J. Biol. Chem., 272:11369-
11377
(1997)]) dexamethasone (See, J.F. Tomes-Roca et al.,'J..Immunol., 165:4822-
4830 [2000]),
or TNFa. (See, H. Albrecht et al., FEBS Lett., 351:45-48 [1994]), induce ROS
early in their
death responses as a means to initiate downstream effector mechanisms such as
caspase
activation. Similarly, the present invention shows that the early OZ response
induced by
interaction of Bz-423 with mitochondria signals an apoptotic program in B
lymphocytes.
Several B cell-specific signaling pathways respond to ROS and, when so
engaged, initiate
apoptosis. .For example, activation of Bruton's tyrosine leinase by ROS (See,
S. Qin et al.,
Proc. Natl. Acad. Sci. U.S.A., 97:7118-7123 [2000]) results in phosphorylation
of
phospholipase Cg (See, M. Takata and T. Kurosaki, J. Exp. Med., 184:31-40
[1996]), which
can lead to Ca2+-dependent apoptosis. This and related ROS-dependent processes
may
contribute to the susceptibility of B cells to Bz-423:
The present invention provides a number of small molecules that increase
intracellular OZ in a variety of ways, including, but not limited to, release
of OZ from
oxygenases, single electron reductions, inhibition of oxido-reductases, and
disruption of
MRC activity. (See, A.G. Siralci et al., Free Radic. Biol. Med., 32:2-10
[2002]).
Traditional chemotherapeutics compromise mitochondrial function indirectly.
However, the some embodiments, of the present invention contemplate directly
targeting
the MRC or MPT pore. The critical elements of the Bz-423 response i~a vivo, OZ
production
and decreased B cell survival, are observed in NZB/W mice after dosing with Bz-
423. In
the NZB/W model of lupus, pathogenic autoantibodies are the products of
activated, class-
switched B cells that emerge from GCs (J.P. Portanova et al., Mol. Immunol.,
32:117-135
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[1987]; M.J. Shlomchilc et al., Nat. Rev. Immunol., 1:147-153 [2001; and Y.
Munakata et
al., Eur. J. Immunol., 28:1435-1444 [1998]). In one preferred embodiment,
after dosing for
12 wk, Bz-423 dramatically reduced the number and size of GCs and increased
apoptosis in
remaining GCs. Moreover, no significant decreases in other splenic lymphocyte
populations, evidence of lymphopenia, or changes in cytol~ines was detected
(unpublished
data). Thus, the present invention contemplates that.this B cell population is
am important
target for SLE intervention. (See e.g., M.J. Shlomchik et al., infra.).
The survival and apoptotic threshold GC B cells in normal mice is tightly
regulated
by c-FLIP and signaling through the BCR, CD40, CD80, and Fas (M.V. Eijk et
al., Trends
Immunol., 22:677-682 [2001]; and V.K. Tsiagbe.et al., Crit. Rev. Immunol.,
16:381-421
[1996]). Although an understanding of the mechanism is not necessary to
practice the
present invention and the present invention is not so limited, it is
contemplated that the
longevity of NZB/W GC B cells is tied to increased expression of co-
stimulatory molecules
like CD81, down regulation of inhibitory receptors such as FcgRIIBI (See e.g.,
Y. Jiang et
al., Int. Irrununol., 11:1685-1691 [1999]), and hypo-responsiveness to
persistent BCR
stimulation (e.g., defective activation induced B cell death) (See e.g., Y.
Kozono et al., J.
T_mmunol., 156:4498-4503 [1999]). In this context, the present invention also
notes that
BCR stimulation also sensitizes B cells to cytotoxic agents. (See e.g., C.E.
Lin et al., Exp.
Cell. Res., 244:1-13 [1998]). Hence, in some embodiments it is contemplated
that receptor
ligation contributes to the selectivity of Bz-423. For example, studies show
that anti-IgM
sensitizes B cells to Bz-423. While the molecular basis for BCR sensitization
is not fully
understood, it is lmown that cross-linlcing BCRs can itself elevate
intracellular ROS (See
e.g., W. Fang et al., J. Immunol., 155:66-75 [1995]) and that lymphocytes from
lupus
patients have decreased stores of reduced glutathione and increased ROS
relative to normal
cells (See e.g., P. Gergely Jr. et al., Arthritis Rheum., 46:175-190 [2002]).
Thus, the
vulnerability of NZB/W GC B cells may, in part, result from an additive effect
of Bz-423-
induced OZ and endogenously generated ROS, such that radicals from all sources
combine
to overwhelm the limited reducing potential of these lymphocytes and trigger
apoptosis.
Bz-423 is a pro-apoptotic molecule that engages the cell-death machinery in an
Oz
dependent manner. The present invention provides,a new structure-function
relationship for
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benzodiazepines and points to a new molecular target and pharmacological
mechanism
valuable for the management of SLE.
The present invention further contemplates that Bz-423 kills Ramos B cells in
a
dose-dependent fashion (Figure SB). The activity of Bz-423 was compared to
ligands of the
peripheral benzodiazepine receptor (PBR), an 18 KDa transmembrane protein
located in the
mitochondrial membrane, because some ligands of the PBR are thought to
modulate death
signals from mitochondria. (See e.g., A. Beurdeley-Thomas et al., J.
Neurooncol.~ 46:45-56
[2000]). Both 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-
isoquinolinecarboxamide (PK11195) and 4'-chlorodiazepam bind tightly to the
PBR (KD =
0.3 and 30 nM, respectively; but are only cytotoxic at > 10-times the ED50 of
Bz-423.
Competition assays for the PBR demonstrate that a 1000-fold excess of Bz-423
is necessary
to reduce [3H]-PK11195 binding by 50%, and pre-incubation of cells with excess
PK11195
(>20 ~,M) does not block the activity of Bz-423. These data indicate that cell
killing by Bz-
423 does not result from binding to the PBR. The activity of Bz-423 depends on
its specific
structure, since deleting either the napthyl (DNAP) or the phenolic hydroxyl
(DOH) groups,
elements that distinguish Bz-423 from diazepam, dramatically reduces cytotoxic
activity
(Figure SB). Bz-423-induced cytotoxicity is characterized by cell shrinkage,
nuclear
condensation, cytoplasmic vacuolization, membrane blebbing, and DNA
fragmentation
(hypodiploid DNA; Figures SC and SD), consistent with apoptosis. To probe the
role of
caspases in Bz-423 killing, cells were pre-incubated with z-VAD, an
irreversible caspase
inhibitor. z-VAD completely prevents Bz-423-mediated apoptosis as measured by
DNA
fragmentation. Less than 5% of cells treated with z-VAD and Bz-423 have
hypodiploid
DNA, compared to 69% of cells treated with Bz-423 alone (Figure SD). However,
inubiting caspase activity does not protect against cell death; cellular
morphology
demonstrates that Bz-423 still kills >80% of cells in.cultures containing z-
VAD (See, Figure
SC). Similar results have been reported for other apoptotic stimuli, where
blocking caspase
activity prevents DNA fragmentation but does not inhibit overall cell death
(A.S. Belzacq et
al., Cancer Res., 61:1260-1264 [2001]).
In yet other embodiments, in experiments with isolated mitochondria under
conditions supporting state 3 respiration demonstrate an Oz response analogous
to that in
whole cells. Mitochondria do not respond to Bz-423 in state 4 where energy is
supplied in
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the absence of ADP. Although an understanding of the mechanism is not
necessary to
practice the present invention and the present invention is not so limited, it
is contemplated
that, in transitioning from state 3 to 4, the proton motive force becomes
sufficiently high
that intermediates competent in one electron reduction reactions (e.g.,
ubiquinol) have
extended half lives, and these conditions favor reduction of OZ to OZ at
complex III. (See,
V.P. Slculachev, Mol. Aspects Med:, 20:139-184 [1999]). Hence, in some
embodiments, it
is possible that Bz-423 generates Oz- by inducing a state 3 to 4 conversion.
Oligomycin, a
macrolide natural product that binds to complex V, induces a state 3 to 4
transition and
generates OZ- like Bz-423. These similarities indicate that complex V is also
the molecular
target for Bz-423. Complex V is a large mufti-protein assembly and can be
inhibited
by small molecules in a number of different ways. However, it is contemplated
in some
embodiments that the similarities to oligomycin suggests that Bz-423 may
inhibit the
ATPase activity of complex V by binding to an element of complex V that
includes the
oligomycin sensitivity conferring protein, which is in the F1 domain.
In additional embodiments, to further characterize the death mechanism engaged
by
Bz-423, intracellular ROS, ~~I'"" cytochrome c release, caspase activation,
and DNA
fragmentation were measured over time. In preferred embodiments, the present
invention
used endpoints as previously implicated in B cell apoptosis. (See e.g., T. Doi
et al., Int.
hmnunol., 11:933-941 [1999]). The first event detected after exposure to Bz-
423 is an
increase in the fraction of cells that stain with DHE, a,redox-sensitive agent
that reacts
specifically with OZ (a dose-dependent increase in the mean fluorescence
intensity was also
observed; Figure 6A). Levels of OZ diminish after an,early maximum at 1 h and
then
increase again after 4 h of continued treatment (Figure' 6B). This bimodal
pattern points to a
cellular mechanism limiting Oz and suggests that the "early" and "late" Oz
maxima result
from different processes. Collapse of O~I'", was detected using DiOC~(3), a
mitochondria-
selective potentiometric probe (N. Zamzami et al., J. Exp. Med. 182:367-377
[1995]). The
gradient change begins after the early OZ response and is observed in >90% of
cells by 5 h.
Cytochrome c release from mitochondria, a key step enabling apoptosome
formation and
caspase activation (P. Li et al., Cell, 91:479-489 [1997]), was studied by
immunoblotting
cytosolic .fractions. Levels of cytosolic cytochrome c above amounts in cells
treated with
vehicle are detected by 5 h (inset, Figure 6B). This release is coincident
with the disruption
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of ~~I'"" and together, these results are consistent with opening of the
mitochondria)
permeability transition (MPT) pore. Indeed, the late increase in Oz closely
tracks with the
D~I'", collapse and the release of cytochrome c, suggesting that the secondary
rise in OZ
results from these processes. (See e.g., C.M. Luetjens et al., J. Neurosci.,
20:5715-5723
[2000]). Caspase activation, measured by processing of the pan-caspase
sensitive
fluorescent substrate FAM-VAD-fluoromethyll~etone; tracks the gradient
changes, whereas
the appearance of hypodiploid DNA is slightly delayed with respect to caspase
activation
(Figure 6B). Although an understanding of the mechanism is not necessary to
practice the
present invention and the present invention is not so limited, it is
contemplated that Bz-423
induces a mitochondria) dependent apoptotic pathway.
In some embodiments of the present invention, Bz-423 is contemplated to
directly
target mitochondria. Since early OZ precedes caspase~activation, collapse of
O~f"" and DNA
fragmentation, it is possible that this ROS has a regulatory role. In non-
phagocytic cells,
redox enzymes, along with the MRC, are the primary sources of ROS. (See e.g.,
T. Finkel,
J. Leukoc. Biol., 65:337-340 [1999]). Therefore, in some embodiments, the
present
invention assayed inhibitors of these systems for their ability to regulate Bz-
423-induced OZ
(at 1 h) to determine the basis for this response (Table.2). Of these
reagents, only NaN3,
which acts primarily on complex IV of the MRC (E. Fosslien, Ann. Clin. Lab.
Sci., 31:25-
67 [2001 ]), and micromolar amounts of FK-506, which block the formation of OZ
at
complex III (R. Zini et al., Life Sci., 63:357-368 [1998]), modulate Bz-423.
Other agents
that inhibit components of the MRC (e.g., rotenone,~inyxothiazol,
thenoyltrifluoroacetone,
antimycin A, stigmatellin, and oligomycin), along with the flavoenzyme
inhibitor
diphenyleneiodonium, were less informative as they independently generate
significant
ROS in Ramos cells. Collectively, these findings suggested that mitochondria
are the
source of Bz-423-induced OZ and that a component of the MRC may be involved in
the
response. Although the inhibition by FK506 could result from binding to either
calcineurin
or FK506-binding proteins, natural products that bind tightly to these
proteins (e.g.,
rapamycin .and cyclosporin A, respectively) do not diminish the Bz-423 OZ
response (Table
2). Table 2 shows the effect of ROS inhibitors on the activity of Bz-423.
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Table 2
Target ~~~~ Inhibitor % DHE positive: % DHE positive: itelarive
Inhibitor alone , Bz-423 plus response~
inhibitor
DMSO control 7 72 100 (~13)
MRC NaN3 0 0 1 (~l)
( 1 mM)
MRC/Calcineurin FK506 1 10 14 (~10)
( 1 ~M)
FK506-binding proteins Rapamycin 1 ~ 60 83 (~2)
(1 ~M)
Calcineurin Cyclosporin A 2 60 83 (~2)
(0.5~M)
Cytochrome P450s Benzylimidazole 14 , , ' 90 ' 126 (~7)
(100 ~M)
Cytochrome P450s SKF525A 12 92 128 (~6)
(5 ~M)
Cytochrome P450 Cimetidine 6 83 116 (~7)
(CYP231) (100 ~M)
Cyclooxygenase Indomethacin (100 4 69 97 (~4)
~M)
Monoaminoxidase Phenelzine 5 78 109 (~4)
( 10 ~.M)
Xanthine Oxidase Allopurinol 6 79 111 (~7)
( 100 ~.M)
A. Data are normalized to the response of Bz-423 alone.
Although an understanding of the mechanism;is not necessary to practice the
present
invention and the present invention is not so limited, it is contemplated that
the way in
which Bz-423 produces Oz involves binding to a protein within mitochondria or
a target in
another compartment that signals mitochondria to generate OZ . To distinguish
between
these alternatives, isolated rat liver mitochondria were assayed for ROS
production in the
presence and absence of Bz-423. ROS were detected by monitoring the oxidation
of
DCFH-DA to DCF as previously described in Esposti. (See, M.D. Esposti, Methods
Cell.
Biol., 65:75-96 [2001 ]). In this assay, the rate of DCF production increases
after a lag
period of ca. 15 min, during which time endogenous reducing equivalents within
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mitochondria are consumed and acetate moieties on the probe are hydrolyzed to
yield
DCFH, which is the redox-active species. Under aerobic conditions supporting
state 3
respiration (succinate plus ADP), both antimycin A, which generates Oz by
inhibiting
ubiquinol-cytochrome c reductase witlun complex III, and Bz-423 increase the
rate of ROS
production nearly two-fold relative to solvent control (See, Esposti, ifzf
°a.) (Figure 7A).
Mitochondria) swelling is not observed demonstrating that Bz-423 does not
directly target
the MPT pore. (A.S. Belzacq et al., Cancer Res., 61:1260-1264 [2001]). (Figure
7B).
Neither Bz-423 nor antimycin A generate substantial ROS in the subcellular S
15 fraction
(cytosol and microsomes; Figure 7C), and Bz-423 does not stimulate ROS if
mitochondria
are in state 4 (succinate plus oligomycin), even though antimycin A is active
under these
conditions (Figure 7D). Together, these experiments 3emonstrate that
mitochondria contain
a molecular target for Bz-423, and state 3 respiration is required for the OZ
response.
To relate data obtained with mitochondria to observations in whole cells,
isolated
mitochondria were also probed with DHE and DIOCG(3). In both state 3 and 4,
mitochondria fluoresce brightly with DIOC~(3), indicating that O~I'", is
intact (Figure 7E).
Addition of Bz-423 does not alter the DIOC~(3) signal over the course of
measurement,
indicating that O~I'", has not collapsed. In contrast, the protonophore CCCP
disrupts O~I'""
which abolishes the green fluorescence. Oxidation of DHE by endogenous OZ
produces red
fluorescence within mitochondria. Bz-423 does not induce Oz over control in
state 4.
However, DHE fluorescence is markedly increased after incubating mitochondria
with Bz-
423 in buffer favoring state 3 respiration and reflects elevated levels of 02
. Collectively,
these experiments confirm the results of the DCF, assay; demonstrate an ROS
response in
mitochondria consistent with that seen in whole cells, and provide evidence to
support the
hypothesis that the early OZ does not result from,collapse of the
mitochondria)
transmembrane gradient (See, Figure 6B).
In some embodiments, the present invention contemplates that Bz-423-induced
apoptosis depends on the early OZ response. To determine if OZ is needed for
Bz-423-
induced apoptosis, some embodiments of the present invention probed the
dependency of
the apoptotic process on ROS. Pre-treating cells with FI~506, which prevents
formation of
Bz-423-induced OZ , significantly inhibits caspase activation, mitochondria)
depolarization,
DNA fragmentation, and cell death (Figure ~A). Moreover, after cells are
incubated with
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Bz-423 for ca. 1 h, which is the point at which the early OZ response is
maximal (See,
Figure 6B), addition of FK506 provides significantly less protection against
cell death
(Figure 8B). Pre-incubating cells with vitamin E, an antioxidant that
scavenges ROS, or
MnTBAP, an OZ dismutase mimetic, also attenuates each of these endpoints
(Figure 8A).
MnTBAP is somewhat less effective at influencing the downstream effectors
engaged by
Bz-423 because dismutation of OZ yields H202, which also triggers the same
effectors (T.
Ohse et al., J. W or. Biochem., 85:201-208 [2001]) (Figure 8A). These data
demonstrate that
cell billing by Bz-423 depends on Oz .
Still further embodiments of the present invention provide methods of using Bz-
423
that induce ROS and kill primary B lymphocytes in~vivo. In the NZB/W model of
lupus, ,
splenic GC B cells are hyperactivated and pathologically expanded. Somatic
hypermutation
within this compartment produces anti-DNA and other autoantibodies, a subset
of which are
pathogenic and contribute to the development of glomerulonephritis (See e.g.,
J.P.
Portanova et al., Mol. Immunol., 32:117-135 [1987]; Y. Munakata et al., Eur.
J. hnmunol.,
28:1435-1444 [1998]; and A.N. Theofilopoulos and F.J. Dixon, Adv. Immunol.,
37:269-390
[1985]). Because Ramos cell behavior parallels many of the responses of GC B
cells (See,
C.T. Gregory et al., J. Immunol., 139:313-318 [1987]),,preferred embodiments
of the
present invention contemplate that Bz-423 has activity in NZB/W mice that
could be
therapeutically useful. To characterize the effect of Bz-423 on NZB/W
lymphocytes, mice
were given a single dose of either Bz-423 (60 mg/lcg; n = 4) or vehicle (n =
4) and sacrificed
after 2 h. This dose provides peak serum levels of ca. ,5 ~,M 1 h post-
injection, which is near
the ECSO ira vitro (See, Figure SB). Bz-423 induces robust ROS production in
the spleen
compared to control. Based on these findings, Bz-423~was administered (60
mg/kg/d) for 7
d to determine whether it was lymphotoxic in the murine model. A short time
frame was
chosen so that cellular recruitment or proliferation would not significantly
alter splenocyte
populations. Flow cytometric analysis after sacrifice shows that lymphocyte
viability in the
,.,
Bz-423-treated animals is decreased (81 ~ 1 vs. 85 ~ 1; P = 0.006) and B cell
apoptosis is
increased versus controls (32 ~ 2 vs. 25 ~ 2; P = 0.02). The decrease in
viability is
relatively small because early apoptotic cells are rapidly cleared by the
reticuloendothelial
system in vivo (N. Zamzami et al., J. Exp. Med. 182:367-377 [19,95]). An
increase in the
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frequency of T cell apoptosis was not observed (12 ~ 4 for both control and
treatment; P =
0.3).
Bz-423 reduces lymphoproliferative and autoimmune disease in NZB/W mice. In
certain embodiments, additional studies were conducted to determine if Bz-423
alters the
progression of disease in NZB/W mice. The endpoints of these studies were
measures of
GC B cell hyperplasia, glomerulonephritis, and autoantibody titers,
respectively. In one
protocol, female NZB/W mice were given Bz-423 (60 mg/lcg/eveiy other day; n =
25) or
vehicle (aqueous DMSO; n = 20) from 6.5 to 9.5 months of age. This time frame
begins
after the histological onset of disease and continues to when severe nephritis
is usually
observed. (See e.g., A.N. Theofilopoulos and F.J. Dixon, Adv, linmunol.,
37:269-390
[1985]). Disease-related GC hyperplasia in NZB/W mice leads to an expansion of
the white
pulp that distorts normal splenic architecture. Analyzing spleen sections from
all treatment
and control animals revealed a reduction in the white pulp in mice receiving
Bz-423 (1-2+
vs. 3-4+; P = 0.018). Mice dosed with Bz-423 have 40% fewer GCs relative to
controls (10
+ 2 vs. 17 + 1 per 10 mm2, P = 0.009), and the GCs in treated mice are 40%
smaller than in
the controls (20 + 2 vs. 35 + 5 x103 mm2, P = 0.013). Furthermore, spleens
from Bz-423-
treated animals have more TLJNEL-positive B cells within GCs (ca. 3+ vs. 1+, P
= 0.038)
than controls, and such differences in TUNEL staining are not observed in
other areas of the
spleen. These observations suggest that the decrease in GCs may result from
increased
apoptosis within this compartment. In some embodiments, the changes in
splenocyte
populations were also investigated by flow cytometry. At the end of the study,
a specific
effect on B cells was observed (Bz-423: 59 ~ 3, control:, 67~ 2%; P = 0.03)
with no
measurable effect on T cells (Bz-423: 16 ~ 2, control: 17~ 2%; P = 0.5), which
is
consistent with the histochemical findings.
Based on histology, 60% of controls (12 of 20) had severe nephritis (?2+),
while
only 16% of Bz-423-treated mice (4 of 25) had disease (c2: P = 0.003). Disease
in the
control animals is characterized by diffuse, proliferative glomerulonephritis
with expansion
of all cellular elements and occasional wire-loop formation, consistent with
an average
histopathological score of 3+. Iii contrast, Bz-423 mice have milder changes
(1+, P =
0.002) and less glomerular deposition of IgG (1+ for Bz-423 vs. 3+ in
controls, P = 0.002).
At the end of the study, 83% of controls had abnormally high BLTN (? 30
mg/dL), compared
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to 27% of the treatment group (c2: P = 0.001). A similar trend was observed
with
proteinuria: 49% of controls had significant proteinuria, (>100 mg/dL)
compared to 18% of
Bz-423-treated mice (c2: P = 0.1). Collectively, these data show that mice
administered Bz-
423 had less disease than controls.
Treating NZB/W mice with lymphotoxic drugs life azathioprine and
methylprednisolone can reduce nephritis without significantly altering total
serum anti-
DNA levels. (See e.g., M.C. Gelfand and A.D. Steinberg, Arthritis Rheum.,
15:247-255
[1972]). Similarly, anti-DNA and IgG titers were measured after terminating
the study and
siguficant differences between the groups were not observed (e.g., Bz-423 anti-
dsDNA:
789 ~ 145, control: 733 ~ 198 IT/mL; P = 0.5; Bz-423 IgG: 6.7 ~ 0.8, control:
5.5 ~ 1.0
mg/mL; P = 0.3). Although autoantibodies are produced by several B cell
subtypes, Bz-423
reduces GCs, the site of pathogenic autoantibody development.
III. Synthesis of exemplary benzodiazepine derivatives
The compounds of the present invention are.benzodiazepine compounds. In some
aspects, the benzodiazepine compounds have the following structure:
or
R~
O
N
Ra I ~ R2
~N\
I/) R3
O
or its enantiomer, wherein, R, is aliphatic or aryl; RZ is aliphatic, aryl, -
NH2, -NHC(=O)-R5;
or a moiety that participates in hydrogen bonding, wherein RS is aryl,
heterocyclic, -R6 NH-
CA 02457405 2004-02-11
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C(=O)-R, or -R~ C(=O)-NH-R,, wherein R6 is an aliphatic linker of 1-6 carbons
and R, is
aliphatic, aryl, or heterocyclic, each of R3 and R4 is independently a
hydroxy, alkoxy, halo,
amino, lower-allcyl-substituted-amino, acetylamino, hydroxyamino, an aliphatic
group
having 1-8 carbons and 1-20 hydrogens, aryl, or heterocyclic; or a
pharmaceutically
acceptable salt, prodrug or derivative thereof.
In the above structures, R, is a hydrocarbyl group of 1-20 carbons and 1-20
hydrogens. Preferably, RI has 1-15 carbons, and more preferably, has 1-12
carbons.
Preferably, R, has 1-12 hydrogens, and more preferably, 1-10 hydrogens. Thus
R, can be an
aliphatic group or an aryl group.
The term "aliphatic" represents the groups commonly known as alkyl, alkenyl,
allcynyl, alicyclic. The teen "aryl" as used herein represents a single
aromatic ring such as a
phenyl ring, or two or more aromatic rings that are connected to each other
(e.g., bisphenyl)
or fused together (e.g., naphthalene or anthracene). The aryl group can be
optionally
substituted with a lower aliphatic group (e.g., C,-C4 all~yl, allcenyl,
allcynyl, or C3-C6
alicyclic). Additionally, the aliphatic and aryl groups can be further
substituted by one or
more functional groups such as -NHz, -NHCOCH3, -OH, lower alkoxy (C,-C4), halo
(-F, -Cl,
-Br, or -I). It is preferable that Rl is primarily a nonpolar moiety.
In the above structures, RZ can be aliphatic, aryl, -NH2, -NHC(=O)-R5, or a
moiety
that participates in hydrogen bonding, wherein R5, is aryl, heterocyclic, R~
NH-C(=O)-R~ or
-R~ C(=O)-NH-R~, wherein RG is an aliphatic linker of 1-6 carbons and R~ is an
aliphatic,
t
aryl, or heterocyclic. The terms "aliphatic" and "aryl" are as defined above.
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 alleoxy, lower carbonyl, lower carboxyl, lower
ethers and
phenolic groups. The qualifier "lower" as used herein refers to lower
aliphatic groups (C1-
C4) to which the respective oxygen-containing functional group is attached.
Thus, for example, the term "lower carbonyl" refers to ihtey~ alia,
formaldehyde,
acetaldehyde.
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Some nonlimiting examples of nitrogen-containing groups that pa~.-ticipate 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 vitro, N-hydroxy and nitrous
groups.
It is also possible that the hydrogen-bond acceptor in the present invention
can be
the ~ electrons of an aromatic ring. However, the hydrogen bond participants
of this
invention do not include those groups containing metal atoms such as boron.
Further the
hydrogen bonds formed within the scope of practicing this invention do not
include those
formed between two hydrogens, known as "dihydrogen bonds." (See, R.H.
Crabtree,
Science, 282:2000-2001 [1998], for further description of such dihydrogen
bonds).
The term "heterocyclic" represents, for example, a 3-6 membered aromatic or
nonaromatic ring containing one or more heteroatoms: ,The heteroatoms can be
the same or
different from each other. Preferably, at least one of the heteroatom's is
nitrogen. Other
heteroatoms that can be present on the heterocyclic ring include oxygen and
sulfur.
Aromatic and nonaromatic heterocyclic rings axe 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 axe 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.
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, vitro, acetyl (i.e., -C(=O)-CH3), or aryl groups.
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Each of R3 and R4 can be independently a hydroxy, alkoxy, halo, amino, or
substituted amino (such as lower-allcyl-substituted-amino, or acetylamino or
hydroxyaxnino), or an aliphatic group having 1-8 carbons and 1-20 hydrogens.
When each
of R3 and R4 is an aliphatic group, it can be further substituted with one or
more functional
groups such as a hydroxy, alkoxy, halo, amino or substituted amino groups as
described
above. The terms "aliphatic" is defined above. Alternatively, each of R3 and
R4 can be
hydrogen.
It is well-known that many 1,4-benzodiazepines exist as optical isomers due to
the
clurality introduced into the heterocyclic ring at tile C3 position. The
optical isomers are
sometimes described as L- or D-isomers in the literature. Alternatively, the
isomers are also
referred to as R- and S- enantiomorphs. For the sake of simplicity, these
isomers are
referred to as enantiomorphs or enantiomers. The 1,4-benzodiazepine compounds
described
herein include their enantiomeric forms as well as racemic mixtures. Thus, the
usage
"benzodiazepine or its enantiomers" herein refers to the benzodiazepine as
described or
depicted, including all its enantiomorphs as well as their racemic mixture.
From the above description, it is apparent that many specific examples are
represented by the generic formulas presented above. Thus, in one example, Rl
is aliphatic,
RZ is aliphatic, whereas in another example, Rl is aryl and Rz is a moiety
that participates in
hydrogen bond formation. Alternatively, R, can be aliphatic, and RZ can be an -
NHC(=O)-
R5, or a moiety that participates in hydrogen bonding, wherein RS is aryl,
heterocyclic, -R6
NH-C(=O)-R, or -R~ C(=O)-NH-R~, wherein R6 is an aliphatic linker of 1-6
carbons and R~
is an aliphatic, aryl, or heterocyclic. 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.
Further, it should be understood that the numerical ranges given throughout
this
disclosure should be construed as a flexible range that contemplates any
possible subrange
within that range. For example, the description of a group having the range of
1-10 carbons
would also contemplate a group possessing a subrange of, for example, 1-3, 1-
5, 1-8, or 2-3,
2-5, 2-8, 3-4, 3-5, 3-7, 3-9, 3-10, etc., carbons. Thus, the range 1-10 should
be understood
to represent the outer boundaries of the range within which many possible
subranges are
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clearly contemplated. Additional examples contemplating ranges in other
contexts can be
found throughout this disclosure wherein such ranges include analogous
subranges within.
Some specific examples of the benzodiazepine compounds of this invention
include:
10
Hy;
G
1
N
N N
CI '~ .i' N
O O
OH
64
Image
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N!
fi
N
Q NOz
In summary, a large number of benzodiazepine compounds are presented herein.
Any one or more of these benzodiazepine compounds can be used to treat a
variety of
dysregulatory disorders related to cellular death as described elsewhere
herein. The above-
described benzodiazepines can also be used in drug screening assays and other
diagnostic
methods.
IV. Pharmaceutical compositions, formulations, and exemplary administration
routes and dosing considerations
Exemplary embodiments of various contemplated medicaments and pharmaceutical
compositions are provided below.
A. Benzodiazepine derivatives as compounds for preparing medicaments
The benzodiazepine compounds of the present invention useful in the
preparation of
medicaments to treat a variety of conditions associated with dysregulation of
cell death,
aberrant cell growth and hyperproliferation.
In addition, the compounds are also useful for preparing medicaments for
treating
other disorders wherein the effectiveness of the benzodiazepines are known or
predicted.
Such disorders may include, but are not limited to, neurological (e.g.,
epilepsy) or
neuromuscular disorders. The methods and techniques for preparing medicaments
of a
compound are well-known in the art. Exemplary pharmaceutical formulations and
routes of
delivery are described below.
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
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standard pharmaceutical manufacturing procedures. Such medicaments can be
delivered to
the subj ect by using delivery methods that are well-lmown in the
pharmaceutical arts.
B. Exemplary pharmaceutical compositions and formulation
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
(e.g.,
benzodiazepine derivative), as defined above, together with a solid support or
alternatively,
together with one or more pharmaceutically acceptable carriers and optionally
other
therapeutic agents. Each Garner must be "acceptable" in the sense that it is
compatible with
the other ingredients of the formulation and not injurious to the subject.
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.
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.
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),
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lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch
glycolate, cross-linked
povidone, cross-linlced 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.
m 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 coiriprising the active
ingredient in a
suitable liquid carrier.
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 alternatively
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 compounds) that
enhances
absorption or penetration of the active agents) through the slcin or other
affected areas.
Examples of such dermal penetration eWancers include dimethylsulfoxide (DMSO)
and
related analogues.
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, maimitol, sorbitol, glycerol and
polyethylene glycol
and mixtures thereof.
In some embodiments, oily phase emulsions of this invention are constituted
from
known ingredients in an known manner. This phase typically comprises an lone
emulsifier
(otherwise known as an emulgent), it is also desirable in some embodiments for
this phase
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to further comprises a mixture of at least one emulsifier with a fat or an oil
or with both a fat
and an oil.
Preferably, a hydrophilic emulsifier is included together with a lipophilic
emulsifier
so as to act as a stabilizer. It some embodiments it is also preferable to
include both an oil
and a fat. Together, the emulsifiers) with or without stabilizers) male 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.
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 lau~yl sulfate.
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 palmitat~ 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.
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.
Formulations for rectal administration may be presented as a suppository with
suitable base comprising, for example, cocoa butter or a salicylate.
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 l~nown in the art to be appropriate.
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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 admiiustered 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 Garner 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.
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
Garner, 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.
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.
It should be understood that in addition to the ingredients particularly
mentioned
above, the formulations of this invention may ixiclude 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, thiclceners 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 linoleic acid), extenders, and
stabilizers, etc.
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C. Exemplary administration routes and dosing considerations
Various delivery systems are known and can be used to administer a therapeutic
agents (e.g., benzodiazepine derivatives) 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, infra-arterial, infra-
muscular,
intravenous, intranasal, and oral routes. In specific embodiments, it may be
desirable to
achninister 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.
The agents identified herein as effective for their intended purpose can be
administered to subjects or individuals susceptible to or at risk of
developing pathological
growth of target cells and condition correlated with this. 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 determine 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.
When delivered
to an animal, the method is useful to further confirm efficacy of the agent.
One example of
an animal model is MLRlMpJ-lprllpr ("MLR-lpr") (available from Jacl~son
Laboratories,
Bal Harbor, Maine ). MLR-lp~ mice develop systemic autoimmune disease.
Alternatively,
other animal models can be developed by inducing tumor growth, for example, by
subcutaneously inoculating nude mice with about 105 to about 10~
hyperproliferative, cancer
or target cells as defined herein. When the tumor is established, the
compounds described
herein are administered, for example, by subcutaneous injection around the
tumor. Tumor
measurements to determine reduction of tumor size are made in two dimensions
using
venier calipers twice a week. Other animal models may also be employed as
appropriate.
Such animal models for the above-described diseases and conditions are well-
l~nown in the
art.
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In some embodiments, ira 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.
Suitable dosage formulations and methods of administering the agents are
readily
determined by those of sleill 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/lcg to about
100 mg/kg,
even more preferably at about 0.5 mg/lcg to about 50 mg/lcg. 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.
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 talce
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.
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,
intrasnuscular, 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.
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.
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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.
D. Exemplary co-administration routes and dosing considerations
The present invention also includes methods involving co-adminstration 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-admiustering 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.
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
autoirninune disorder, the additional agent can be an immunosuppressant or an
anti-
inflarmnatory 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-lcnown 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 slcill in the art or can be determined with relative ease.
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 l~nown therapeutic agents. In certain cancers, for example,
resistance to
chemicals and radiation therapy has been shown to be associated with
inhibition of
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apoptosis. Some therapeutic agents have deleterious side effects, including
non-specific
lymphotoxicity, renal and bone marrow toxicity.
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 appear to sensitize target cells to known agents and,
accordingly, less of
these agents are needed to achieve a therapeutic benefit.
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.
V. Mitochondria) ATP synthase (mitochondria) FoF, ATPase) activity modulators
hl particularly preferred embodiments, the compositions (e.g., benzodiazepine
derivatives) of the present invention 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, etc.) by modulating (e.g., inhibiting or
promoting) the
activity of the mitochondria) ATP synthase (as referred to as mitochondria)
FoF, ATPase)
complexes in affected cells or tissues. In particularly preferred embodiments,
the
compositions of the present invention inhibit the activity of mitochondria)
ATP synthase
complex by binding to a specific subunit of this multi-subunit protein
complex. While the
present invention is not limited to any particular mechanism, nor to any
understanding of
the action of the agents being administered, in some embodiments, the
compositions of the
present invention bind to the oligomycin sensitivity conferring protein (OSCP)
portion of
the mitochondria) ATP synthase complex. Lilcewise, it is further contemplated
that when
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the compositions of the present invention bind to the OSCP the initial affect
is overall
inhibition of the mitochondria) ATP synthase complex, and that the downstream
consequence of binding is a change in ATP level and the production of reactive
oxygen
species (e.g., OZ-). In still other preferred embodiments, while the present
invention is not
limited to any particular mechanism, nor to any understanding of the action of
the agents
being adminstered, it is contemplated that the generation of free radicals
ultimately results
in cell billing. In yet other embodiments, while the present invention is not
limited to any
particular mechanism, nor to any understanding of the action of the agents
being
administered, it is contemplated that the inhibiting mitochondria) ATP
synthase complex
using the compositions and methods of the present invention provides
therapeutically useful
inhibition of cell proliferation.
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) the activity of the mitochondria) ATP
synthase
complexes in affected cells or tissues via binding to the oligomycin
sensitivity confernng
protein (OSCP) portion of the mitochondria) ATP synthase complex. Importantly,
by itself
the OSCP has no biological activity.
Thus, in one broad sense, preferred embodiments of the present invention are
directed to the discovery that many diseases characterized by dysregulation of
necrosis
and/or apoptosis processes in a cell or tissue, or diseases characterized by
aberrant cell
growth and/or hyperproliferation, ete., can be treated by modulating the
activity of the
mitochondria) ATP synthase complex including, but not limited to, by binding
to the
oligomycin sensitivity conferring protein (OSCP) component thereof. The
present
invention is not intended to be limited, however, to the practice of the
compositions and
methods explicitly described herein. Indeed, those sbilled in the art will
appreciate that a
number of additional compounds not specifically recited herein (e.g., non-
benzodiazepine
derivatives) are suitable for use in the methods disclosed herein of
modulating the activity
of mitochondria) ATP synthase.
The present invention thus specifically contemplates that any number of
suitable
compomds presently brown in the art, or developed later, can optionally find
use in the
methods of the present invention. For example, compounds including, but not
limited to,
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oligomycin, ossamycin, cytovaricin, apoptolidin, bafilomyxcin, and
dicyclohexylcarbodiimide (DCCD), and the like, find use in the methods of the
present
invention. The present invention is not intended, however, to be limited to
the methods or
compounds specified above. In one embodiment, that compounds potentially
useful in the
methods of the present invention may be selected from those suitable as
described in the
scientific literature. (See e.g., K.B. Wallace and A.A. Starkov, Annu. Rev.
Pharmacol.
Toxicol., 40:353-388 [2000]; A.R. Solomon et al., Proc. Nat. Acad. Sci.
U.S.A.,
97(26):14766-14771 [2000]).
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. Moncs 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.
In one aspect, derivatives (e.g., pharmaceutically acceptable salts, analogs,
stereoisomers, and the lilce) of the exemplary compounds or other suitable
compounds are
also contemplated as being useful in the methods of the present invention.
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 ark not limited to, the
toxicity, safety,
efficacy, availability, and cost of the particular compounds.
VI. Drug screens
In preferred embodiments of the present invention, the compounds of the
present
invention, and other potentially useful compounds, are screened for their
binding affinity to
the oligomycin sensitivity conferring protein (OSCP) portion of the
mitochondrial ATP
synthase complex. In particularly preferred embodiments, compounds are
selected for use
in the methods of the present invention by measuring their biding affinity to
recombinant
OSCP protein. 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 iya vivo or ex vivo systems. While in some embodiments
quantifying the
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intracellular level of ATP following administration of the compounds of the
present
invention provides an indication of the efficacy of the methods, preferred
embodiments of
the present invention do not require intracellular ATP 'level quantification.
Additional embodiments are directed to measuring levels (e.g., intracellular)
of
superoxide in cells and/or tissues to measure the effectiveness of particular
contemplated
methods and compounds of the present invention. In this regard, those skilled
in the art will
appreciate and be able to provide a number of assays and methods useful for
measuring
superoxide levels in cells and/or tissues
In some embodiments, structure-based virtual screening methodologies are
contemplated for predicting the binding affinity of compounds of the present
invention with
OSCP. .
Any suitable assay that allows for a measureyent of the rate of binding or the
affinity of a benzodiazepine or other compound to the. OSCP may be utilized.
Examples
include, but are not limited to, surface plasma resonaoe.(SPR) and radio-
immunopreciptiation assays (Lowman et al., J. Biol.Chem. 266:10982 [1991]).
Surface
Plasmon Resonance techniques involve a surface coated with a thin film of a
conductive
metal, such as gold, silver, chrome or aluminum, in which electromagnetic
waves, called
Surface Plasmons, can be induced by a beam of light incident on the metal
glass interface at
a specific angle called the Surface Plasmon Resonance angle. Modulation of the
refractive
index of the interfacial region between the solution and the metal surface
following binding
of the captured macromolecules causes a change in the SPR angle which can
either be
measured directly or which causes the amount of light reflected from the
underside of the
metal surface to change. Such changes can be directly,related to the mass and
other optical
properties of the molecules binding to the SPR device surface. Several
biosensor systems
based on such principles have been disclosed (See e.g., WO 90/05305). There
are also
several commercially available SPR biosensors (e.g., BiaCore, Uppsala,
Sweden).
In some embodiments, benzodiazepine copmpounds are screened in cell culture or
ira
vivo (e.g., non-human or human mammals) for their ability to modulate
mitochondrial ATP
synthase activity. Any suitable assay may be utilized, including, but not
limited to, cell
proliferation assays (Commercially available from, e.g., Promega, Madison, WI
and
Stratagene, La Jolla, CA) and cell based dimerization, assays. (See e.g., Fuh
et al., Science,
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256:1677 [1992]; Colosi et al., J. Biol. Chem., 268:12617 [1993]). Additional
assay
formats that find use with the present invention include, but are not limited
to, assays for
measuring cellular ATP levels, and cellular superoxide levels.
The present invention also provides methods of modifying and derivatizing the
compositions of the present invention to increase desirable properties (e.g.,
binding affinity,
activity, and the life), or to minimize undesirable properties (e.g.,
nonspecific reactivity,
toxicity, and the life). The principles of chemical derivatization are well
understood. In
some embodiments, iterative design and chemical synthesis approaches are used
to produce
a library of derivatized child compounds from a parent compound. In other
embodiments,
rational design methods are used to predict and model i~. silico ligand-
receptor interactions
prior to confirming results by routine experimentation.
EXAMPLES
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 ,
Preparation of Compounds
The benzodiazepine compounds are prepared using either solid-phase or soluble-
phase combinatorial synthetic methods as well as on an individual basis from
well-
established techniques. (See e.g., C.J. Boojamra et al.., J. Org. Chem.,
62:1240-1256
[1996]); B.A. Bunin et al., Proc. Natl. Acad. Sci.~USA, 91:4708-4712 [1994];
S.Y. Stevens
et al., J. Am. Chem. Soc., 118:10650-10651 [1996]; E.M. Gordon et al., J. Med.
Chem.,
37:51385-1401 [1994]; and U.S. 4,110,337 and 4,076,823, which are all
incorporated by
reference herein in their entirety. For illustration, the following general
methodologies are
provided.
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Preparation of 1,4-benzodiazepine-2-one compounds
Improved solid-phase synthetic methods for the preparation of a variety of 1,4-
benzodiazepine-2-one derivatives with very high overall yields have been
reported in the
literature. (See e.g., Bunin and Ellinan, J. Am. Chem. Soc., 114:10997-10998
[1992]).
Using these improved methods, the 1,4-benzodiazepine-2-ones is constructed on
a solid
support from three separate components: 2-aminobenzophenones, a-amino acids,
and
(optionally) alkylating agents.
Preferred 2-aminobenzophenones include the substituted 2-aminobenzophenones,
for example, the halo-, hydroxy-, and halo-hydroxy-substituted 2-
aminobenzophenones,
such as 4-halo-4'-hydroxy-2-aminobenzophenones. A preferred substituted 2-
aminobenzophenone is 4-chloro-4'-hydroxy-2-aminobenzophenone. Preferred a-
amino
acids include the 20 common naturally occurring, a-amino acids as well as a-
amino acid
mimicl~ing structures, such as homophenylalanine, homotyrosine, and thyroxine.
Alkylating agents include both activated and inactivated electrophiles, of
which a
wide variety are well lcnown in the art. Preferred allcylating agents include
the activated
electrophiles p-bromobenzyl bromide and t-butyl-bromoacetate.
W the first step of such a synthesis, the 2-amiriobenzophenone derivative is
attached
to a solid support, such as a polystyrene solid support, through either a
hydroxy or
carboxylic acid functional group using well known methods and employing an
acid-
cleavable liner, such as the commercially available [4-
(hydroxymethyl)phenoxy]acetic
acid, to yield the supported 2-aminobenzophenone. '(See e.g., Sheppard and
Williams, Intl.
,.
J. Peptide Protein Res., 20:451-454 [1982]). The 2-amino group of the
aminobenzophenone
is preferably protected prior to reaction with the linking reagent, for
example, by reaction
with FMOC-Cl (9-fluorenylmethyl chloroformate) to yield the protected amino
group 2'-
NHFMOC.
In the second step, the protected 2-amino group is deprotected (for example,
the -
NHFMOC group may be deprotected by treatment with piperidine in
dimethylformamide
(DMF)), and the unprotected 2-aminobenzophenone is then coupled via an amide
linkage to
an a-amino acid (the amino group of which has itself been protected, for
example, as an -
NHFMOC group) to yield the intermediate. Standard activation methods used for
general
solid-phase peptide synthesis are used (such as the use of carbodiimides and
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hydroxybentzotriazole or pentafluorophenyl active esters) to facilitate
coupling. However, a
preferred activation method employs treatment of the 2-aminobenzophenone with
a
methylene chloride solution of the of a,-N-FMOC-amino acid fluoride in the
presence of the
acid scavenger 4-methyl-2,6-di-tert-butylpyridine yields complete coupling via
an anode
linkage. This preferred coupling method has been found to be effective even
for unreactive
aminobenzophenone derivatives, yielding essentially complete coupling for
derivatives
possessing both 4-chloro and 3-carboxy deactivating substituents.
In the third step, the protected amino group (which originated with the amino
acid)
is first deprotected (e.g., -NHFMOC may be converted ~to -NHZ with piperidine
in DMF),
and the deprotected Bz-423s reacted with acid, for example, 5% acetic acid in
DMF at
60°C, to yield the supported 1,4-benzodiazepine derivative. Complete
cyclization has been
reported using this method for a variety of 2-aminobenzophenone derivatives
with widely
differing steric and electronic properties.
In an optional fourth step, the 1,4-benzodiazepine derivative is alkylated, by
reaction
with a suitable alkylating agent and a base, to yield the supported fully
derivatized 1,4-
benzodiazepine. Standard alkylation methods, for eXample, an excess of a
strong base such
as LDA (lithium diisopropylamide) or NaH, is used; however, such methods may
result in
undesired deprotonation of other acidic functionalities and over-alkylation.
Preferred bases,
which may prevent over-alkylation of the benzodiazepine derivatives (for
example, those
with ester and carbamate functionalities), are those which are basic enough to
completely
deprotonate the anilide functional group, but not basic enough to deprotonate
amide,
carbamate or ester functional groups. An example of such a base is lithiated 5-
(phenyhnethyl)-2-oxaxolidinone, which is reacted with the 1,4-benzodiazepine
in
tetrahydrofuran (THF) at -78 °C. Following deprotonation, a suitable
allcylating agent, as
described above, is added.
In the final step, the fully derivatized 1,4-benzodiazepine is cleaved from
the solid
support. This is achieved (along with concomitant removal of acid-labile
protecting
groups), for example, by exposure to a suitable acid, such as a mixture of
trifluoroacetic
acid, water, and dimethylsulfide (85:5:10, by volume). Alternatively, the
above
benzodiazepines is prepared in soluble phase. The synthetic methodology was
outlined by
Gordon et al., J. Med. Chem., 37:1386-1401 [1994]) which is hereby
incorporated by
CA 02457405 2004-02-11
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reference. Briefly, the methodology comprises trans-imidating an amino acid
resin with
appropriately substituted 2-aminobenzophenone imines to form resin-bound
imines. These
imines are cyclized and tethered by procedures similar to those in solid-phase
synthesis
described above. The general purity of benzodiazepines prepared using the
above
methodology is about 90% or higher.
Preparation of 1,4-benzodiazepine-2,5-diones
A general method for the solid-phase synthesis of 1,4-benzodiazepine-2,5-
diones has
been reported in detail by C.J. Boojamra et al., J. Org. Chem., 62:1240-1256
[1996]). This
method is used to prepare the compounds of the present invention.
A Merrifield resin, for example, a (chloromethyl)polystyrene is derivatized by
allcylation with 4-hydroxy-2,6-dimethoxybenzaldehyde sodium to provide resin-
bound
aldehyde. An oc-amino ester is then attached to the elerivatized support by
reductive
amination using NaBH(OAc)3 in 1% acetic acid in DMF. This reductive amination
results
in the formation of a resin-bound secondary amine.
The secondary amine is acylated with a wide variety of unprotected anthranilic
acids
result in support-bound tertiary amides. Acylation is best achieved by
performing the
coupling reaction in the presence of a carbodiimide and the hydrochloride salt
of a tertiary
amine. One good coupling agent is 1-ethyl-8-[8-(dimethylamino)propyl]
carbodiimide
hydrochloride. The reaction is typically performed in the presence of
anhydrous 1-methyl
2-pyrrolidinone. The coupling procedure is typically repeated once more to
ensure
complete acylation.
Cyclization of the acyl derivative is accomplished through base-catalyzed
lactamation through the formation of an anilide anion which would react with
an alkylhalide
for simultaneous introduction of the substituent at the 1-position on the
nitrogen of the
heterocyclic ring of the benzodiazepine. The lithium salt of acetanilide is a
good base to
catalyze the reaction. Thus, the Bz-423s reacted with.lithium acetanilide in
DMF/THF (1:1)
for 30 hours followed by reaction with appropriate all~ylating agent provides
the fully
derivatized support-bound benzodiazepine. The compounds are cleaved from the
support in
good yield and high purity by using TFA/DMS/HZO (90:5:5).
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Some examples of the oc-amino ester starting-materials, alkylating agents, and
anthranilic acid derivatives that are used in the present invention are listed
by C.J. Boojamra
et al., J. Org. Chem., 62:1240-1256 [1996], supra at 1246. Additional reagents
are readily
determined and either are commercially obtained or readily prepared by one of
ordinary
slcill in the art to arrive at the novel substituents disclosed in the present
invention.
For example, from Boojamra, supra, one realizes that: all~ylating agents
provide the
Rl substituents; a-amino ester starting materials provide the RZ substituents,
and anthranilic
acids provide the R4 substituents. By employing these starting materials that
are
appropriately substituted, one arrives at the desired 1,4-benzodiazepine-2,5-
dione. The R3
substituent is obtained by appropriately substituting the amine of the a-
aminoester starting
material. If steric crowding becomes a problem, ,the R3 substituent is
attached through
conventional methods after the 1,4-benzodiazepine-2,5-dione is isolated.
Example 2
Chirality
It should be recognized that many of the benzodiazepines of the present
invention
exist as optical isomers due to chirality wherein the stereocenter is
introduced by the a-
amino acid and its ester starting materials. The above-described general
procedure
preserves the chirality of the a-amino acid or ester starting materials. In
many cases, such
preservation of chirality is desirable. However, when the desired optical
isomer of the oc-
amino acid or ester starting material is unavailable or expensive, a racemic
mixture is
produced which is separated into the correspondiyg optical isomers and the
desired
benzodiazepine enantiomer is isolated.
For example, in the case of the 2,5-dione compounds, Boojamra, supra,
discloses
that complete racemization is accomplished by preequilibrating the
hydrochloride salt of the
enantiomerically pure oc-amino ester starting material with 0.3 equivalents of
i-Pr2EtN and
the resin-bound aldehyde for 6 hours before the addition of NaBH(OAc)3. The
rest of the
above-described synthetic procedure remains the same. : Similar steps are
employed, if
needed, in the case of the 1,4-benzodiazepine-2-dione compounds as well.
Methods to prepare individual benzodiazepines are well-known in the art. (See
e.g.,
U.S. 3,415,814; 3,384,635; and 3,261,828, which are hereby incorporated by
reference). By
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selecting the appropriately substituted starting materials in any of the above-
described
methods, the benzodiazepines of this invention are prepared with relative
ease.
Example 3
Reagents
Bz-423 is synthesized as described above. FK506 is obtained from Fujisawa
(Osaka, Japan). N benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD) is
obtained
from Enzyme Systems (Livermore, CA). Dihydroethidium (DHE) and 3,3'-
dihexyloxacarbocyanine iodide (DiOC~(3)) are obtained from Molecular Probes
(Eugene,
OR). FAM-VAD-fmk is obtained from Intergen (Purchase, NJ). Manganese(III)meso-
tetrakis(4-benzoic acid)porphyrin (MnTBAP) is purchased from Alexis
Biochemicals (San
Diego, CA). Benzodiazepines is synthesized as described (See, B.A. Bunin et
al., Proc.
Natl. Acad: Sci. U.S.A., 91:4708-4712 [1994]). Other reagents were obtained
from Sigma
(St. Louis, MO).
Example 4 ,
Animals and drug delivery
Female NZB/W mice (Jackson Labs, Bar Harbor, ME) are randomly distributed into
treatment and control groups. Control mice receive vehicle (SO p,L aqueous
DMSO) and
treatment mice receive Bz-423 dissolved in vehicle (60 mg/kg) through
intraperitoneal
injections. Peripheral blood is obtained from the tail veins for the
preparation of serum.
Samples of the spleen and kidney are preserved in either 10% buffered-formalin
or by
freezing in OCT. An additional section of spleen from each animal is reserved
for the
preparation of single cell suspensions.
Example 5
Primary splenocytes, cell lines, and culture conditions
Primary splenocytes are obtained from 6 month old mice by mechanical
disruption
of spleens with isotonic lysis of real blood cells. B cell-rich fractions are
prepared by
negative selection using magnetic cell sorting with CD4, CDBa and CD1 1b
coated
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microbeads (Miltenyi Biotec, Auburn, California). The Ramos line is purchased
from the
ATCC (Monassis, Georgia). Cells are maintained in RPMI supplemented with 10%
heat-
inactivated fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100
~,g/ml) and
L-glutamine (290 ~,g/ml). Media for primary cells also contains 2-
mercaptoethanol (50
~M). All iyt vivo studies are performed with 0.5% DMSO and 2% FBS. Ih vitro
experiments are conducted in media containing 2%°FBS. Organic compounds
are dissolved
in media containing 0.5% DMSO.
Example 6
Histology
Formalin-fixed kidney sections were stained with hematoxylin and eosin (H&E)
and
glomerular immune-complex deposition is detected by~direct immunofluorescence
using
frozen tissue stained with FITC-conjugated goat anti-mouse IgG (Southern
Biotechnology,
Birmingham, AL). Sections are analyzed in a blinded fashion for nephritis and
IgG
deposition using a 0-4+ scale. The degree of lymphoid hyperplasia is scored on
a 0-4+ scale
using spleen sections stained with H&E. To identify B cells, sections are
stained with
biotinylated-anti-B220 (Pharmingen; 1 ~,g/mL) followed by streptavidin-Alexa
594
(Molecular Probes; 5 ~.g/mL). Frozen spleen sections are analyzed for TUNEL
positive
cells using an In situ Cell Death Detection kit (Roche) and are evaluated
using a 0-4+ scale.
Example 7
TiJNEL staining
Frozen spleen sections are analyzed using an Ira situ Cell Death Detection lit
(Roche
Molecular Biochemicals, Indianapolis, III. Sections are blindly evaluated and
assigned a
score (0-4+) on the basis of the amount of TUNEL-positive staining. B cells
are identified
by staining with biotinylated-anti-B220 (Pharmingen,,San Diego, CA; 1 ~.g/mL,
1 h, 22 °C)
followed by streptavidin-Alexa 594 (Molecular Probes, Eugene, Oregon; 5
~,g/mL, 1 h, 22
°C).
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Example 8
Flow cytometric analysis of spleen cells from treated animals
Surface markers are detected (15 m, 4 °C) with fluorescent-conjugated
anti-Thy 1.2
(Phanningen, 1 ~,g/mL) and/or anti-B220 (Pharmingen, 1 ~.g/mL). To detect
outer-
membrane phosphatidyl serine, cells are incubated with FITC-conjugated Annexin
V and
propidium iodide (PI) according to manufacturer protocols (Roche Molecular
Biochemicals). Detection of TUNEL-positive cells by flow cytometry uses the
APO-BRDU
lit (Pharmingen). Superoxide and MPT are assessed by incubation of cells for
30 m at 27
°G with 10 ~,M dihydroethidium and 2 ~M 3,3'-dihexyloxacarbocyanine
iodide (DIOC~(3))
(Molecular Probes). Prodidium idodie is used to determine viability and DNA
content.
Samples are analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San
Diego,
CA).
Example 9
B cell stimulation
Ramos cells are activated with soluble goat Fab2 anti-human IgM (Southern
Biotechnology Associates, 1 ~,g/ml) and/or purified anti-human CD40
(Phanningen, clone
SC3, 2.5 ~,g/ml). Mouse B cells are activated with affinity purified goat anti-
mouse IgM
(ICN, Aurora, Ohio; 20 ~,g/ml) immobilized in culture wells, and/or soluble
purified anti-
mouse CD40 (Phanningen, clone HM40-3, 2.5 ~,g/ml). LPS is used at 10 ~,g/ml.
Bz-423 is
added to cultures immediately after stimuli are applied.. Inhibitors are added
30 m prior to
Bz-423.
Example 10
Statistical analysis
Statistical analysis is conducted using the SPSS software package. Statistical
significance is assessed using the Mann-Whitney U test, and correlation
between variables is
assessed by two-way ANOVA. Allp-values reported are one-tailed and data are
presented
as mean ~ SEM.
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Example llv
Detection of cell death and hypodiploid DNA
Cell viability is assessed by staining with propidium iodide (PI, 1 ~,g/mL).
PI
fluorescence is measured using a FACScalibur flow cytometer (Becton Dickinson,
San
Diego, CA). Measurement of hypodiploid DNA is conducted after incubating cells
in
DNA-labeling solution (50 pg/mL of PI in PBS containing 0.2% Triton and 10
~g/mL
RNAse A) ovenught at 4 °C. The data is analyzed using the CellQuest
software excluding
aggregates.
Example 12
Detection of Oi , 0'I'm, and caspase activation
To detect OZ , cells are incubated with DHE (10.~M) for 30 min at 37 °C
and are
analyzed by flow cytometry to measure etludium fluorescence. Flow analysis of
mitochondrial transmembrane potential (~'IJ "~ is, conducted by labeling cells
with DiOC6(3)
(20 nM) for 15 min at 37 °C. A positive control for,disruption of ~'I'
", is established using
carbonyl cyanide m-chlorophenylhydrazone (CCCP, 50 p,M). Caspase activation
assays are
performed with FAM-VAD-fluoromethylketone. Processing of the substrate is
evaluated by
flow cytometry.
Example 13
Subcellular fractionation and cytochrome c detection
Ramos cells (250 x 10~ cells/sample) are treated with Bz-423 (10 ~M) or
vehicle for
1 to 5 h. Cells are pelleted, re-suspended in buffer (68 mM sucrose, 220 mM
mannitol, 10
mM HEPES-NaOH, pH 7.4, 10 mM KCI, 1 mM EDTA, 1 mM EGTA, 10 wg/mL leupeptin,
10 ~.g/mL aprotinin, 1 mM PMSF), incubated on ice for 10 min, and homogenized.
The
homogenate is centrifuged twice for 5 min at 4 °C (.800g) to pellet
nuclei and debris and for
15 min at 4 °C (16,000g) to pellet mitochondria. The supernatant is
concentrated,
electrophoresed on 12% SDS-PAGE gels, and transferred to Hybond ECL membranes
(Amersham, Piscataway, N~. After blocking (PBS containing 5% dried milk and
0.1%
Tween), the membranes are probed with an anti-cytochrome c monoclonal antibody
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(Pharmingen, San Diego, CA; 2 ~,g/mL) followed by an anti-mouse horseradish
peroxidase-
conjugated secondary with detection by chemiluminescence (Amersham).
Example 14
ROS production in isolated mitochondria
Male Long Evans rats are starved overnight and sacrificed by decapitation.
Liver
samples are homogenized in ice cold buffer A (250 mlVI sucrose, 10 mM Tris,
0.1 mM
EGTA, pH 7.4), and nuclei and cellular debris are pelleted (10 min, 830g, 4
°C).
Mitochondria are collected by centrifugation (10 min, 15,000g, 4 °C),
and the supernatant is
collected as the S 15 fraction. The mitochondria) pellet is washed three times
with buffer B
(250 mM sucrose, 10 mM Tris, pH 7.4), and re-suspended in buffer B at 20-30
mg/mL.
Mitochondria are diluted (0.5 mg/mL) in buffer C (200 mM sucrose, 10 mM Tris,
pH 7.4, 1
mM I~IHZP04, 10 ~M EGTA, 2.5 ~M rotenone, 5 mM succinate) containing 2',7'-
dichlorodihydrofluorescin diacetate (DCFH-DA, 1 ~.M). For state 3
measurements, ADP (2
mM) is included in the buffer, and prior to the addition of Bz-423,
mitochondria are allowed
to charge for 2 min. To induce state 4, oligomycin (1,0 ~,M) is added to
buffer C. The
oxidation of DCFH to 2',T-dichlorofluorescein (DCF) is moutored at 37
°C with a
spectrofluorimeter (?~eX: 503 nm; ~,en,: 522 nm). To detect effects on OZ and
0'hn"
mitochondria are incubated for 15 min at 37 °C in buffer C with
vehicle, Bz-423, or CCCP
containing DHE (5 ~,M) or DIOC~(3) (20 nM), and aliquots are removed for
analysis by
fluorescence microscopy.
Example 15 ,
Flow cytometric analysis of splenocytes
Splenocytes are prepared by mechanical disruption and red blood cells removed
by
isotonic lysis. Cells are stained at 4 °C with fluorescent-conjugated
anti-Thy 1.2
(Pharmingen; 1 ~g/mL) and/or anti-B220 (Pharmingen; 1 ~,g/mL) for 15 min. To
detect
outer-membrane phosphatidyl serine, cells are incubated with FITC-conjugated
Annexin V
and PI (Roche Molecular Biochemicals, Indianapolis, IN; 1 ~g/mL).
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Example 16
In vivo determination of ROS
Spleens are removed from 4-mo old NZB/W mice treated with Bz-423 or vehicle
and frozen in OCT. ROS production is measured using manganese(II)3,3,9-
diaminobenzidine as described in E.D. Kerver et al. (See, E.D. Kerver et al.,
Histochem. J.,
29:229-237 [1997).
Example 17
IgG titers, BUN, and proteinuria
Anti-DNA and IgG titers are determined by ELISA as described in P.C. Swanson
et
al. (See, P.C. Swanson et al., Biochemistry, 35:1624-1633 [1996]). Serum BUN
is
measured by the University of Michigan Hospital's clinical laboratory.
Proteinuria is
,.
monitored using ChemStrip 6 (Boehringer Mannheim).
Example 18
Benzodiazepine studies
Geh.e~al Methods
Cell Ps°epa~ation:
Cell lines were cultured in complete media (RPMI or DMEM containing 10% fetal
bovine serum supplemented with penicillin, streptomycin, and L-glutamine) at
37 °C, 5%
CO2. For activity assays, cells in log-phase growth were removed and diluted
to a
concentration between 100,000 and 300,000/mL.' Some cells were lcept in
complete media,
while an identical aliquot was exchanged into reduced serum media (RPMI or
DMEM
containing 0.2% fetal bovine serum supplemented with penicillin, streptomycin,
and L-
glutamine) by centrifugation.
Activity Assays:
Cells in both complete media and reduced serum media were dispensed into 96
well
plates in 100 ~,L aliquots giving 10,000 to 30,000 cells/well. Compound was
then added to
appropriate wells in the plate (2 ~.L of each SOX stock) at concentrations
between 1 nM to
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20 ~,M. Cells were then cultured overnight 37 °C, 5% COZ). Relative
cell number/cell
viability was measured using standard techniques (trypan blue
exclusion/hemocytometry,
MTT dye conversion assay).
Example 18.1: Ability to Induce Cell Death
Several individual representative compounds that induce apoptosis have been
shown
above. Of these, the most potent Bz-423s identified as Bz-423, which is shown
below.
Bz-423 was used to induce cell death in a variety of cells by using the above-
described materials and methods. Table 3 shows cell viability data after 18
hours of culture
with Bz-423 in reduced serum media as described above.
Table 3
Cell Line Sounce~ ~ Type 4 ~M Bz; 6 ~M Bz 10 ~M Bz
Jurkat Human T-cell 0 0 0
IMR Human Neuroblastoma100 0 0
SHSY-SY Human NeuroblastomaNd 0
Shep Human NeuroblastomaNd 80 0
293T Human Embryonic Nd Nd 30
fibroblast
RAW 246.7 Murine monocytic 70% 0 0
NIH 3T3 Murine fibroblast Nd Nd 25
(lower numbers equal mcreasea killing). lva = mot aeiermmea
Example 18.2
MRL/MpJ-lpr/lpr (MRL-lp~) mice develop similar serological and histological
manifestations of autoimmune disease as human SLE: These mice were developed
by a
series of cross-breeding of inbred strains until an autoimmune phenotype
appeared. (A.N.
Theofilopoulos and F.J. Dixon, Adv. Immunol., 37:269-390 [19850. The MRL-lpr
mice
are characterized by the spontaneous development of systemic autoimmune
disease. This
disease is manifested in several physiological locations .and resembles a
variety of human
diseases. For instance, the leidney damage in these mice is associated with
high serological
titers of anti-DNA as in human SLE. They also develop an erosive arthropathy
and a
lymphocytic infiltration of the salivary glands, similar to the human diseases
rheumatoid
arthritis (RA) and Sjorgen's disease, respectively (Theofilopoulos, supra).
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In general MRL-lpn mice have a profound defect in apoptosis due the mutation
of
the lpY gene locus. (K. Salcata et al., Clin. Tm_m__uriol. IW munopathol. 87:1-
7 [1998]). The
defect has been linked to a mutation in the Fas receptor gene, important in
the signaling of
apoptosis in activated lymphocytes. (R. Watanabe-Fukunaga et al., Nature,
356:314-317
[1992]). Consequently, these mice show a profound lymphoproliferation
resulting in
massive enlargement of the lymph nodes and spleen. Grossly, these mice
demonstrate
swollen footpads and erythematous skin lesions. Histologically,
glomerulonephritis,
arthritis, and inflammatory infiltration of the salivary glands are notable.
Methods
Mice:
Six weelc old, female MRL-lpn mice were purck~ased from Jackson Laboratories
(Bar
Harbor, ME). The animals were allowed to adapt to their enviromnent for one
week prior to
commencement of the treatment study. The mice were~housed in a climate
controlled
specific pathogen-free environment on a 12 hour light,darlc cycle with food
and water ad
libitmn. Once a week, weights were measured and proteinuria, was examined
using a
colorimetric reaction (Boehringer Mannheim ChemStrip 6).
Treatment Regimen:
Mice were randomized into three groups: controls receiving PBS (50 ~,L,
qod),controls receiving DMSO (50 ~,L, qod), and mice receiving Bz-423 in 50
~,L of DMSO
(60 mg/lcg qod ip for 20 mice and 30 mg/kg qod ip for l0 mice).
Intraperitoneal injections
were given with a 30 G needle and glass syringes (Hamilton) on an every other
day dosing
schedule. Treatment started at 7 weeks of age for the control mice(those
receiving PBS and
DMSO) and at 8 or 9 weelcs for the treatment mice. , At.the end of the study,
blood was
collected by tail bleeds. The mice were subsequently;anesthetized by metophane
inhalation
and were sacrificed by exsanguination by axillary dissection. Sample organs
were then
removed for histological analysis.
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Statistical Analysis:
Analysis of statistical significance was done using the computer program SFSS.
Unless otherwise noted, the Mann-Whitney U test (one-tailed) was used and
probability
values > 5% were considered insignificant.
Results
Disease Progr-essiou:
MRL-lp~ mice are known to develop a kidney disease very similar to that seen
in the
human autoimmune disease Systemic Lupus Erythematosus (SLE). The hallmark of
this
disease is a glomeuulonephritis that results in loss of kidney function and
eventual death due
to kidney failure. A marlcer for the development of kidney disease is the
amount of protein
present in the urine. As kidney function deteriorates, the glomerular
filtration mechanism
fails and proteinuria increases. Unlike the periodicity of the human disease,
the marine
form of lupus is progressive; thus, once a mouse develops nephritis and the
ensuing
proteinuria the disease progresses on a continuum until death. This allows the
use of
proteinuria measurements to follow the progression of kidney disease in the
MRL-lpr mice.
The development of disease in our study was followed by weekly measurement of
the proteinuria. Any given mouse was determined to have lupus if she had
proteinuria
values > 2+ (>100 mg/dL) for 2 or more consecutive weelcs. No mice meeting
this criterion
were ever found to have drops in proteinuria below 2+. Furthermore, the mice
that died
with > 2+ proteinuria were found to have very significant glomerulonephritis
and the mice
that died with values <2+ had healthy kidneys and causes of death unrelated to
the
autoiminune disease.
Figure 9 provides disease progression analysis for mice treated 60 mglkg qod.
Similar data are obtained with the 30 mg/lcg qod dosing schedule. As seen in
Figure 9,
treating mice with Bz-423 significantly delays the onset of and effectively
treats the lupus-
like disease relative to the control mice (p = 0.0043). These data are
supported by the
observation that BUN values for the treated animals are normal whereas those
receiving
vehicle alone are in renal distress.
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Laboratory Diagnostics:
The blood from the animals was analyzed for alterations in total numbers of
white
blood cells (WBC) as well as differential representation of subtypes. Mice
receiving Bz-
423 (60 mg/l~g) have nearly identical values for hematocrit, platelet count,
and WBC
relative to those receiving vehicle alone (Table 4).
Table '4
Group HCT PLAT WBC POLYS LYMPHS MONO EOS BASO
Control46.70 809 13.22 31.50 65.07 2.35 0.88 0.13
(0.91) (129) (4.65) (8.38) (9.61) (2.03) (0.42) (0.06)
Treatment42.60 895 13.68 43.00 53.66 2.01 1.15 0.17
'
Group HCT PLAT WBC POLYS LYMPHS MONO EOS BASO
(7.57) (148) (7.27) (16.37)(16.81)(1.05) (1.95) (0.27)
P value0.267 0.183 0.5 0.069 0.0,69 .0473 0.147 0.267
HCT, hematocrit (%); YLA'1', platelets (K/~.L); VVJ3C:, white blood cells
(K/~.L); YUL Y ~,
polymorphonuclear cells(%); LY1V11'HS, lymphocytes (%); MONO, monocytes (%);
EOS,
eosinophils BASO, basophils (%).
Autoa~ztibodies:
Serum samples from all of the mice were analyzed to determine the titer of
antibodies to several autoantigens (Table 5). These antibodies are total serum
polyclonal
antibodies. At the termination of the study, the mice receiving Bz-423 showed
significantly
lower titers of antibodies to ssDNA (p = 0.019), histones (p =0.0056), and La
antigen (p =
0.0265). Anti-dsDNA titers were lower in the treatment mice but not
statistically different
from those in the control animals (p = 0.082). There was also no difference in
antibodies to
Ro antigen between the two groups of mice; however, the actual absorbance
measurements
were very low for these ELISAs and any differences may have been mashed by the
sensitivity of the assay. Anti-dsDNA titers were only observed in a few of the
animals in
both groups and no conclusions could be made regarding differences between the
groups.
These anti-Sm findings are consistent with the literature, which reports that
only 10% of
MRL-lpr mice are expected to be positive for antibodies against Sm antigen
(Murphy, E.D.
(1981). For data only lymphoproliferation lp~) and other single-locus models
for marine
lupus. (See, Iminunologic Defects in Laboratory Animals (E.M. Gershwin and B.
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Merchant, eds.); Vol. 2, pp. 143-173 (Plenum, New Yorlc). The observed
differences in
autoantibody levels are found in a baclcground of very high total IgG
concentrations which
do not differ statistically between the control and the treatment groups (p =
0.3312).
Table 5
Anti-ssDNAAnti-dsDNAAnti- TotalIgG Anti-Rho Anti-La
(U/xnl) (U/ml) Histone*(mglml) (U/ml) (U/ml)
Drug Group508 ~ 247 ~ 0.613 23.3 ~ 304 t 226 ~
193 101 ~ . 6.2 256 162
0.526
Control 887 ~ 650 ~ 1.387 25.8 ~ 456 ~ 529 ~
328 454 ~ 8.7 328 462
Group 0.537
P value 0.019 0.082 0.0056 0.3312 0.1588 0.0265
Titers were not available at the time of tries report. . Anri-tnstone revels
are reportea as
OD4os values at a 1/400 dilution of serum. ,
,Ioiyat Histology:
In addition to the lupus syndrome, NIRL-lpf° mice spontaneously develop
a nerosive
arthropathy that resembles human rheumatoid arthritis, both histologically and
serologically. The arthritic lesions in these mice are characterized by
inflammatory changes
in the synovium and the periarticular connective tiss~ea'frequently
accompanied by the
presence of circulating rheumatoid factors in the serum. This arthritic
process is progressive
and proceeds through several different stages from a mild synovitis to a
nerosive arthritis,
which can eventually lead to a scarred joint. Histologically, the majority of
5 month old
MRL-lpy° mice demonstrate synovial cell proliferation, destruction of
articular cartilage and
subchondral bone, infiltration of synovial stroma by inflammatory cells,
periarticular
inflammation (vasculitis, myositis, tendinitis, perineuritis), exudates,
pannus formation, and
subcutaneous fibrinoid nodules (L. Hang, et al., J. Exp. Med., 155:1690-1701
[1982]; W.J.
I~oopman and S. Gay, Scand. J. Rheumatology. Suppl., 75:284-289 [1988]).
The paws of all the mice treated with Bz-423 were examined for signs of
arthritis
and synovitis. The control mice (those receiving vehicle alone) have a severe
synovitis
characterized by a marlced thickening of the synovium with occasional
formation of
papillary, vinous configurations. Typically, the synovial pathology was a
result of synovial
cell proliferation and infiltration of the synovial stroma by inflammatory
cells. In a
substantial percentage of the control mice, the disease process was
accompanied by pamius
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formation and erosion of the articular surface (both articular cartilage and
subchondral
bone). In contrast, the treatment mice were found to 'have a milder synovitis
as well as
fewer erosions and limited pannus formation (Table 6). The character of the
disease in the
animals receiving Bz-423 was much less aggressive with less synovial cell
proliferation and
inflammatory infiltration. Of further interest, it was observed that the
treatment mice had a
lessened degree of periarticular inflammation. The combination of these
findings suggest
that Bz-423 is ameliorating the arthritic disease process that typically
destroys the joints of
MPL-lp~ mice.
Table 6
Group Number of Avg. Number Number Number of
of of
mice histologicmice with mice with mice with
score synovitis erosions* pannus
>
synovitis 2* formation*
Control 7 2.1 5 (71%) 4 (57%) 4 (57%)
Treatment 7 1.3 0 (0%) 1 (14%) 1 (14%)
Pvalue p=0.001 p=0.01 p=0.13 p=0.13
p value determined by cross-tabulation and chi-square analysis
Delayed Type Hypersensitivity (DTH):
Mice treated with Bz-423 (60 mg/kg) showed no difference in DTH response to
TNBS on comparison to the control mice. (Figure 10C). Importantly, neither
group of
animals demonstrated a significant footpad swelling following antigen
challenge. This
phenomenon has been documented and old MRL-lp~ mice (>10 weeps) are expected
to
have a diminished iyz vivo T cell response to stimulus ~as evidenced by the
absence of a DTH
response. (See e.g., H. Ol~uyama et al., Clin. Exp: Immunol., 63:87-94 [1986],
C.F. Scott et
al., J. Immunol., 132:633 [1984]). However, suppression of T cells can result
in a rescue of
the DTH response. (See, H. Olcuyama et al., Intl. Arch. Allergy Appl.
Immunol., 1588:394
[1989]). Such a rescue was not observed in this study's treatment protocol.
These data
suggest that Bz-423 does not alter T cell function. Figures 10A-1 OC show the
results of the
Footpad Swelling experiment.
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In2tnuhe Gell Functiott:
Thymidine uptake assays of using both stimulated and unstimulated T and B
cells
was conducted to determine if Bz-423 affects lymphoproliferation in vitro.
At about a concentration of 10 ~.M, no effect on lymphocyte proliferation was
observed.
Example 18.3: Bz-423 as a lymphotoxic agent
Methods
Animals aytd experimental design:
Female NZB/W mice (Jackson Labs) were housed in specific pathogen-free,
environmentally controlled rooms operated by the University of Michigan's Unit
for
Laboratory Animal Medicine with 12 hr light-dark cycles and were given food
and water ad
libitum. Mice were randomly distributed into treatment and control groups. All
mice were
dosed through intraperitoneal injections using a Hamilton repeating dispenser
with glass
microliter syringes and 30 gauge needles. Control mice received vehicle (50
~,L aqueous
DMSO) and treatment mice received Bz-423 dissolved in vehicle. Animal weights
were
determined weekly, and dosing schedules readjusted thereafter.
Collection of BloodlTissues:
Peripheral blood was obtained from the tail veins of all mice for complete
blood
counts analysis and collection of serum. Blood was first allowed to clot at
room temperature
for I h, and then overnight at 4°C. Serum was separated from the formed
clot by
centrifugation (6 min., 16,000 x g). A section of spleen was removed
aseptically for
preparation of single cell suspensions. Samples of the following organs were
preserved in
IO% buffered-formalin: heart, liver, lung, spleen, kidney small intestine,
reproductive
system, salivary glands, thymus, mesenteric and axillary lymph nodes, and
skin. Additional
sections of lcidney and spleen were preserved by snap-freezing in OCT. Bone
marrow
smears were prepared from each femur.
Histology:
All histological determinations were made in a blinded fashion by a
pathologist.
Fonnalin-fixed sections were cut and stained with hematoxylin and eosin (H&E)
using
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standard protocols (L.G. Luna in: Manual of Histological Staining Methods of
the Armed
Forces Institute of Pathology, McGraw-Hill, New York (1960)). Immune-complex
deposition in the l~idneys was evaluated by direct immunofluorescence using
frozen sections
stained with FITC-conjugated goat anti-mouse IgG (Southern Biotechnology
Associates,
S Birmingham, AL) and C3 (Cappel-Organon Teknika, Durham, NC). The degree of
lymphoid hyperplasia was scored 0-4+scale.
TUNEL staining:
Frozen spleen sections (4 ~,m thiclc) were assayed for DNA strand breaks using
the
In situ Cell Death Detection lcit (Roche Molecular Biochemicals) according to
the
manufacturer's protocols. Sections were analyzed using a 0-4+ scale. Sections
were blindly
evaluated and assigned a score on the basis of the amomit of TUNEL-positive
staining.
Fluorescence ayaalysis of lymphocyte populations:
Single cell suspensions were prepared by teasing apart the spleen in media,
followed
by removal red blood cell with isotonic lysis buffer (A.M. Kruisbeek in:
Current Protocols
in Inununology, eds., J.E. Coligan et al., pp. 3.1.2-3.1.5, John Wiley & Sons,
Inc., [1997]).
106 cells were stained at 4°C with fluorescently-conjugated anti-Thy
1.2 (Pharmingen,
clone: 53-2.1, 1, ~,g/mL) and/or anti-B220 (Pharmingen, clone: RA3-6B2 1,
:g/mL) for 15
min. In samples stained to detect outer-membrane phosphatidyl serine, cells
were then
incubated with FITC-conjugated Annexin V and PI according to manufacturer
protocols
(Roche Molecular Biochemicals). Cells were analyzed on a Coulter ELITE
flowcytometer.
For each sample, at least 10,000 events were counted.
Seruy~a Atzti-DNA:
Titers were determined by direct ELISA as previously described (P.C. Swanson,
et
al., J. Clin. Invest., 97:1748-1760 [1996]). Detection of IgG anti-DNA used an
alkaline
phosphatase-conjugated Goat anti-Mouse IgG (H-chain only) secondary antibody
(1/1000
dilution, SIGMA). To convert absorbance readings into titers, pooled serum
from
unmanipulated eight month old female NZB/W was used as a reference standard
which was
arbitrarily assigned a value of I 000 U/mL.
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Serum Immunoglobulih:
Concentrations were determined by capture ELISA. Goat anti-Mouse Ig (Southern
Biotechnology Associates) was diluted to 10 pg/mL in PBS and coated overnight
at 4 °C on
Immulon II microtiter plates. Otherwise, ELISAs were performed as previously
described.
To convert absorbances into concentrations, a standard curve was generated
using a
previously quantified mouse irnmunoglobulin reference serum (ICN Biomedicals,
Aurora,
OH).
Blood us°ea nitrogefz (BLIN) ahd complete.blood coufZts (CBC):
Serum BUN measurements were conducted by the University of Michigan Hospital's
clinical pathology laboratory. CBC analyses were conducted by the diagnostic
laboratory of
University of Michigan's Unit for Laboratory Animal Medicine. Automated counts
determined by a Hemavet 15 OR were confirmed by visual examination of blood
smears.
Serum 1,4-benzodiazepihe levels:
Serum samples from mice injected with Bz-423 were precipitated with acetone
(5X
volume, -20 °C, 10 min). After centrifugation (16,000 x g, 10 min), the
supernatant
containing Bz-423 was concentrated in vacuo, and then extracted from any
remaining
protein using a Sep-pals C18 column (Waters Corp.) running a step gradient
from 1 0%
acetonitrile in water to 100% acetonitrile. Material eluting in the organic
fraction was
concentrated in vacuo, and then analyzed by reversed-phase BPLC using a
Phenomenex
C18 column. Peals areas were determined using a Shimadzu integrator and were
referenced
to a standard curve.
Statistical Analysis:
Statistical analyses was conducted using the SPSS software pacl~age. The Mann-
Whitney U and chi-square tests were used for histological and clinical data.
Student's t-test
was used for flow cytometric data. Correlations were assessed by ANOVA.
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Example 18.4
To model neuroblastoma in mice, the mice were transfected with the human
neuroblastoma cell line SI~NAS, to cause the cells to overexpress the
neuroblastorna
associated human oncogene N-myc. The resulting cell line is designated as D2.
ThesE cells
form tumors when xenografted into T cell-deficient athymic mice; thus
providing a relevant
animal model of human neuroblastoma.
Is~ vitro testing of the D2 cells was conducted to determine their sensitivity
to
benzodiazepine. D2 cells were plated into 96-well tissue culture plates at a
density of
10,000 cells per well in culture media (DMEM, 10% V:V heat inactivated fetal
bovine
serum (FBS), 100 ~hnl penicillin, 100 p./ml streptomycin, 290 p/ml glutamine)
and cultured
(37 °C, 5% CO2) overnight. Subsequently, culture media was exchanged
with media
containing 1% FBS. Solvent control (dimethyl sulfoxide (DMSO); final
concentration 1%
V/V) or benzodiazepine at concentrations of 2.5-20 ~,M was added.
After 18 hours cell viability was assessed using the MTT assay as previously
described in
this application. Figure 11 demonstrates that benzodiazepine bills D2 cells in
a dose-
response fashion.
To test the effect of benzodiazepine on neuroblastoma tumor growth , 1x10' D2
cells were aseptically inoculated into the thigh musculature of each of eight
six-weelc old
nu/nu female mice (Jackson Labs). Beginning one weelc after tumor cell
inoculation, 4 mice
were dosed with DMSO (20 ~, injected into the peritoneal cavity every day) and
4 mice were
dosed with benzodiazepine (2.5 mg dissolved in 20 ~,1 DMSO injected in the
peritoneal
cavity every day). The mice were evaluated regularly for tumor development and
once
present the size of the primary tumor was measured every other day. Table 9
demonstrates
that in mice that formed tumors, treatment with benzodiazepine significantly
decreased the
rate of tumor growth.
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Table 7
Treatment and control mice with___t_umorsDays for tumor volume to increase
~~ 5 fold
Mouse 1 with Bz-423 9
Mouse 2 with Bz-423 12
Mouse 3 with Bz-423 9
Mouse 4 with Bz-423 16
Mouse 5 with DMSO 2 3
Mouse 6 with DMSO 3 3
Administration of Benzodiazepine slows rate of neuroblastoma tumor growth in
nu/nu mice
(p < 0.02).
Specifically, tumors in control mice increased in volume 5-fold over an
average 4
day period, whereas 12 days were required for the same increase in tumor size
in
benzodiazepine-treated animals (p < 0.02). These findings support the claim
that
benzodiazepine is able to treat human malignant disease in a mouse model.
Further,
benzodiazepine has specific activity against human neuroblastoma both ira
vitro and in vivo.
Example 18.5
In another line of experiments, we sought to determine if benzodiazepine is
able to
bill tumor cells that are otherwise resistant to present' standard
chemotherapy drugs.
Ovarian cancer provides an excellent model for studying the problem of
chemoresistance in that treatment failures are commonly ascribed to the
emergence of
chemotherapy resistant cells. The A2780 human ovarian cancer cell line is
known to
contain wild-type p53; express low levels of bcl-2 and bcl-XL survival
factors; and is
sensitive to treatment with cis-platinum(H) diamirie dichloride (CDDP), a
standard
chemotherapeutic for treatment of ovarian cancer. These cells were transfected
with an
expression vector encoding human bcl-XL, a survival factor that when over-
expressed is
linlced to the development of chemotherapy resistance. These transfected cells
are
designated 2B1, and the empty vector transfected controls are designated
vector only. A
third ovarian cancer cell line, designated SI~OV3, was also obtained. This
cell line is
characterized as: 1. Deficient in wild-type p53 expression; 2. Expressing high
levels of
endogenous bcl-XL; and 3. Relatively resistant to the cytotoxic actions of
CDDP.
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Each of these cell lines was maintained using standard tissue culture
conditions in
complete media composed of RPNII, 10% FBS, 100 U/ml penicillin, 100 p/ml
streptomycin, 290 p,/ml glutamine. Each cell type was plated into a series of
separate wells
on 24-well tissue culture plates at 50,000 cells per well. Approximately 24
hours after
plating, media was exchanged to contain the same culture media made with only
2% FBS.
At this point either control solvent (DMSO, 1% V/V), increasing concentrations
of Bz-423
(4-20 p.M), or increasing concentrations of CDDP (6.7-66.7 p,M) was added to
cells. After
twenty-four hours of culture all cells present in each well were removed using
trypsin-
EDTA and mixed with propidium iodide (final concentration 1 p,/ml). After
incubating 20
minutes cells were analyzed by flow cytometry (Coulter FACS Calibur) to
determine cell
death on the basis of plasma membrane integrity measured as the fraction of
cells that had
taken-up propidiumiodide. Experiments demonstrate that the predicted pattern
of
chemosensitivity and resistance towards CDDP (A2780 and vector sensitive; 2B1
and
SI~OV3 resistant) was observed. Figure 12 demonstrates that benzodiazepine
bills each of
these types, irrespective of CDDP resistance. Further, benzodiazepine lcills
ovarian cancer
cells that aa-e resistant to standard chemotherapy., Further, benzodiazepine
kills tumor cells
that express high levels of survival factors (bcl-XL),, as well as those that
are deficient in p53
expression.
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.
100
