U.S. patent application number 11/445010 was filed with the patent office on 2007-02-22 for unsolvated benzodiazepine compositions and methods.
This patent application is currently assigned to The Regents of the University of Michigan. Invention is credited to Gary D. Glick, Adam Matzger.
Application Number | 20070043033 11/445010 |
Document ID | / |
Family ID | 38006350 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070043033 |
Kind Code |
A1 |
Glick; Gary D. ; et
al. |
February 22, 2007 |
Unsolvated benzodiazepine compositions and methods
Abstract
The present invention relates to systems and methods for
generating new forms of benzodiazepine and benzodiazepine related
compounds as well as new compounds and formulations generated by
such methods. In particular, the present invention provides high
throughput systems and methods for generating and identifying new
crystalline benzodiazepine and benzodiazepine related polymorphs
and new unsolvated, solvated, and other forms of the compounds that
find use as improved drugs and drug formations.
Inventors: |
Glick; Gary D.; (Ann Arbor,
MI) ; Matzger; Adam; (Ann Arbor, MI) |
Correspondence
Address: |
Medlen & Carroll, LLP
Suite 350
101 Howard Street
San Francisco
CA
94105
US
|
Assignee: |
The Regents of the University of
Michigan
Ann Arbor
MI
|
Family ID: |
38006350 |
Appl. No.: |
11/445010 |
Filed: |
June 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60686348 |
Jun 1, 2005 |
|
|
|
60704102 |
Jul 29, 2005 |
|
|
|
Current U.S.
Class: |
514/221 ;
540/504 |
Current CPC
Class: |
A61P 17/06 20180101;
A61P 25/14 20180101; A61P 25/28 20180101; A61P 25/00 20180101; A61P
35/02 20180101; A61P 9/12 20180101; A61P 17/02 20180101; A61P 35/00
20180101; A61P 3/10 20180101; C07D 243/24 20130101; A61P 29/00
20180101; A61P 9/04 20180101; A61P 9/10 20180101; A61P 43/00
20180101; A61P 15/00 20180101; A61P 37/02 20180101; A61P 37/06
20180101; A61P 9/00 20180101; A61K 31/5513 20130101; A61P 7/02
20180101; A61P 9/06 20180101; A61P 27/02 20180101; A61P 13/12
20180101 |
Class at
Publication: |
514/221 ;
540/504 |
International
Class: |
A61K 31/5513 20070101
A61K031/5513; C07D 243/12 20060101 C07D243/12 |
Goverment Interests
[0002] This invention was supported in part with NIH grant
GM046831. The United States government may have rights in this
invention.
Claims
1. A composition comprising an unsolvated compound having the
structure described by the following formula: ##STR112## including
both R and S enantiomeric forms and racemic mixtures; wherein R1,
R2, R3 and R4 are selected from the group consisting of: hydrogen;
CH.sub.3; a linear or branched, saturated or unsaturated aliphatic
chain having at least 1 carbon; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one hydroxy subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one thiol subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, wherein said
aliphatic chain terminates with an aldehyde subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one ketone subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; wherein said aliphatic chain terminates with a
carboxylic acid subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one amide subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one acyl group; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one nitrogen containing moiety; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amine subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ether subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one halogen subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one nitronium subgroup; wherein R5 is selected
from the group consisting of: OH; NO.sub.2; OR'; wherein R' is
selected from the group consisting of: a linear or branched,
saturated or unsaturated aliphatic chain having at least one
carbon; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one hydroxyl
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one thiol
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, wherein said aliphatic chain
terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein said aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; wherein R6 is selected from the group
consisting of: Hydrogen; NO.sub.2; Cl; F; Br; I; SR'; and
NR'.sub.2; wherein R' is defined as above in R5; wherein R7 is
selected from the group consisting of: Hydrogen; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; and wherein R8 is an aliphatic cyclic group larger than
benzene; wherein said larger than benzene comprises any chemical
group containing 7 or more non-hydrogen atoms.
2. The composition of claim 1, wherein said compound is:
##STR113##
3. The composition of claim 1, wherein said unsolvated compound is
anhydrous.
4. The composition of claim 1, wherein said unsolvated compound has
an orthorhombic crystal structure.
5. A composition comprising a compound selected from the group
consisting of Bz-423 ethanol solvate, Bz-423 succinic acid, Bz-423
citric acid, BZ-423-acetic acid, BZ-423-CH.sub.3CN,
BZ-423-methanol, BZ-423-ethyl acetate, BZ-423-toluene,
BZ-423-oxalic acid, BZ-423-fumaric acid, BZ-423-octanol,
BZ-423-heptanoic acid, BZ-423-diphenyl ether, Bz-423 1-propanol
solvate, Bz-423 2-propanol solvate, Bz-423 1-butanol solvate,
Bz-423 2-butanol solvate, Bz-423 1-pentanol solvate, Bz-423
propylene glycol, Bz-423 1-octanol solvate, Bz-423 acetone glass,
and BZ-423-trichlorobenzene.
6. A composition comprising an orthorhombic benzodiazepine crystal,
said benzodiazepine having the structure: ##STR114## including both
R and S enantiomeric forms and racemic mixtures; wherein R1, R2, R3
and R4 are selected from the group consisting of: hydrogen;
CH.sub.3; a linear or branched, saturated or unsaturated aliphatic
chain having at least 1 carbon; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one hydroxy subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one thiol subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, wherein said
aliphatic chain terminates with an aldehyde subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one ketone subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; wherein said aliphatic chain terminates with a
carboxylic acid subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one amide subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one acyl group; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one nitrogen containing moiety; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amine subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ether subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one halogen subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one nitronium subgroup; wherein R5 is selected
from the group consisting of: OH; NO.sub.2; OR'; wherein R' is
selected from the group consisting of: a linear or branched,
saturated or unsaturated aliphatic chain having at least one
carbon; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one hydroxyl
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one thiol
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, wherein said aliphatic chain
terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein said aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; wherein R6 is selected from the group
consisting of: Hydrogen; NO.sub.2; Cl; F; Br; I; SR'; and
NR'.sub.2; wherein R' is defined as above in R5; wherein R7 is
selected from the group consisting of: Hydrogen; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; and wherein R8 is an aliphatic cyclic group larger than
benzene; wherein said larger than benzene comprises any chemical
group containing 7 or more non-hydrogen atoms.
7. The composition of claim 6, wherein said compound is:
##STR115##
8. The composition of claim 6, wherein said orthorhombic
benzodiazepine crystal is anhydrous.
9. A composition comprising an oral dose of a benzodiazepine having
the structure: ##STR116## including both R and S enantiomeric forms
and racemic mixtures; wherein R1, R2, R3 and R4 are selected from
the group consisting of: hydrogen; CH.sub.3; a linear or branched,
saturated or unsaturated aliphatic chain having at least 1 carbon;
a linear or branched, saturated or unsaturated aliphatic chain
having at least 2 carbons, and having at least one hydroxy
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one thiol
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, wherein said aliphatic chain
terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein said aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
ether subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; wherein R5 is selected from the group
consisting of: OH; NO.sub.2; OR'; wherein R' is selected from the
group consisting of: a linear or branched, saturated or unsaturated
aliphatic chain having at least one carbon; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one hydroxyl subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one thiol subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
wherein said aliphatic chain terminates with an aldehyde subgroup;
a linear or branched, saturated or unsaturated aliphatic chain
having at least 2 carbons, and having at least one ketone subgroup;
a linear or branched, saturated or unsaturated aliphatic chain
having at least 2 carbons; wherein said aliphatic chain terminates
with a carboxylic acid subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one amide subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one acyl group; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one nitrogen containing moiety; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amine subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one halogen subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one nitronium subgroup; wherein R6 is selected
from the group consisting of: Hydrogen; NO.sub.2; Cl; F; Br; I;
SR'; and NR'.sub.2; wherein R' is defined as above in R5; wherein
R7 is selected from the group consisting of: Hydrogen; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; and wherein R8 is an aliphatic cyclic group larger than
benzene; wherein said larger than benzene comprises any chemical
group containing 7 or more non-hydrogen atoms.
10. The composition of claim 9, wherein said compound is:
##STR117##
11. The composition of claim 9, wherein said benzodiazepine
compound is anhydrous.
12. The composition of claim 9, wherein said benzodiazepine
compound has an orthorhombic crystal structure.
13. A method of treating an autoimmune disorder or
hyperproliferative disorder comprising administering to a subject
an effective amount of a composition comprising the composition of
claim 1.
Description
[0001] This application claims priority to U.S. Provisional
Application Ser. Nos. 60/686,348, filed Jun. 1, 2005, and
60/704,102, filed Jul. 29, 2005, the disclosures of which are
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to systems and methods for
generating new forms of benzodiazepine and benzodiazepine related
compounds as well as new compounds and formulations generated by
such methods. In particular, the present invention provides high
throughput systems and methods for generating and identifying new
crystalline benzodiazepine and benzodiazepine related polymorphs
and new unsolvated, solvated, and other forms of the compounds that
find use as improved drugs and drug formations.
BACKGROUND OF THE INVENTION
[0004] Multicellular organisms exert precise control over cell
number. A balance between cell proliferation and cell death
achieves this homeostasis. Cell death occurs in nearly every type
of vertebrate cell via necrosis or through a suicidal form of cell
death, known as apoptosis. Apoptosis is triggered by a variety of
extracellular and intracellular signals that engage a common,
genetically programmed death mechanism.
[0005] Multicellular organisms use apoptosis to instruct damaged or
unnecessary cells to destroy themselves for the good of the
organism. Control of the apoptotic process therefore is very
important to normal development, for example, fetal development of
fingers and toes requires the controlled removal, by apoptosis, of
excess interconnecting tissues, as does the formation of neural
synapses within the brain. Similarly, controlled apoptosis is
responsible for the sloughing off of the inner lining of the uterus
(the endometrium) at the start of menstruation. While apoptosis
plays an important role in tissue sculpting and normal cellular
maintenance, it is also the primary defense against cells and
invaders (e.g., viruses) which threaten the well being of the
organism.
[0006] Not surprisingly many diseases are associated with
dysregulation of the process of cell death. Experimental models
have established a cause-effect relationship between aberrant
apoptotic regulation and the pathenogenicity of various neoplastic,
autoimmune and viral diseases. For instance, in the cell mediated
immune response, effector cells (e.g., cytotoxic T lymphocytes
"CTLs") destroy virus-infected cells by inducing the infected cells
to undergo apoptosis. The organism subsequently relies on the
apoptotic process to destroy the effector cells when they are no
longer needed. Autoimmunity is normally prevented by the CTLs
inducing apoptosis in each other and even in themselves. Defects in
this process are associated with a variety of autoimmune diseases
such as lupus erythematosus and rheumatoid arthritis.
[0007] Multicellular organisms also use apoptosis to instruct cells
with damaged nucleic acids (e.g., DNA) to destroy themselves prior
to becoming cancerous. Some cancer-causing viruses overcome this
safeguard by reprogramming infected (transformed) cells to abort
the normal apoptotic process. For example, several human papilloma
viruses (HPVs) have been implicated in causing cervical cancer by
suppressing the apoptotic removal of transformed cells by producing
a protein (E6) which inactivates the p53 apoptosis promoter.
Similarly, the Epstein-Barr virus (EBV), the causative agent of
mononucleosis and Burkitt's lymphoma, reprograms infected cells to
produce proteins that prevent normal apoptotic removal of the
aberrant cells thus allowing the cancerous cells to proliferate and
to spread throughout the organism.
[0008] Still other viruses destructively manipulate a cell's
apoptotic machinery without directly resulting in the development
of a cancer. For example, the destruction of the immune system in
individuals infected with the human immunodeficiency virus (HIV) is
thought to progress through infected CD4.sup.+ T cells (about 1 in
100,000) instructing uninfected sister cells to undergo
apoptosis.
[0009] Some cancers that arise by non-viral means have also
developed mechanisms to escape destruction by apoptosis. Melanoma
cells, for instance, avoid apoptosis by inhibiting the expression
of the gene encoding Apaf-1. Other cancer cells, especially lung
and colon cancer cells, secrete high levels of soluble decoy
molecules that inhibit the initiation of CTL mediated clearance of
aberrant cells. Faulty regulation of the apoptotic machinery has
also been implicated in various degenerative conditions and
vascular diseases.
[0010] It is apparent that the controlled regulation of the
apoptotic process and its cellular machinery is vital to the
survival of multicellular organisms. Typically, the biochemical
changes that occur in a cell instructed to undergo apoptosis occur
in an orderly procession. However, as shown above, flawed
regulation of apoptosis can cause serious deleterious effects in
the organism.
[0011] There have been various attempts to control and restore
regulation of the apoptotic machinery in aberrant cells (e.g.,
cancer cells). For example, much work has been done to develop
cytotoxic agents to destroy aberrant cells before they proliferate.
As such, cytotoxic agents have widespread utility in both human and
animal health and represent the first line of treatment for nearly
all forms of cancer and hyperproliferative autoimmune disorders
like lupus erythematosus and rheumatoid arthritis.
[0012] Many cytotoxic agents in clinical use exert their effect by
damaging DNA (e.g., cis-diaminodichroplatanim(II) cross-links DNA,
whereas bleomycin induces strand cleavage). The result of this
nuclear damage, if recognized by cellular factors like the p53
system, is to initiate an apoptotic cascade leading to the death of
the damaged cell.
[0013] However, existing cytotoxic chemotherapeutic agents have
serious drawbacks. For example, many known cytotoxic agents show
little discrimination between healthy and diseased cells. This lack
of specificity often results in severe side effects that can limit
efficacy and/or result in early mortality. Moreover, prolonged
administration of many existing cytotoxic agents results in the
expression of resistance genes (e.g., bcl-2 family or multi-drug
resistance (MDR) proteins) that render further dosing either less
effective or useless. Some cytotoxic agents induce mutations into
p53 and related proteins. Based on these considerations, ideal
cytotoxic drugs should only kill diseased cells and not be
susceptible to chemo-resistance.
[0014] One strategy to selectively kill diseased cells is to
develop drugs that selectively recognize 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
known for most cancers. Moreover, for diseases like lupus, specific
molecular targets for drug development have not been
identified.
[0015] What are needed are improved compositions and methods for
regulating the apoptotic processes in subjects afflicted with
diseases and conditions characterized by faulty regulation of these
processes (e.g., viral infections, hyperproliferative autoimmune
disorders, chronic inflammatory conditions, and cancers).
SUMMARY
[0016] The present invention relates to systems and methods for
generating new forms of benzodiazepine and benzodiazepine related
compounds as well as new compounds and formulations generated by
such methods. In particular, the present invention provides high
throughput systems and methods for generating and identifying new
crystalline benzodiazepine and benzodiazepine related polymorphs
and new unsolvated and other forms of the compounds that find use
as improved drugs and drug formations.
[0017] For example, the present invention provides unsolvated forms
of benzodiazepine compounds and methods of making such compounds.
In some embodiments, the benzodiazepine compounds have orthorhombic
crystalline forms. The unsolvated benzodiazepines of the present
invention find use in pharmaceutical formulations with enhanced
properties (e.g., shelf-life, tabletability, etc.). The present
invention is illustrated with the benzodiazepine, Bz-423. However,
the present invention is not limited to this particular compound.
It will be understood that a variety of related compounds find use
in the compositions and methods of the present invention. In some
embodiments, the benzodiazepines of the present invention have
orthorhombic crystals (e.g., Bz-423). In some preferred
embodiments, the compounds are anhydrous benzodiazepines, an
ethanol solvate of benzodiazepines, a succinic acid (2:1)
formulation of benzoediazepines, a citric acid (2:1) formulation of
benzodiazepines, biphenyl derivate formulations of benzodiazepines,
acetic acid formulations of benzodiazepine, CH.sub.3CN formulations
of benzodiazepine, methanol formulations of benzodiazepines, ethyl
acetate formulations of benzodiazepines, toluene formulations of
benzodiazepines, oxalic acid formulations of benzodiazepines,
fumaric acid formulations of benzodiazepines, octanol formulations
of benzodiazepines, heptanoic acid formulations of benzodiazepines,
diphenyl ether formulations of benzodiazepines, and
trichlorobenzene formulations of benzodiazepines. Other solvated,
unsolvated and salt forms may also be used.
[0018] In certain embodiments, the present invention provides a
composition comprising a solution of dissolved benzodiazepine
(e.g., Bz-423) in contact with a crystal form (e.g., orthorhombic)
of the same benzodiazepine obtained from the solution, wherein the
crystal form, when isolated, is capable of being provided in
unsolvated form. In preferred embodiments, the composition further
comprises a polymer surface in contact with the crystals. In
preferred embodiments, the solution comprises an aqueous
solution.
[0019] In certain embodiments, the present invention provides a
method for producing orthorhombic Bz-423 crystals comprising
providing the above described composition and isolating the
crystals.
[0020] In preferred embodiments, the method further comprises the
step of preparing benzodiazepines to generated the above described
compositions.
[0021] The present invention further provides methods of preparing
a pharmaceutical preparation comprising new benzodiazepine
compositions (alone, or in combination with other drugs or agents).
In preferred embodiments, the pharmaceutical preparation comprises
a tablet.
[0022] In preferred embodiments, the method further comprises the
step of selling the pharmaceutical preparation, prescribing the
pharmaceutical preparation to a patient, and/or administering the
pharmaceutical preparation to a subject (e.g., human).
[0023] In certain embodiments, the present invention provides a
method for producing a crystal form of a benzodiazepine described
above comprising exposing a solution containing a benzodiazepine to
a polymer under conditions that permit crystal formation.
[0024] In certain embodiments, the present invention provides a
method of treating an autoimmune disorder or hyperproliferative
disorder comprising administering to a subject an effective amount
of a composition comprising the new benzodiazepine formulations
described above.
[0025] In preferred embodiments, the composition comprises an oral
dose of the new benzodiazepine formulations described above.
[0026] In certain embodiments, the present invention provides a
composition comprising an unsolvated compound having the structure
described by the following formula: ##STR1## including both R and S
enantiomeric forms and racemic mixtures; wherein R.sub.1, R.sub.2,
R.sub.3 and R.sub.4 are selected from the group consisting of:
hydrogen; CH.sub.3; a linear or branched, saturated or unsaturated
aliphatic chain having at least 1 carbon; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one hydroxy subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one thiol subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
wherein the aliphatic chain terminates with an aldehyde subgroup; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 2 carbons, and having at least one ketone subgroup; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 2 carbons; wherein the aliphatic chain terminates with a
carboxylic acid subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one amide subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one acyl group; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one nitrogen containing moiety; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amine subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ether subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one halogen subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one nitronium subgroup; wherein R.sub.5 is
selected from the group consisting of: OH; NO.sub.2; OR'; wherein
R' is selected from the group consisting of: a linear or branched,
saturated or unsaturated aliphatic chain having at least one
carbon; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one hydroxyl
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one thiol
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, wherein the aliphatic chain
terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein the aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; wherein R.sub.6 is selected from the group
consisting of: Hydrogen; NO.sub.2; Cl; F; Br; I; SR'; and
NR'.sub.2; wherein R' is defined as above in R.sub.5; wherein
R.sub.7 is selected from the group consisting of: Hydrogen; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 2 carbons; and wherein R.sub.8 is an aliphatic cyclic
group larger than benzene; wherein the larger than benzene
comprises any chemical group containing 7 or more non-hydrogen
atoms.
[0027] In preferred embodiments, the compound is: ##STR2##
[0028] In preferred embodiments, the unsolvated compound is
anhydrous. In preferred embodiment, the unsolvated compound has an
orthorhombic crystal structure.
[0029] In certain embodiments, the present invention provides a
composition comprising a compound selected from the group
consisting of Bz-423 ethanol solvate, Bz-423 succinic acid, Bz-423
citric acid, Bz-423 biphenyl derivate, BZ-423-acetic acid,
BZ-423-CH.sub.3CN, BZ-423-methanol, BZ-423-ethyl acetate,
BZ-423-toluene, BZ-423-oxalic acid, BZ-423-fumaric acid,
BZ-423-octanol, BZ-423-heptanoic acid, BZ-423-diphenyl ether,
Bz-423 1-propanol solvate, Bz-423 2-propanol solvate, Bz-423
1-butanol solvate, Bz-423 2-butanol solvate, Bz-423 1-pentanol
solvate, Bz-423 propylene glycol, Bz-423 1-octanol solvate, Bz-423
acetone glass, and BZ-423-trichlorobenzene.
[0030] In certain embodiments, the present invention provides a
composition comprising an orthorhombic benzodiazepine crystal, the
benzodiazepine having the structure: ##STR3## including both R and
S enantiomeric forms and racemic mixtures; wherein R.sub.1,
R.sub.2, R.sub.3 and R.sub.4 are selected from the group consisting
of: hydrogen; CH.sub.3; a linear or branched, saturated or
unsaturated aliphatic chain having at least 1 carbon; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one hydroxy subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one thiol subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, wherein the aliphatic chain terminates with an aldehyde
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one ketone
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons; wherein the aliphatic chain
terminates with a carboxylic acid subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amide subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one acyl group; a linear or branched, saturated
or unsaturated aliphatic chain having at least 2 carbons, and
having at least one nitrogen containing moiety; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one amine subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one ether subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one halogen subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one nitronium subgroup; wherein
R.sub.5 is selected from the group consisting of: OH; NO.sub.2;
OR'; wherein R' is selected from the group consisting of: a linear
or branched, saturated or unsaturated aliphatic chain having at
least one carbon; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
hydroxyl subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
thiol subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, wherein the aliphatic
chain terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein the aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; wherein R.sub.6 is selected from the group
consisting of: Hydrogen; NO.sub.2; Cl; F; Br; I; SR'; and
NR'.sub.2; wherein R' is defined as above in R.sub.5; wherein
R.sub.7 is selected from the group consisting of: Hydrogen; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 2 carbons; and wherein R.sub.8 is an aliphatic cyclic
group larger than benzene; wherein the larger than benzene
comprises any chemical group containing 7 or more non-hydrogen
atoms.
[0031] In preferred embodiments, the compound is: ##STR4##
[0032] In preferred embodiments, the orthorhombic benzodiazepine
crystal is anhydrous.
[0033] In certain embodiments, the present invention provides a
composition comprising an oral dose of a benzodiazepine having the
structure: ##STR5## including both R and S enantiomeric forms and
racemic mixtures; wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are
selected from the group consisting of: hydrogen; CH.sub.3; a linear
or branched, saturated or unsaturated aliphatic chain having at
least 1 carbon; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
hydroxy subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
thiol subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, wherein the aliphatic
chain terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein the aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
ether subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; wherein R.sub.5 is selected from the group
consisting of: OH; NO.sub.2; OR'; wherein R' is selected from the
group consisting of: a linear or branched, saturated or unsaturated
aliphatic chain having at least one carbon; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one hydroxyl subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one thiol subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
wherein the aliphatic chain terminates with an aldehyde subgroup; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 2 carbons, and having at least one ketone subgroup; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 2 carbons; wherein the aliphatic chain terminates with a
carboxylic acid subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one amide subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one acyl group; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one nitrogen containing moiety; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amine subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one halogen subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one nitronium subgroup; wherein R.sub.6 is
selected from the group consisting of: Hydrogen; NO.sub.2; Cl; F;
Br; I; SR'; and NR'.sub.2; wherein R' is defined as above in R5;
wherein R.sub.7 is selected from the group consisting of: Hydrogen;
a linear or branched, saturated or unsaturated aliphatic chain
having at least 2 carbons; and wherein R.sub.8 is an aliphatic
cyclic group larger than benzene; wherein the larger than benzene
comprises any chemical group containing 7 or more non-hydrogen
atoms.
[0034] In preferred embodiments, the compound is: ##STR6##
[0035] In preferred embodiments, the benzodiazepine compound is
anhydrous. In some embodiments, the benzodiazepine compound has an
orthorhombic crystal structure.
[0036] In certain embodiments, the present invention provides a
method of treating an autoimmune disorder or hyperproliferative
disorder comprising administering to a subject an effective amount
of a composition comprising an unsolvated compound.
DESCRIPTION OF THE FIGURES
[0037] FIG. 1 shows structural data of anhydrous Bz-423.
[0038] FIG. 2 shows powder x-ray diffraction data for anhydrous
Bz-423.
[0039] FIG. 3 shows Raman spectroscopy data for anhydrous
Bz-423.
[0040] FIG. 4 shows structural data of Bz-423 ethanol solvate.
[0041] FIG. 5 shows powder x-ray diffraction data for Bz-423
ethanol solvate.
[0042] FIG. 6 shows Raman spectroscopy data for Bz-423 ethanol
solvate.
[0043] FIG. 7 shows Raman spectroscopy data for ball milled Bz-423
succinic acid (2:1).
[0044] FIG. 8 shows Raman spectroscopy data for ball milled Bz-423
citric acid (2:1).
[0045] FIG. 9 shows the structural data of Bz-423 biphenyl
derivate.
[0046] FIG. 10 shows solubility (e.g., absorbance) as a function of
time for unsolvated Bz-423, anhydrous Bz-423, Bz-423 acetic acid,
and Bz-423 citric acid.
[0047] FIG. 11 shows a comparison of ATP hydrolysis between
unsolvated Bz-423 and solvated Bz-423.
[0048] FIG. 12 shows a comparison of ATP synthesis between
unsolvated Bz-423 and solvated Bz-423.
[0049] FIG. 13 shows a comparison of cell viability between
unsolvated Bz-423 and solvated Bz-423.
[0050] FIG. 14 shows a UV-vis spectrum of Bz-423 in simulated
gastric fluid.
[0051] FIG. 15 shows a UV-vis spectrum of Bz-423 in simulated
gastric fluid before and after addition of K.sub.2CO.sub.3.
[0052] FIG. 16 shows Raman spectroscopy data for Bz-423 ethanol
solvate.
[0053] FIG. 17 shows Raman spectroscopy data for Bz-423 1-propanol
solvate.
[0054] FIG. 18 shows Raman spectroscopy data for Bz-423 2-propanol
solvate.
[0055] FIG. 19 shows Raman spectroscopy data for Bz-423 1-butanol
solvate.
[0056] FIG. 20 shows Raman spectroscopy data for Bz-423 2-butanol
solvate.
[0057] FIG. 21 shows Raman spectroscopy data for Bz-423 1-pentanol
solvate.
[0058] FIG. 22 shows Raman spectroscopy data for Bz-423 1-octanol
solvate.
[0059] FIG. 23 shows Raman spectroscopy data for Bz-423 propylene
glycol solvate.
[0060] FIG. 24 shows Raman spectroscopy data for Bz-423 acetone
glass.
DEFINITIONS
[0061] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below.
[0062] As used herein, the term "benzodiazepine" refers to a seven
membered non-aromatic heterocyclic ring fused to a phenyl ring
wherein the seven-membered ring has two nitrogen atoms, as part of
the heterocyclic ring. In some aspects, the two nitrogen atoms are
in the 1 and 4 positions or the 1 and 5 positions, as shown in the
general structures below: ##STR7##
[0063] The term "larger than benzene" refers to any chemical group
containing 7 or more non-hydrogen atoms.
[0064] As used herein, the term "polymorph" refers to a crystalline
phase of a substance. Many substances feature polymorphism, which
is the ability of a substance to exist as two or more crystalline
phases that have different arrangements and/or conformations of the
molecules in the crystal lattice. As used herein, the term
polymorph includes amorphous phases and solvents/hydrates (i.e.,
psuedopolymorphs).
[0065] As used herein, the term "polymer library" refers to a
composition comprising a plurality of different polymers positioned
in particular locations so as to allow reactions to occur on the
polymers at the particular locations. For example, containers or
solid surfaces (e.g., plate, glass, metal, or ceramic surfaces,
multi-well plates, dishes, vials, tubes, flasks, etc.) with a
plurality of different polymers contained in discrete locations of
the surface are polymer libraries. For example, a multi-well plate
that contains a first polymer in a first well and a second polymer
in a second well, etc. provides a polymer library.
[0066] As used herein, the term "tabletability" refers to the
capacity of a powdered material to be transformed into a tablet of
specified strength under the effect of compaction pressure (Joiris
et al., Pharm. Res., 15:1122 (1998); herein incorporated by
reference in its entirety). Tabletability describes the
effectiveness of the applied pressure in increasing the tensile
strength of the tablet and demonstrates the relationship between
the cause, the compaction pressure, and the effect, the strength of
the compact.
[0067] As used herein, the term "compressibility" refers to the
ability of a material to undergo a reduction in volume as a result
of an applied pressure (Joiris et al., Pharm. Res., 15:1122 (1998);
herein incorporated by reference in its entirety). Compressibility
indicates the ease with which a power bed undergoes volume
reduction under compaction pressure and is often represented by a
plot showing the reduction of tablet porosity with increasing
compaction pressure.
[0068] As used herein, the term "compactibility" refers to the
ability of a material to produce tablets with sufficient strength
under the effect of densification (Joiris et al., Pharm. Res.,
15:1122 (1998); herein incorporated by reference in its entirety).
Compactibility shows the tensile strength of tablets normalized by
tablet porosity. In many cases, the tensile strength decreases
exponentially with increasing porosity (Ryshkewitch, J. Am. Cer.
Soc., 36:65 (1953); herein incorporated by reference in its
entirety).
[0069] As used herein, the term "substituted aliphatic" refers to
an alkane 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 nitro, 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.
[0070] 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 nitro, 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). Examples of such
include, but are not limited to, hydroxyphenyl and the like.
[0071] 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
like.
[0072] As used herein, the term "substituted cycloaliphatic" refers
to a cycloalkane possessing less than 10 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 nitro, a thio, an amino, a hydroxy, a ketone, an
aldehyde, an ester, an amide, a lower aliphatic, a substituted
lower aliphatic, or a ring (aryl, substituted aryl, cycloaliphatic,
or substituted cycloaliphatic). Examples of such include, but are
not limited to, 1-chlorodecalyl, bicyclo-heptanes, octanes, and
nonanes (e.g., nonrbornyl) and the like.
[0073] As used herein, the term "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. Examples of such include,
but are not limited to, morpholino and the like.
[0074] 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 ketone, an aldehyde, an ester,
an amide, a lower aliphatic, a substituted lower aliphatic, or a
ring (aryl, substituted aryl, cycloaliphatic, or substituted
cycloaliphatic). Examples of such include, but are not limited to
2-chloropyranyl.
[0075] 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.
[0076] 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.
[0077] As used herein, the term "lower-alkyl-substituted-halogen"
refers to any alkyl chain containing up to and including eight
carbon atoms where one of the aliphatic hydrogen atoms is replaced
by a halogen. Examples of such include, but are not limited to,
chlorethyl and the like.
[0078] As used herein, the term "acetylamino" shall mean any
primary or secondary amino that is acetylated. Examples of such
include, but are not limited to, acetamide and the like.
[0079] 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.
[0080] The term "epidermal hyperplasia," as used herein, refers to
an abnormal multiplication or increase in the number of normal
cells in normal arrangement in epidermal tissue. Epidermal
hyperplasia is a characteristic of numerous disorders, including
but not limited to, psoriasis.
[0081] The term "keratinocyte" as used herein, refers to a skin
cell of the keratinized layer of the epidermis.
[0082] The term "fibroblast" as used herein, refers to mesodermally
derived resident cells of connective tissue that secrete fibrillar
procollagen, fibronectin and collegenase.
[0083] The term "pigment disorder" as used herein, refers to
disorders involving skin pigment (e.g., melanin). Examples of
pigment disorders include, but are not limited to, all forms of
albinism, melasma, pigment loss after skin damage, and
vitiligo.
[0084] The term "stent" or "drug-eluting stent," as used herein,
refers to any device which when placed into contact with a site in
the wall of a lumen to be treated, will also place fibrin at the
lumen wall and retain it at the lumen wall. This can include
especially devices delivered percutaneously to treat coronary
artery occlusions and to seal dissections or aneurysms of splenic,
carotid, iliac and popliteal vessels. The stent can also have
underlying polymeric or metallic structural elements onto which the
fibrin is applied or the stent can be a composite of fibrin
intermixed with a polymer. For example, a deformable metal wire
stent such as that disclosed in U.S. Pat. No. 4,886,062, herein
incorporated by reference, could be coated with fibrin as set forth
above in one or more coats (i.e., polymerization of fibrin on the
metal framework by application of a fibrinogen solution and a
solution of a fibrinogen-coagulating protein) or provided with an
attached fibrin preform such as an encircling film of fibrin. The
stent and fibrin could then be placed onto the balloon at a distal
end of a balloon catheter and delivered by conventional
percutaneous means (e.g. as in an angioplasty procedure) to the
site of the restriction or closure to be treated where it would
then be expanded into contact with the body lumen by inflating the
balloon. The catheter can then be withdrawn, leaving the fibrin
stent of the present invention in place at the treatment site. The
stent may therefore provide both a supporting structure for the
lumen at the site of treatment and also a structure supporting the
secure placement of fibrin at the lumen wall. Generally, a
drug-eluting stent allows for an active release of a particular
drug at the stent implementation site.
[0085] As used herein, the term "catheter" refers generally to a
tube used for gaining access to a body cavity or blood vessel.
[0086] As used herein, the term "valve" or "vessel" refers to any
lumen within a mammal. Examples include, but are not limited to,
arteries, veins, capillaries, and biological lumen.
[0087] As used herein, the term "restenosis" refers to any valve
which is narrowed. Examples include, but are not limited to, the
reclosure of a peripheral or coronary artery following trauma to
that artery caused by efforts to open a stenosed portion of the
artery, such as, for example, by balloon dilation, ablation,
atherectomy or laser treatment of the artery.
[0088] As used herein, "angioplasty" or "balloon therapy" or
"balloon angioplasty" or "percutaneous transluminal coronary
angioplasty" refers to a method of treating blood vessel disorders
that involves the use of a balloon catheter to enlarge the blood
vessel and thereby improve blood flow.
[0089] As used herein, "cardiac catheterization" or "coronary
angiogram" refers to a test used to diagnose coronary artery
disease using a catheterization procedure. Such a procedure may
involve, for example, the injection of a contrast dye into the
coronary arteries via a catheter, permitting the visualization of a
narrowed or blocked artery.
[0090] 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.
[0091] The term "diagnosed," as used herein, refers to the
recognition of a disease by its signs and symptoms (e.g.,
resistance to conventional therapies), or genetic analysis,
pathological analysis, histological analysis, and the like.
[0092] As used herein, the terms "anticancer agent," or
"conventional anticancer agent" refer to any chemotherapeutic
compounds, radiation therapies, or surgical interventions, used in
the treatment of cancer.
[0093] As used herein the term, "in vitro" refers to an artificial
environment and to processes or reactions that occur within an
artificial environment. In vitro environments include, but are not
limited to, test tubes and cell cultures. The term "in vivo" refers
to the natural environment (e.g., an animal or a cell) and to
processes or reaction that occur within a natural environment.
[0094] As used herein, the term "host cell" refers to any
eukaryotic or prokaryotic cell (e.g., mammalian cells, avian cells,
amphibian cells, plant cells, fish cells, and insect cells),
whether located in vitro or in vivo.
[0095] As used herein, the term "cell culture" refers to any in
vitro culture of cells. Included within this term are continuous
cell lines (e.g., with an immortal phenotype), primary cell
cultures, finite cell lines (e.g., non-transformed cells), and any
other cell population maintained in vitro, including oocytes and
embryos.
[0096] 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.
Granulocycles include eosinophils and macrophages. In some
embodiments, target cells are continuously cultured cells or
uncultered cells obtained from patient biopsies.
[0097] Cancer cells include tumor cells, neoplastic cells,
malignant cells, metastatic cells, and hyperplastic cells.
Neoplastic cells can be benign or malignant. Neoplastic cells are
benign if they do not invade or metastasize. A malignant cell is
one that is able to invade and/or metastasize. Hyperplasia is a
pathologic accumulation of cells in a tissue or organ, without
significant alteration in structure or function.
[0098] 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.
[0099] As used herein, the term "un-activated target cell" refers
to a cell that is either in the G.sub.o phase or one in which a
stimulus has not been applied.
[0100] As used herein, the term "activated target lymphoid cell"
refers to a lymphoid cell that has been primed with an appropriate
stimulus to cause a signal transduction cascade, or alternatively,
a lymphoid cell that is not in G.sub.o phase. Activated lymphoid
cells may proliferate, undergo activation induced cell death, or
produce one or more of cytotoxins, cytokines, and other related
membrane-associated proteins characteristic of the cell type. 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.
[0101] As used herein, the term "activated cancer cell" refers to a
cancer cell that has been primed with an appropriate stimulus to
cause a signal transduction. An activated cancer cell may or may
not be in the G.sub.O phase.
[0102] An activating agent is a stimulus that upon interaction with
a target cell results in a signal transduction cascade. Examples of
activating stimuli include, but are not limited to, small
molecules, radiant energy, and molecules that bind to cell
activation cell surface receptors. Responses induced by activation
stimuli can be characterized by changes in, among others,
intracellular Ca.sup.2+, superoxide, or hydroxyl radical levels;
the activity of enzymes like kinases or phosphatases; or the energy
state of the cell. For cancer cells, activating agents also include
transforming oncogenes.
[0103] 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, but not limited to, a T cell receptor
ligand, a B cell activating factor, a TNF, a Fas ligand, a
proliferation inducing ligand, a cytokine, a chemokine, a hormone,
an amino acid, 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. Antibodies include monoclonal
or polyclonal or a mixture thereof.
[0104] Examples of a T cell ligand include, but are not limited to,
a peptide that binds to an MHC molecule, a peptide MHC complex, or
an antibody that recognizes components of the T cell receptor.
[0105] 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.
[0106] Examples of agents or conditions that enhance cell stress
include heat, radiation, oxidative stress, or growth factor
withdrawal and the like. Examples of growth factors include, but
are not limited to serum, IL-2, platelet derived growth factor
("PDGF"), and the like.
[0107] 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.
[0108] As used herein, the term "dysregulation of the process of
cell death" refers to any aberration in the ability of (e.g.,
predisposition) a cell to undergo cell death via either necrosis or
apoptosis. Dysregulation of cell death is associated with or
induced by a variety of conditions, including for example,
autoimmune disorders (e.g., systemic lupus erythematosus,
rheumatoid arthritis, 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.
[0109] It should be noted that when the dysregulation is induced by
or associated with a viral infection, the viral infection may or
may not be detectable at the time dysregulation occurs or is
observed. That is, viral-induced dysregulation can occur even after
the disappearance of symptoms of viral infection.
[0110] A "hyperproliferative disorder," as used herein refers to
any condition in which a localized population of proliferating
cells in an animal is not governed by the usual limitations of
normal growth. Examples of hyperproliferative disorders include
tumors, neoplasms, lymphomas and the like. A neoplasm is said to be
benign if it does not undergo, invasion or metastasis and malignant
if it does either of these. A metastatic cell or tissue means that
the cell can invade and destroy neighboring body structures.
Hyperplasia is a form of cell proliferation involving an increase
in cell number in a tissue or organ, without significant alteration
in structure or function. Metaplasia is a form of controlled cell
growth in which one type of fully differentiated cell substitutes
for another type of differentiated cell. Metaplasia can occur in
epithelial or connective tissue cells. A typical metaplasia
involves a somewhat disorderly metaplastic epithelium.
[0111] The pathological growth of activated lymphoid cells often
results in an autoimmune disorder or a chronic inflammatory
condition. As used herein, the term "autoimmune disorder" refers to
any condition in which an organism produces antibodies or immune
cells which recognize the organism's own molecules, cells or
tissues. Non-limiting examples of autoimmune disorders include
autoimmune hemolytic anemia, autoimmune hepatitis, Berger's disease
or IgA nephropathy, Celiac Sprue, chronic fatigue syndrome, Crohn's
disease, dermatomyositis, fibromyalgia, graft versus host disease,
Grave's disease, Hashimoto's thyroiditis, idiopathic
thrombocytopenia purpura, lichen planus, multiple sclerosis,
myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,
scleroderma, Sjorgren syndrome, systemic lupus erythematosus, type
1 diabetes, ulcerative colitis, vitiligo, and the like.
[0112] As used herein, the term "chronic inflammatory condition"
refers to a condition wherein the organism's immune cells are
activated. Such a condition is characterized by a persistent
inflammatory response with pathologic sequelae. This state is
characterized by infiltration of mononuclear cells, proliferation
of fibroblasts and small blood vessels, increased connective
tissue, and tissue destruction. Examples of chronic inflammatory
diseases include, but are not limited to, Crohn's disease,
psoriasis, chronic obstructive pulmonary disease, inflammatory
bowel disease, multiple sclerosis, and asthma. Autoimmune diseases
such as rheumatoid arthritis and systemic lupus erythematosus can
also result in a chronic inflammatory state.
[0113] As used herein, the term "co-administration" refers to the
administration of at least two agent(s) (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 skill in the art understand that the
formulations and/or routes of administration of the various
agents/therapies used may vary. The appropriate dosage for
co-administration can be readily determined by one skilled in the
art. In some embodiments, when agents/therapies are
co-administered, the respective agents/therapies are administered
at lower dosages than appropriate for their administration alone.
Thus, co-administration is especially desirable in embodiments
where the co-administration of the agents/therapies lowers the
requisite dosage of a known potentially harmful (e.g., toxic)
agent(s).
[0114] 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.
[0115] As used herein, the term "pharmaceutical composition" refers
to the combination of an active agent with a carrier, inert or
active, making the composition especially suitable for diagnostic
or therapeutic use in vivo, in vivo or ex vivo.
[0116] 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]).
[0117] As used herein, the term "pharmaceutically acceptable salt"
refers to any pharmaceutically acceptable salt (e.g., acid or base)
of a compound of the present invention which, upon administration
to a subject, is capable of providing a compound of this invention
or an active metabolite or residue thereof. As is known to those of
skill in the art, "salts" of the compounds of the present invention
may be derived from inorganic or organic acids and bases. Examples
of acids include, but are not limited to, hydrochloric,
hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic,
phosphoric, glycolic, lactic, salicylic, succinic,
toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,
ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,
benzenesulfonic acid, and the like. Other acids, such as oxalic,
while not in themselves pharmaceutically acceptable, may be
employed in the preparation of salts useful as intermediates in
obtaining the compounds of the invention and their pharmaceutically
acceptable acid addition salts.
[0118] Examples of bases include, but are not limited to, alkali
metals (e.g., sodium) hydroxides, alkaline earth metals (e.g.,
magnesium), hydroxides, ammonia, and compounds of formula
NW.sub.4.sup.+, wherein W is C.sub.1-4 alkyl, and the like.
[0119] Examples of salts include, but are not limited to: acetate,
adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
palmoate, pectinate, persulfate, phenylpropionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate,
undecanoate, and the like. Other examples of salts include anions
of the compounds of the present invention compounded with a
suitable cation such as Na.sup.+, NH.sub.4.sup.+, and
NW.sub.4.sup.+ (wherein W is a C.sub.1-4 alkyl group), and the
like.
[0120] 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.
[0121] 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, solid phase supports may refer to resins such as
polystyrene (e.g., PAM-resin obtained from Bachem, Inc., Peninsula
Laboratories, etc.), POLYHIPE) 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).
[0122] 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.
[0123] The terms "bacteria" and "bacterium" refer to all
prokaryotic organisms, including those within all of the phyla in
the Kingdom Procaryotae. It is intended that the term encompass all
microorganisms considered to be bacteria including Mycoplasma,
Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of
bacteria are included within this definition including cocci,
bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included
within this term are prokaryotic organisms which are gram negative
or gram positive. "Gram negative" and "gram positive" refer to
staining patterns with the Gram-staining process which is well
known in the art. (See e.g., Finegold and Martin, Diagnostic
Microbiology, 6th Ed., C V Mosby St. Louis, pp. 13-15 [1982]).
"Gram positive bacteria" are bacteria which retain the primary dye
used in the Gram stain, causing the stained cells to appear dark
blue to purple under the microscope. "Gram negative bacteria" do
not retain the primary dye used in the Gram stain, but are stained
by the counterstain. Thus, gram negative bacteria appear red.
[0124] As used herein, the term "microorganism" refers to any
species or type of microorganism, including but not limited to,
bacteria, archaea, fungi, protozoans, mycoplasma, and parasitic
organisms. The present invention contemplates that a number of
microorganisms encompassed therein will also be pathogenic to a
subject.
[0125] As used herein, the term "fungi" is used in reference to
eukaryotic organisms such as the molds and yeasts, including
dimorphic fungi.
[0126] As used herein, the term "virus" refers to minute infectious
agents, which with certain exceptions, are not observable by light
microscopy, lack independent metabolism, and are able to replicate
only within a living host cell. The individual particles (i.e.,
virions) typically consist of nucleic acid and a protein shell or
coat; some virions also have a lipid containing membrane. The term
"virus" encompasses all types of viruses, including animal, plant,
phage, and other viruses.
[0127] The term "sample" as used herein is used in its broadest
sense. A sample suspected of indicating a condition characterized
by the dysregulation of apoptotic function may comprise a cell,
tissue, or fluids, chromosomes isolated from a cell (e.g., a spread
of metaphase chromosomes), genomic DNA (in solution or bound to a
solid support such as for Southern blot analysis), RNA (in solution
or bound to a solid support such as, for Northern blot analysis),
cDNA (in solution or bound to a solid support) and the like. A
sample suspected of containing a protein may comprise a cell, a
portion of a tissue, an extract containing one or more proteins and
the like.
[0128] 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.
[0129] As used herein, the term "antigen binding protein" refers to
proteins which bind to a specific antigen. "Antigen binding
proteins" include, but are not limited to, immunoglobulins,
including polyclonal, monoclonal, chimeric, single chain, and
humanized antibodies, Fab fragments, F(ab')2 fragments, and Fab
expression libraries. Various procedures known in the art are used
for the production of polyclonal antibodies. For the production of
antibody, various host animals can be immunized by injection with
the peptide corresponding to the desired epitope including but not
limited to rabbits, mice, rats, sheep, goats, etc. In a preferred
embodiment, the peptide is conjugated to an immunogenic carrier
(e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole
limpet hemocyanin [KLH]). Various adjuvants are used to increase
the immunological response, depending on the host species,
including but not limited to Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum.
[0130] For preparation of monoclonal antibodies, any technique that
provides for the production of antibody molecules by continuous
cell lines in culture may be used (See e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.). These include, but are not
limited to, the hybridoma technique originally developed by Kohler
and Milstein (Kohler and Milstein, Nature, 256:495-497 [1975]), as
well as the trioma technique, the human B-cell hybridoma technique
(See e.g., Kozbor et al., Immunol. Today, 4:72 [1983]), and the
EBV-hybridoma technique to produce human monoclonal antibodies
(Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96 [1985]).
[0131] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778;
herein incorporated by reference) can be adapted to produce
specific single chain antibodies as desired. An additional
embodiment of the invention utilizes the techniques known in the
art for the construction of Fab expression libraries (Huse et al.,
Science, 246:1275-1281 [1989]) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity.
[0132] 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.
[0133] Genes encoding antigen binding proteins can be isolated by
methods known in the art. In the production of antibodies,
screening for the desired antibody can be accomplished by
techniques known in the art (e.g., radioimmunoassay, ELISA
(enzyme-linked immunosorbant assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitin reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), Western Blots,
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays, etc.), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.) etc.
[0134] As used herein, the term "immunoglobulin" or "antibody"
refer to proteins that bind a specific antigen. Immunoglobulins
include, but are not limited to, polyclonal, monoclonal, chimeric,
and humanized antibodies, Fab fragments, F(ab').sub.2 fragments,
and includes immunoglobulins of the following classes: IgG, IgA,
IgM, IgD, IbE, and secreted immunoglobulins (sIg). Immunoglobulins
generally comprise two identical heavy chains and two light chains.
However, the terms "antibody" and "immunoglobulin" also encompass
single chain antibodies and two chain antibodies.
[0135] 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.
[0136] The terms "specific binding" or "specifically binding" when
used in reference to the interaction of an antibody and a protein
or peptide means that the interaction is dependent upon the
presence of a particular structure (i.e., the antigenic determinant
or epitope) on the protein; in other words the antibody is
recognizing and binding to a specific protein structure rather than
to proteins in general. For example, if an antibody is specific for
epitope "A," the presence of a protein containing epitope A (or
free, unlabelled A) in a reaction containing labeled "A" and the
antibody will reduce the amount of labeled A bound to the
antibody.
[0137] 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).
[0138] 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.
[0139] 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 conferring protein in
mitochondrial 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 mitochondrial ATP
synthase, etc.). 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
(K.sub.D) for binding to the substrate may be different for the two
molecules.
[0140] As used herein, the term "instructions for administering
said 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 (e.g., providing dosing,
route of administration, decision trees for treating physicians for
correlating patient-specific characteristics with therapeutic
courses of action). 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.
[0141] The term "test compound" refers to any chemical entity,
pharmaceutical, drug, and the like, that can be used to treat or
prevent a disease, illness, sickness, or disorder of bodily
function, or otherwise alter the physiological or cellular status
of a sample (e.g., the level of dysregulation of apoptosis in a
cell or tissue). Test compounds comprise both known and potential
therapeutic compounds. A test compound can be determined to be
therapeutic by using the screening methods of the present
invention. A "known therapeutic compound" refers to a therapeutic
compound that has been shown (e.g., through animal trials or prior
experience with administration to humans) to be effective in such
treatment or prevention. In preferred embodiments, "test compounds"
are agents that modulate apoptosis in cells.
[0142] 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 compound for
treating conditions characterized by the dysregulation of apoptotic
processes.
GENERAL DESCRIPTION OF THE INVENTION
[0143] Pharmaceutical companies expend much of their resources in
attempts to find new blockbuster drugs (greater than $1
billion/year sales) (D. Eric Walters, BC 5220, Techniques in
Biomedical Research, "The Rational Basis of Drug Design"). In order
to be successful, a new drug should satisfy several criteria: safe
to use; effective for the intended use; stable (chemically and
metabolically); good solubility profile; synthetically feasible;
and novel (i.e., patentable). An important aspect of drug
development is the identification of leads. A lead is any chemical
compound that shows the biological activity sought. A lead is not
the same as a drug however--as it should meet the criteria listed
above prior to use as a drug. There are two broad tasks in drug
discovery. The first is lead-finding. Here the task is to find a
chemical compound that has a desired bioactivity. The second is
lead-optimization, modifying the lead structure to build in the
other desirable properties (safety, solubility, stability,
etc.).
[0144] There are many ways to find lead compounds. In the
beginning, plants and other natural products were the source of
most medicinal substances. As the science of medicinal chemistry
evolved, it was discovered that the plants and natural products
contained specific compounds that are responsible for the
therapeutic effect. It became possible to isolate the active
components, so that dosage could be more accurately regulated.
[0145] Other medicines came about because of accidental
observations and discoveries (e.g., penicillin). The discovery of
penicillin led to a large-scale screening effort, in which
thousands of soil microorganisms were grown and tested to see
whether they could produce other substances that kill bacteria.
Antibiotics such as streptomycin, neomycin, gentamicin,
erythromycin, and the tetracyclines resulted from these
efforts.
[0146] Chemical modification of known drugs can often lead to
improved drugs. For example, naturally occurring penicillin G is
broken down by bacterial beta-lactamases. Addition of two
--OCH.sub.3 groups produces methicillin, which is resistant to
lactamase. Another example of chemical modification is found in the
opiate analgesics. The parent compound is morphine, which occurs in
opium poppies. Morphine is a powerful analgesic, but it has serious
side effects: respiratory depression, constipation, and dependence
liability. Thousands of analogs (related chemical structures) have
been synthesized in an effort to find analgesics with lower
incidence of side effects. In some cases, small changes in chemical
structure may have a big influence on the activity. For example,
nalorphine is a partial agonist (shows some morphine-like activity,
and at higher concentration, antagonizes morphine effects), and
naloxone is an antagonist. Considerable simplification of the
molecule is possible. For example, meperidine has only two rings
instead of four, but it maintains strong analgesic activity. It has
better oral absorption than morphine, and shows less GI side
effects. Methadone is an analgesic in which the original piperidine
ring (6-membered ring containing a nitrogen atom) is completely
absent. It retains analgesic activity, has good oral activity, and
has a long half-life in the body. Dextromethorphan is constructed
on a mirror image of the morphine ring system. It has no opiate
analgesic effects or side effects, but is a useful anti-tussive
agent.
[0147] Some drugs are discovered by observing side effects of
existing drugs. For example, minoxidil was found to grow hair on
bald men as a side effect in a study of its antihypertensive
effects. Viagra's effect on penile dysfunction was discovered in
clinical trials for treatment of angina; it had originally been
designed as an antihypertensive drug.
[0148] In the modern era, most leads are discovered using various
screening processes. For example, over a couple of decades, the
National Cancer Institute has put hundreds of thousands of
different chemical compounds through a battery of anti-cancer
assays. Current screening assays often employ miniaturization and
automation with robots for high throughput screening, allowing
hundreds of thousands of compounds to be screened in a short period
of time.
[0149] Structure-based molecular design is yet another method to
identify lead molecules for drug design. This method is based on
the premise that desired drug candidates possess significant
structural and chemical complementarity with their target
molecules. This design method can create molecules with specific
properties that make them conducive for binding to the target site.
The molecular structures that are designed by the structure-based
design process are meant to interact with biochemical targets, for
example, whose three-dimensional structures are known.
[0150] Even with the extensive resources expended in drug discovery
and design, there are no systematic methods for generating drugs
with desired properties. Thus, the art is in need of additional
systems and methods to facilitate the discovery and optimization of
therapeutic and other useful compounds.
[0151] As a class of drugs, benzodiazepine compounds have been
widely studied and reported to be effective medicaments for
treating a number of disease. For example, U.S. Pat. Nos.
4,076,823, 4,110,337, 4,495,101, 4,751,223 and 5,776,946, each
incorporated herein by reference in its entirety, report that
certain benzodiazepine compounds are effective as analgesic and
anti-inflammatory agents. Similarly, U.S. Pat. No. 5,324,726 and
U.S. Pat. No. 5,597,915, each incorporated by reference in its
entirety, report that certain benzodiazepine compounds are
antagonists of cholecystokinin and gastrin and thus might be useful
to treat certain gastrointestinal disorders.
[0152] Other benzodiazepine compounds have been studied as
inhibitors of human neutrophil elastase in the treating of human
neutrophil elastase-mediated conditions such as myocardial
ischemia, septic shock syndrome, among others (See e.g., U.S. Pat.
No. 5,861,380 incorporated herein by reference in its entirety).
U.S. Pat. No. 5,041,438, incorporated herein by reference in its
entirety, reports that certain benzodiazepine compounds are useful
as anti-retroviral agents.
[0153] Despite the attention benzodiazepine compounds have drawn,
it will become apparent from the description below, that the
present invention provides novel uses for benzodiazepine compounds
and related and other compounds and methods of using the compounds,
as well as known compounds, for treating a variety of diseases.
[0154] Benzodiazepine compounds are known to bind to benzodiazepine
receptors in the central nervous system (CNS) and thus have been
used to treat various CNS disorders including anxiety and epilepsy.
Peripheral benzodiazepine receptors have also been identified,
which receptors may incidentally also be present in the CNS. The
present invention demonstrates that benzodiazepines and related
compounds have pro-apoptotic and cytotoxic properties. The route of
action of these compounds is not through the previously identified
benzodiazepine receptors.
[0155] Thus, in some embodiments, the present invention provides a
number of compounds. In other embodiments, the present invention
provides methods for using such compounds to regulate biological
processes. The present invention also provides drug-screening
methods to identify and optimize compounds. In preferred
embodiments, the present invention provides unsolvated
benzodiazepine structures and benzodiazepine related structures
with long term storage capability, and stability under high
pressures (e.g., storage pressures necessary in generating drug
tablets). These and other research and therapeutic utilities are
described below.
DETAILED DESCRIPTION OF THE INVENTION
[0156] Exemplary compositions and methods of the present invention
are described in more detail in the following sections: I.
Modulators of Cell Death; II. Modulators of Cell Growth and
Proliferation; III. Benzodiazepine and Benzodiazepine Related
Crystal Forms; IV. Pharmaceutical compositions, formulations, and
exemplary administration routes and dosing considerations; V. Drug
screens; VI. Therapeutic Applications; and VII. ATPase Inhibitors
And Methods For Identifying Therapeutic Inhibitors.
[0157] The present invention herein incorporates by reference U.S.
Provisional Patent Nos. 60/131,761, 60/165,511, 60/191,855,
60/312,560, 60/313,689, 60/396,670, 60/565,788, 60/607,599,
60/641,040, and U.S. patent application Ser. Nos. 11/324,419,
11/176,719, 11/110,228, 10/935,333, 10/886,450, 10/795,535,
10/634,114, 10/427,211, 10/427,212, 10/217,878, 09/767,283,
09/700,101, and related applications. All compounds and uses
described in the above mentioned cases are contemplated to be part
of the present invention. Additionally, all other known uses of
benzodiazepines may be used with the new formulations of the
invention. Additional references include, but are not limited to,
Otto, M. W., et al., (2005) J. Clin. Psychiatry 66 Suppl. 2:34-38;
Yoshii, M., et al., (2005) Nippon Yakurigaku Zasshi 125(1):33-36;
Yasuda, K. (2004) Nippon Rinsho. 62 Suppl. 12:360-363; Decaudin, D.
(2004) 15(8):737-745; Bonnot, O., et al. (2003) Encephale.
29(6):553-559; Sugiyama, T. (2003) Ryoikibetsu Shokogun Shirizu.
40:489-492; Lacapere, J. J., et al., (2003) Steroids.
68(7-8):569-585; Galiegue, S., et al., (2003) Curr. Med. Chem.
10(16):1563-1572; Papadopoulo, V. (2003) Ann. Pharm. Fr.
61(1):30-50; Goethals, I., et al., (2002) Eur. J. Nucl. Med. Mol.
Imaging 30(2):325-328; Castedo, M., et al., (2002) J. Exp. Med.
196(9):1121-1125; Buffett-Jerrott, S. E., et al., (2002) Curr.
Pham. Des. 8(1):45-58; Beurdeley-Thomas, A., et al., (2000) J.
Nuerooncol. 46(1):45-56; Smyth, W. F., et al., (1998)
Electrophoresis 19(16-17):2870-2882; Yoshii, M., et al., (1998)
Nihon Shinkei Seishin Yakurigaku Zasshi. 18(2):49-54; Trimble, M.
and Hindmarch, I. (2000) Benzodiazepines, published by Wrighton
Biomedical Publishing; and Salamone, S. J. (2001) Benzodiazepines
and GHB--Detection and Pharmacology, published by Humana Press;
each herein incorporated by reference in their entireties.
[0158] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of organic chemistry,
pharmacology, molecular biology (including recombinant techniques),
cell biology, biochemistry, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature, such as, "Molecular cloning: a laboratory manual"
Second Edition (Sambrook et al., 1989); "Oligonucleotide synthesis"
(M. J. Gait, ed., 1984); "Animal cell culture" (R. I. Freshney,
ed., 1987); the series "Methods in enzymology" (Academic Press,
Inc.); "Handbook of experimental immunology" (D. M. Weir & C.
C. Blackwell, eds.); "Gene transfer vectors for mammalian cells"
(J. M. Miller & M. P. Calos, eds., 1987); "Current protocols in
molecular biology" (F. M. Ausubel et al., eds., 1987, and periodic
updates); "PCR: the polymerase chain reaction" (Mullis et al.,
eds., 1994); and "Current protocols in immunology" (J. E. Coligan
et al., eds., 1991), each of which is herein incorporated by
reference in its entirety.
I. Modulators of Cell Death
[0159] In preferred embodiments, the compounds of the present
invention regulate apoptosis through the exposure of cells to
compounds. The effect of compounds can be measured by detecting any
number of cellular changes. Cell death may be assayed as described
herein and in the art. In preferred embodiments, cell lines are
maintained under appropriate cell culturing conditions (e.g., gas
(CO.sub.2), temperature and media) for an appropriate period of
time to attain exponential proliferation without density dependent
constraints. Cell number and or viability are measured using
standard techniques, such as trypan blue exclusion/hemo-cytometry,
or MTT dye conversion assay. Alternatively, the cell may be
analyzed for the expression of genes or gene products associated
with aberrations in apoptosis or necrosis.
[0160] In preferred embodiments, exposing the compounds of the
present invention to a cell induces apoptosis. In some embodiments,
the present invention causes an initial increase in cellular ROS
levels (e.g., O.sub.2.sup.-). In further embodiments, exposure of
the compounds of the present invention to a cell causes an increase
in cellular O.sub.2.sup.- levels. In still further embodiments, the
increase in cellular O.sub.2.sup.- levels resulting from the
compounds of the present invention is detectable with a
redox-sensitive agent that reacts specifically with O.sub.2.sup.-
(e.g., dihyroethedium (DHE)).
[0161] In other embodiments, increased cellular O.sub.2.sup.-
levels resulting from compounds of the present invention diminish
after a period of time (e.g., 10 minutes). In other embodiments,
increased cellular O.sub.2.sup.- levels resulting from the
compounds of the present invention diminish after a period of time
and increase again at a later time (e.g., 10 hours). In further
embodiments, increased cellular O.sub.2.sup.- levels resulting from
the compounds of the present invention diminish at 1 hour and
increase again after 4 hours. In preferred embodiments, an early
increase in cellular O.sub.2.sup.- levels, followed by a
diminishing in cellular O.sub.2.sup.- levels, followed by another
increase in cellular O.sub.2.sup.- levels resulting from the
compounds of the present invention is due to different cellular
processes (e.g., bimodal cellular mechanisms).
[0162] In some embodiments, the compounds of the present invention
cause a collapse of a cell's mitochondrial .DELTA..PSI..sub.m. In
preferred embodiments, a collapse of a cell's mitochondrial
.DELTA..PSI..sub.m resulting from the present invention is
detectable with a mitochondria-selective potentiometric probe
(e.g., DiOC.sub.6). In further embodiments, a collapse of a cell's
mitochondrial .DELTA..PSI..sub.m resulting from the present
invention occurs after an initial increase in cellular
O.sub.2.sup.- levels.
[0163] In some embodiments, the compounds of the present invention
enable caspace activation. In other embodiments, the compounds of
the present invention cause the release of cytochrome c from
mitochondria. In further embodiments, the compounds of the present
invention alter cystolic cytochrome c levels. In still other
embodiments, altered cystolic cytochrome c levels resulting from
the compounds of the present invention are detectable with
immunoblotting cytosolic fractions. In preferred embodiments,
diminished cystolic cytochrome c levels resulting from the
compounds of the present invention are detectable after a period of
time (e.g., 10 hours). In further preferred embodiments, diminished
cystolic cytochrome c levels resulting from the compounds of the
present invention are detectable after 5 hours.
[0164] In other embodiments, the compounds of the present invention
cause the opening of the mitochondrial PT pore. In preferred
embodiments, the cellular release of cytochrome c resulting from
the compounds of the present invention are consistent with a
collapse of mitochondrial .DELTA..PSI..sub.m. In still further
preferred embodiments, the compounds of the present invention cause
an increase in cellular O.sub.2.sup.- levels after a mitochondrial
.DELTA..PSI..sub.m collapse and a release of cytochrome c. In
further preferred embodiments, a rise in cellular O.sub.2.sup.-
levels is caused by a mitochondrial .DELTA..PSI..sub.m collapse and
release of cytochrome c resulting from the compounds of the present
invention.
[0165] In other embodiments, the compounds of the present invention
cause cellular caspase activation. In preferred embodiments,
caspase activation resulting from the compounds of the present
invention is measurable with a pan-caspase sensitive fluorescent
substrate (e.g., FAM-VAD-fink). In still further embodiments,
caspase activation resulting from the compounds of the present
invention tracks with a collapse of mitochondrial
.DELTA..PSI..sub.m. In other embodiments, the compounds of the
present invention cause an appearance of hypodiploid DNA. In
preferred embodiments, an appearance of hypodiploid DNA resulting
from the compounds of the present invention is slightly delayed
with respect to caspase activation.
[0166] In some embodiments, the molecular target for the compounds
of the present invention is found within mitochondria. In further
embodiments, the molecular target of the compounds of the present
invention involves the mitochondrial ATPase. The primary sources of
cellular ROS include redox enzymes and the mitochondrial
respiratory chain (hereinafter MRC). In preferred embodiments,
cytochrome c oxidase (complex IV of the MRC) inhibitors (e.g.,
NaN.sub.3) preclude a dependent increase in cellular ROS levels for
the compounds of the present invention. In other preferred
embodiments, the ubiquinol-cytochrome c reductase component of MRC
complex III inhibitors (e.g., FK506) preclude a dependent increase
in ROS levels for the compounds of the present invention.
[0167] In some embodiments, an increase in cellular ROS levels due
to the compounds of the present invention result from the binding
of the compounds of the present invention to a target within
mitochondria. In preferred embodiments, the compounds of the
present invention oxidizes 2',7'-dichlorodihydrofluorescin
(hereinafter DCF) diacetate to DCF. DCF is a redox-active species
capable of generating ROS. In further embodiments, the rate of DCF
production resulting from the present invention increases after a
lag period.
[0168] Antimycin A generates O.sub.2.sup.- by inhibiting
ubiquinol-cytochrome c reductase. In preferred embodiments, the
compounds of the present invention increase the rate of ROS
production in an equivalent manner to antimycin A. In further
embodiments, the compounds of the present invention increase the
rate of ROS production in an equivalent manner to antimycin A under
aerobic conditions supporting state 3 respiration. In further
embodiments, the compounds of the present invention do not directly
target the MPT pore. In additional embodiments, the compounds of
the present invention do not generate substantial ROS in the
subcellular S15 fraction (e.g., cytosol; microsomes). In even
further embodiments, the compounds of the present invention do not
stimulate ROS if mitochondria are in state 4 respiration.
[0169] MRC complexes I-III are the primary sources of ROS within
mitochondria. In preferred embodiments, the primary source of an
increase in cellular ROS levels resulting from the dependent
invention emanates from these complexes as a result of inhibiting
the mitochondrial F.sub.1F.sub.0-ATPase. Indeed, in still further
embodiments, the present invention inhibits mitochondrial ATPase
activity of bovine sub-mitochondrial particles (hereinafter SMPs).
In particularly preferred embodiments, the compounds of the present
invention bind to the OSCP component of the mitochondrial
F.sub.1F.sub.0-ATPase.
[0170] Oligomycin is a macrolide natural product that binds to the
mitochondrial F.sub.1F.sub.0-ATPase, induces a state 3 to 4
transition, and as a result, generates ROS (e.g., O.sub.2.sup.-).
In preferred embodiments, the compounds of the present invention
bind the OSCP component of the mitochondrial F.sub.1F.sub.0-ATPase.
In preferred embodiments, the compounds of the present invention
bind the junction between the OSCP and the F.sub.1 subunit of the
mitochondrial F.sub.1F.sub.0-ATPase. In some embodiments, the
compounds of the present invention bind the F, subunit. In certain
embodiments, screening assays of the present invention permit
detection of binding partners of the OSCP, F.sub.1, or OSCP/F.sub.1
junction. OSCP is an intrinsically fluorescent protein. In certain
embodiments, titrating a solution of test compounds of the present
invention into an E. Coli sample overexpressed with OSCP results in
quenching of the intrinsic OSCP fluorescence. In other embodiments,
fluorescent or radioactive test compounds can be used in direct
binding assays. In other embodiments, competition binding
experiments can be conducted. In this type of assay, test compounds
are assessed for their ability to compete with Bz-423 for binding
to the OSCP. In some embodiments, the compounds of the present
invention cause a reduced increase in cellular ROS levels and
reduced apoptosis in cells through regulation of the OSCP gene
(e.g., altering expression of the OSCP gene). In further
embodiments, the present invention functions by altering the
molecular motions of the ATPase motor.
II. Modulators of Cellular Proliferation and Cell Growth
[0171] In some embodiments, the compounds and methods of the
present invention cause descreased cellular proliferation. In other
embodiments, the compounds and methods of the present invention
cause decreased cellular proliferation and apoptosis. For example,
cell culture cytotoxicity assays conducted during the development
of the present invention demonstrated that the compounds and
methods of the present invention prevents cell growth after an
extended period in culture (e.g., 3 days).
III. Benzodiazepine and Benzodiazepine Related Crystal Forms
[0172] The present invention relates to systems and methods for
generating new formulations of benzodiazepine compounds and
benzodiazepine related compounds. The present invention also
provides high throughput systems and methods for generating and
identifying new crystalline benzodiazepine and benzodiazepine
related compounds.
[0173] For example, the present invention provides libraries of
polymers from which crystals are nucleated by exposing solutions
(e.g., supersaturated solutions), the melt or vapor of the compound
to the polymers. Growth of crystals on a plurality of polymers
provides new methods for obtaining desired polymorphs of compounds
and for generating previously unidentified polymorphs of compounds.
For example, the systems and methods of the present invention have
been used to identify novel polymorphs of benzodiazepine compounds.
For example, the systems and methods have also been used to
generate efficient methods for producing orthorhombic
benzodiazepine compounds from solution. The novel polymorphs
identified by the systems and methods of the present invention find
use in identifying drugs with enhanced properties, compared to
previously available polymorphs of the compound. Thus, the systems
and methods of the present invention provide means for finding drug
leads and/or optimizing existing drugs (see, e.g., U.S. patent
application Ser. No. 10/269,190; herein incorporated by reference
in its entirety).
[0174] Many pharmaceutical solids exhibit polymorphism (e.g., the
ability of a substance to exist as two or more crystalline phases
that have different arrangements and/or conformations of the
molecules in the crystal lattice). Because of their structural
differences, polymorphs have different solid-state properties.
Consequently, polymorphism can exert profound effects on
pharmaceutical processing, including, but not limited to, milling,
granulation, and tableting (Conte et al., II Farmaco (Ed. Pr.)
30:194 (1974); Otsuka et al., Chem. Pharm. Bull., 45:894 (1997);
Otsuka et al., J. Pharm. Sci., 84:614 (1995); Tuladhar et al., J.
Pharm. Pharmacol., 35:269 (1982); and Wong and Mitchell, Int.; J.
Pharm., 88:261 (1992)).
[0175] Despite the fact that upon dissolution, two polymorphs will
yield identical solutions, the crystalline form affects the rate of
dissolution, equilibrium solubility, shelf life and ultimately
bioavailabilty. This has implications for isolation, clinical
trials, and mass production and is therefore an important aspect of
creating a viable therapeutic. With a greater number of polymorphs
to choose between for a solid dosage, it is more likely that an
optimal mixture of properties can be achieved leading to more
efficacious drugs.
[0176] In its most simple form, the process of crystallization can
be considered to start from a supersaturated solution, produced by
evaporation, cooling, or addition of a nonsolvent, by formation of
nuclei. These species must achieve a sufficient size in order to
proceed on to bulk crystals and it is the arrangement of the
molecules in these nanometer-sized structures that leads to the
macroscopic crystal. Thus the formation of unstable polymorphs can
be attributed to their success in forming viable nuclei, a kinetic
effect. Additives designed by consideration of functional groups
and lattice parameters (derived from diffraction methods) can also
interact with these nuclei to stabilize or destabilize them, and
this approach of using designed additives has met with success in
some cases (Weissbuch et al., Acta Crystallogr. Sect. B-Struct.
Sci. 51:115 (1995); Chen et al., J. Cryst. Growth 144:297 (1994);
and Davey et al., J. Am. Chem. Soc., 119:1767 (1997)). However,
this method is best suited for modifying the crystallization
behavior of known polymorphs and is not readily adapted to the
generation of new forms with unknown lattice parameters.
[0177] Even in crystallizations where no additives are used, it is
recognized that spontaneous (homogenous) nucleation is not very
common, and in most cases impurities on vessel walls function as
heteronuclei to induce crystal formation. The reluctance of
saturated solutions to undergo homogeneous nucleation can be
explained by the energetic barrier to building a species with a
high surface area to volume ratio where many of the molecules do
not experience the full stabilization of the bulk. A heteronucleus
reduces this barrier by providing stabilization of a growing face
of the crystal.
[0178] The present invention provides systems and methods for
utilizing a combinatorial library of functionalized polymers for
crystal formation. Both the types of functional groups and the
spacing of these groups are altered to produce surfaces that
facilitate polymorph generation. By varying these parameters (e.g.,
systematically) throughout the library, these polymers produce
crystal forms without prior knowledge of the polymorph's structure
and allow the discovery of new forms of compounds (e.g.,
pharmaceutical compounds, etc.) with improved properties over
previously available structures. Properties that differ among
polymorphs include, but are not limited to: packing properties
(e.g., molar volume and density, refractive index, electrical
conductivity, thermal conductivity, hygroscopicity); thermodynamic
properties (e.g., melting and sublimation temperatures, internal
[e.g., structural] energy, enthalpy, heat capacity, entropy, free
energy and chemical potential, thermodynamic activity, vapor
pressure, solubility); spectroscopic properties (e.g., electronic
transitions such as ultraviolet to visible absorption spectra,
vibrational transitions such as infrared absorption and Raman
spectra, rotational transitions such as far infrared and microwave
absorption spectra, nuclear spin transitions such as nuclear
magnetic resonance spectra); kinetic properties (e.g., dissolution
rate, rates of solid state reactions, and stability); surface
properties (e.g., surface free energy, interfacial tensions,
habit); and mechanical properties (e.g., hardness, tensile
strength, compactibility, tableting, handling, flow, and blending)
(See e.g., "Polymorphism in Pharmaceutical Solids," ed. Harry G.
Brittain, Marcel Dekker, Inc., New York (1999)).
[0179] In some embodiments of the present invention, a plurality of
polymers are provided with (e.g., placed onto or into) a solid
surface or vessel to facilitate high throughput crystal growth and
analysis. In some embodiments, the solid surface or vessel is a
multi-chamber plate (e.g., a 96-well or 384-well plate). However,
the present invention is not limited by the solid surface or vessel
employed. As used herein, the terms "solid support" or "support"
refer to any material that provides a solid or semi-solid structure
with which another material (e.g., a polymer) can be associated.
Such materials include smooth supports (e.g., metal, glass,
plastic, silicon, and ceramic surfaces) as well as textured and
porous materials. Such materials also include, but are not limited
to, gels, rubbers, polymers, and other non-rigid materials. Solid
supports need not be flat. Supports include any type of shape
including spherical shapes (e.g., beads). Materials associated with
the solid support may be associated with any portion of the solid
support (e.g., may be attached, enclosed, or in contact with an
interior portion of a porous solid support material).
[0180] The present invention is not limited by the nature of the
polymer used to promote crystal growth. In preferred embodiments,
the plurality of polymers used in screening methods of the present
invention comprise two or more polymers (e.g., three or more, four
or more, five or more, . . . , ten or more, . . . , twenty or more,
. . . , fifty or more different polymers). The maximum number of
polymers employed in the systems and methods of the present
invention is constrained only by the availability of polymer
materials (i.e., any of the thousands of known polymers may be
employed, as well as new polymers that are identified in the
future) and by physical and space limitations of the testing area.
However, the systems and methods of the present invention may be
employed at very large scales. For example, in some embodiments,
384-well plates are used wherein the bottom surface of each well
contains a different polymer material. Dozens of such plates may be
arranged on shelves and dozens of shelves may be placed in racks. A
single laboratory space can hold hundreds of racks. Thus, a single
room can house tens of millions of different polymers, wherein a
solution with a candidate compound is applied to each of the
polymers and crystals are grown and analyzed to identify the
properties of the crystals. Further miniaturization allows even
more reactions to be run simultaneously in a single run.
[0181] While the present invention is not limited by the nature of
the polymer, commercially available polymers that find use with the
present invention include, but are not limited to,
acrylonitrile/butadiene/styrene resin, alginic acid (sodium salt),
butyl/isobutyl methacrylate copolymer, cellulose acetate, cellulose
acetate butyrate, cellulose propionate, cellulose triacetate, ethyl
cellulose, ethylene/acrylic acid copolymer, ethylene/ethyl acrylate
copolymer, ethylene/propylene copolymer, ethylene/vinyl acetate
(14, 18, 25, 28, 33% and 40% VA) copolymer, hydroxybutyl methyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
methyl cellulose, methyl vinyl ether/maleic acid copolymer, methyl
vinyl ether/maleic anhydride copolymer, nylon 6, nylon 6/6, nylon
6/9, nylon 6/10, nylon 6/12, nylon 6(3)T, nylon 11, nylon 12,
phenoxy resin, polyacetal, polyacrylamide, polyacrylamide carboxyl
modified (low), polyacrylamide carboxyl modified (high),
poly(acrylic acid), polyamide resin, 1,2-polybutadiene,
poly(1-butene) isotactic, poly(n-butyl methacrylate), polycarbonate
resin, poly(diallyl isophthalate), poly(diallyl phthalate),
poly(2,6-dimethyl-p-phenylene oxide),
poly(4,4-dipropoxy-2,2-diphenyl propane fumarate), poly(ethyl
methacrylate), polyethylene high density, polyethylene low density,
polyethylene chlorinated (25, 36, 42, and 48% chlorine),
polyethylene chlorosulfonated, poly(ethylene oxide), polyethylene
oxidized, poly(ethylene terephthalate), poly(2-hydroxyethyl
methacrylate), poly(isobutyl methacrylate), polyisoprene
chlorinated, poly(methyl methacrylate), poly(4-methyl-1-pentene),
poly(alpha-methylstyrene), poly(p-phenylene ether-sulphone),
poly(phenylene sulfide), polypropylene isotactic chlorinated,
polypropylene isotactic, polystyrene, polysulfone resin,
poly(tetrafluoroethylene), poly(2,4,6-tribromostyrene), poly(vinyl
acetate), poly(vinyl alcohol) 100% hydrolyzed, poly(vinyl alcohol)
98% hydrolyzed, poly(vinyl buyral), poly(vinyl chloride),
poly(vinyl chloride) 1.8% carboxylated, poly(vinyl formal),
polyvinylpyrrolidone, poly(vinyl stearate), poly(vinylidene
fluoride), styrene/acrylonitrile copolymer (75/25),
styrene/acrylonitrile copolymer (70/30), styrene/allyl alcohol
copolymer, styrene/butadiene ABA block copolymer, styrene/butyl
methacrylate copolymer, styrene/ethylene-butylene ABA block
copolymer, styrene/maleic anhydride copolymer, vinyl alcohol/vinyl
butyral copolymer, vinyl chloride/vinyl acetate (10, 12, and 19%
VA) copolymer, vinyl chloride/vinyl acetate copolymer 1%
carboxylated, vinyl chloride/vinyl acetate/hydroxypropyl acrylate
terpolymer, and vinyl chloride/vinyl acetate/vinyl alcohol
terpolymer, as well as, functionalized polybutadienes,
poly(ethylene-co-propylene-co-5-methylene-2-norbornene),
poly(perfluoropropylene oxide)-co-poly(perfluoroformaldehyde),
metaldehyde, pectic acid, polyethylenimine, poly(ethylene-co-carbon
monoxide), poly(3-hydroxybutyric acid) and copolymers with valeric
acid, polylactide, polyaminoacids, polyacenaphthylene,
poly(dimethylsiloxane), poly[(dibenzo-18-crown-6)-co-formaldehyde]
and other polymers containing metal chelating groups, poly[(phenyl
isocyanate)-co-formaldehyde], poly(vinylsulfonic acid),
poly(melamine-co-formaldehyde), polyphosphates, polyphosphazenes,
tributyltin fluoride, polysaccharides, and other organic and
inorganic polymers.
[0182] Polymers at each location in the library can comprise a
mixture of two or more different polymers in one or more different
locations. The combination of polymers in different ratios
dramatically expands the diversity of conditions available in the
libraries.
[0183] Solutions containing the compound to be screened are applied
to the polymers and incubated under conditions that facilitate
crystal growth. The present invention is not limited by the manner
in which the compounds are applied to the polymers. In some
embodiments, a solution is used to supply each region of a polymer
library. Solution may be delivered by pouring, transfer through
tubing, injection, or any other means. Where thousands to millions
of individual polymers are used, in preferred embodiments, an
automated delivery system is used. The present invention is not
limited to the use of solutions. Melts of materials and vapors onto
the polymers also find use in the system and methods of the present
invention.
[0184] The solvent used to solubilize any particular compound may
be varied. In some embodiments, a variety of solvents are used for
each compound, wherein each different solvent type is exposed to
each type of polymer to increase the range of crystallization
conditions used in the library. In such embodiments, multiple
regions (e.g., zones) of each polymer are created in the library to
allow each solvent type to be combined with each polymer type. In
addition to different solvent types, a variety of different
ingredients (e.g., salts) may be placed in the solution, yet
further expanding the array of choices for library analysis. Such
applications find use in the generation and isolation of new
pseudopolymorphs (solvates and salts) that may be used as
drugs.
[0185] Crystals formed on each polymer are analyzed using any
suitable method. In some embodiments, analysis is conducted
directly on the polymer surface, without removing the crystals. In
other embodiments, crystals are removed and analyzed. Analysis
includes, but is not limited to, crystal structure analysis,
analysis of spectroscopic, packing, density, thermodynamic,
properties, kinetic, surface, and mechanical properties. In some
embodiments, analysis includes functional analysis such as testing
bioavailability or biological activity after administration to a
test organism (e.g., an animal or plant). For example, in some
embodiments, rapid screening is conducted using the D8 Discover
with GADDS X-ray diffraction system (Bruker AXS, Madison, Wis.) or
similar systems.
[0186] Polymorphs identified in the screening method are compared
to existing polymorphs. Where a new polymorph is identified, the
polymorph is characterized to identify properties that differ from
previously known polymorphs (e.g., to identify improved drugs).
Known polymorphs generated using the systems and methods of the
present invention also are compared to existing production methods
to identify whether the polymer-based method of the present
invention provides advantages over existing production methods
(e.g., less expensive or easier to produce, greater purity,
superior crystals, ability to produce from aqueous solution,
etc.).
[0187] Polymorphs identified by the present invention can be
produced in large quantities. In some embodiments, crystals are
collected and used to seed further solutions of the compound.
However, in some cases the presence of the polymer surface may be
required to generate crystals. In such embodiments, large or
multiple surfaces or vessels are provided with the polymer known to
generate the crystal to allow large-scale production.
[0188] Polymorphs produced by the methods of the present invention
may be used in the generation of pharmaceutical formulations. The
novel polymorphs identified increase the available choices for
designing drugs with desired properties, both in biological
activity and in handling. For example, in order for many drugs to
take action, they must dissolve in the gut and be absorbed in the
blood stream. In many cases the rate at which the drug dissolves
can limit its effectiveness. The polymorphs of the present
invention, either alone, or in combination with other forms of the
drug find use in optimizing effectiveness, generally, or for
particular patients or patient groups (e.g., age groups, genders,
species, etc.). In some cases, the novel polymorphs provide
advantages in shelf-life or the ability of the compound to be
included in tablets (See e.g., Sun and Grant, Pharm. Res., 18:274
(2001)).
[0189] Exemplary benzodiazepine compounds provided by the present
invention include crystal forms and formulations of Bz-423:
##STR8##
[0190] 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")
weak (K.sub.d ca. 1 .mu.M) and prevents binding to the central
benzodiazepine receptor so that Bz-423 is not a sedative.
Additionally, compostions of the present invention comprising
benzodiazepine compounds (e.g., Bz-423) have been shown to bind to
the oligomycin sensitivity conferring protein (OSCP) portion of the
mitochondrial F.sub.0F.sub.1 ATPase synthase complex, to the OSCP
junction, or to the F.sub.1 subunit (see, e.g., U.S. Provisional
Patent Nos. 60/131,761, 60/165,511, 60/191,855, 60/312,560,
60/313,689, 60/396,670, 60/565,788, 60/607,599, 60/641,040, and
U.S. patent application Ser. Nos. 11/324,419, 11/176,719,
11/110,228, 10/935,333, 10/886,450, 10/795,535, 10/634,114,
10/427,211, 10/427,212, 10/217,878, 09/767,283, 09/700,101, and
related applications; each herein incorporated by reference in
their entireties).
[0191] Exemplary crystal forms of Bz-423 and related compounds
provided by the present invention include, but are not limited to,
anhydrous Bz-423, Bz-423 ethanol solvate, Bz-423 succinic acid
(2:1), Bz-423 citric acid (2:1), Bz-423 biphenyl derivate,
BZ-423-acetic acid, BZ-423-CH.sub.3CN, BZ-423-methanol,
BZ-423-ethyl acetate, BZ-423-toluene, BZ-423-oxalic acid,
BZ-423-fumaric acid, BZ-423-octanol, BZ-423-heptanoic acid,
BZ-423-diphenyl ether, Bz-423-1-propanol solvate, Bz-423 2-propanol
solvate, Bz-423 1-butanol solvate, Bz-423 2-butanol solvate, Bz-423
1-pentanol solvate, Bz-423 propylene glycol, Bz-423 acetone glass,
and BZ-423-trichlorobenzene.
[0192] Additional exemplary compounds of the present invention also
include, but are not limited to, crystal forms and formulations of:
##STR9## or its enantiomer, wherein, R.sub.1 is aliphatic or aryl;
R.sub.2 is aliphatic, aryl, --NH.sub.2, --NHC(.dbd.O)--R.sub.5; or
a moiety that participates in hydrogen bonding, wherein R.sub.5 is
aryl, heterocyclic, --R.sub.6--NH--C(.dbd.O)--R.sub.7 or
--R.sub.6--C(.dbd.O)--NH--R.sub.7, wherein R.sub.6 is an aliphatic
linker of 1-6 carbons and R.sub.7 is aliphatic, aryl, or
heterocyclic, each of R.sub.3 and R.sub.4 is independently a
hydroxy, alkoxy, halo, amino, lower-alkyl-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.
[0193] In the above structures, R.sub.1 is a hydrocarbyl group of
1-20 carbons and 1-20 hydrogens. Preferably, R.sub.1 has 1-15
carbons, and more preferably, has 1-12 carbons. Preferably, R.sub.1
has 1-12 hydrogens, and more preferably, 1-10 hydrogens. Thus
R.sub.1 can be an aliphatic group or an aryl group.
[0194] The term "aliphatic" represents the groups commonly known as
alkyl, alkenyl, alkynyl, alicyclic. The term "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.sub.1-C.sub.4 alkyl, alkenyl, alkynyl, or
C.sub.3-C.sub.6 alicyclic). Additionally, the aliphatic and aryl
groups can be further substituted by one or more functional groups
such as --NH.sub.2, --NHCOCH.sub.3, --OH, lower alkoxy
(C.sub.1-C.sub.4), halo (--F, --Cl, --Br, or --I). It is preferable
that R.sub.1 is primarily a nonpolar moiety.
[0195] In the above structures, R.sub.2 can be aliphatic, aryl,
--NH.sub.2, --NHC(.dbd.O)--R.sub.5, or a moiety that participates
in hydrogen bonding, wherein R.sub.5, is aryl, heterocyclic,
R.sub.6--NH--C(.dbd.O)--R.sub.7 or
--R.sub.6--C(.dbd.O)--NH--R.sub.7, wherein R.sub.6 is an aliphatic
linker of 1-6 carbons and R.sub.7 is an aliphatic, aryl, or
heterocyclic. The terms "aliphatic" and "aryl" are as defined
above.
[0196] 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.
[0197] Some specific non-limiting examples of moieties that
participate in hydrogen bonding include a fluoro, oxygen-containing
and nitrogen-containing groups that are well-known in the art. Some
examples of oxygen-containing groups that participate in hydrogen
bonding include: hydroxy, lower alkoxy, lower carbonyl, lower
carboxyl, lower ethers and phenolic groups. The qualifier "lower"
as used herein refers to lower aliphatic groups (C.sub.1-C.sub.4)
to which the respective oxygen-containing functional group is
attached.
[0198] Thus, for example, the term "lower carbonyl" refers to inter
alia, formaldehyde, acetaldehyde.
[0199] Some nonlimiting examples of nitrogen-containing groups that
participate in hydrogen bond formation include amino and amido
groups. Additionally, groups containing both an oxygen and a
nitrogen atom can also participate in hydrogen bond formation.
Examples of such groups include nitro, N-hydroxy and nitrous
groups.
[0200] It is also possible that the hydrogen-bond acceptor in the
present invention can be the .PI. 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).
[0201] 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.
[0202] Aromatic and nonaromatic heterocyclic rings are well-known
in the art. Some nonlimiting examples of aromatic heterocyclic
rings include pyridine, pyrimidine, indole, purine, quinoline and
isoquinoline. Nonlimiting examples of nonaromatic heterocyclic
compounds include piperidine, piperazine, morpholine, pyrrolidine
and pyrazolidine. Examples of oxygen containing heterocyclic rings
include, but not limited to furan, oxirane, 2H-pyran, 4H-pyran,
2H-chromene, and benzofuran. Examples of sulfur-containing
heterocyclic rings include, but are not limited to, thiophene,
benzothiophene, and parathiazine.
[0203] 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.
[0204] Examples of heterocyclic rings containing two different
heteroatoms include, but are not limited to, phenothiazine,
morpholine, parathiazine, oxazine, oxazole, thiazine, and
thiazole.
[0205] The heterocyclic ring is optionally further substituted with
one or more groups selected from aliphatic, nitro, acetyl (i.e.,
--C(.dbd.O)--CH.sub.3), or aryl groups.
[0206] Each of R.sub.3 and R.sub.4 can be independently a hydroxy,
alkoxy, halo, amino, or substituted amino (such as
lower-alkyl-substituted-amino, or acetylamino or hydroxyamino), or
an aliphatic group having 1-8 carbons and 1-20 hydrogens. When each
of R.sub.3 and R.sub.4 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
R.sub.3 and R.sub.4 can be hydrogen.
[0207] It is well-known that many 1,4-benzodiazepines exist as
optical isomers due to the chirality introduced into the
heterocyclic ring at tile C.sub.3 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.
[0208] From the above description, it is apparent that many
specific examples are represented by the generic formulas presented
above. Thus, in one example, R.sub.1 is aliphatic, R.sub.2 is
aliphatic, whereas in another example, R.sub.1 is aryl and R.sub.2
is a moiety that participates in hydrogen bond formation.
Alternatively, R.sub.1 can be aliphatic, and R.sub.2 can be an
--NHC(.dbd.O)--R.sub.5, or a moiety that participates in hydrogen
bonding, wherein R.sub.5 is aryl, heterocyclic,
--R.sub.6--NH--C(.dbd.O)--R.sub.7 or
--R.sub.6--C(.dbd.O)--NH--R.sub.7, wherein R.sub.6 is an aliphatic
linker of 1-6 carbons and R.sub.7 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.
[0209] Additional exemplary compounds of the present invention also
include, but are not limited to, crystal forms and formulations of:
##STR10## [0210] enantiomers and pharmaceutically acceptable salts
thereof: [0211] wherein R.sub.1 is an aliphatic group having 1 to
20 carbon atoms and 1 to 20 hydrogen atoms or an aryl group having
up to 20 carbon atoms and up to 20 hydrogen atoms; [0212] wherein
each of R.sub.2 and R.sub.3 is independently selected from the
group consisting of hydrogen, hydroxy, C.sub.4alkoxy, halo, amino,
C.sub.1-4alkyl-substituted-amino, acetylamino, hydroxyamino, an
aliphatic group having 1-8 carbons and 1-20 hydrogens, an aryl
group having from 6 to 14 carbon atoms, and a heterocyclic group
having a 3-6 membered aromatic or nonaromatic ring containing one
or more heteroatoms selected from nitrogen, oxygen, and sulfur;
[0213] wherein R.sub.4 is aliphatic or aryl; [0214] wherein R.sub.5
is selected from the group consisting of an aryl group having from
6 to 14 carbon atoms, a heterocyclic group having a 3-6 membered
aromatic or nonaromatic ring containing one or more heteroatoms
selected from nitrogen, oxygen, and sulfur,
--R.sub.6--NH--C(.dbd.O)--R.sub.7, and
--R.sub.6--C(.dbd.O)--NH--R.sub.7; [0215] wherein R.sub.6 is an
aliphatic linker group of 1-6 carbons; and, [0216] wherein R.sub.7
is selected from the group consisting of an aliphatic group having
1-8 carbons and 1-20 hydrogens, an aryl group having from 6 to 14
carbon atoms and a heterocyclic group having a 3-6 membered
aromatic or nonaromatic ring containing one or more heteroatoms
selected from nitrogen, oxygen, and sulfur.
[0217] In some preferred embodiments, crystal forms and
formulations of the following exemplary compound are provided:
##STR11##
[0218] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR12## or its enantiomer, wherein, R.sub.1 is aliphatic or aryl;
R.sub.2 is aliphatic, aryl, --NH.sub.2, --NHC(.dbd.O)--R.sub.5; or
a moiety that participates in hydrogen bonding, wherein R.sub.5 is
aryl, heterocyclic, --R.sub.6--NH--C(.dbd.O)--R.sub.7 or
--R.sub.6--C(.dbd.O)--NH--R.sub.7, wherein R.sub.6 is an aliphatic
linker of 1-6 carbons and R.sub.7 is aliphatic, aryl, or
heterocyclic, each of R.sub.3 and R.sub.4 is independently a
hydroxy, alkoxy, halo, amino, lower-alkyl-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.
[0219] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR13##
[0220] and dimethylphenyl (all isomers) and ditrifluoromethyl (all
isomers).
[0221] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR14##
[0222] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR15## or a stereoisomer, a pharmaceutically-acceptable salt,
hydrate, or prodrug thereof, wherein: R.sub.1 and R.sub.5 are
attached to any available carbon atom of phenyl rings A and B
respectively, and at each occurrence are independently selected
from alkyl, substituted alkyl, halogen, cyano, nitro, OR.sub.8,
NR.sub.8R.sub.9, C(.dbd.O)R.sub.8, CO.sub.2R.sub.8,
C(.dbd.O)NR.sub.8R.sub.9, NR.sub.8C(.dbd.O)R.sub.9,
NR.sub.8C(.dbd.O)OR.sub.9, S(O)OR.sub.9, NR.sub.8SO.sub.2R.sub.9,
SO.sub.2NR.sub.8R.sub.9, cycloalkyl, heterocycle, aryl, and
heteroaryl, and/or two of R.sub.1 and/or two of R.sub.5 join
together to form a fused benzo ring; R.sub.2, R.sub.3 and R.sub.4
are independently selected from hydrogen, alkyl, and substituted
alkyl, or one of R.sub.2, R.sub.3 and R.sub.4 is a bond to R, T or
Y and the other of R.sub.2, R.sub.3 and R.sub.4 is selected from
hydrogen, alkyl, and substituted alkyl; Z and Y are independently
selected from C(.dbd.O), --CO.sub.2--, --SO.sub.2--, CH.sub.2--,
--CH.sub.2C(.dbd.O), and --C(.dbd.O)C(.dbd.O)--, or Z may be
absent; R and T are selected from --CH.sub.2--, --C(.dbd.O), and
--CH[(CH.sub.2).sub.p(Q)]-, wherein Q is NR.sub.10R.sub.11,
OR.sub.10 or CN; R.sub.6 is selected from alkyl, alkenyl,
substituted alkyl, substituted alkenyl, aryl, cycloalkyl,
heterocyclo, and heteroaryl; provided that where R.sub.2 is
hydrogen, Z-R.sub.6 together are not --SO.sub.2-Me or ##STR16##
R.sub.7 is selected from hydrogen, alkyl, substituted alkyl,
alkenyl, substituted alkenyl, aminoalkyl, halogen, cyano, nitro,
keto (.dbd.O), hydroxy, alkoxy, alkylthio, C(.dbd.O)H, acyl,
CO.sub.2H, alkoxycarbonyl, carbamyl, sulfonyl, sulfonamidyl,
cycloalkyl, heterocycle, aryl, and heteroaryl; R.sub.8 and R.sub.9
are independently selected from hydrogen, alkyl, substituted alkyl,
cycloalkyl, heterocycle, aryl, and heteroaryl, or R.sub.8 and
R.sub.9 taken together to form a heterocycle or heteroaryl, except
R.sub.9 is not hydrogen when attached to a sulfonyl group as in
SO.sub.2R.sub.9; R.sub.10 and R.sub.1, are independently selected
from hydrogen, alkyl, and substituted alkyl; m and n are
independently selected from 0, 1, 2 and 3; o, p and q are
independently 0, 1 or 2; and r and t are 0 or 1.
[0223] In further exemplary compounds, Z-R.sub.6 taken together are
selected from: i. thiophenyl optionally substituted with R.sub.14;
ii. imidazolyl optionally substituted with R.sub.14; iii.
--CH(aryl)(CO.sub.2C.sub.1-6alkyl); iv. --CO.sub.2-alkyl; v.
--SO.sub.2-alkyl optionally substituted with up to three of halogen
and/or phenyl; vi. --SO.sub.2-alkenyl optionally substituted with
phenyl; and vii. ##STR17## wherein R.sub.15 is halogen, alkyl,
nitro, cyano, hydroxy, alkoxy, NHC(.dbd.O)alkyl, and/or two
R.sub.15 groups are taken together to form a fused benzo ring or a
five to six membered heteroaryl; R.sub.16 is selected from
hydrogen, halogen, alkyl, nitro, cyano, hydroxy, alkoxy,
NHC(.dbd.O)alkyl, and phenyloxy or benzyloxy in turn optionally
substituted with 1 to 3 of halogen, cyano, and C.sub.4alkoxy;
R.sub.17 is selected from alkyl, alkoxy, CO.sub.2C.sub.1-6alkyl,
and SO.sub.2phenyl; and u and v are independently 0, 1 or 2.
[0224] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR18## or a stereoisomer, a pharmaceutically-acceptable salt,
hydrate, or prodrug thereof, in which: R.sub.1 and R.sub.5 are
attached to any available carbon atom of phenyl ring A and phenyl
ring B, respectively, and at each occurrence are independently
selected from C.sub.1-6alkyl, substituted C.sub.1-6alkyl, halogen,
cyano, O(C.sub.1-6alkyl), O(phenyl), O(benzyl), NH.sub.2,
NH(C.sub.1-6alkyl), N(C.sub.1-6alkyl).sub.2, C(.dbd.O)H,
C(.dbd.O)(C.sub.1-6alkyl), CO.sub.2H, CO.sub.2(C.sub.1-6alkyl),
C(.dbd.O)NH.sub.2, C(.dbd.O)NH(C.sub.1-6alkyl),
C(.dbd.O)N(C.sub.1-6alkyl).sub.2, NHC(.dbd.O)(C.sub.1-6alkyl),
S(O).sub.2(C.sub.1-6alkyl), NHSO.sub.2(C.sub.1-6alkyl),
SO.sub.2NH.sub.2, SO.sub.2NH(C.sub.1-6alkyl),
SO.sub.2N(C.sub.1-6alkyl).sub.2, C.sub.3-7cycloalkyl, phenyl, five
or six membered heteroaryl, or four to seven membered heterocyclo,
and/or two of R.sub.1 and/or two of R.sub.5 join together to form a
fused benzo ring; R.sub.2 and R.sub.3 are independently selected
from hydrogen and C.sub.1-4alkyl; Z is --CO.sub.2--, --SO.sub.2--,
or is absent; R.sub.6 is selected from optionally-substituted
alkyl, alkenyl, aryl, and heteroaryl; m and n are independently
selected from 0, 1, and 2; and q is 0 or 1.
[0225] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR19## or a stereoisomer, a pharmaceutically-acceptable salt,
hydrate, or prodrug thereof, wherein: R.sub.1 and R.sub.5 are
attached to any available carbon atom of phenyl ring A and phenyl
ring B, respectively, and at each occurrence are independently
selected from alkyl, substituted alkyl, halogen, cyano, nitro,
hydroxy, alkoxy, alkylthio, alkylamino, C(.dbd.O)H, acyl,
CO.sub.2H, alkoxycarbonyl, carbamyl, sulfonyl, sulfonamidyl,
cycloalkyl, heterocycle, aryl, and heteroaryl, and/or two of
R.sub.1 and/or two of R.sub.5 join together to form a fused benzo
ring; R.sub.2, R.sub.3 and R.sub.4 are independently selected from
hydrogen and alkyl; Z is --CO.sub.2--, --SO.sub.2--, or is absent;
R.sub.6 is selected from: a) C.sub.1-4alkyl or C.sub.1-4alkenyl
optionally substituted with up to three of halogen, aryl and
CO.sub.2C.sub.1-6alkyl; b) phenyl optionally substituted with up to
three R.sub.12 and/or having fused thereto a benzo-ring or a five
to six membered heteroaryl; c) heteroaryl selected from thiophenyl,
imidazolyl, pyrazolyl, and isoxazolyl, wherein said heteroaryl is
optionally substituted with up to two R.sub.12, provided that where
R.sub.2 is hydrogen, Z-R.sub.6 together are not --SO.sub.2-Me or
##STR20## R.sub.7 is selected from hydrogen, keto (.dbd.O),
C.sub.1-6alkyl, substituted C.sub.1-6alkyl, halogen, cyano,
O(C.sub.1-6alkyl), O(phenyl), O(benzyl), NH.sub.2,
NH(C.sub.1-6alkyl), N(C.sub.1-6alkyl).sub.2, C(.dbd.O)H,
C(.dbd.O)(C.sub.1-6alkyl), CO.sub.2H, CO.sub.2(C.sub.1-6alkyl-);
R.sub.12 at each occurrence is independently selected from each
other R.sub.12 from the group consisting of C.sub.1-6alkyl,
halogen, nitro, cyano, hydroxy, alkoxy, NHC(.dbd.O)alkyl,
--CO.sub.2alkyl, --SO.sub.2phenyl, five to six membered monocyclic
heteroaryl, and phenyloxy or benzyloxy in turn optionally
substituted with halogen, C.sub.1-4alkyl, and/or O(C.sub.1-4alkyl);
and m and n are independently selected from 0, 1, or 2.
[0226] In further exemplary compounds, Z is --SO.sub.2--; R.sub.6
is selected from C.sub.1-4alkyl, trifluoromethyl, benzyl,
C.sub.2-3alkenyl substituted with phenyl, ##STR21## R.sub.15 is
halogen, alkyl, nitro, cyano, hydroxy, alkoxy, NHC(.dbd.O)alkyl,
and/or two R.sub.15 groups are taken together to form a fused benzo
ring or a five to six membered heteroaryl; R.sub.16 is selected
from hydrogen, halogen, alkyl, nitro, cyano, hydroxy, alkoxy,
NHC(.dbd.O)alkyl, and phenyloxy or benzyloxy in turn optionally
substituted with 1 to 3 of halogen, cyano, and C.sub.1-4alkoxy;
R.sub.17 is selected from alkyl, alkoxy, CO.sub.2C.sub.1-6alkyl,
and SO.sub.2phenyl; and u and v are independently 0, 1 or 2.
[0227] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR22## or a stereoisomer, a pharmaceutically-acceptable salt,
hydrate, or prodrug thereof, wherein: R.sub.1 is selected from the
group consisting of H, CN and SO.sub.2-piperidine; R.sub.2 is
selected from the group consisting of H, 4-Cl-Ph, Ph, and
2-Me-imidazole; R.sub.3 is selected from the group consisting of H,
CH.sub.2-2-imidazole, and CH.sub.2-2-oxazole.
[0228] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR23## or a stereoisomer, a pharmaceutically-acceptable salt,
hydrate, or prodrug thereof, wherein: R.sub.1 is selected from the
group consisting of H, 2,4-C.sub.12, 2-4-Me.sub.2, and
2,5-(CF.sub.3).sub.2; R.sub.2 is selected from the group consisting
of H, 4-Cl, 4-Me, 2,4-C.sub.12, 2,4-Me.sub.2, 3-Cl; X is selected
from the group consisting of O and NH; Y is selected from the group
consisting of S, O, NCN, CO(3-CN-Ph), CO(4-CN-Ph), CO(4-Cl-Ph), and
COEt.
[0229] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR24## ##STR25##
[0230] Additional exemplary compounds of the present invention are
described in U.S. Provisional Patent Nos. U.S. Provisional Patent
Nos. 60/131,761, 60/165,511, 60/191,855, 60/312,560, 60/313,689,
60/396,670, 60/565,788, 60/607,599, 60/641,040, and U.S. patent
application Ser. Nos. 11/324,419, 11/176,719, 11/110,228,
10/935,333, 10/886,450, 10/795,535, 10/634,114, 10/427,211,
10/427,212, 10/217,878, 09/767,283, 09/700,101, and related
applications; each herein incorporated by reference in their
entireties.
[0231] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR26## ##STR27## ##STR28## ##STR29## ##STR30## ##STR31##
##STR32## R1 is H or hydroxy Each of R2 through R6 may be the same
or different and is selected from hydrogen, a hydroxy, an alkoxy, a
halo, an amino, a lower-alkyl-a substituted-amino, an acetylamino,
a hydroxyamino, an aliphatic group having 1-8 carbons and 1-20
hydrogens, a substituted aliphatic group of similar size, a
cycloaliphatic group consisting of <10 carbons, a substituted
cycloaliphatic group, an aryl, and a heterocyclic ##STR33## Each of
R2 through R10 may be the same or different and is selected from
hydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-a
substituted-amino, an acetylamino, a hydroxyamino, an aliphatic
group having 1-8 carbons and 1-20 hydrogens, a substituted
aliphatic group of similar size, a cycloaliphatic group consisting
of <10 carbons, a substituted cycloaliphatic group, an aryl, and
a heterocyclic ##STR34## Each of R1 through R11 may be the same or
different and is selected from hydrogen, a hydroxy, an alkoxy, a
halo, an amino, a lower-alkyl-a substituted-amino, an acetylamino,
a hydroxyamino, an aliphatic group having 1-8 carbons and 1-20
hydrogens, a substituted aliphatic group of similar size, a
cycloaliphatic group consisting of <10 carbons, a substituted
cycloaliphatic group, an aryl, and a heterocyclic ##STR35## Each of
R1 through R10 may be the same or different and is selected from
hydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-a
substituted-amino, an acetylamino, a hydroxyamino, an aliphatic
group having 1-8 carbons and 1-20 hydrogens, a substituted
aliphatic group of similar size, a cycloaliphatic group consisting
of <10 carbons, a substituted cycloaliphatic group, an aryl, and
a heterocyclic ##STR36## Each of R1 through R10 may be the same or
different and is selected from hydrogen, a hydroxy, an alkoxy, a
halo, an amino, a lower-alkyl-a substituted-amino, an acetylamino,
a hydroxyamino, an aliphatic group having 1-8 carbons and 1-20
hydrogens, a substituted aliphatic group of similar size, a
cycloaliphatic group consisting of <10 carbons, a substituted
cycloaliphatic group, an aryl, and a heterocyclic ##STR37## Each of
R1 through R6 may be the same or different and is selected from
hydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-a
substituted-amino, an acetylamino, a hydroxyamino, an aliphatic
group having 1-8 carbons and 1-20 hydrogens, a substituted
aliphatic group of similar size, a cycloaliphatic group consisting
of <10 carbons, a substituted cycloaliphatic group, an aryl, and
a heterocyclic ##STR38##
[0232] wherein R.sub.1 is selected from napthalalanine; phenol;
1-Napthalenol; ##STR39##
[0233] In some preferred embodiments, compositions comprising
crystal forms and formulations of the following exemplary compounds
are provided: ##STR40## wherein R.sub.1 is selected from: ##STR41##
The stereochemistry of all derivatives embodied in the present
invention is R, S, or racemic.
[0234] In some preferred embodiments, compositions comprising
crystal forms and formulations of the following exemplary compound
are provided: ##STR42##
[0235] In some preferred embodiments, compositions comprising
crystal forms and formulations of the following exemplary compounds
are provided: ##STR43## wherein R1, R2, R3 and R4 are selected from
the group consisting of: hydrogen; CH.sub.3; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
a linear or branched, saturated or unsaturated aliphatic chain
having at least 2 carbons, and having at least one hydroxy
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one thiol
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, wherein said aliphatic chain
terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein said aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety (e.g., nitro, nitrile, etc.); a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one amine subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one ether subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one halogen subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one nitronium subgroup; wherein R5
is selected from the group consisting of: OH; NO.sub.2; NR'; OR';
wherein R' is selected from the group consisting of: a linear or
branched, saturated or unsaturated aliphatic chain having at least
one carbon; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
hydroxyl subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
thiol subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, wherein said aliphatic
chain terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein said aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety (e.g., nitro, nitrile, etc.); a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one amine subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one halogen subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one nitronium subgroup; wherein R6
is selected from the group consisting of: Hydrogen; NO.sub.2; Cl;
F; Br; I; SR'; and NR'.sub.2; wherein R' is defined as above in R5;
wherein R7 is selected from the group consisting of: Hydrogen; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 2 carbons; and wherein R8 is an aliphatic cyclic group
larger than benzene; wherein said larger than benzene comprises any
chemical group containing 7 or more non-hydrogen atoms, and is an
aryl or aliphatic cyclic group. In some embodiments, R' is any
functional group that protects the oxygen of R5 from metabolism in
vivo, until the compound reaches its biological target (e.g.,
mitochondria). In some embodiments, R' protecting group(s) is
metabolized at the target site, converting R5 to a hydroxyl
group.
[0236] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR44## including both R and S enantiomeric forms and racemic
mixtures; wherein R1 comprises a chemical moiety comprising a
hydrogen bonding proton donor (e.g., a hydroxyl group, a phenol
group, an amide group, a sulfonamide group, an amine group, an
aniline group, a benzimidizalone group, a carbamate group, and an
imidizole group); and R2 comprises a hydrophobic chemical
moiety.
[0237] In preferred embodiments, R1 is selected from the group
consisting of: ##STR45## wherein R1', R2, R3 and R4 are selected
from the group consisting of: hydrogen; CH.sub.3; a linear or
branched, saturated or unsaturated aliphatic chain having at least
1 carbon; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons and at least one hydroxy subgroup;
a linear or branched, saturated or unsaturated, substituted or
non-substituted, aliphatic chain having at least 2 carbons and
having at least one thiol subgroup; a linear or branched, saturated
or unsaturated, substituted or non-substituted, aliphatic chain
having at least 2 carbons wherein the aliphatic chain terminates
with an aldehyde subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one ketone subgroup; a linear or branched, saturated or
unsaturated, substituted or non-substituted, aliphatic chain having
at least 2 carbons; wherein the aliphatic chain terminates with a
carboxylic acid subgroup; a linear or branched, saturated or
unsaturated, substituted or non-substituted, aliphatic chain having
at least 2 carbons, and having at least one amide subgroup; a
linear or branched, saturated or unsaturated, substituted or
non-substituted, aliphatic chain having at least 2 carbons, and
having at least one acyl group; a linear or branched, saturated or
unsaturated, substituted or non-substituted, aliphatic chain having
at least 2 carbons, and having at least one nitrogen containing
moiety; a linear or branched, saturated or unsaturated, substituted
or non-substituted, aliphatic chain having at least 2 carbons, and
having at least one amine subgroup; a linear or branched, saturated
or unsaturated, substituted or non-substituted, aliphatic chain
having at least 2 carbons, and having at least one ether subgroup;
a linear or branched, saturated or unsaturated, substituted or
non-substituted, aliphatic chain having at least 2 carbons, and
having at least one halogen subgroup; a linear or branched,
saturated or unsaturated, substituted or non-substituted, aliphatic
chain having at least 2 carbons, and having at least one nitronium
subgroup; and R5 is OH.
[0238] In preferred embodiments, R2 is selected from group
consisting of: napthalalanine; phenol; 1-Napthalenol;
2-Napthalenol; ##STR46## ##STR47##
[0239] In some preferred embodiments, R1 is selected from the group
consisting of: ##STR48##
[0240] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR49## wherein R3 is selected from the group consisting of
Hydrogen; amino; and a linear or branched, saturated or
unsaturated, substituted (e.g., substituted with amines, esters,
amides or phosphatases) or non-substituted, aliphatic chain having
at least 2 carbons;
[0241] R4 is selected from the group consisting of H, a ketone, and
a nitrogen; and
[0242] R5 is selected from H, a hydroxy, an alkoxy, a carboxylic
acid, a carboxylic ester, a halogen, a nitro, a sulfonamide, an
amide, a carbamate, an amino, a lower-alkyl, a substituted-amino,
an acetylamino, a hydroxyamino, an aliphatic group having 1-8
carbons and 1-20 hydrogens, a substituted aliphatic group of
similar size, a cycloaliphatic group consisting of less than 10
carbons, a substituted cycloaliphatic group, an aryl, a
heterocyclic, NO.sub.2; SR'; and NR'.sub.2, wherein R' is defined
as a linear or branched, saturated or unsaturated aliphatic chain
having at least one carbon; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one hydroxyl subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one thiol subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, wherein the
aliphatic chain terminates with an aldehyde subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one ketone subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; wherein the aliphatic chain terminates with a carboxylic
acid subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amide subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
acyl group; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitrogen containing moiety; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one amine subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one halogen subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one nitronium subgroup.
[0243] In other preferred embodiments R2 is any chemical group that
permits the compound to bind to OSCP. In some such embodiments, R2
comprises a hydrophobic aromatic group. In preferred embodiments R2
comprises a hydrophobic aromatic group larger than benzene (e.g., a
benzene ring with non-hydrogen substituents, a moiety having two or
more aromatic rings, a moiety with 7 or more carbon atoms,
etc.).
[0244] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR50## and dimethylphenyl (all isomers) and ditrifluoromethyl
(all isomers).
[0245] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR51##
[0246] wherein R2 is selected from the group consisting of
Hydrogen, alkyl, substituted alkyl, and (CH.sub.2).sub.n wherein
n=1-6;
[0247] wherein R3 is selected from the group consisting of
hydrogen, halogen, alkyl, substituted alkyl, carboxylic acid, amide
SO.sub.2NH.sub.2, NHSO.sub.2alkyl, and NO.sub.2;
[0248] wherein X is selected from the group consisting of ##STR52##
alkyl, substituted alkyl, sulfolamide, SO.sub.2alkyl, NHSO.sub.2,
CH.sub.2, CH.sub.2CH.sub.2, SO.sub.2, CH.sub.2SO.sub.2,
SO.sub.2CH.sub.2, OCH.sub.2CH.sub.2O, SO, CH.sub.2CH.sub.2SO,
SOCH.sub.2CH.sub.2; and
[0249] wherein L, M and N are present or absent, and are selected
from the group consisting of alkyl, NO.sub.2, halogen, OH, O-Alkyl,
methyl ester, propyl ester, ethyl ester, CO.sub.2H, CF.sub.3,
aniline, nitro, heterocycle, mono-substituted alkyl, di-substituted
alkyl, and tri-substituted alkyl, hydrogen, SO.sub.2NH.sub.2,
SO.sub.2NH-alkyl, SOalkyl, NHSO.sub.2alkyl; and
[0250] wherein Y is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, halogen, OH, O-Alkyl, methyl ester,
propyl ester, ethyl ester, CO.sub.2H, nitro, heterocycle,
mono-substituted alkyl, di-substituted alkyl, and tri-substituted
alkyl, hydrogen, SOalkyl, SO.sub.2NH.sub.2, SO.sub.2NH-alkyl,
NHSO.sub.2alkyl, and ##STR53##
[0251] wherein WW, XX, YY and ZZ are present or absent, and are
selected from the group consisting of alkyl, halogen, OH, O-Alkyl,
methyl ester, propyl ester, ethyl ester, CO.sub.2H, aniline, nitro,
heterocycle, mono-substituted alkyl, di-substituted alkyl, and
tri-substituted alkyl, hydrogen, SO.sub.2NH.sub.2,
SO.sub.2NH-alkyl, NHSO.sub.2alkyl; and wherein Z is selected from
the group consisting of ##STR54## wherein R5 is selected from the
group consisting of alkyl, mono-substituted alkyl, di-substituted
alkyl, and tri-substituted alkyl.
[0252] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR55##
[0253] In some preferred embodiments, crystal forms and
formulations of the following exemplary compound is provided:
##STR56##
[0254] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR57## including both R and S enantiomeric forms and racemic
mixtures; wherein R1 is selected from the group consisting of:
##STR58## wherein X is selected from the group consisting of
heteroatom, alkyl, and substituted alkly; ##STR59## wherein Z and Y
are separately selected from the group consisting of O, N and S;
##STR60## wherein R2 is selected from the group consisting of
methyl, H, alkyl, and (CH.sub.2).sub.n-morpholino wherein n=1-6;
and wherein R3 is selected from the group consisting of hydrogen,
halogen, alkyl, substituted alkyl, carboxylic acid, amide
SO.sub.2NH.sub.2, NHSO.sub.2alkyl, and NO.sub.2.
[0255] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR61## wherein R1 is selected from the group consisting of
methyl, hydrogen, alkyl, and (CH.sub.2).sub.n-morpholino wherein
n=1-6; wherein R2 is selected from the group consisting of
##STR62##
[0256] wherein R3 is selected from the group consisting of
hydrogen, halogen, alkyl, substituted alkyl, carboxylic acid,
amide, SO.sub.2NH.sub.2, NHSO.sub.2alkyl, and NO.sub.2; wherein BB,
CC, DD, and R4 are present or absent, and are selected from the
group consisting of hydrogen, CF.sub.3, NO.sub.2, alkyl, halogen,
OH, O-alkyl, nitro, OCH.sub.2CH.sub.2OH, SO.sub.2H,
mono-substituted alkyl, di-substituted alkyl, tri-substituted
alkyl, CO.sub.2H, heterocycle, SO.sub.2NH.sub.2, SO.sub.2NH-alkyl,
NHSO.sub.2alkyl, methyl ester, propyl ester, and ethyl ester; and
wherein R5 is selected from the group consisting of NHSO.sub.2,
CH.sub.2NHSO.sub.2, CH.sub.2CH.sub.2NHSO.sub.2,
CH.sub.2CH.sub.2CH.sub.2NHSO.sub.2, SO.sub.2NH, SO.sub.2NHCH.sub.2,
SO.sub.2NHCH.sub.2CH.sub.2, SO.sub.2NHCH.sub.2CH.sub.2CH.sub.2,
CH.sub.2, CH.sub.2CH.sub.2, CH.sub.2CH.sub.2CH.sub.2, SO.sub.2,
CH.sub.2SO, SOCH.sub.2, OCH.sub.2CH.sub.2O, SO, CH.sub.2CH.sub.2SO,
and SOCH.sub.2CH.sub.2.
[0257] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR63##
[0258] wherein R.sub.1 is selected from: ##STR64##
[0259] In certain preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR65## including both R and S enantiomeric forms and racemic
mixtures; wherein R1 is a nitrogen atom or a carbon atom; wherein
R2 is comprises a chemical moiety comprising a heterocyclic group
containing 3 or more carbon atoms; wherein R3 comprises a chemical
moiety comprising a heterocyclic group containing 3 or more carbon
atoms; and wherein R4 and R5 are separately selected from the group
consisting of: hydrogen; halogen; CH.sub.3; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
a chemical moiety comprising a halogen; a chemical moiety
comprising Sulfur; a chemical moiety comprising Nitrogen; an
aromatic chemical moiety; a hydrophilic chemical moiety; and a
hydrophobic chemical moiety.
[0260] In preferred embodiments, the compound comprises the
formula: ##STR66## wherein R6 is selected from the group consisting
of H and a ketone; and wherein R7 is selected from the group
consisting of H and a ketone.
[0261] In preferred embodiments, the compound comprises the
formula: ##STR67## In such preferred embodiments, R8 is carbon or
nitrogen and R9 is selected from H, a hydroxy, an alkoxy, a
halogen, an amino, a lower-alkyl, a substituted-amino, an
acetylamino, a hydroxyamino, an aliphatic group having 1-8 carbons
and 1-20 hydrogens, a substituted aliphatic group of similar size,
a cycloaliphatic group consisting of less than 10 carbons, a
substituted cycloaliphatic group, an aryl, a heterocyclic,
NO.sub.2; SR'; and NR'.sub.2, wherein R' is defined as a linear or
branched, saturated or unsaturated aliphatic chain having at least
one carbon; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
hydroxyl subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
thiol subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, wherein the aliphatic
chain terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein the aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup.
[0262] In preferred embodiments, the compound comprises the
formula: ##STR68## wherein R9 is selected from H, a hydroxy, an
alkoxy, a halo, an amino, a lower-alkyl, a substituted-amino, an
acetylamino, a hydroxyamino, an aliphatic group having 1-8 carbons
and 1-20 hydrogens, a substituted aliphatic group of similar size,
a cycloaliphatic group consisting of less than 10 carbons, a
substituted cycloaliphatic group, an aryl, a heterocyclic,
NO.sub.2; SR'; and NR'.sub.2, wherein R' is defined as a linear or
branched, saturated or unsaturated aliphatic chain having at least
one carbon; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
hydroxyl subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
thiol subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, wherein the aliphatic
chain terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein the aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup.
[0263] In preferred embodiments, the compound comprises the
formula: ##STR69## wherein R10 is selected from the group
consisting of: hydrogen; halogen; CH.sub.3; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
a chemical moiety comprising a halogen; a chemical moiety
comprising Sulfur; a chemical moiety comprising Nitrogen; an
aromatic chemical moiety; a hydrophilic chemical moiety; and a
hydrophobic chemical moiety; and wherein R7 is selected from the
group consisting of H and a ketone.
[0264] In other preferred embodiments, R3 is selected from the
group consisting of: ##STR70## ##STR71## wherein R12, R13, R14 and
R15 are selected from the group consisting of: hydrogen; CH.sub.3;
a linear or branched, saturated or unsaturated aliphatic chain
having at least 1 carbon; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one hydroxy subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one thiol subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, wherein the
aliphatic chain terminates with an aldehyde subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one ketone subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; wherein the aliphatic chain terminates with a carboxylic
acid subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amide subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
acyl group; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitrogen containing moiety; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one amine subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one ether subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one halogen subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one nitronium subgroup; and R11 is OH.
[0265] In yet other preferred embodiments, R4 or R5 are selected
from group consisting of: napthalalanine; phenol; 1-Napthalenol;
2-Napthalenol; ##STR72## quinolines, and all aromatic
regioisomers.
[0266] In other preferred embodiments, R4 or R5 is selected from
the group consisting of: ##STR73## wherein R16 is carbon or
nitrogen; wherein R17 is selected from the group consisting of
hydrogen; halogen; CH.sub.3; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons; a chemical
moiety comprising a halogen; a chemical moiety comprising Sulfur; a
chemical moiety comprising Nitrogen; an aromatic chemical moiety; a
hydrophilic chemical moiety; and a hydrophobic chemical moiety;
wherein R18 is carbon or nitrogen; wherein R19 is selected from the
group consisting of hydrogen; halogen; CH.sub.3; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; a chemical moiety comprising a halogen; a chemical
moiety comprising Sulfur; a chemical moiety comprising Nitrogen; an
aromatic chemical moiety; a hydrophilic chemical moiety; and a
hydrophobic chemical moiety; and wherein R20 is carbon or
nitrogen.
[0267] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR74## including both R and S enantiomeric forms and racemic
mixtures. In such preferred is embodiments, R1 is a nitrogen atom
or a carbon atom; R2 is carbon or nitrogen; R3 comprises a chemical
moiety comprising a heterocyclic group containing 3 or more carbon
atoms; R4 and R5 are separately selected from the group consisting
of: hydrogen; halogen; CH.sub.3; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons; a chemical
moiety comprising a halogen; a chemical moiety comprising Sulfur; a
chemical moiety comprising Nitrogen; an aromatic chemical moiety; a
hydrophilic chemical moiety; and a hydrophobic chemical moiety; and
R6 is selected from H, a hydroxy, an alkoxy, a halogen, an amino, a
lower-alkyl, a substituted-amino, an acetylamino, a hydroxyamino,
an aliphatic group having 1-8 carbons and 1-20 hydrogens, a
substituted aliphatic group of similar size, a cycloaliphatic group
consisting of less than 10 carbons, a substituted cycloaliphatic
group, an aryl, a heterocyclic, NO.sub.2; SR'; and NR'.sub.2,
wherein R' is defined as a linear or branched, saturated or
unsaturated aliphatic chain having at least one carbon; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one hydroxyl subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one thiol subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, wherein the aliphatic chain terminates with an aldehyde
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one ketone
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons; wherein the aliphatic chain
terminates with a carboxylic acid subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amide subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one acyl group; a linear or branched, saturated
or unsaturated aliphatic chain having at least 2 carbons, and
having at least one nitrogen containing moiety; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one amine subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one halogen subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one nitronium subgroup.
[0268] In preferred embodiments, R3 is selected from the group
consisting of: ##STR75## ##STR76## wherein R12, R13, R14 and R15
are selected from the group consisting of: hydrogen; CH.sub.3; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 1 carbon; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
hydroxy subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
thiol subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, wherein the aliphatic
chain terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein the aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
ether subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; and R10 is OH.
[0269] In preferred embodiments, R4 or R5 are selected from group
consisting of: napthalalanine; phenol; 1-Napthalenol;
2-Napthalenol; ##STR77## quinolines, and all aromatic
regioisomers.
[0270] In other preferred embodiments, R4 or R5 is selected from
the group consisting of: ##STR78## wherein R16 is carbon or
nitrogen; wherein R17 is selected from the group consisting of
hydrogen; halogen; CH.sub.3; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons; a chemical
moiety comprising a halogen; a chemical moiety comprising Sulfur; a
chemical moiety comprising Nitrogen; an aromatic chemical moiety; a
hydrophilic chemical moiety; and a hydrophobic chemical moiety;
wherein R18 is carbon or nitrogen; wherein R19 is selected from the
group consisting of hydrogen; halogen; CH.sub.3; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; a chemical moiety comprising a halogen; a chemical
moiety comprising Sulfur; a chemical moiety comprising Nitrogen; an
aromatic chemical moiety; a hydrophilic chemical moiety; and a
hydrophobic chemical moiety; and wherein R20 is carbon or
nitrogen.
[0271] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR79##
[0272] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR80## including both R and S enantiomeric forms and racemic
mixtures.
[0273] In such preferred embodiments, R1 is a nitrogen atom or a
carbon atom; R2 is selected from H, a hydroxy, an alkoxy, a halo,
an amino, a lower-alkyl, a substituted-amino, an acetylamino, a
hydroxyamino, an aliphatic group having 1-8 carbons and 1-20
hydrogens, a substituted aliphatic group of similar size, a
cycloaliphatic group consisting of less than 10 carbons, a
substituted cycloaliphatic group, an aryl, a heterocyclic,
NO.sub.2; SR'; and NR'.sub.2, wherein R' is defined as a linear or
branched, saturated or unsaturated aliphatic chain having at least
one carbon; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
hydroxyl subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
thiol subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, wherein the aliphatic
chain terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein the aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; R3 comprises a chemical moiety comprising a
heterocyclic group containing 3 or more carbon atoms; and R4 and R5
are separately selected from the group consisting of: hydrogen;
halogen; CH.sub.3; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons; a chemical moiety
comprising a halogen; a chemical moiety comprising Sulfur; a
chemical moiety comprising Nitrogen; an aromatic chemical moiety; a
hydrophilic chemical moiety; and a hydrophobic chemical moiety.
[0274] In preferred embodiments, R3 is selected from the group
consisting of: ##STR81## ##STR82## wherein R12, R13, R14 and R15
are selected from the group consisting of: hydrogen; CH.sub.3; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 1 carbon; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
hydroxy subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
thiol subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, wherein the aliphatic
chain terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein the aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
ether subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; and R11 is OH.
[0275] In preferred embodiments, R4 or R5 are selected from group
consisting of: ##STR83## ##STR84##
[0276] quinolines, and all aromatic regioisomers.
[0277] In other preferred embodiments, R4 or R5 is selected from
the group consisting of: ##STR85## wherein R16 is carbon or
nitrogen; wherein R17 is selected from the group consisting of
hydrogen; halogen; CH.sub.3; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons; a chemical
moiety comprising a halogen; a chemical moiety comprising Sulfur; a
chemical moiety comprising Nitrogen; an aromatic chemical moiety; a
hydrophilic chemical moiety; and a hydrophobic chemical moiety;
wherein R18 is carbon or nitrogen; wherein R19 is selected from the
group consisting of hydrogen; halogen; CH.sub.3; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; a chemical moiety comprising a halogen; a chemical
moiety comprising Sulfur; a chemical moiety comprising Nitrogen; an
aromatic chemical moiety; a hydrophilic chemical moiety; and a
hydrophobic chemical moiety; and wherein R20 is carbon or
nitrogen.
[0278] In some preferred embodiments, crystal forms and
formulations of the following exemplary compounds are provided:
##STR86## including both R and S enantiomeric forms and racemic
mixtures.
[0279] In such preferred embodiments, R1 is carbon or nitrogen; R2
is selected from H, a hydroxy, an alkoxy, a halogen, an amino, a
lower-alkyl, a substituted-amino, an acetylamino, a hydroxyamino,
an aliphatic group having 1-8 carbons and 1-20 hydrogens, a
substituted aliphatic group of similar size, a cycloaliphatic group
consisting of less than 10 carbons, a substituted cycloaliphatic
group, an aryl, a heterocyclic, NO.sub.2; SR'; and NR'.sub.2,
wherein R' is defined as a linear or branched, saturated or
unsaturated aliphatic chain having at least one carbon; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one hydroxyl subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one thiol subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, wherein the aliphatic chain terminates with an aldehyde
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one ketone
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons; wherein the aliphatic chain
terminates with a carboxylic acid subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amide subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one acyl group; a linear or branched, saturated
or unsaturated aliphatic chain having at least 2 carbons, and
having at least one nitrogen containing moiety; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one amine subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one halogen subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one nitronium subgroup; R3 comprises
a chemical moiety comprising a heterocyclic group containing 3 or
more carbon atoms; R4 is selected from the group consisting of:
hydrogen; halogen; CH.sub.3; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons; a chemical
moiety comprising a halogen; a chemical moiety comprising Sulfur; a
chemical moiety comprising Nitrogen; an aromatic chemical moiety; a
hydrophilic chemical moiety; and a hydrophobic chemical moiety; and
R5 is selected from the group consisting of: hydrogen; halogen;
CH.sub.3; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons; a chemical moiety comprising a
halogen; a chemical moiety comprising Sulfur; a chemical moiety
comprising Nitrogen; an aromatic chemical moiety; a hydrophilic
chemical moiety; and a hydrophobic chemical moiety.
[0280] In preferred embodiments, R3 is selected from the group
consisting of: ##STR87## ##STR88## wherein R12, R13, R14 and R15
are selected from the group consisting of: hydrogen; CH.sub.3; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 1 carbon; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
hydroxy subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
thiol subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, wherein the aliphatic
chain terminates with an aldehyde subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ketone subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons;
wherein the aliphatic chain terminates with a carboxylic acid
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one amide
subgroup; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one acyl
group; a linear or branched, saturated or unsaturated aliphatic
chain having at least 2 carbons, and having at least one nitrogen
containing moiety; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
amine subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
ether subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
halogen subgroup; a linear or branched, saturated or unsaturated
aliphatic chain having at least 2 carbons, and having at least one
nitronium subgroup; and R11 is OH.
[0281] In preferred embodiments, R4 or R5 are selected from group
consisting of: ##STR89## ##STR90## quinolines, and all aromatic
regioisomers.
[0282] In other preferred embodiments, R4 or R5 is selected from
the group consisting of: ##STR91## wherein R16 is carbon or
nitrogen; wherein R17 is selected from the group consisting of
hydrogen; halogen; CH.sub.3; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons; a chemical
moiety comprising a halogen; a chemical moiety comprising Sulfur; a
chemical moiety comprising Nitrogen; an aromatic chemical moiety; a
hydrophilic chemical moiety; and a hydrophobic chemical moiety;
wherein R18 is carbon or nitrogen; wherein R19 is selected from the
group consisting of hydrogen; halogen; CH.sub.3; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; a chemical moiety comprising a halogen; a chemical
moiety comprising Sulfur; a chemical moiety comprising Nitrogen; an
aromatic chemical moiety; a hydrophilic chemical moiety; and a
hydrophobic chemical moiety; and wherein R20 is carbon or
nitrogen.
[0283] In certain embodiments, crystal forms and formulations of
the following exemplary compounds comprising the following formula
are provided: A-B--C; wherein A is a chemical moiety comprising a
hydroxyl group (e.g., a phenolic ring); wherein B is a chemical
moiety (e.g., scaffold molecule) separating A and C by at least 1
atom; and wherein C is a hydrophobic chemical moiety (e.g., naphyl
group).
[0284] In some embodiments, A is selected from the group consisting
of: is selected from the group consisting of: ##STR92## wherein
R1', R2, R3 and R4 are selected from the group consisting of:
hydrogen; CH.sub.3; a linear or branched, saturated or unsaturated
aliphatic chain having at least 1 carbon; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one hydroxy subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one thiol subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
wherein the aliphatic chain terminates with an aldehyde subgroup; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 2 carbons, and having at least one ketone subgroup; a
linear or branched, saturated or unsaturated aliphatic chain having
at least 2 carbons; wherein the aliphatic chain terminates with a
carboxylic acid subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one amide subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one acyl group; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one nitrogen containing moiety; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amine subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ether subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one halogen subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one nitronium subgroup; and R5 is OH.
[0285] In some embodiments, C is selected from group consisting of:
napthalalanine; phenol; 1-Napthalenol; 2-Napthalenol; ##STR93##
##STR94## and aromatic regioisomers.
[0286] In some embodiments, C comprises an aryl group and/or an
aliphatic group.
[0287] In some embodiments, B is a benzodiazepine structure
described by the following formula: ##STR95##
[0288] In some embodiments, A is located at position 5 of the
benzodiazepine structure. In some preferred embodiments, C is
located at position 3 of the benzodiazepine structure. In other
preferred embodiments, A is located at a position of the
benzodiazepine structure selected from the group consisting of
position 1, position 2, position 3, position 4, position 5,
position 6, position 7, position 8, position 9, and position
10.
[0289] In some embodiments, crystal forms and formulations of the
following exemplary compounds are provided: ##STR96##
[0290] Certain embodiments of the present invention include crystal
forms and formulations of exemplary compounds with the following
formula: ##STR97## including both R and S enantiomeric forms and
racemic mixtures, wherein R1 is an electron rich heterocycle.
[0291] In preferred embodiments, R1 is selected from the group
consisting of: ##STR98## ##STR99##
[0292] In some embodiments, R2 is a halogen. In some embodiments,
R2 is Chlorine.
[0293] In certain embodiments, examples of crystal forms and
formulations of the exemplary 1,4-benzodiazepine-2,5-dione
compounds include but are not limited to: ##STR100##
[0294] Certain embodiments of the present invention include crystal
forms and formulations of benzodiazepine (and benzodiazepine
related) compounds having a chemical moiety that causes the
benzodiazepine to lack a chiral center associated with the third
carbon position of the benzodiazepine ring. Certain embodiments of
the present invention include crystal forms and formulations of
exemplary compounds with the following formula: ##STR101##
including both R and S enantiomeric forms and racemic mixtures.
[0295] In some embodiments, A - - - B is selected from the group
consisting of N--CH.sub.2 and C.dbd.N.
[0296] In some embodiments, R1 is selected from the group
consisting of ##STR102## R1' is selected from the group consisting
of halogen; alkyl; substituted alkyl; aryl; substituted aryl;
amino; carbonyl; sulfone; sulfonamide; ether; OH; a chemical moiety
comprising Sulfur; a chemical moiety comprising Nitrogen; CH.sub.3;
a linear or branched, saturated or unsaturated aliphatic chain
having at least 1 carbon; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one hydroxy subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one thiol subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, wherein said
aliphatic chain terminates with an aldehyde subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons, and having at least one ketone subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least
2 carbons; wherein said aliphatic chain terminates with a
carboxylic acid subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one amide subgroup; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one acyl group; a linear or branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having
at least one nitrogen containing moiety; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one amine subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one ether subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one halogen subgroup; a linear or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at least one nitronium subgroup.
[0297] In some embodiments, R2 is an aliphatic cyclic group larger
than benzene, wherein said larger than benzene comprises any
chemical group containing 7 or more non-hydrogen atoms.
[0298] In some embodiments, R2 is selected from group consisting
of: napthalalanine; phenol; 1-Napthalenol; 2-Napthalenol;
##STR103## quinolines, and all aromatic regioisomers.
[0299] In some embodiments, R2 is: ##STR104## wherein X is selected
from the group consisting of ##STR105## alkyl, substituted alkyl,
sulfolamide, SO.sub.2alkyl, NHSO.sub.2, CH.sub.2, CH.sub.2CH.sub.2,
SO.sub.2, CH.sub.2SO.sub.2, SO.sub.2CH.sub.2, OCH.sub.2CH.sub.2O,
SO, CH.sub.2CH.sub.2SO, SOCH.sub.2CH.sub.2; and wherein L, M and N
are present or absent, and are selected from the group consisting
of alkyl, NO.sub.2, halogen, OH, O-Alkyl, methyl ester, propyl
ester, ethyl ester, CO.sub.2H, CF.sub.3, aniline, nitro,
heterocycle, mono-substituted alkyl, di-substituted alkyl, and
tri-substituted alkyl, hydrogen, SO.sub.2NH.sub.2,
SO.sub.2NH-alkyl, SOalkyl, NHSO.sub.2alkyl; wherein Y is selected
from the group consisting of hydrogen, alkyl, substituted alkyl,
halogen, OH, O-Alkyl, methyl ester, propyl ester, ethyl ester,
CO.sub.2H, nitro, heterocycle, mono-substituted alkyl,
di-substituted alkyl, and tri-substituted alkyl, hydrogen, SOalkyl,
SO.sub.2NH2, SO.sub.2NH-alkyl, NHSO.sub.2alkyl, and ##STR106##
wherein WW, XX, YY and ZZ are present or absent, and are selected
from the group consisting of alkyl, halogen, OH, O-Alkyl, methyl
ester, propyl ester, ethyl ester, CO.sub.2H, aniline, nitro,
heterocycle, mono-substituted alkyl, di-substituted alkyl, and
tri-substituted alkyl, hydrogen, SO.sub.2NH.sub.2,
SO.sub.2NH-alkyl, and NHSO.sub.2alkyl.
[0300] In some embodiments, R3 is an isostere of OH. In some
embodiments, R3 is selected from the group consisting of hydrogen;
halogen; OH; MnO4; a linear or branched, saturated or unsaturated,
substituted or non-substituted, aliphatic chain having at least 2
carbons; a chemical moiety comprising Sulfur; a chemical moiety
comprising Nitrogen. In some embodiments, R3 is selected from the
group consisting of alkyl; mono-substituted alkyl; di-substituted
alkyl; tri-substituted alkyl; (CH.sub.2).sub.n wherein n=1-6; CN;
N.sub.3; CNO; NH.sub.2; SH; CF.sub.3; OCH.sub.3;
NCH.sub.2CH(CH.sub.2)N(CH.sub.3).sub.2;
NCH.sub.2CHCH.sub.2N(CH.sub.3).sub.2; phenyl; 2-pyridyl; 3-pyridyl;
4-pyridyl; NCH.sub.3; NCONHCH.sub.3; CH.sub.2OH; NHCONH.sub.2;
NHCOCH.sub.3; NHSO.sub.2CH.sub.3; NHCN; NHCHO; SOCH.sub.3;
SO.sub.2CH.sub.3; CHNOH; CHNOCH.sub.3; SCH.sub.3; CH.sub.2CO;
CH.sub.2SO.sub.2; CONH; CH.sub.2C(NOH); CH.sub.2C(NOMe);
NHSO.sub.2PH; NHCS; CH.sub.2NHCO; COCH.sub.2; NHCO.sub.2; and
NHCOS. In some embodiments, R3 is described by any of the isosteres
described in, for example, Patani, G. and LaVoie, E. J., 1996,
Chem. Rev. 96:3147-3176; herein incorporated by reference in its
entirety.
[0301] In some embodiments, R4 is a chemical moiety that causes the
benzodiazepine to lack a chiral center. In some embodiments, R4 is
hydrogen, ##STR107## wherein R4' is a linear or branched, saturated
or unsaturated, substituted or non-substituted, aliphatic chain
having at least 2 carbons.
[0302] Certain embodiments of the present invention include crystal
forms and formulations of exemplary compounds with the following
formula: ##STR108##
[0303] In some embodiments, the present invention includes crystal
forms and formulations of the following exemplary compound:
##STR109##
[0304] Additional exemplary compounds and uses useful in the
present invention are described at U.S. Patent Publication
2004/0009972, published Jan. 15, 2004, herein incorporated by
reference in its entirety. Additional exemplary compounds and uses
useful in the present invention are described in U.S. Provisional
Patent Nos. 60/131,761, 60/165,511, 60/191,855, 60/312,560,
60/313,689, 60/396,670, 60/565,788, 60/607,599, 60/641,040, and
U.S. patent application Ser. Nos. 11/324,419, 11/176,719,
11/110,228, 10/935,333, 10/886,450, 10/795,535, 10/634,114,
10/427,211, 10/427,212, 10/217,878, 09/767,283, 09/700,101, and
related applications; each herein incorporated by reference in
their entireties. Additional exemplary compounds and uses useful in
the present invention are described in Atwal, et al., Bioorg. Med.
Chem. Lett. 14, 1027-1030 (2004) and Atwal, et al., J. Med. Chem.
47, 1081-1084 (2004); each herein incorporated by reference in
their entireties.
[0305] 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 clearly contemplated. Additional examples
contemplating ranges in other contexts can be found throughout this
disclosure wherein such ranges include analogous subranges
within.
[0306] In summary, a large number of compounds are presented
herein. Any one or more of these compounds can be used to treat a
variety of dysregulatory disorders related to cellular death as
described elsewhere herein. Additionally, any one or more of these
compounds can be used to inhibit ATP Hydrolysis while not affecting
cell synthesis or cell viability. Additionally, any one or more of
these compounds can be used in combination with at least one other
therapeutic agent (e.g., potassium channel openers, calcium channel
blockers, sodium hydrogen exchanger inhibitors, antiarrhythmic
agents, antiatherosclerotic agents, anticoagulants, antithrombotic
agents, prothrombolytic agents, fibrinogen antagonists, diuretics,
antihypertensive agents, ATPase inhibitors, mineralocorticoid
receptor antagonists, phospodiesterase inhibitors, antidiabetic
agents, anti-inflammatory agents, antioxidants, angiogenesis
modulators, antiosteoporosis agents, hormone replacement therapies,
hormone receptor modulators, oral contraceptives, antiobesity
agents, antidepressants, antianxiety agents, antipsychotic agents,
antiproliferative agents, antitumor agents, antiulcer and
gastroesophageal reflux disease agents, growth hormone agents
and/or growth hormone secretagogues, thyroid mimetics,
anti-infective agents, antiviral agents, antibacterial agents,
antifungal agents, cholesterol/lipid lowering agents and lipid
profile therapies, and agents that mimic ischemic preconditioning
and/or myocardial stunning, antiatherosclerotic agents,
anticoagulants, antithrombotic agents, antihypertensive agents,
antidiabetic agents, and antihypertensive agents selected from ACE
inhibitors, AT-1 receptor antagonists, ET receptor antagonists,
dual ET/AII receptor antagonists, and vasopepsidase inhibitors, or
an antiplatelet agent selected from GPIIb/IIIa blockers, P2Y.sub.1
and P2Y.sub.12 antagonists, thromboxane receptor antagonists, and
aspirin) in along with a pharmaceutically-acceptable carrier or
diluent in a pharmaceutical composition. Additionally, any one or
more of these compounds can be used to treat a mitochondrial
F.sub.1F.sub.0 ATP hydrolase associated disorder (e.g., myocardial
infarction, ventricular hypertrophy, coronary artery disease, non-Q
wave MI, congestive heart failure, cardiac arrhythmias, unstable
angina, chronic stable angina, Prinzmetal's angina, high blood
pressure, intermittent claudication, peripheral occlusive arterial
disease, thrombotic or thromboembolic symptoms of thromboembolic
stroke, venous thrombosis, arterial thrombosis, cerebral
thrombosis, pulmonary embolism, cerebral embolism, thrombophilia,
disseminated intravascular coagulation, restenosis, atrial
fibrillation, ventricular enlargement, atherosclerotic vascular
disease, atherosclerotic plaque rupture, atherosclerotic plaque
formation, transplant atherosclerosis, vascular remodeling
atherosclerosis, cancer, surgery, inflammation, systematic
infection, artificial surfaces, interventional cardiology,
immobility, medication, pregnancy and fetal loss, and diabetic
complications comprising retinopathy, nephropathy and neuropathy)
in a patient. The above-described compounds can also be used in
drug screening assays and other diagnostic methods.
IV. Pharmaceutical Compositions, Formulations, and Exemplary
Administration Routes and Dosing Considerations
[0307] Exemplary embodiments of various contemplated medicaments
and pharmaceutical compositions are provided below.
[0308] A. Preparing Medicaments
[0309] The compounds of the present invention are useful in the
preparation of medicaments to treat a variety of conditions
associated with dysregulation of cell death, aberrant cell growth
and hyperproliferation.
[0310] In addition, the compounds are also useful for preparing
medicaments for treating other disorders wherein the effectiveness
of the compounds are known or predicted. Such disorders include,
but are not limited to, 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.
[0311] One of skill in the art will appreciate that any one or more
of the compounds described herein, including the many specific
embodiments, are prepared by applying standard pharmaceutical
manufacturing procedures. Such medicaments can be delivered to the
subject by using delivery methods that are well-known in the
pharmaceutical arts.
[0312] B. Exemplary Pharmaceutical Compositions and Formulation
[0313] 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 crystal forms and
formulations and benzodiazepine related crystal forms and
formulations), as defined above, together with a solid support or
alternatively, together with one or more pharmaceutically
acceptable carriers and optionally other therapeutic agents. Each
carrier should be "acceptable" in the sense that it is compatible
with the other ingredients of the formulation and not injurious to
the subject.
[0314] 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.
[0315] 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.
[0316] In some embodiments, tablets comprise at least one active
ingredient and optionally one or more accessory agents/carriers are
made by compressing or molding the respective agents. In preferred
embodiments, compressed tablets are prepared by compressing in a
suitable machine the active ingredient in a free-flowing form such
as a powder or granules, optionally mixed with a binder (e.g.,
povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert
diluent, preservative, disintegrant (e.g., sodium starch glycolate,
cross-linked povidone, cross-linked sodium carboxymethyl cellulose)
surface-active or dispersing agent. Molded tablets are made by
molding in a suitable machine a mixture of the powdered compound
(e.g., active ingredient) moistened with an inert liquid diluent.
Tablets may optionally be coated or scored and may be formulated so
as to provide slow or controlled release of the active ingredient
therein using, for example, hydroxypropylmethyl cellulose in
varying proportions to provide the desired release profile. Tablets
may optionally be provided with an enteric coating, to provide
release in parts of the gut other than the stomach.
[0317] Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavored basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0318] 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
compound(s) that enhances absorption or penetration of the active
agent(s) through the skin or other affected areas. Examples of such
dermal penetration enhancers include dimethylsulfoxide (DMSO) and
related analogues.
[0319] If desired, the aqueous phase of a cream base includes, for
example, at least about 30% w/w of a polyhydric alcohol, i.e., an
alcohol having two or more hydroxyl groups such as propylene
glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and
polyethylene glycol and mixtures thereof.
[0320] 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
to further comprises a mixture of at least one emulsifier with a
fat or an oil or with both a fat and an oil.
[0321] 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 emulsifier(s) with or without stabilizer(s) make up
the so-called emulsifying wax, and the wax together with the oil
and/or fat make up the so-called emulsifying ointment base which
forms the oily dispersed phase of the cream formulations.
[0322] Emulgents and emulsion stabilizers suitable for use in the
formulation of the present invention include Tween 60, Span 80,
cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and
sodium lauryl sulfate.
[0323] The choice of suitable oils or fats for the formulation is
based on achieving the desired properties (e.g., cosmetic
properties), since the solubility of the active compound/agent in
most oils likely to be used in pharmaceutical emulsion formulations
is very low. Thus creams should preferably be a non-greasy,
non-staining and washable products with suitable consistency to
avoid leakage from tubes or other containers. Straight or branched
chain, mono- or dibasic alkyl esters such as di-isoadipate,
isocetyl stearate, propylene glycol diester of coconut fatty acids,
isopropyl myristate, decyl oleate, isopropyl palmitate, butyl
stearate, 2-ethylhexyl palmitate or a blend of branched chain
esters known as Crodamol CAP may be used, the last three being
preferred esters. These may be used alone or in combination
depending on the properties required. Alternatively, high melting
point lipids such as white soft paraffin and/or liquid paraffin or
other mineral oils can be used.
[0324] 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.
[0325] Formulations for rectal administration may be presented as a
suppository with suitable base comprising, for example, cocoa
butter or a salicylate.
[0326] Formulations suitable for vaginal administration may be
presented as pessaries, creams, gels, pastes, foams or spray
formulations containing in addition to the agent, such carriers as
are known in the art to be appropriate.
[0327] Formulations suitable for nasal administration, wherein the
carrier is a solid, include coarse powders having a particle size,
for example, in the range of about 20 to about 500 microns which
are administered in the manner in which snuff is taken, i.e., by
rapid inhalation (e.g., forced) through the nasal passage from a
container of the powder held close up to the nose. Other suitable
formulations wherein the carrier is a liquid for administration
include, but are not limited to, nasal sprays, drops, or aerosols
by nebulizer, an include aqueous or oily solutions of the
agents.
[0328] Formulations suitable for parenteral administration include
aqueous and non-aqueous isotonic sterile injection solutions which
may contain antioxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents, and liposomes
or other microparticulate systems which are designed to target the
compound to blood components or one or more organs. In some
embodiments, the formulations are presented/formulated in unit-dose
or multi-dose sealed containers, for example, ampoules and vials,
and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for
example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules and tablets of the kind previously
described.
[0329] 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.
[0330] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention
may include other agents conventional in the art having regard to
the type of formulation in question, for example, those suitable
for oral administration may include such further agents as
sweeteners, thickeners and flavoring agents. It also is intended
that the agents, compositions and methods of this invention be
combined with other suitable compositions and therapies. Still
other formulations optionally include food additives (suitable
sweeteners, flavorings, colorings, etc.), phytonutrients (e.g.,
flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, and
other acceptable compositions (e.g., conjugated linoelic acid),
extenders, and stabilizers, etc.
[0331] C. Exemplary Administration Routes and Dosing
Considerations
[0332] Various delivery systems are known and can be used to
administer a therapeutic agents (e.g., benzodiazepine crystal forms
and formulations and benzodiazepine related crystal forms and
formulations) of the present invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, receptor-mediated
endocytosis, and the like. Methods of delivery include, but are not
limited to, intra-arterial, intramuscular, intravenous, intranasal,
and oral routes. In specific embodiments, it may be desirable to
administer the pharmaceutical compositions of the invention locally
to the area in need of treatment; this may be achieved by, for
example, and not by way of limitation, local infusion during
surgery, injection, or by means of a catheter.
[0333] 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.
[0334] 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 MLR/MpJ-lpr/lpr ("MLR-lpr")
(available from Jackson Laboratories, Bal Harbor, Me.). MLR-lpr
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
10.sup.5 to about 10.sup.9 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-known in the art.
[0335] In some embodiments, in vivo administration is effected in
one dose, continuously or intermittently throughout the course of
treatment. Methods of determining the most effective means and
dosage of administration are well known to those of skill in the
art and vary with the composition used for therapy, the purpose of
the therapy, the target cell being treated, and the subject being
treated. Single or multiple administrations are carried out with
the dose level and pattern being selected by the treating
physician.
[0336] Suitable dosage formulations and methods of administering
the agents are readily determined by those of skill in the art.
Preferably, the compounds are administered at about 0.01 mg/kg to
about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100
mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg.
When the compounds described herein are co-administered with
another agent (e.g., as sensitizing agents), the effective amount
may be less than when the agent is used alone.
[0337] The pharmaceutical compositions can be administered orally,
intranasally, parenterally or by inhalation therapy, and may take
the form of tablets, lozenges, granules, capsules, pills, ampoules,
suppositories or aerosol form. They may also take the form of
suspensions, solutions and emulsions of the active ingredient in
aqueous or nonaqueous diluents, syrups, granulates or powders. In
addition to an agent of the present invention, the pharmaceutical
compositions can also contain other pharmaceutically active
compounds or a plurality of compounds of the invention.
[0338] More particularly, an agent of the present invention also
referred to herein as the active ingredient, may be administered
for therapy by any suitable route including, but not limited to,
oral, rectal, nasal, topical (including, but not limited to,
transdermal, aerosol, buccal and sublingual), vaginal, parental
(including, but not limited to, subcutaneous, intramuscular,
intravenous and intradermal) and pulmonary. It is also appreciated
that the preferred route varies with the condition and age of the
recipient, and the disease being treated.
[0339] 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.
[0340] 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.
[0341] D. Exemplary Co-Administration Routes and Dosing
Considerations
[0342] The present invention also includes methods involving
co-administration of the compounds described herein with one or
more additional active agents. Indeed, it is a further aspect of
this invention to provide methods for enhancing prior art therapies
and/or pharmaceutical compositions by co-administering a compound
of this invention. In co-administration procedures, the agents may
be administered concurrently or sequentially. In one embodiment,
the compounds described herein are administered prior to the other
active agent(s). The pharmaceutical formulations and modes of
administration may be any of those described above. In addition,
the two or more co-administered chemical agents, biological agents
or radiation may each be administered using different modes or
different formulations.
[0343] The agent or agents to be co-administered depends on the
type of condition being treated. For example, when the condition
being treated is cancer, the additional agent can be a
chemotherapeutic agent or radiation. When the condition being
treated is an autoimmune disorder, the additional agent can be an
immunosuppressant or an anti-inflammatory agent. When the condition
being treated is chronic inflammation, the additional agent can be
an anti-inflammatory agent. The additional agents to be
co-administered, such as anticancer, immunosuppressant,
anti-inflammatory, and can be any of the well-known agents in the
art, including, but not limited to, those that are currently in
clinical use. The determination of appropriate type and dosage of
radiation treatment is also within the skill in the art or can be
determined with relative ease.
[0344] Treatment of the various conditions associated with abnormal
apoptosis is generally limited by the following two major factors:
(1) the development of drug resistance and (2) the toxicity of
known therapeutic agents. In certain cancers, for example,
resistance to chemicals and radiation therapy has been shown to be
associated with inhibition of apoptosis. Some therapeutic agents
have deleterious side effects, including non-specific
lymphotoxicity, renal and bone marrow toxicity.
[0345] 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 vice
versa) and, accordingly, less of these agents are needed to achieve
a therapeutic benefit.
[0346] 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. Drug Screens
[0347] In preferred embodiments of the present invention, the
compounds of the present invention, and other potentially useful
compounds, are screened for their biological activity (e.g.,
ability to initiate cell death alone or in combination with other
compounds). 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 in vivo or ex vivo systems. While in some
embodiments quantifying the 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 or pH
level quantification.
[0348] 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.
[0349] In some embodiments, structure-based virtual screening
methodologies are contemplated for predicting the binding affinity
of compounds of the present invention with OSCP.
[0350] In some embodiments, compounds are screened in cell culture
or in 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, Wis.
and Stratagene, La Jolla, Calif.) and cell based dimerization
assays. (See e.g., Fuh et al., Science, 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.
[0351] Any suitable assay that allows for a measurement 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,
competition binding using Bz-423, surface plasma resonace (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).
[0352] In some embodiments, compounds are screened in cell culture
or in 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, Wis.
and Stratagene, La Jolla, Calif.) and cell based dimerization
assays. (See e.g., Fuh et al., Science, 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.
[0353] 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
like), or to minimize undesirable properties (e.g., nonspecific
reactivity, toxicity, and the like). 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 in silico ligand-receptor interactions prior to confirming
results by routine experimentation.
VI. Therapeutic Application
[0354] A. General Therapeutic Application
[0355] In particularly preferred embodiments, the compositions 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 mitochondrial
ATP synthase (as referred to as mitochondrial F.sub.0F.sub.1
ATPase) complexes in affected cells or tissues (e.g., myocardial
infarction, ventricular hypertrophy, coronary artery disease, non-Q
wave MI, congestive heart failure, cardiac arrhythmias, unstable
angina, chronic stable angina, Prinzmetal's angina, high blood
pressure, intermittent claudication, peripheral occlusive arterial
disease, thrombotic or thromboembolic symptoms of thromboembolic
stroke, venous thrombosis, arterial thrombosis, cerebral
thrombosis, pulmonary embolism, cerebral embolism, thrombophilia,
disseminated intravascular coagulation, restenosis, atrial
fibrillation, ventricular enlargement, atherosclerotic vascular
disease, atherosclerotic plaque rupture, atherosclerotic plaque
formation, transplant atherosclerosis, vascular remodeling
atherosclerosis, cancer, surgery, inflammation, systematic
infection, artificial surfaces, interventional cardiology,
immobility, medication, pregnancy and fetal loss, and diabetic
complications comprising retinopathy, nephropathy and neuropathy).
In further preferred embodiments, the compositions of the present
invention are used to treat autoimmune/chronic inflammatory
conditions (e.g., psoriasis). In even further embodiments, the
compositions of the present invention are used in conjunction with
stenosis therapy to treat compromised (e.g., occluded) vessels.
Indeed, any application where benzodiazepines find use is
contemplated by the present invention.
[0356] In particularly preferred embodiments, the compositions of
the present invention inhibit the activity of mitochondrial ATP
synthase complex by binding to a specific subunit or subunits 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, it is contemplated that the compositions of the
present invention bind to the oligomycin sensitivity conferring
protein (OSCP) portion of the mitochondrial ATP synthase complex,
to the OSCP/F1 junction, or to the F1 subunit. Likewise, it is
further contemplated that when the compositions of the present
invention bind to the OSCP the initial affect is overall inhibition
of the mitochondrial ATP synthase complex, and that the downstream
consequence of binding is a change in ATP or pH level and the
production of reactive oxygen species (e.g., O.sub.2--). 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 administered, it is contemplated
that the generation of free radicals ultimately results in cell
killing. 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 mitochondrial ATP synthase complex using the
compositions and methods of the present invention provides
therapeutically useful inhibition of cell proliferation.
[0357] 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 mitochondrial ATP synthase
complexes in affected cells or tissues via binding to the
oligomycin sensitivity conferring protein (OSCP) portion of the
mitochondrial ATP synthase complex. Importantly, by itself the
OSCP, the OSCP/F1 junction, or the F1 subunit has no biological
activity.
[0358] 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, etc., can be
treated by modulating the activity of the mitochondrial ATP
synthase complex including, but not limited to, by binding to the
oligomycin sensitivity conferring protein (OSCP)/F1 components
thereof. The present invention is not intended to be limited,
however, to the practice of the compositions and methods explicitly
described herein. Indeed, those skilled 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
mitochondrial ATP synthase.
[0359] The present invention thus specifically contemplates that
any number of suitable compounds presently known 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, oligomycin, ossamycin, cytovaricin, apoptolidin,
bafilomyxcin, resveratrol, piceatannol, 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]).
[0360] In some embodiments, compounds potentially useful in methods
of the present invention are screened against the National Cancer
Institute's (NCI-60) cancer cell lines for efficacy. (See e.g., A.
Monks et al., J. Natl. Cancer Inst., 83:757-766 [1991]; and K. D.
Paull et al., J. Natl. Cancer Inst., 81:1088-1092 [1989]).
Additional screens suitable screens (e.g., autoimmunity disease
models, etc.) are within the skill in the art.
[0361] In one aspect, derivatives (e.g., pharmaceutically
acceptable salts, analogs, stereoisomers, and the like) of the
exemplary compounds or other suitable compounds are also
contemplated as being useful in the methods of the present
invention.
[0362] In other preferred embodiments, the compositions of the
present invention are used in conjunction with stenosis therapy to
treat compromised (e.g., occluded) vessels. In further embodiments,
the compositions of the present invention are used in conjunction
with stenosis therapy to treat compromised cardiac vessels.
[0363] Vessel stenosis is a condition that develops when a vessel
(e.g., aortic valve) becomes narrowed. For example, aortic valve
stenosis is a heart condition that develops when the valve between
the lower left chamber (left ventricle) of the heart and the major
blood vessel called the aorta becomes narrowed. This narrowing
(e.g., stenosis) creates too small a space for the blood to flow to
the body. Normally the left ventricle pumps oxygen-rich blood to
the body through the aorta, which branches into a system of
arteries throughout the body. When the heart pumps, the 3 flaps, or
leaflets, of the aortic valve open one way to allow blood to flow
from the ventricle into the aorta. Between heartbeats, the flaps
close to form a tight seal so that blood does not leak backward
through the valve. If the aortic valve is damaged, it may become
narrowed (stenosed) and blood flow may be reduced to organs in the
body, including the heart itself. The long-term outlook for people
with aortic valve stenosis is poor once symptoms develop. People
with untreated aortic valve stenosis who develop symptoms of heart
failure usually have a life expectancy of 3 years or less.
[0364] Several types of treatment exist for treating compromised
valves (e.g., balloon dilation, ablation, atherectomy or laser
treatment). One type of treatment for compromised cardiac valves is
angioplasty. Angioplasty involves inserting a balloon-tipped tube,
or catheter, into a narrow or blocked artery in an attempt to open
it. By inflating and deflating the balloon several times,
physicians usually are able to widen the artery.
[0365] A common limitation of angioplasty or valve expansion
procedures is restenosis. Restenosis is the reclosure of a
peripheral or coronary artery following trauma to that artery
caused by efforts to open a stenosed portion of the artery, such
as, for example, by balloon dilation, ablation, atherectomy or
laser treatment of the artery. For these angioplasty procedures,
restenosis occurs at a rate of about 20-50% depending on the
definition, vessel location, lesion length and a number of other
morphological and clinical variables. Restenosis is believed to be
a natural healing reaction to the injury of the arterial wall that
is caused by angioplasty procedures. The healing reaction begins
with the thrombotic mechanism at the site of the injury. The final
result of the complex steps of the healing process can be intimal
hyperplasia, the uncontrolled migration and proliferation of medial
smooth muscle cells, combined with their extracellular matrix
production, until the artery is again stenosed or occluded.
[0366] In an attempt to prevent restenosis, metallic intravascular
stents have been permanently implanted in coronary or peripheral
vessels. The stent is typically inserted by catheter into a
vascular lumen told expanded into contact with the diseased portion
of the arterial wall, thereby providing mechanical support for the
lumen. However, it has been found that restenosis can still occur
with such stents in place. Also, the stent itself can cause
undesirable local thrombosis. To address the problem of thrombosis,
persons receiving stents also receive extensive systemic treatment
with anticoagulant and antiplatelet drugs.
[0367] To address the restenosis problem, it has been proposed to
provide stents which are seeded with endothelial cells (Dichek, D.
A. et al Seeding of Intravascular Stents With Genetically
Engineered Endothelial Cells; Circulation 1989; 80: 1347-1353). In
that experiment, sheep endothelial cells that had undergone
retrovirus-mediated gene transfer for either bacterial
beta-galactosidase or human tissue-type plasminogen activator were
seeded onto stainless steel stents and grown until the stents were
covered. The cells were therefore able to be delivered to the
vascular wall where they could provide therapeutic proteins. Other
methods of providing therapeutic substances to the vascular wall by
means of stents have also been proposed such as in international
patent application WO 91/12779 "Intraluminal Drug Eluting
Prosthesis" and international patent application WO 90/13332 "Stent
With Sustained Drug Delivery". In those applications, it is
suggested that antiplatelet agents, anticoagulant agents,
antimicrobial agents, anti-inflammatory agents, antimetabolic
agents and other drugs could be supplied in stents to reduce the
incidence of restenosis. Further, other vasoreactive agents such as
nitric oxide releasing agents could also be used.
[0368] An additional cause of restenosis is the over-proliferation
of treated tissue. In preferred embodiments, the anti-proliferative
properties of the present invention inhibit restenosis.
Drug-eluting stents are well known in the art (see, e.g., U.S. Pat.
No. 5,697,967; U.S. Pat. No. 5,599,352; and U.S. Pat. No.
5,591,227; each of which are herein incorporated by reference). In
preferred embodiments, the compositions of the present invention
are eluted from drug-eluting stents in the treatment of compromised
(e.g., occluded) vessels. In further embodiments, the compositions
of the present invention are eluted from drug-eluting stents in the
treatment of compromised cardiac vessels.
[0369] Those skilled in the art of preparing pharmaceutical
compounds and formulations will appreciate that when selecting
optional compounds for use in the methods disclosed herein, that
suitability considerations include, but are not limited to, the
toxicity, safety, efficacy, availability, and cost of the
particular compounds.
[0370] In preferred embodiments, pharmaceutical compositions
comprise compounds of the invention and, for example, therapeutic
agents (e.g., antiatherosclerotic agents, anticoagulants,
antithrombotic agents, antihypertensive agents, and antidiabetic
agents). Antihypertensive agents include, but are not limited to,
ACE inhibitors, AT-1 receptor antagonists, ET receptor antagonists,
dual ET/AII receptor antagonists, and vasopepsidase inhibitors, or
an antiplatelet agent selected from GPIIb/IIIa blockers, P2Y.sub.1
and P2Y.sub.12 antagonists, thromboxane receptor antagonists, and
aspirin.
[0371] In preferred embodiments, the compounds of the present
invention are useful in treating a mitochondrial F.sub.1F.sub.0 ATP
hydrolase associated disorder (e.g., myocardial infarction,
ventricular hypertrophy, coronary artery disease, non-Q wave MI,
congestive heart failure, cardiac arrhythmias, unstable angina,
chronic stable angina, Prinzmetal's angina, high blood pressure,
intermittent claudication, peripheral occlusive arterial disease,
thrombotic or thromboembolic symptoms of thromboembolic stroke,
venous thrombosis, arterial thrombosis, cerebral thrombosis,
pulmonary embolism, cerebral embolism, thrombophilia, disseminated
intravascular coagulation, restenosis, atrial fibrillation,
ventricular enlargement, atherosclerotic vascular disease,
atherosclerotic plaque rupture, atherosclerotic plaque formation,
transplant atherosclerosis, vascular remodeling atherosclerosis,
cancer, surgery, inflammation, systematic infection, artificial
surfaces, interventional cardiology, immobility, medication,
pregnancy and fetal loss, and diabetic complications comprising
retinopathy, nephropathy and neuropathy) in a patient.
[0372] B. Autoimmune Disorder and Chronic Inflammatory Disorder
Therapeutic Application
[0373] Autoimmune disorders and chronic inflammatory disorders
often result from dysfunctional cellular proliferation regulation
and/or cellular apoptosis regulation. Mitochondria perform a key
role in the control and execution of cellular apoptosis. The
mitochondrial permeability transition pore (MPTP) is a pore that
spans the inner and outer mitochondrial membranes and functions in
the regulation of proapoptotic particles. Transient MPTP opening
results in the release of cytochrome c and the apoptosis inducing
factor from the mitochondrial intermembrane space, resulting in
cellular apoptosis.
[0374] The oligomycin sensitivity conferring protein (OSCP) is a
subunit of the F.sub.0F.sub.1 mitochondrial ATP synthase/ATPase and
functions in the coupling of a proton gradient across the F.sub.0
sector of the enzyme in the mitochondrial membrane. In preferred
embodiments, compounds of the present invention binds the OSCP, the
OSCP/F1 junction, or the F1 subunit increases superoxide and
cytochrome c levels, increases cellular apoptosis, and inhibits
cellular proliferation. The adenine nucleotide translocator (ANT)
is a 30 kDa protein that spans the inner mitochondrial membrane and
is central to the mitochondrial permeability transition pore
(MPTP). Thiol oxidizing or alkylating agents are powerful
activators of the MPTP that act by modifying one or more of three
unpaired cysteines in the matrix side of the ANT.
4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide, ##STR110##
inhibits the ANT.
[0375] The compounds and methods of the present invention are
useful in the treatment of autoimmune disorders and chronic
inflammatory disorders. In such embodiments, the present invention
provides a subject suffering from an autoimmune disorder and/or a
chronic inflammatory disorder, and a composition comprising, for
example, ##STR111## Additionally, in preferred embodiments, the
compositions may comprise any of the compounds described in the
present invention, and any of the compounds described in U.S.
Provisional Patent Nos. 60/131,761, 60/165,511, 60/191,855,
60/312,560, 60/313,689, 60/396,670, 60/565,788, 60/607,599,
60/641,040, and U.S. patent application Ser. Nos. 11/324,419,
11/176,719, 11/110,228, 10/935,333, 10/886,450, 10/795,535,
10/634,114, 10/427,211, 10/427,212, 10/217,878, 09/767,283,
09/700,101, and related applications; each herein incorporated by
reference in their entireties.
[0376] C. Treatment of Epidermal Hyperplasia
[0377] Epidermal hyperplasia (e.g., excessive keratinocyte
proliferation) leading to a significant thickening of the epidermis
in association with shedding of the thickened epidermis, is a
feature of diseases such as psoriasis (see, e.g., Krueger G C, et
al., (1984) J. Am. Acad. Dermatol. 11: 937-947; Fry L. (1988),
Brit. J. Dermatol. 119:445-461; each herein incorporated by
reference in their entireties) and also occurs under physiological
conditions (e.g., during wound-healing).
[0378] Topical treatment of the skin with all-trans retinoic acid
(RA) or its precursor, all-trans retinol (ROL) also results in
epidermal hyperplasia (see, e.g., Varani J, et al., (2001) J.
Invest. Dermatol, 117:1335-1341; herein incorporated by reference
in its entirety). While the underlying etiologies are different,
all of these hyperplasias have in common the activation of the
epidermal growth factor (EGF) receptor in the proliferating
keratinocytes (see, e.g., Varani J, et al., (2001) J. Invest.
Dermatol 117:1335-1341; Baker B S, et al., (1992) Brit. J.
Dermatol. 126:105-110; Gottlieb A B, et al., (1988) J. Exp. Med.
167:670-675; Elder J T, et al., (1989) Science 243:811-814;
Piepkorn M, et al., (1998) J Invest Dermatol 111:715-721; Piepkorn
M, et al., (2003) Arch Dermatol Res 27:27; Cook P W, et al., (1992)
Cancer Res 52:3224-3227; each herein incorporated by reference in
their entireties). Normal epidermal growth does not appear to be as
dependent on EGF receptor function as hyperplastic growth (see,
e.g., Varani J, et al., (2001) J. Invest. Dermatol 117:1335-1341;
Varani J, et al., (1998) Pathobiology 66:253-259; each herein
incorporated by reference in their entireties). Likewise, function
of the dermis in intact skin does not depend on EGF receptor
function (see, e.g., Varani J, et al., (2001) J. Invest. Dermatol
117:1335-1341; herein incorporated by reference in its
entirety).
[0379] The central role of the EGF receptor in regulating
hyperplastic epithelial growth makes the EGF receptor tyrosine
kinase a target for antiproliferative agents. Likewise, the series
of signaling molecules engaged downstream of this receptor are
additional points at which keratinocyte growth can be interrupted.
The mitogen activated protein kinase (MAPK) cascade is activated by
the EGF receptor (see, e.g., Marques, S. A., et al., (2002) J
Pharmacol Exp Ther 300, 1026-1035; herein incorporated by reference
in its entirety). In hyperproliferative epidermis, but not in
normal epidermis, extracellular signal-regulated kinases 1/2 (Erk
1/2) are activated in basal and suprabasal keratinocytes and
contribute to epidermal hyperproliferation (see, e.g., Haase, I.,
et al., (2001) J Clin Invest 108, 527-536; Takahashi, H., et al.,
(2002) J Dermatol Sci-30, 94-99; each herein incorporated by
reference in their entireties). In culture models, keratinocyte
growth regulation through the EGF receptor results in increased
MAPK activity. In keratinocytes, growth factor-stimulated MAPK
activity is also dependent on integrin engagement and extracellular
matrix molecules that bind integrins are capable of independently
activating MAPKs and increasing keratinocyte proliferation (see,
e.g., Haase, I., et al., (2001) J Clin Invest 108, 527-536; herein
incorporated by reference in its entirety). The proliferation of
other skin cells, including fibroblasts, is less dependent on Erk
1/2 activity, making Erk inhibition a potentially useful
characteristic to evaluate lead compounds for potential utility
against epidermal hyperplasia.
[0380] In preferred embodiments, compounds of the present invention
are used for treating epidermal hyperplasias.
[0381] In preferred embodiments, compounds of the present invention
are used in treating psoriasis. Psoriasis is common and chronic
epidermal hyperplasia. Plaque psoriasis is the most common type of
psoriasis and is characterized by red skin covered with silvery
scales and inflammation. Patches of circular to oval shaped red
plaques that itch or burn are typical of plaque psoriasis. The
patches are usually found on the arms, legs, trunk, or scalp but
may be found on any part of the skin. The most typical areas are
the knees and elbows. Psoriasis is not contagious and can be
inherited. Environmental factors, such as smoking, sun exposure,
alcoholism, and HIV infection, may affect how often the psoriasis
occurs and how long the flares up last.
[0382] Treatment of psoriasis includes topical steroids, coal tar,
keratolytic agents, vitamin D-3 analogs, and topical retinoids.
Topical steroids are agents used to reduce plaque formation.
Topical steroid agents have anti-inflammatory effects and may cause
profound and varied metabolic activities. In addition, topical
steroid agents modify the body's immune response to diverse
stimuli. Examples of topical steroids include, but are not limited
to, triamcinolone acetonide (Artistocort, Kenalog) 0.1% cream, and
betamethasone diproprionate (Diprolene, Diprosone) 0.05% cream.
Coal tar is an inexpensive treatment available over the counter in
shampoos or lotions for use in widespread areas of involvement.
Coal tar is particularly useful in hair-bearing areas. An example
of coal tar is coal tar 2-10% (DHS Tar, Doctar, Theraplex
T)--antipruitic. Keratolytic agents are used to remove scale,
smooth the skin, and to treat hyperkeratosis. An example of a
keratolytic agent is anthralin 0.1-1% (Drithocreme, Anthra-Derm).
Vitamin D-3 analogs are used in patients with lesions resistant to
older therapy or with lesions on the face or exposed areas where
thinning of the skin would pose cosmetic problems. An example of a
vitamin D-3 analog is calcipotriene (Dovonex). Topical retinoids
are agents that decrease the cohesiveness of follicular epithelial
cells and stimulate mitotic activity, resulting in an increase in
turnover of follicular epithelial cells. Examples of topical
retinoids include, but are not limited to, tretinoin (Retin-A,
Avita), and tazarotene (Tazorac).
[0383] Approximately 1-2% of people in the United States, or about
5.5 million, have plaque psoriasis. Up to 30% of people with plaque
psoriasis also have psoriatic arthritis. Individuals with psoriatic
arthritis have inflammation in their joints and may have other
arthritis symptoms. Sometimes plaque psoriasis can evolve into more
severe disease, such as pustular psoriasis or erythrodermic
psoriasis. In pustular psoriasis, the red areas on the skin contain
blisters with pus. In erythrodermic psoriasis, a wide area of red
and scaling skin is typical, and it may be itchy and painful. The
present invention is useful in treating additional types of
psoriasis, including but not limited to, guttate psoriasis, nail
psoriasis, inverse psoriasis, and scalp psoriasis.
[0384] In some embodiments, the compounds of the present invention
are useful in treating pigmentation disorders (e.g., albinism,
melasma, and vitiligo). The present invention is not limited to a
particular mechanism for treating pigment disorders. In preferred
embodiments, pigment disorders are treated through targeting of the
F.sub.1F.sub.0-ATPase by the compounds of the present invention. In
further embodiments, pigment disorders are treated through the
rerouting of tyrosinase by the compounds of the present invention.
In further embodiments, pigment disorders are treated through
targeting of prohibition by the compounds of the present
invention.
VII. ATPase Inhibitors and Methods for Identifying Therapeutic
Inhibitors
[0385] The present invention provides compounds that target the
F.sub.1F.sub.0-ATPase. In addition, the present invention provides
compounds that target the F.sub.1F.sub.0-ATPase as a treatment for
autoimmune disorders, and in particular, compounds with low
toxicity. The present invention further provides methods of
identifying compounds that target the F.sub.1F.sub.0-ATPase.
Additionally, the present invention provides therapeutic
applications for compounds targeting the F.sub.1F.sub.0-ATPase.
[0386] A majority of ATP within eukaryotic cells is synthesized by
the mitochondrial F.sub.1F.sub.o-ATPase (see, e.g., C. T. Gregory
et al., J. Immunol., 139:313-318 [1987]; J. P. Portanova et al.,
Mol. Immunol., 32:117-135 [1987]; M. J. Shlomchik et al., Nat. Rev.
Immunol., 1: 147-153 [2001]; each herein incorporated by reference
in their entireties). Although the F.sub.1F.sub.0-ATPase
synthesizes and hydrolyzes ATP, during normal physiologic
conditions, the F.sub.1F.sub.o-ATPase only synthesizes ATP (see,
e.g., Nagyvary J, et al., Biochem. Educ. 1999; 27:193-99; herein
incorporated by reference in its entirety). The mitochondrial
F.sub.1F.sub.o-ATPase is composed of three major domains: F.sub.o,
F.sub.1 and the peripheral stator. F.sub.1 is the portion of the
enzyme that contains the catalytic sites and it is located in the
matrix (see, e.g., Boyer, P D, Annu Rev Biochem. 1997; 66:717-49;
herein incorporated by reference in its entirety). This domain is
highly conserved and has the subunit composition
.alpha..sub.3.beta..sub.3.gamma..delta..epsilon.. The landmark
X-ray structure of bovine F.sub.1 revealed that
.alpha..sub.3.beta..sub.3 forms a hexagonal cylinder with the
.gamma. subunit in the center of the cylinder. F.sub.o is located
within the inner mitochondrial membrane and contains a proton
channel. Translocation of protons from the inner-membrane space
into the matrix provides the energy to drive ATP synthesis. The
peripheral stator is composed of several proteins that physically
and functionally link F.sub.o with F.sub.1. The stator transmits
conformational changes from F.sub.o into in the catalytic domain
that regulate ATP synthesis (see, e.g., Cross R L, Biochim Biophys
Acta 2000; 1458:270-75; herein incorporated by reference in its
entirety).
[0387] Mitochondrial F.sub.1F.sub.o-ATPase inhibitors are
invaluable tools for mechanistic studies of the
F.sub.1F.sub.o-ATPase (see, e.g., James A M, et al., J Biomed Sci
2002; 9:475-87; herein incorporated by reference in its entirety).
Because F.sub.1F.sub.o-ATPase inhibitors are often cytotoxic, they
have been explored as drugs for cancer and other hyperproliferative
disorders. Macrolides (e.g., oligomycin and apoptolidin) are
non-competitive inhibitors of the F.sub.1F.sub.o-ATPase (see, e.g.,
Salomon A R, et al., PNAS 2000; 97:14766-71; Salomon A R, et al.,
Chem Biol 2001; 8:71-80; herein incorporated by reference in its
entirety). Macrolides bind to F.sub.o which blocks proton flow
through the channel resulting in inhibition of the
F.sub.1F.sub.o-ATPase. Macrolides are potent (e.g., the IC.sub.50
for oligomycin=10 nM) and lead to large decreases in [ATP]. As
such, macrolides have an unacceptably narrow therapeutic index and
are highly toxic (e.g., the LD.sub.50 for oligomycin in rodents is
two daily doses at 0.5 mg/kg) (see, e.g., Kramar R, et al., Agents
& Actions 1984, 15:660-63; herein incorporated by reference in
its entirety). Other inhibitors of F.sub.1F.sub.o-ATPase include
Bz-423, which binds to the OSCP in F, (as described elsewhere
herein). Bz-423 has an K.sub.i.about.9 .mu.M. Bz-423 is described
in U.S. patent application Ser. Nos. 10/634,114, 10/427,211,
10/427,212, 10/217,878, 09/767,283, 09/700,101, and related
applications, each herein incorporated by reference in their
entireties.
[0388] In cells that are actively respiring (known as state 3
respiration), inhibiting F.sub.1F.sub.o-ATPase blocks respiration
and places the mitochondria in a resting state (known as state 4).
In state 4, the MRC is reduced relative to state 3, which favors
reduction of O.sub.2 to O.sub.2.sup.- at complex III (see, e.g., N.
Zamzami et al., J. Exp. Med., 181:1661-1672 [1995]; herein
incorporated by reference in its entirety). For example, treating
cells with either oligomycin leads to a rise of intracellular
O.sub.2.sup.- as a consequence of inhibiting complex V. In the case
of oligomycin, supplementing cells with ATP protects against death
whereas antioxidants do not, indicating that cell death results
from the drop in ATP (see, e.g., Zhang J G, et al., Arch Biochem
Biophys 2001; 393:87-96; McConkey D J, et al., The ATP switch in
apoptosis. In: Nieminen La, ed. Mitochondria in pathogenesis. New
York: Plenum, 2001:265-77; each herein incorporated by reference in
their entireties). Bz-423-induced cell death is blocked by
antioxidants and is not affected by supplementing cells with ATP,
indicating that Bz-423 engages an ROS-dependent death response
(see, e.g., N. B. Blatt, et al., J. Clin. Invest., 2002, 110, 1123;
herein incorporated by reference in its entirety). As such,
F.sub.1F.sub.o-ATPase inhibitors are either toxic (e.g.,
oligomycin) or therapeutic (e.g., Bz-423).
[0389] The present invention provides a method of distinguishing
toxic F.sub.1F.sub.o-ATPase inhibitors from therapeutic
F.sub.1F.sub.o-ATPase inhibitors. F.sub.1F.sub.o-ATPase inhibitors
with therapeutic potential present a novel mode of inhibition.
Specifically, F.sub.1F.sub.o-ATPase inhibitors with beneficial
properties are uncompetitive inhibitors that only bind
enzyme-substrate complexes at high substrate concentration and do
not alter the k.sub.cat/K.sub.m ratio. This knowledge forms the
basis to identify and distinguish F.sub.1F.sub.o-ATPase inhibitors
with therapeutic potential from toxic compounds.
[0390] The present invention provides compounds that target the
F.sub.1F.sub.o-ATPase as an autoimmune disorder treatment. In
particular, the present invention provides methods of identifying
compounds that target the F.sub.1F.sub.0-ATPase while not altering
the k.sub.cat/K.sub.m ratio. Additionally, the present invention
provides therapeutic applications for compounds targeting the
F.sub.1F.sub.o-ATPase.
A. ATPase Inhibiting Compounds
[0391] The present invention provides compounds that inhibit the
F.sub.1F.sub.o-ATPase. In some embodiments, the compounds do not
bind free F.sub.1F.sub.o-ATPase, but rather bind to an
F.sub.1F.sub.o-ATPase-substrate complex. The compounds show maximum
activity at high substrate concentration and minimal activity
(e.g., F.sub.1F.sub.o-ATPase inhibiting) at low substrate
concentration. In preferred embodiments, the compounds do not alter
the k.sub.cat/K.sub.m ratio of the F.sub.1F.sub.o-ATPase. The
properties of the F.sub.1F.sub.o-ATPase inhibitors of the present
invention are in contrast with oligomycin, which is a
F.sub.1F.sub.o-ATPase inhibitor that is acutely toxic and lethal.
Oligomycin is a noncompetitive inhibitor, which binds to both free
F.sub.1F.sub.o-ATPase and F.sub.1F.sub.o-ATPase-substrate complexes
and alters the k.sub.cat/K.sub.m ratio.
[0392] The compounds of the present invention that inhibit
F.sub.1F.sub.o-ATPase while not altering the k.sub.cat/K.sub.m
ratio, in some embodiments, have the structure described elsewhere
herein. However, compounds of other structures that are identified
as therapeutic inhibitors by the methods of the present invention
are also encompassed by the present invention.
B. Identifying ATPase Inhibitors
[0393] The present invention provides methods of identifying (e.g.,
screening) compounds useful in treating autoimmune disorders. The
present invention is not limited to a particular type compound. In
preferred embodiments, compounds of the present invention include,
but are not limited to, pharmaceutical compositions, small
molecules, antibodies, large molecules, synthetic molecules,
synthetic polypeptides, synthetic polynucleotides, synthetic
nucleic acids, aptamers, polypeptides, nucleic acids, and
polynucleotides. The present invention is not limited to a
particular method of identifying compounds useful in treating
autoimmune disorders. In preferred embodiments, compounds useful in
treating autoimmune disorders are identified as possessing an
ability to inhibit an F.sub.1F.sub.o-ATPase while not altering the
k.sub.cat/K.sub.m ratio.
C. Therapeutic Applications With F.sub.1F.sub.o-ATPase
Inhibitors
[0394] The present invention provides methods for treating
disorders (e.g., neurodegenerative diseases, Alzheimers, ischemia
reprofusion injury, neuromotor disorders, non-Hodgkin's lymphoma,
lymphocytic leukemia, cutaneous T cell leukemia, an autoimmune
disorder, cancer, solid tumors, lymphomas, leukemias, and
tuberculosis). The present invention is not limited to a particular
form of treatment. In preferred embodiments, treatment includes,
but is not limited to, symptom amelioration, symptom prevention,
disorder prevention, and disorder amelioration. The present
invention provides methods of treating autoimmune disorders
applicable within in vivo, in vitro, and/or ex vivo settings.
[0395] In some embodiments, the present invention treats autoimmune
disorders through inhibiting of target cells. The present invention
is not limited to a particular form of cell inhibition. In
preferred embodiments, cell inhibition includes, but is not limited
to, cell growth prevention, cell proliferation prevention, and cell
death. In preferred embodiments, inhibition of a target cell is
accomplished through contacting a target cell with an
F.sub.1F.sub.0-ATPase inhibitor of the present invention. In
further embodiments, target cell inhibition is accomplished through
targeting of the F.sub.1F.sub.o-ATPase with an
F.sub.1F.sub.0-ATPase inhibitor of the present invention. The
present invention is not limited to a particular
F.sub.1F.sub.0-ATPase inhibitor. In preferred embodiments, the
F.sub.1F.sub.0-ATPase inhibitor possesses the ability to inhibit an
F.sub.1F.sub.0-ATPase while not altering the k.sub.cat/K.sub.m
ratio.
[0396] The present invention further provides methods for
selectively inhibiting the pathology of target cells in a subject
in need of therapy. The present invention is not limited to a
particular method of inhibition target cell pathology. In preferred
embodiments, target cell pathology is inhibited through
administration of an effective amount of a compound of the
invention. The present invention is not limited to a particular
compound. In preferred embodiments, the compound is an
F.sub.1F.sub.o-ATPase inhibitor. In further preferred embodiments,
the compound inhibits the F.sub.1F.sub.o-ATPase while not altering
the k.sub.cat/K.sub.m ratio.
EXAMPLES
[0397] 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. Examples illustrating the various uses and applications of
benzodiazepine compounds and benzodiazepine related compounds are
described, for example, in U.S. Provisional Patent Nos. 60/131,761,
60/165,511, 60/191,855, 60/312,560, 60/313,689, 60/396,670,
60/565,788, 60/607,599, 60/641,040, and U.S. patent application
Ser. Nos. 11/324,419, 11/176,719, 11/110,228, 10/935,333,
10/886,450, 10/795,535, 10/634,114, 10/427,211, 10/427,212,
10/217,878, 09/767,283, 09/700,101, and related applications; each
herein incorporated by reference in their entireties.
Example 1
[0398] This example describes the formation of benzodiazepine
crystal forms and formulations. Bz-423 (15.8 mg) was added to a 1
mL vial and dissolved in polyethylene glycol dimethyl ether 500
(0.01 mL) at 88.degree. C. The vial was kept at 88.degree. C. for
16 hours, during which time crystals formed. The vial was then
cooled to room temperature and washed with cold ethyl ether
(5.times.2 mL) to yield 4.2 mg of anhydrous Bz-423 crystals, a 27%
yield. FIG. 1 shows the structural data of anhydrous Bz-423, FIG. 2
shows powder x-ray diffraction data for anhydrous Bz-423, and FIG.
3 shows Raman spectroscopy data for anhydrous Bz-423.
[0399] Bz-423 ethanol solvate was crystallized by evaporation of
ethanol at room temperature. FIG. 4 shows the structural data of
Bz-423 ethanol solvate, FIG. 5 shows powder x-ray diffraction data
for Bz-423 ethanol solvate, and FIG. 6 shows Raman spectroscopy
data for Bz-423 ethanol solvate.
[0400] Ball milled Bz-423 succinic acid (2:1) was crystallized from
tetraethylene glycol dimethyl ether at 88.degree. C. FIG. 7 shows
Raman spectroscopy data for ball milled Bz-423 succinic acid
(2:1).
[0401] Ball milled Bz-423 citric acid (2:1) was generated through
ball milling a 2:1 mixture of Bz-423 and citric acid. FIG. 8 shows
Raman spectroscopy ball milled Bz-423 citric acid (2:1).
[0402] Bz-423 biphenyl derivate was crystallized from methanol at
room temperature. FIG. 9 shows the structural data of Bz-423
biphenyl derivate.
[0403] BZ-423-acetic acid was crystallized by evaporation of acetic
acid at room temperature. BZ-423-CH.sub.3CN was crystallized by
evaporation of CH.sub.3CN at room temperature. BZ-423-methanol was
crystallized by evaporation of methanol at room temperature.
BZ-423-ethyl acetate was crystallized by evaporation of ethyl
acetate at room temperature. BZ-423-toluene was crystallized by
evaporation of toluene at room temperature. BZ-423-oxalic acid was
crystallized from tetraethylene glycol dimethyl ether at 88.degree.
C. BZ-423-fumaric acid was crystallized from tetraethylene glycol
dimethyl ether at 88.degree. C. BZ-423-octanol was crystallized
from a supersaturated solution of octanol. BZ-423-heptanoic acid
was crystallized from a supersaturated solution of heptanoic acid.
BZ-423-diphenyl ether was crystallized from a supersaturated
solution of diphenyl ether. BZ-423-trichlorobenzene was
crystallized from a supersaturated solution of
trichlorobenzene.
Example 2
[0404] This example demonstrates that anhydrous Bz-423 is more
soluble than solvated Bz-423. Water solubility at 37.degree. C.
analyses were conducted for solvated Bz-423, anhydrous Bz-423,
Bz-423 acetic acid, and Bz-423 citric acid. Table 1 shows the
solubility results for solvated Bz-423, anhydrous Bz-423, Bz-423
acetic acid, Bz-423 citric acid, and Bz-423 micronized.
TABLE-US-00001 TABLE 1 as supplied BZ-423 ball milled 1.00
anhydrous BZ-423 ball milled 1.4 BZ-423 acetic acid 1.4 BZ-423
citric acid 1.4 Bz-423 micronized 0.85
FIG. 10 shows solubility (e.g., absorbance) as a function of time
for solvated Bz-423, anhydrous Bz-423, Bz-423 acetic acid, and
Bz-423 citric acid.
Example 3
[0405] This example demonstrates that unsolvated Bz-423 is capable
of inhibiting ATP hydrolysis, not inhibiting cell synthesis, not
affecting cell viability, and its activity is related to binding of
the OSCP, along or with other components of the mitochondrial F0F1
ATPase (e.g., F1 subunit). In particular, FIG. 11 shows a
comparison of ATP hydrolysis between unsolvated Bz-423 and,
solvated Bz-423, FIG. 12 shows a comparison of ATP synthesis
between unsolvated Bz-423 and solvated Bz-423, and FIG. 13 shows a
comparison of cell viability between unsolvated Bz-423 and solvated
Bz-423.
Example 4
[0406] This example demonstrates that unsolvated Bz-423 is more
soluble in simulated gastric fluid than solvated Bz-423. Simulated
gastric fluid solubility at 37.degree. C. analyses were conducted
for solvated Bz-423, and anhydrous Bz-423. Anhydrous Bz-423 was
more soluble than solvated Bz-423. FIG. 14 shows a UV-vis spectrum
of Bz-423 in simulated gastric fluid. FIG. 15 shows a UV-vis
spectrum of Bz-423 in simulated gastric fluid before and after
addition of K.sub.2CO.sub.3.
Example 5
[0407] This example describes the formation of benzodiazepine
formulations. FIG. 16 shows Raman spectroscopy data for Bz-423
ethanol solvate. BZ-423 (12.3 mg) was dissolved completely in 0.5
mL of ethanol at 70.degree. C. Upon cooling to room temperature
crystallization of the solvate occurred. The Raman spectrum above
was obtained from the crystals. The characteristic Raman shifts for
the ethanol solvate occur at about 1673 and 1560 cm.sup.-1.
[0408] FIG. 17 shows Raman spectroscopy data for Bz-423 1-propanol
solvate. BZ-423 (46.6 mg) was dissolved completely in 0.5 mL of
1-propanol at 70.degree. C. Upon cooling to room temperature
crystallization of the solvate occurred. The Raman spectrum above
was obtained from the crystals. The characteristic Raman shifts for
the 1-propanol solvate occur at about 1671 and 1561 cm.sup.-1.
[0409] FIG. 18 shows Raman spectroscopy data for Bz-423 2-propanol
solvate. BZ-423 (53.2 mg) was dissolved completely in 0.5 mL of
2-propanol at 70.degree. C. Upon cooling to room temperature
crystallization of the solvate occurred. The Raman spectrum above
was obtained from the crystals. The characteristic Raman shifts for
the 2-propanol solvate occur at about 1667 and 1562 cm.sup.-1.
[0410] FIG. 19 shows Raman spectroscopy data for Bz-423 1-butanol
solvate. BZ-423 (105.8 mg) was dissolved completely in 0.5 mL of
1-butanol at 70.degree. C. Upon cooling to room temperature
crystallization of the solvate occurred. The Raman spectrum above
was obtained from the crystals. The characteristic Raman shifts for
the 1-butanol solvate occur at about 1661 and 1556 cm.sup.-1.
[0411] FIG. 20 shows Raman spectroscopy data for Bz-423 2-butanol
solvate. BZ-423 (97.3 mg) was dissolved completely in 0.5 mL of
2-butanol at 70.degree. C. Upon cooling to room temperature
crystallization of the solvate occurred. The Raman spectrum above
was obtained from the crystals. The characteristic Raman shifts for
the 2-butanol solvate occur at about 1666 and 1562 cm.sup.-1.
[0412] FIG. 21 shows Raman spectroscopy data for Bz-423 1-pentanol
solvate. BZ-423 (99.3 mg) was dissolved completely in 0.5 mL of
1-pentanol at 70.degree. C. Upon cooling to room temperature
crystallization of the solvate occurred. The Raman spectrum above
was obtained from the crystals. The characteristic Raman shifts for
the 1-pentanol solvate occur at about 1646 and 1563 cm.sup.-1.
[0413] FIG. 22 shows Raman spectroscopy data for Bz-423 1-octanol
solvate. BZ-423 (5.2 mg) was dissolved completely in 0.5 mL of
1-octanol at 70.degree. C. Upon cooling to room temperature
crystallization of the solvate occurred. The Raman spectrum above
was obtained from the crystals. The characteristic Raman shifts for
the 1-octanol solvate occur at about 1669 and 1559 cm.sup.-1.
[0414] FIG. 23 shows Raman spectroscopy data for Bz-423 propylene
glycol solvate. BZ-423 (16.8 mg) was dissolved completely in 0.5 mL
of propylene glycol at 70.degree. C. Upon cooling to room
temperature crystallization of the solvate occurred. The Raman
spectrum above was obtained from the crystals. The characteristic
Raman shifts for the propylene glycol solvate occur at about 1660
and 1556 cm.sup.-1.
[0415] FIG. 24 shows Raman spectroscopy data for Bz-423 acetone
glass. BZ-423 (14.3 mg) was dissolved completely in 0.5 mL of
acetone at room temperature. Upon evaporation a glass was formed.
The Raman spectrum above was obtained from the glass. The
characteristic Raman shifts for the acetone glass occur at about
1674 and 1559 cm.sup.-1.
[0416] 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.
* * * * *