U.S. patent application number 13/323630 was filed with the patent office on 2012-09-20 for treatment of cancer.
This patent application is currently assigned to BiPar Sciences, Inc.. Invention is credited to John BURNIER, Bruce KEYT, Jerome MOORE, Valeria S. OSSOVSKAYA, Barry SHERMAN, Max TOTROV.
Application Number | 20120238601 13/323630 |
Document ID | / |
Family ID | 39158003 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120238601 |
Kind Code |
A1 |
MOORE; Jerome ; et
al. |
September 20, 2012 |
TREATMENT OF CANCER
Abstract
The present invention provides compositions of matter, kits and
methods for their use in the treatment of cancer. In particular,
the invention provides compositions and methods for treating cancer
in a subject by inhibiting a poly-ADP-ribose polymerase, as well as
providing formulations and modes of administering such
compositions.
Inventors: |
MOORE; Jerome; (Issaquah,
WA) ; KEYT; Bruce; (Hillsborough, CA) ;
BURNIER; John; (Pacifica, CA) ; SHERMAN; Barry;
(Hillsborough, CA) ; TOTROV; Max; (San Diego,
CA) ; OSSOVSKAYA; Valeria S.; (San Francisco,
CA) |
Assignee: |
BiPar Sciences, Inc.
Bridgewater
NJ
|
Family ID: |
39158003 |
Appl. No.: |
13/323630 |
Filed: |
December 12, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11850626 |
Sep 5, 2007 |
8143447 |
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13323630 |
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60842474 |
Sep 5, 2006 |
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Current U.S.
Class: |
514/309 ;
514/364; 514/365; 514/374; 514/415; 514/438; 514/562; 514/618 |
Current CPC
Class: |
C07C 239/18 20130101;
C07C 237/30 20130101; C07C 323/62 20130101; C07C 233/65 20130101;
A61P 35/02 20180101; A61P 43/00 20180101; A61P 35/00 20180101; C07C
235/46 20130101 |
Class at
Publication: |
514/309 ;
514/562; 514/618; 514/365; 514/364; 514/374; 514/438; 514/415 |
International
Class: |
A61K 31/166 20060101
A61K031/166; A61K 31/472 20060101 A61K031/472; A61K 31/426 20060101
A61K031/426; A61P 35/00 20060101 A61P035/00; A61K 31/421 20060101
A61K031/421; A61K 31/381 20060101 A61K031/381; A61K 31/404 20060101
A61K031/404; A61K 31/197 20060101 A61K031/197; A61K 31/4245
20060101 A61K031/4245 |
Claims
1-3. (canceled)
4. A method of treating a cancer comprising administering to a
subject in need thereof an effective amount of a composition
comprising an isolated compound of formula (IIa): ##STR00013##
wherein at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 substituent is always sulfur, and where R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are independently selected from the
group consisting of hydrogen, hydroxy, amino, nitro, iodo, bromo,
fluoro, chloro, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkoxy,
(C.sub.3-C.sub.7) cycloalkyl, and phenyl, wherein at least two of
the five R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5
substituents are always hydrogen, and wherein the sulfur-containing
substituent is not a thiol, or a pharmaceutically acceptable salt
thereof.
5. A method of treating a cancer comprising administering to a
subject in need thereof an effective amount of a composition
comprising one or more isolated compound(s) selected from a group
consisting of: ##STR00014## ##STR00015## or a salt thereof, and a
compound of the formula MS213 ##STR00016## wherein R.sub.6 is
selected from the group consisting of alkyl (C.sub.1-C.sub.8),
alkoxy (C.sub.1-C.sub.8), isoquinolinones, indoles, thiazole,
oxazole, oxadiazole, thiophene, and phenyl or a salt thereof.
6. The method of claim 4, further comprising administering an
effective amount of buthionine sulfoximine.
7. (canceled)
8. The method of claim 4, wherein a tumor cell undergoes apoptosis,
cell cycle arrest, and/or necrosis in a patient.
9. (canceled)
10. (canceled)
11. The method of claim 4, wherein the compound is an isolated
compound of the structural formula (IIa): ##STR00017## wherein at
least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5
substituents is always a sulfur-containing substituent and where
R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently
selected from the group consisting of hydrogen, hydroxy, amino,
nitro, iodo, bromo, fluoro, (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkoxy, (C.sub.3-C.sub.7) cycloalkyl, and phenyl,
wherein at least two of the five R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 substituents are always hydrogen, and wherein
the sulfur-containing substituent is selected from the group
consisting of: ##STR00018## or a pharmaceutically acceptable salt
thereof.
12. The method of claim 4, wherein the compound is an isolated
compound selected from the group consisting of: ##STR00019##
##STR00020## or a salt thereof, and a compound of the formula MS213
##STR00021## wherein R.sub.6 is selected from the group consisting
of alkoxy (C.sub.1-C.sub.8), isoquinolinones, thiazole, oxazole,
oxadiazole, and thiophene, or a salt thereof.
13. The method of claim 12, wherein said compound is in the form of
a pharmaceutically acceptable salt thereof.
14. The method of claim 4, wherein the compound is an isolated
compound of the formula MS213: ##STR00022## wherein R.sub.6 is
selected from the group consisting of alkoxy (C.sub.1-C.sub.8),
isoquinolinones, thiazole, oxazole, oxadiazole, and thiophene, or a
salt thereof.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/842,474, filed Sep. 5, 2006, which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Cancer is a serious threat to modern society. Malignant
cancerous growths, due to their unique characteristics, pose
significant challenges for modern medicine. Their characteristics
include uncontrollable cell proliferation resulting in unregulated
growth of malignant tissue, an ability to invade local and even
remote tissues, lack of differentiation, lack of detectable
symptoms and most significantly, the lack of effective therapy and
prevention.
[0003] Cancer can develop in any tissue of any organ or tissue at
any age. The etiology of cancer is not clearly defined but
mechanisms such as genetic susceptibility, chromosome breakage
disorders, viruses, environmental factors and immunologic disorders
have all been linked to malignant cell growth and transformation.
Cancer encompasses a large category of medical conditions,
affecting millions of individuals worldwide. Cancer develops when
cells in a part of the body begin to undifferentiate or grow out of
control. All cancer types begin with the out-of-control growth of
abnormal cells.
[0004] There are many types of cancer, including breast, lung,
ovarian, bladder, prostate, pancreatic, cervical and leukemia.
Currently, some of the main treatments available are surgery,
radiation therapy, and chemotherapy. Surgery is often a drastic
measure and can have serious consequences. For example, all
treatments for ovarian cancer may result in infertility. Some
treatments for cervical cancer and bladder cancer may cause
infertility and/or sexual dysfunction. Surgical procedures to treat
pancreatic cancer may result in partial or total removal of the
pancreas and can carry significant risks to the patient. Breast
cancer surgery invariably involves removal of part or the entire
breast, and in severe cases, surrounding tissue. Some surgical
procedures for prostate cancer carry the risk of urinary
incontinence and impotence. Surgical procedures for lung cancer
patients often give rise to significant post-operative pain, as the
ribs must be cut through to access and remove the cancerous lung
tissue. In addition, patients who have both lung cancer and another
lung disease, such as emphysema or chronic bronchitis, typically
experience an increase in their shortness of breath following the
surgery.
[0005] Radiation therapy has the advantage of killing cancer cells
but it also damages non-cancerous tissue at the same time.
Chemotherapy involves the administration of various anti-cancer
drugs to a patient but often is accompanied by adverse side
effects.
[0006] Worldwide, more than 10 million people are diagnosed with
cancer every year and it is estimated that this number will grow to
15 million new cases every year by 2020. Cancer causes six million
deaths every year or 12% of the deaths worldwide. There remains a
need for methods that can treat cancer. These methods can provide
the basis for pharmaceutical compositions useful in the prevention
and treatment of cancer in humans and other mammals.
[0007] A series of anti-tumor drugs have been identified. These
drugs include nitro and nitroso compounds and their metabolites,
which are the subject of U.S. Pat. No. 5,464,871 issued on Nov. 7,
1995 entitled "Aromatic Nitro and Nitroso Compounds and their
Metabolites Useful as Anti-viral and Anti-tumor Agents," U.S. Pat.
No. 5,670,518 issued on Sep. 23, 1997 entitled "Aromatic Nitro and
Nitroso Compounds and their Metabolites Useful as Anti-viral and
Anti-tumor Agents," U.S. Pat. No. 6,004,978 issued on Dec. 21, 1999
entitled "Methods of Treating Cancer with Aromatic Nitro and
Nitroso Compounds and their Metabolites" the disclosures of which
are incorporated herein by reference. The use of these compounds
has been described in the art as useful in treating mammary gland
adenocarcinomas, mammary gland duct carcinomas, lymphocytic
leukemia, Kaposi's sarcoma in immunosuppressed patients with AIDS,
and neoplastic growths such as non-Hodgkin's lymphoma, and primary
lymphomas. However, there is a need to identify additional
compounds with anti-tumor activity. Metabolites isolated from
precursor compounds provide a source of anti-tumor agents that can
be used individually or in combination with other compounds to
target various cancerous cells. Metabolites isolated from benzamide
precursor compounds provide one such source.
SUMMARY OF THE INVENTION
[0008] The present invention relates generally to methods of
treatment of tumorigenic diseases using aromatic nitrobenzamide
metabolite compounds. More specifically, it relates to the
nitrobenzamide metabolite compounds derived from the nitro compound
benzamide precursor molecules and the use of said metabolites or a
salt, solvate, isomer, tautomer, metabolite, analog, or prodrug
thereof, in suppressing and inhibiting tumor growth in a
mammal.
[0009] In one aspect of the invention, a method for treatment of
cancer and disorders associated with cancer is provided comprising
the administration of pharmaceutical compositions comprising a
compound of formula (Ia) with one or more additional
pharmacologically active agents. In another aspect, a method for
treatment of cancer and disorders associated with cancer is
provided comprising the administration of a combination of a
metabolite compound derived from a compound of formula (Ia) and
buthionine sulfoximine (BSO). Said metabolite(s) can also be
administered in combination with a benzopyrone compound of formula
(II), with or without BSO.
[0010] Embodiments of these aspects include methods of treating
various cancers, including leukemia, breast cancer, ovarian cancer,
lung cancer, bladder cancer, prostate cancer, pancreatic cancer,
and cervical cancer, as well as other cancer types described
herein.
[0011] This invention relates to compositions of matter and
pharmaceutical compositions, and to methods for their use in the
treatment of cancer. For example, a composition of the invention
can be a combination of two or more compounds described herein
and/or a combination of two or more forms of a compound described
herein. A pharmaceutical composition of the invention may be a
composition suitable for administration to a subject.
INCORPORATION BY REFERENCE
[0012] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0014] FIG. 1 illustrates (FACS dot plots and histograms)
cell-cycle analysis in HTC116 cells treated with PARP-1 inhibitor
(4-iodo-3-nitrobenzamide or "BA") for 19 hours. BA treatment caused
an increase in the number of cells in the G1 phase (48.6%) as
compared to control (18.6%) with a concomitant decrease of S phase
cell cycle.
[0015] FIG. 2 (FACS dot plots and histograms) illustrates
cell-cycle analysis of Hela cells treated with PARP-1 inhibitors
BA, 4-iodo-3-nitrosobenzamide (BNO) and
4-iodo-3-hydroxyaminobenzamide (BNHOH) for 24 hours. BrdU staining
label cells in S-phase of cell cycle. GF7 staining label cells in
mitosis.
[0016] FIG. 3 (FACS dot plots and histograms) illustrates
cell-cycle analysis of Hela cells treated with PARP-1 inhibitors
BA, 4-iodo-3-nitrosobenzamide (BNO) and
4-iodo-3-hydroxyaminobenzamide (BNHOH) for 72 hours. BrdU staining
label cells in S-phase of cell cycle. GF7 staining label cells in
mitosis.
[0017] FIG. 4 shows BA activity in the human OVCAR-3 ovarian
adenoma xenograft in female SCID mice. BA was dosed orally b.i.d.
at 50 mg/kg/dose or s.c. via osmotic pumps at a dose of 25
mg/kg/week.
[0018] FIG. 5 shows that BA targets multi-drug resistant cells that
up-regulate MDR1 as well as MDR1-negative cells. FIG. 5A shows the
level of MDR1 expression in KB 3-1 cells. FIG. 5B shows the level
of MDR1 expression in KB V-1 cells. FACS histogram illustrates
overexpression of MDR1 in KB V-1 cells. As overexpression of MDR1
is associated with multidrug resistance, it is notable that, as
shown in FIG. 5C, BA results in dose-dependent cell death in both
KB 3-1 and KB V-1 cells.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0019] "Nitrobenzamide precursor compound(s)" means a compound of
the formula (Ia)
##STR00001##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are,
independently selected from the group consisting of hydrogen,
hydroxy, amino, nitro, iodo, (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkoxy, (C.sub.3-C.sub.7) cycloalkyl, and phenyl,
wherein at least two of the five R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 substituents are always hydrogen, at least one
of the five substituents are always nitro, and at least one
substituent positioned adjacent to a nitro is always iodo, and
pharmaceutically acceptable salts, solvates, isomers, tautomers,
metabolites, analogs, or prodrugs thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 can also be a halide such as chloro,
fluoro, or bromo. "Precursor compound" is a compound that undergoes
one or more chemical or biochemical processes (e.g., in a cell or
in an organism) that result in a metabolite compound. The terms
"precursor", "precursor compound", "benzamide precursor" or
"nitrobenzamide precursor" are used interchangeably herein.
[0020] "Metabolite" means a compound produced through any in vitro
or in vivo metabolic process which results in a product that is
different in structure than that of the starting compound. The term
"metabolite" includes nitrobenzamide metabolite compounds. A
metabolite can include a varying number or types of substituents
that are present at any position relative to a precursor compound,
such as the precursor compound depicted in the formula (Ia). In
addition, a metabolite can vary in the number of types of
substituents that are present at any position relative to the
compounds depicted in herein. In addition, the terms "metabolite",
"metabolite compound", "benzamide metabolite compound" or
"nitrobenzamide metabolite compound" are used interchangeably
herein.
[0021] "Surgery" means any therapeutic or diagnostic procedure that
involves methodical action of the hand or of the hand with an
instrument, on the body of a human or other mammal, to produce a
curative, remedial, or diagnostic effect.
[0022] "Radiation therapy" means exposing a patient to high-energy
radiation, including without limitation x-rays, gamma rays, and
neutrons. This type of therapy includes without limitation
external-beam therapy, internal radiation therapy, implant
radiation, brachytherapy, systemic radiation therapy, and
radiotherapy.
[0023] "Chemotherapy" means the administration of one or more
anti-cancer drugs and/or other agents to a cancer patient by
various methods, including intravenous, oral, intramuscular,
intraperitoneal, intravesical, subcutaneous, transdermal, buccal,
or inhalation or in the form of a suppository. Chemotherapy may be
given prior to surgery to shrink a large tumor prior to a surgical
procedure to remove it, after surgery or radiation therapy to
prevent the growth of any remaining cancer cells in the body.
[0024] The terms "effective amount" or "pharmaceutically effective
amount" refer to a non-toxic but sufficient amount of the agent to
provide the desired biological, therapeutic, and/or prophylactic
result. That result can be reduction and/or alleviation of the
signs, symptoms, or causes of a disease, or any other desired
alteration of a biological system. For example, an "effective
amount" for therapeutic uses is the amount of a nitrobenzamide
metabolite compound as disclosed herein per se or a composition
comprising the nitrobenzamide metabolite compound herein required
to result in a clinically significant decrease in a disease. An
appropriate effective amount in any individual case may be
determined by one of ordinary skill in the art using routine
experimentation.
[0025] By "pharmaceutically acceptable" or "pharmacologically
acceptable" is meant a material which is not biologically or
otherwise undesirable, i.e., the material may be administered to an
individual without causing any undesirable biological effects or
interacting in a deleterious manner with any of the components of
the composition in which it is contained.
[0026] The term "treating" and its grammatical equivalents as used
herein include achieving a therapeutic benefit and/or a
prophylactic benefit. By therapeutic benefit is meant eradication
or amelioration of the underlying disorder being treated. For
example, in a cancer patient, therapeutic benefit includes
eradication or amelioration of the underlying cancer. Also, a
therapeutic benefit is achieved with the eradication or
amelioration of one or more of the physiological symptoms
associated with the underlying disorder such that an improvement is
observed in the patient, notwithstanding the fact that the patient
may still be afflicted with the underlying disorder. For
prophylactic benefit, a method of the invention may be performed
on, or a composition of the invention administered to a patient at
risk of developing cancer, or to a patient reporting one or more of
the physiological symptoms of such conditions, even though a
diagnosis of the condition may not have been made.
[0027] As used herein "BA" means 4-iodo-3-nitrobenzamide; "BNO"
means 4-iodo-3-nitrosobenzamide; "BNHOH" means
4-iodo-3-hydroxyaminobenzamide.
[0028] (i) Nitrobenzamide Metabolite Compounds
[0029] Precursor compounds useful in the present invention are of
Formula (Ia)
##STR00002##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are,
independently selected from the group consisting of hydrogen,
hydroxy, amino, nitro, iodo, (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkoxy, (C.sub.3-C.sub.7) cycloalkyl, and phenyl,
wherein at least two of the five R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 substituents are always hydrogen, at least one
of the five substituents are always nitro, and at least one
substituent positioned adjacent to a nitro is always iodo, and
pharmaceutically acceptable salts, solvates, isomers, tautomers,
metabolites, analogs, or pro-drugs thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 can also be a halide such as chloro,
fluoro, or bromo substituents.
[0030] A preferred precursor compound of formula Ia is:
##STR00003##
[0031] Metabolites useful in the present invention are of the
Formula (IIa):
##STR00004##
wherein either: (1) at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 substituent is always a sulfur-containing
substituent, and the remaining substituents R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are independently selected from the
group consisting of hydrogen, hydroxy, amino, nitro, iodo, bromo,
fluoro, chloro, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkoxy,
(C.sub.3-C.sub.7) cycloalkyl, and phenyl, wherein at least two of
the five R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5
substituents are always hydrogen; or (2) at least one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 substituents is not a
sulfur-containing substituent and at least one of the five
substituents R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
always iodo, and wherein said iodo is always adjacent to a R.sub.1,
R.sub.2, R.sub.3, R.sub.4, or R.sub.5 group that is either a nitro,
a nitroso, a hydroxyamino, hydroxy or an amino group; and
pharmaceutically acceptable salts, solvates, isomers, tautomers,
metabolites, analogs, or pro-drugs thereof. In some embodiments,
the compounds of (2) are such that the iodo group is always
adjacent a R.sub.1, R.sub.2, R.sub.3, R.sub.4 or R.sub.5 group that
is a nitroso, hydroxyamino, hydroxy or amino group. In some
embodiments, the compounds of (2) are such that the iodo group is
always adjacent a R.sub.1, R.sub.2, R.sub.3, R.sub.4 or R.sub.5
group that is a nitroso, hydroxyamino, or amino group.
[0032] The following compositions are preferred metabolite
compounds, each represented by a chemical formula:
##STR00005## ##STR00006## ##STR00007## ##STR00008##
[0033] While not being limited to any one particular mechanism, the
following provides an example for MS292 metabolism via a
nitroreductase or glutathione conjugation mechanism:
[0034] Nitroreductase Mechanism
##STR00009##
[0035] BA Glutathione Conjugation and Metabolism:
##STR00010##
[0036] The present invention provides for the use of the aforesaid
nitrobenzamide metabolite compounds for the treatment of other
breast cancers including a ductal carcinoma in a mammary gland,
other forms of leukemia including acute pro-myelocytic leukemia in
peripheral blood, ovarian cancer, lung cancer, bladder cancer,
prostate cancer, pancreatic cancer, and cervical cancer, as well as
other cancer types described herein.
[0037] It has been reported that nitrobenzamide metabolite
compounds have selective cytotoxicity upon malignant cancer cells
but not upon non-malignant cancer cells. See Rice et al., Proc.
Natl. Acad. Sci. USA 89:7703-7707 (1992). In one embodiment, the
nitrobenzamide metabolite compounds utilized in the methods of the
present invention may exhibit more selective toxicity towards tumor
cells than non-tumor cells.
[0038] It has been reported that the tumorigenicity of
nitrobenzamide and nitrososbenzamide compounds is enhanced when BSO
is co-administered to cancer cells. See Mendeleyev et al.,
Biochemical Pharmacol. 50(5):705-714 (1995). Buthionine sulfoximine
(BSO) inhibits gamma-glutamylcysteine synthetase, a key enzyme in
the biosynthesis of glutathione, which is responsible in part for
cellular resistance to chemotherapy. See Chen et al., Chem. Biol.
Interact. April 24; 111-112:263-75 (1998). The invention also
provides a method for treating cancer comprising the administration
of a nitrobenzamide metabolite compound and/or benzopyrone compound
in combination with BSO. Alternatively, metabolite compounds can be
administered with precursor compounds and/or benzopyrone.
[0039] In addition to BSO, other inhibitors of
gamma-glutamylcysteine synthetase can be used in combination with
nitrobenzamide and/or benzopyrone compounds. Other suitable analogs
of BSO include, but are not limited to, proprothionine sulfoximine,
methionine sulfoximine, ethionine sulfoximine, methyl buthionine
sulfoximine, .gamma.-glutamyl-.alpha.-aminobutyrate and
.gamma.-glutamylcysteine.
Benzopyrone Compounds
[0040] In some embodiments, the metabolite(s) compounds are
administered in combination with benzamide compounds and/or
benzopyrone compounds of formula II. The benzopyrone compounds of
formula II are,
##STR00011##
wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently
selected from the group consisting of H, halogen, optionally
substituted hydroxy, optionally substituted amine, optionally
substituted lower alkyl, optionally substituted phenyl, optionally
substituted C.sub.4-C.sub.10 heteroaryl and optionally substituted
C.sub.3-C.sub.8 cycloalkyl or a salt, solvate, isomer, tautomers,
metabolite, or pro-drug thereof.
[0041] In a preferred embodiment, the invention relates to the
following benzopyrone compound of formula II
##STR00012##
[0042] (ii) Mechanism of Nitrobenzamide Metabolite Compounds
[0043] Not intending to be limited by one mechanism of action, the
compounds described herein are believed to have anti-cancer
properties via the modulation of a poly (ADP-ribose) polymerase
enzyme. The drugs' mechanism of action is related to their ability
to act as a ligand for the nuclear enzyme poly (ADP-ribose)
polymerase (PARP-1). See Mendeleyev et al., supra, (1995). PARP-1
is expressed in the nucleus and catalyzes the conversion of
(3-nicotinamide adenine dinucleotide (NAD.sup.+) into nicotinamide
and poly-ADP-ribose (PAR). The role of PARP-1 in homeostatic
conditions seems to be limited to DNA transcription and repair.
However, when cellular stress causes DNA damage, PARP-1 activity
increases dramatically, which appears to be necessary for genomic
integrity. Shall et al., Mutat Res. June 30; 460(1):1-15 (2000). In
addition, while PARP-1 is the best known member of the PARP family
of enzymes, the mechanism of action is equally applicable to any
member of the PARP family (e.g., PARP-2 through PARP-18, or
additional members discovered to operate by similar mechanisms of
action, or sharing similar structures to PARP-1).
[0044] One function of PARP-1 is the synthesis of the biopolymer,
poly (ADP-ribose). Both poly (ADP-ribose) and PARP-1 have been
linked to the repair of DNA repair, apoptosis, the maintenance of
genomic stability, and carcinogenesis. See Masutani et al., Genes,
Chromosomes, and Cancer 38:339-348 (2003). PARP-1 plays a role in
DNA repair, specifically base excision repair (BER). BER is a
protection mechanism in mammalian cells for single-base DNA
breakage. PARP-1 binds to the ends of DNA fragments through its
zinc finger domains with great affinity and thereby acts as a DNA
damage sensor. Gradwohl et al., Proc. Natl. Acad. Sci. USA
87:2990-2994 (1990); Murcia et al., Trends Biochem. Sci 19: 172-176
(1994). A breakage in the DNA triggers a binding response by PARP-1
to the site of the break. PARP-1 then increases its catalytic
activity several hundred fold (See Simonin et al., J. Biol. Chem.
278: 13454-13461 (1993)) and begins to convert poly
ADP-ribosylation of itself (Desmarais et al., Biochim. Biophys.
Acta 1078: 179-186 (1991)) and BER proteins, such as DNA-dependent
protein kinase (DNA-PKcs) and the molecular scaffold protein
XRCC-1. See Ruscetti et al., J. Biol. Chem. June 5;
273(23):14461-14467 (1998) and Masson et al., Mol Cell Biol. June,
18(6):3563-71 (1998). BER proteins are rapidly recruited to the
site of DNA damage. El-Kaminsy et al., Nucleic Acid Res.
31(19):5526-5533 (2003); Okano et al., Mol Cell Biol.
23(11):3974-3981 (2003). PARP-1 dissociates from the DNA breakage
site but remains in the vicinity of the DNA repair event.
[0045] Inhibiting the activity of a PARP molecule includes reducing
the activity of these molecules. The term "inhibits" and its'
grammatical conjugations, such as "inhibitory," is not intended to
require complete reduction in PARP activity. Such reduction is
preferably by at least about 50%, at least about 75%, at least
about 90%, and more preferably by at least about 95% of the
activity of the molecule in the absence of the inhibitory effect,
e.g., in the absence of an inhibitor, such as a nitrobenzamide
metabolite compound of the invention. Most preferably, the term
refers to an observable or measurable reduction in activity. In
treatment scenarios, preferably the inhibition is sufficient to
produce a therapeutic and/or prophylactic benefit in the condition
being treated. The phrase "does not inhibit" and its' grammatical
conjugations does not require a complete lack of effect on the
activity. For example, it refers to situations where there is less
than about 20%, less than about 10%, and preferably less than about
5% of reduction in PARP activity in the presence of an inhibitor
such as a nitrobenzamide metabolite compound of the invention.
[0046] Another mechanism of action can include inhibition of
inosine monophosphate dehydrogenase (IMPDH). IMPDH is the
rate-limiting enzyme of the branched purine nucleotide synthetic
pathway that provides guanylates including GTP and dGTP. There are
two isoforms of IMPDH, type I that is constitutively present in all
cells, and type II that is inducible and is present in highly
proliferating cells such as cancer. Inhibition of the latter enzyme
brings about a profound depletion of intracellular guanosine
nucleotides essential for tumor cell growth and replication. One or
more metabolite compounds of the present invention or can target
IMPDH thus inhibiting tumor cell growth.
[0047] Yet another mechanism of action can include inhibition of
histone deacytelase (HDAC). Tumor-associated alterations in
transcription factor pools may lead to misregulation of genes
important in normal growth and development. Chimeric transcription
factors cause transcriptional repression of growth regulatory
target genes by the aberrant recruitment of transcriptional
corepressors and their associated HDAC activity. In human acute
promyelocytic leukemia, chimeric transcription factors involving
retinoic acid receptor (PML-RAR.alpha. and PLZF-RAR.alpha.) have
been found to repress transcription of target genes such as the
RAR.beta. gene. Transcriptional corepressor complexes contain HDAC
activity and transcriptional coactivator complexes contain histone
acetyltransferase activity. HDAC inhibitors such as trichostatinA
or NaBu are able to relieve the transcriptional repression caused
by the chimeric transcription factors PML-RAR.alpha. and
PLZFRAR.alpha.. HDAC inhibitors alone or in combination with
retinoids have been shown to induce leukemia remission and
prolonged survival in an animal model of acute promyelocytic
leukemia without apparent side effects.
[0048] Acetylation and deacetylation of histones alter higher order
chromatin structure by influencing histone interaction with DNA.
Transcription factors may also be acetylated, and the acetylated
status of these proteins may influence their interaction with DNA,
as well as their ability to interact with other transcriptional
coregulatory proteins. For example, acetylation of p53 enhances its
sequence specific DNA binding activity. Deacetylated histones are
associated with cell growth, whereas hyperacetylated histones are
associated with cell growth arrest, differentiation, and/or
apoptosis.
[0049] For example, Saito et al. evaluated the efficacy of
MS-27-275, a synthetic benzene derivative that inhibits HDAC. Saito
et al. Proc. Natl. Acad. Sci., 1999; 96: 4592-4597. In a number of
adult tumor cell lines, MS-27-275 inhibited tumor cell growth with
an IC50 in the submicromolar range. The inhibition of cell growth
was accompanied by a cell cycle arrest and an induction of the cell
cycle inhibitor p21. MS-27-275 administered p.o. inhibited the
growth of established adult tumor lines s.c. implanted in nude mice
with minimal toxicities. See also, Jaboin et al. Cancer Research,
2002; 62:6108-6115.
[0050] As such the compounds of the present invention, as HDAC
inhibitors, can modulate transcriptional activity. Therefore, the
compounds of the present invention, can also act to block
angiogenesis and cell cycling, and promote apoptosis and
differentiation. By targeting these key components of tumor
proliferation, HDAC inhibitors have the potential to occupy an
indomitable position in the fast-moving cytostatic market. Two
major reasons why HDAC inhibitors could play such a key role
because they can be used concurrent to other treatment regimes,
such as to improve the efficacy of existing cytostatics (such as
the retinoids) and moreover, they are able to target the
transcription of specific disease-causing genes, conferring
unprecedented therapeutic windows to cancer therapy.
Uses of the Benzamide Metabolite Compounds
Cancer Types
[0051] The invention provides methods to treat several specific
cancers or tumors. For example, cancer types include adrenal
cortical cancer, anal cancer, aplastic anemia, bile duct cancer,
bladder cancer, bone cancer, bone metastasis, Adult CNS brain
tumors, Children CNS brain tumors, breast cancer, Castleman's
Disease, cervical cancer, childhood Non-Hodgkin's lymphoma, colon
and rectum cancer, endometrial cancer, esophagus cancer, Ewing's
family of tumors, eye cancer, gallbladder cancer, gastrointestinal
carcinoid tumors, gastrointestinal stromal tumors, gestational
trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney
cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic
leukemia, acute myeloid leukemia, children's leukemia, chronic
lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung
cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast
cancer, malignant mesothelioma, multiple myeloma, myelodysplastic
syndrome, nasal cavity and paranasal cancer, nasopharyngeal cancer,
neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma,
ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor,
prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland
cancer, sarcoma (adult soft tissue cancer), melanoma skin cancer,
non-melanoma skin cancer, stomach cancer, testicular cancer, thymus
cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar
cancer, and Waldenstrom's macroglobulinemia.
[0052] Carcinoma of the thyroid gland is the most common malignancy
of the endocrine system. Carcinoma of the thyroid gland includes
differentiated tumors (papillary or follicular) and poorly
differentiated tumors (medullary or anaplastic). Carcinomas of the
vagina include squamous cell carcinoma, adenocarcinoma, melanoma
and sarcoma. Testicular cancer is broadly divided into seminoma and
non-seminoma types.
[0053] Thymomas are epithelial tumors of the thymus, which may or
may not be extensively infiltrated by non-neoplastic lymphocytes.
The term thymoma is customarily used to describe neoplasms that
show no overt atypia of the epithelial component. A thymic
epithelial tumor that exhibits clear-cut cytologic atypia and
histologic features no longer specific to the thymus is known as a
thymic carcinoma (also known as type C thymoma).
[0054] The methods provided by the invention may comprise the
administration of the benzamide metabolite compounds in combination
with other therapies. The choice of therapy that can be
co-administered with the compositions of the invention will depend,
in part, on the condition being treated. For example, for treating
acute myeloid leukemia, a benzamide compound of some embodiments of
the invention can be used in combination with radiation therapy,
monoclonal antibody therapy, chemotherapy, bone marrow
transplantation, gene therapy, immunotherapy, or a combination
thereof.
[0055] Breast Cancer
[0056] In one aspect, the invention provides a method of treating
breast cancer, preferably a ductal carcinoma in duct tissue in a
mammary gland.
[0057] Several types of breast cancer exist that may be treated by
the methods provided by the invention. A lobular carcinoma in situ
and a ductal carcinoma in situ are breast cancers that have
developed in the lobules and ducts, respectively, but have not
spread to the fatty tissue surrounding the breast or to other areas
of the body. An infiltrating (or invasive) lobular and a ductal
carcinoma are cancers that have developed in the lobules and ducts,
respectively, and have spread to either the breast's fatty tissue
and/or other parts of the body. Other cancers of the breast that
would benefit from treatment by the methods provided by the
invention are medullary carcinomas, colloid carcinomas, tubular
carcinomas, and inflammatory breast cancer.
[0058] Treatments available for breast cancer patients are surgery,
immunotherapy, radiation therapy, chemotherapy, endocrine therapy,
or a combination thereof. A lumpectomy and a mastectomy are two
possible surgical procedures available for breast cancer
patients.
[0059] Chemotherapy utilizes anti-tumor agents to prevent cancer
cells from multiplying, invading, metastasizing and killing a
patient. Several drugs are available to treat breast cancer,
including cytotoxic drugs such as doxorubicin, cyclophosphamide,
methotrexate, paclitaxel, thiotepa, mitoxantrone, vincristine, or
combinations thereof. Endocrine therapy may be an effective
treatment where the remaining breast tissue retains endocrine
sensitivity. Agents administered for this therapy include
tamoxifen, megestrol acetate, aminoglutethimide, fluoxymesterone,
leuprolide, gosserelin, and prednisone.
[0060] The methods provided by the invention can provide a
beneficial effect for breast cancer patients, by administration of
a nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, chemotherapy, or endocrine therapy.
[0061] In some embodiments, the invention provides for treatment of
so-called "triple negative" breast cancer. There are several
subclasses of breast cancer identified by classic biomarkers such
as estrogen receptor (ER) and/or progesterone receptor (PR)
positive tumors, HER2-amplified tumors, and ER/PR/HER2-negative
tumors. These three subtypes have been reproducibly identified by
gene expression profiling in multiple breast cancer and exhibit
basal-like subtype expression profiles and poor prognosis. Triple
negative breast cancer is characterized by ER/PR/HER2-negative
tumors.
Ovarian Cancer
[0062] In another aspect, the invention provides a method of
treating ovarian cancer, including epithelial ovarian tumors.
Preferably, the invention provides a method of treating an ovarian
cancer selected from the following: an adenocarcinoma in the ovary
and an adenocarcinoma that has migrated from the ovary into the
abdominal cavity. Surgery, immunotherapy, chemotherapy, hormone
therapy, radiation therapy, or a combination thereof, are some
possible treatments available for ovarian cancer. Some possible
surgical procedures include debulking, and a unilateral or
bilateral oophorectomy and/or a unilateral or bilateral
salpigectomy.
[0063] Anti-cancer drugs that may be used include cyclophosphamide,
etoposide, altretamine, and ifosfamide. Hormone therapy with the
drug tamoxifen may be used to shrink ovarian tumors. Radiation
therapy may be external beam radiation therapy and/or
brachytherapy.
[0064] The methods provided by the invention can provide a
beneficial effect for ovarian cancer patients, by administration of
a nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, chemotherapy endocrine therapy, or a combination
thereof.
Cervical Cancer
[0065] In another aspect, the invention provides a method of
treating cervical cancer, preferably an adenocarcinoma in the
cervical epithelium. Two main types of this cancer exist: squamous
cell carcinoma and adenocarcinomas. The former constitutes about
80-90% of all cervical cancers and develops where the ectocervix
(portion closest to the vagina) and the endocervix (portion closest
to the uterus) join. The latter develop in the mucous-producing
gland cells of the endocervix. Some cervical cancers have
characteristics of both of these and are called adenosquamous
carcinomas or mixed carcinomas.
[0066] The chief treatments available for cervical cancer are
surgery, immunotherapy, radiation therapy and chemotherapy. Some
possible surgical options are cryosurgery, a hysterectomy, and a
radical hysterectomy. Radiation therapy for cervical cancer
patients includes external beam radiation therapy or brachytherapy.
Anti-cancer drugs that may be administered as part of chemotherapy
to treat cervical cancer include cisplatin, carboplatin,
hydroxyurea, irinotecan, bleomycin, vincrinstine, mitomycin,
ifosfamide, fluorouracil, etoposide, methotrexate, and combinations
thereof.
[0067] The methods provided by the invention can provide a
beneficial effect for cervical cancer patients, by administration
of a nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, chemotherapy, or a combination thereof.
Prostate Cancer
[0068] In one other aspect, the invention provides methods to treat
prostate cancer, preferably a prostate cancer selected from the
following: an adenocarcinoma or an adenocarcinoma that has migrated
to the bone. Prostate cancer develops in the prostate organ in men,
which surrounds the first part of the urethra. The prostate has
several cell types but 99% of tumors are adenocarcinomas that
develop in the glandular cells responsible for generating seminal
fluid.
[0069] Surgery, immunotherapy, radiation therapy, cryosurgery,
hormone therapy, and chemotherapy are some treatments available for
prostate cancer patients. Possible surgical procedures to treat
prostate cancer include radical retro-pubic prostatectomy, a
radical perineal prostatectomy, and a laparoscopic radical
prostatectomy. Some radiation therapy options are external beam
radiation, including three dimensional conformal radiation therapy,
intensity modulated radiation therapy, and conformal proton beam
radiation therapy. Brachytherapy (seed implantation or interstitial
radiation therapy) is also an available method of treatment for
prostate cancer. Cryosurgery is another possible method used to
treat localized prostate cancer cells.
[0070] Hormone therapy, also called androgen deprivation therapy or
androgen suppression therapy, may be used to treat prostate cancer.
Several methods of this therapy are available including an
orchiectomy in which the testicles, where 90% of androgens are
produced, are removed. Another method is the administration of
luteinizing hormone-releasing hormone (LHRH) analogs to lower
androgen levels. The LHRH analogs available include leuprolide,
goserelin, triptorelin, and histrelin. An LHRH antagonist may also
be administered, such as abarelix.
[0071] Treatment with an anti-androgen agent, which blocks androgen
activity in the body, is another available therapy. Such agents
include flutamide, bicalutamide, and nilutamide. This therapy is
typically combined with LHRH analog administration or an
orchiectomy, which is termed a combined androgen blockade
(CAB).
[0072] Chemotherapy may be appropriate where a prostate tumor has
spread outside the prostate gland and hormone treatment is not
effective. Anti-cancer drugs such as doxorubicin, estramustine,
etoposide, mitoxantrone, vinblastine, paclitaxel, docetaxel,
carboplatin, and prednisone may be administered to slow the growth
of prostate cancer, reduce symptoms and improve the quality of
life.
[0073] The methods provided by the invention can provide a
beneficial effect for prostate cancer patients, by administration
of a nitrobenzamide metabolite compound or a combination
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, chemotherapy, hormone therapy, or a combination
thereof.
Pancreatic Cancer
[0074] In another aspect, the invention provides methods of
treating pancreatic cancer, preferably a pancreatic cancer selected
from the following: an epitheloid carcinoma in the pancreatic duct
tissue and an adenocarcinoma in a pancreatic duct.
[0075] The most common type of pancreatic cancer is an
adenocarcinoma, which occurs in the lining of the pancreatic duct.
The possible treatments available for pancreatic cancer are
surgery, immunotherapy, radiation therapy, and chemotherapy.
Possible surgical treatment options include a distal or total
pancreatectomy and a pancreaticoduodenectomy (Whipple
procedure).
[0076] Radiation therapy may be an option for pancreatic cancer
patients, specifically external beam radiation where radiation is
focused on the tumor by a machine outside the body. Another option
is intra-operative electron beam radiation administered during an
operation.
[0077] Chemotherapy may be used to treat pancreatic cancer
patients. Appropriate anti-cancer drugs include 5-fluorouracil
(5-FU), mitomycin, ifosfamide, doxorubicin, streptozocin,
chlorozotocin, and combinations thereof.
[0078] The methods provided by the invention can provide a
beneficial effect for pancreatic cancer patients, by administration
of a nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, or chemotherapy.
Bladder Cancer
[0079] In another aspect, the invention provides methods of
treating bladder cancer, preferably a transitional cell carcinoma
in urinary bladder. Bladder cancers are urothelial carcinomas
(transitional cell carcinomas) or tumors in the urothelial cells
that line the bladder. The remaining cases of bladder cancer are
squamous cell carcinomas, adenocarcinomas, and small cell cancers.
Several subtypes of urothelial carcinomas exist depending on
whether they are non-invasive or invasive and whether they are
papillary, or flat. Non-invasive tumors are in the urothelium, the
innermost layer of the bladder, while invasive tumors have spread
from the urothelium to deeper layers of the bladder's main muscle
wall. Invasive papillary urothelial carcinomas are slender
finger-like projections that branch into the hollow center of the
bladder and also grow outward into the bladder wall. Non-invasive
papillary urothelial tumors grow towards the center of the bladder.
While a non-invasive, flat urothelial tumor (also called a flat
carcinoma in situ) is confined to the layer of cells closest to the
inside hollow part of the bladder, an invasive flat urothelial
carcinoma invades the deeper layer of the bladder, particularly the
muscle layer.
[0080] To treat bladder cancer, surgery, radiation therapy,
immunotherapy, chemotherapy, or a combination thereof may be
applied. Some possible surgical options are a transurethral
resection, a cystectomy, or a radical cystectomy. Radiation therapy
for bladder cancer may include external beam radiation and
brachytherapy.
[0081] Immunotherapy is another method that may be used to treat a
bladder cancer patient. Typically this is accomplished
intravesically, which is the administration of a treatment agent
directly into the bladder by way of a catheter. One method is
Bacillus Calmete-Guerin (BCG) where a bacterium sometimes used in
tuberculosis vaccination is given directly to the bladder through a
catheter. The body mounts an immune response to the bacterium,
thereby attacking and killing the cancer cells.
[0082] Another method of immunotherapy is the administration of
interferons, glycoproteins that modulate the immune response.
Interferon alpha is often used to treat bladder cancer.
[0083] Anti-cancer drugs that may be used in chemotherapy to treat
bladder cancer include thitepa, methotrexate, vinblastine,
doxorubicin, cyclophosphamide, paclitaxel, carboplatin, cisplatin,
ifosfamide, gemcitabine, or combinations thereof.
[0084] The methods provided by the invention can provide a
beneficial effect for bladder cancer patients, by administration of
a nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, immunotherapy, chemotherapy, or a combination
thereof.
Blood Cancer
[0085] Lymphoma
[0086] B-Cell Lymphomas
[0087] Non-Hodgkin's Lymphomas caused by malignant (cancerous)
B-Cell lymphocytes represent a large subset (about 85% in the US)
of the known types of lymphoma (the other 2 subsets being T-Cell
lymphomas and lymphomas where the cell type is the Natural Killer
Cell or unknown). Cells undergo many changes in their life cycle
dependent on complex signaling processes between cells and
interaction with foreign substances in the body. Various types of
lymphoma or leukemia can occur in the B-Cell life cycle.
Acute Myeloid Leukemia
[0088] In another aspect, the invention provides methods of
treating acute myeloid leukemia (AML), preferably acute
promyelocytic leukemia in peripheral blood. AML begins in the bone
marrow but can spread to other parts of the body including the
lymph nodes, liver, spleen, central nervous system, and testes. It
is acute, meaning it develops quickly and may be fatal if not
treated within a few months. AML is characterized by immature bone
marrow cells usually granulocytes or monocytes, which continue to
reproduce and accumulate.
[0089] AML may be treated by immunotherapy, radiation therapy,
chemotherapy, bone marrow or peripheral blood stem cell
transplantation, or a combination thereof. Radiation therapy
includes external beam radiation and may have side effects.
Anti-cancer drugs that may be used in chemotherapy to treat AML
include cytarabine, anthracycline, anthracenedione, idarubicin,
daunorubicin, idarubicin, mitoxantrone, thioguanine, vincristine,
prednisone, etoposide, or a combination thereof.
[0090] Monoclonal antibody therapy may be used to treat AML
patients. Small molecules or radioactive chemicals may be attached
to these antibodies before administration to a patient in order to
provide a means of killing leukemia cells in the body. The
monoclonal antibody, gemtuzumab ozogamicin, which binds CD33 on AML
cells, may be used to treat AML patients unable to tolerate prior
chemotherapy regimens.
[0091] Bone marrow or peripheral blood stem cell transplantation
may be used to treat AML patients. Some possible transplantation
procedures are an allogenic or an autologous transplant.
[0092] The methods provided by the invention can provide a
beneficial effect for leukemia patients, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, chemotherapy, or transplantation therapy.
[0093] There are other types of leukemia's that can also be treated
by the methods provided by the invention including but not limited
to, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Chronic
Lymphocytic Leukemia, Chronic Myeloid Leukemia, Hairy Cell
Leukemia, Myelodysplasia, and Myeloproliferative Disorders.
Lung Cancer
[0094] In another aspect, the invention provides methods to treat
lung cancer. The most common type of lung cancer is non-small cell
lung cancer (NSCLC), which accounts for approximately 80-85% of
lung cancers and is divided into squamous cell carcinomas,
adenocarcinomas, and large cell undifferentiated carcinomas. Small
cell lung cancer accounts for 15-20% of lung cancers.
[0095] Treatment options for lung cancer include surgery,
immunotherapy, radiation therapy, chemotherapy, photodynamic
therapy, or a combination thereof. Some possible surgical options
for treatment of lung cancer are a segmental or wedge resection, a
lobectomy, or a pneumonectomy. Radiation therapy may be external
beam radiation therapy or brachytherapy.
[0096] Some anti-cancer drugs that may be used in chemotherapy to
treat lung cancer include cisplatin, carboplatin, paclitaxel,
docetaxel, gemcitabine, vinorelbine, irinotecan, etoposde,
vinblastine, gefitinib, ifosfamide, methotrexate, or a combination
thereof. Photodynamic therapy (PDT) may be used to treat lung
cancer patients.
[0097] The methods provided by the invention can provide a
beneficial effect for lung cancer patients, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, chemotherapy, photodynamic therapy, or a
combination thereof.
Skin Cancer
[0098] In another aspect, the invention provides methods to treat
skin cancer. There are several types of cancer that start in the
skin. The most common types are basal cell carcinoma and squamous
cell carcinoma, which are non-melanoma skin cancers. Actinic
keratosis is a skin condition that sometimes develops into squamous
cell carcinoma. Non-melanoma skin cancers rarely spread to other
parts of the body. Melanoma, the rarest form of skin cancer, is
more likely to invade nearby tissues and spread to other parts of
the body. Different types of treatment are available for patients
with non-melanoma and melanoma skin cancer and actinic keratosis
including surgery, radiation therapy, chemotherapy and photodynamic
therapy. Some possible surgical options for treatment of skin
cancer are Mohs micrographic surgery (MMS), simple excision,
electrodesiccation and curettage, cryosurgery, laser surgery.
Radiation therapy may be external beam radiation therapy or
brachytherapy. Other types of treatments that are being tested in
clinical trials are biologic therapy or immunotherapy,
chemoimmunotherapy, topical chemotherapy with fluorouracil and
photodynamic therapy.
[0099] The methods provided by the invention can provide a
beneficial effect for skin cancer patients, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, chemotherapy, photodynamic therapy, or a
combination thereof.
Eye Cancer, Retinoblastoma
[0100] In another aspect, the invention provides methods to treat
eye retinoblastoma. Retinoblastoma is a malignant tumor of the
retina. Although retinoblastoma may occur at any age, it most often
occurs in younger children, usually before the age of 5 years. The
tumor may be in one eye only or in both eyes. Retinoblastoma is
usually confined to the eye and does not spread to nearby tissue or
other parts of the body. Treatment options that attempt to cure the
patient and preserve vision include enucleation (surgery to remove
the eye), radiation therapy, cryotherapy, photocoagulation,
immunotherapy, thermotherapy and chemotherapy. Radiation therapy
may be external beam radiation therapy or brachytherapy.
[0101] The methods provided by the invention can provide a
beneficial effect for eye retinoblastoma patients, by
administration of a nitrobenzamide metabolite compound or a
combination of administration of a nitrobenzamide metabolite
compound and surgery, radiation therapy, cryotherapy,
photocoagulation, thermotherapy and chemotherapy, or a combination
thereof.
Eye Cancer, Intraocular Melanoma
[0102] In another aspect, the invention provides methods to treat
intraocular (eye) melanoma. Intraocular melanoma, a rare cancer, is
a disease in which cancer cells are found in the part of the eye
called the uvea. The uvea includes the iris, the ciliary body, and
the choroid. Intraocular melanoma occurs most often in people who
are middle aged. Treatments for intraocular melanoma include
surgery, immunotherapy, radiation therapy and laser therapy.
Surgery is the most common treatment of intraocular melanoma. Some
possible surgical options are iridectomy, iridotrabeculectomy,
iridocyclectomy, choroidectomy, enucleation and orbital
exenteration. Radiation therapy may be external beam radiation
therapy or brachytherapy. Laser therapy may be an intensely
powerful beam of light to destroy the tumor, thermotherapy or
photocoagulation.
[0103] The methods provided by the invention can provide a
beneficial effect for intraocular melanoma patients, by
administration of a nitrobenzamide metabolite compound or a
combination of administration of a nitrobenzamide metabolite
compound and surgery, radiation therapy and laser therapy, or a
combination thereof.
Endometrium Cancer
[0104] In another aspect, the invention provides methods to treat
endometrium cancer. Endometrial cancer is a cancer that starts in
the endometrium, the inner lining of the uterus. Some of the
examples of the cancer of uterus and endometrium include, but are
not limited to, adenocarcinomas, adenoacanthomas, adenosquamous
carcinomas, papillary serous adenocarcinomas, clear cell
adenocarcinomas, uterine sarcomas, stromal sarcomas, malignant
mixed mesodermal tumors, and leiomyosarcomas.
[0105] The methods provided by the invention can provide a
beneficial effect for endometrium cancer patients, by
administration of a nitrobenzamide compound or a combination of
administration of a nitrobenzamide compound and surgery, radiation
therapy, chemotherapy, gene therapy, photodynamic therapy,
antiangiogenesis therapy, and immunotherapy, or a combination
thereof.
Liver Cancer
[0106] In another aspect, the invention provides methods to treat
primary liver cancer (cancer that begins in the liver). Primary
liver cancer can occur in both adults and children. Different types
of treatments are available for patients with primary liver cancer.
These include surgery, immunotherapy, radiation therapy,
chemotherapy and percutaneous ethanol injection. The types of
surgery that may be used are cryosurgery, partial hepatectomy,
total hepatectomy and radiofrequency ablation. Radiation therapy
may be external beam radiation therapy, brachytherapy,
radiosensitizers or radiolabel antibodies. Other types of treatment
include hyperthermia therapy and immunotherapy.
[0107] The methods provided by the invention can provide a
beneficial effect for liver cancer patients, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, chemotherapy, percutaneous ethanol injection,
hyperthemia therapy and immunotherapy, or a combination
thereof.
Kidney Cancer
[0108] In another aspect, the invention provides methods to treat
kidney cancer. Kidney cancer (also called renal cell cancer or
renal adenocarcinoma) is a disease in which malignant cells are
found in the lining of tubules in the kidney. Kidney cancer may be
treated by surgery, radiation therapy, chemotherapy and
immunotherapy. Some possible surgical options to treat kidney
cancer are partial nephrectomy, simple nephrectomy and radical
nephrectomy. Radiation therapy may be external beam radiation
therapy or brachytherapy. Stem cell transplant may be used to treat
kidney cancer.
[0109] The methods provided by the invention can provide a
beneficial effect for kidney cancer patients, by administration of
a nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
radiation therapy, chemotherapy, immunotherapy and stem cell
transplant, or a combination thereof.
Thyroid Cancer
[0110] In another aspect, the invention provides methods to treat
thyroid cancer. Thyroid cancer is a disease in which cancer
(malignant) cells are found in the tissues of the thyroid gland.
The four main types of thyroid cancer are papillary, follicular,
medullary and anaplastic. Thyroid cancer may be treated by surgery,
immunotherapy, radiation therapy, hormone therapy and chemotherapy.
Surgery is the most common treatment of thyroid cancer. Some
possible surgical options for treatment of thyroid cancer are
lobectomy, near-total thyroidectomy, total thyroidectomy and lymph
node dissection. Radiation therapy may be external radiation
therapy or may required intake of a liquid that contains
radioactive iodine. Hormone therapy uses hormones to stop cancer
cells from growing. In treating thyroid cancer, hormones can be
used to stop the body from making other hormones that might make
cancer cells grow.
[0111] The methods provided by the invention can provide a
beneficial effect for thyroid cancer patients, by administration of
a nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
surgery, radiation therapy, hormone therapy and chemotherapy, or a
combination thereof.
AIDS Related Cancers
AIDS-Related Lymphoma
[0112] In another aspect, the invention provides methods to treat
AIDS-related lymphoma. AIDS-related lymphoma is a disease in which
malignant cells form in the lymph system of patients who have
acquired immunodeficiency syndrome (AIDS). AIDS is caused by the
human immunodeficiency virus (HIV), which attacks and weakens the
body's immune system. The immune system is then unable to fight
infection and diseases that invade the body. People with HIV
disease have an increased risk of developing infections, lymphoma,
and other types of cancer. Lymphomas are cancers that affect the
white blood cells of the lymph system. Lymphomas are divided into
two general types: Hodgkin's lymphoma and non-Hodgkin's lymphoma.
Both Hodgkin's lymphoma and non-Hodgkin's lymphoma may occur in
AIDS patients, but non-Hodgkin's lymphoma is more common. When a
person with AIDS has non-Hodgkin's lymphoma, it is called an
AIDS-related lymphoma. Non-Hodgkin's lymphomas may be indolent
(slow-growing) or aggressive (fast-growing). AIDS-related lymphoma
is usually aggressive. The three main types of AIDS-related
lymphoma are diffuse large B-cell lymphoma, B-cell immunoblastic
lymphoma and small non-cleaved cell lymphoma.
[0113] Treatment of AIDS-related lymphoma combines treatment of the
lymphoma with treatment for AIDS. Patients with AIDS have weakened
immune systems and treatment can cause further damage. For this
reason, patients who have AIDS-related lymphoma are usually treated
with lower doses of drugs than lymphoma patients who do not have
AIDS. Highly-active antiretroviral therapy (HAART) is used to slow
progression of HIV. Medicine to prevent and treat infections, which
can be serious, is also used. AIDS-related lymphomas may be treated
by chemotherapy, immunotherapy, radiation therapy and high-dose
chemotherapy with stem cell transplant. Radiation therapy may be
external beam radiation therapy or brachytherapy. AIDS-related
lymphomas can be treated by monoclonal antibody therapy.
[0114] The methods provided by the invention can provide a
beneficial effect for AIDS-related lymphoma patients, by
administration of a nitrobenzamide metabolite compound or a
combination of administration of a nitrobenzamide metabolite
compound and chemotherapy, radiation therapy and high-dose
chemotherapy, or a combination thereof.
Kaposi's Sarcoma
[0115] In another aspect, the invention provides methods to treat
Kaposi's sarcoma. Kaposi's sarcoma is a disease in which cancer
cells are found in the tissues under the skin or mucous membranes
that line the mouth, nose, and anus. Classic Kaposi's sarcoma
usually occurs in older men of Jewish, Italian, or Mediterranean
heritage. This type of Kaposi's sarcoma progresses slowly,
sometimes over 10 to 15 years. Kaposi's sarcoma may occur in people
who are taking immunosuppressants. Kaposi's sarcoma in patients who
have Acquired Immunodeficiency Syndrome (AIDS) is called epidemic
Kaposi's sarcoma. Kaposi's sarcoma in people with AIDS usually
spreads more quickly than other kinds of Kaposi's sarcoma and often
is found in many parts of the body. Kaposi's sarcoma may be treated
with surgery, chemotherapy, radiation therapy and immunotherapy.
External radiation therapy is a common treatment of Kaposi's
sarcoma. Some possible surgical options to treat Kaposi's Sarcoma
are local excision, electrodessiccation and curettage, and
cryotherapy.
[0116] The methods provided by the invention can provide a
beneficial effect for Kaposi's sarcoma, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and surgery,
chemotherapy, radiation therapy and immunotherapy, or a combination
thereof.
Viral-Induced Cancers
[0117] In another aspect, the invention provides methods to treat
viral-induced cancers. Several common viruses are clearly or
probable causal factors in the etiology of specific malignancies.
These viruses either normally establish latency or few can become
persistent infections. Oncogenesis is probably linked to an
enhanced level of viral activation in the infected host, reflecting
heavy viral dose or compromised immune control. The major
virus-malignancy systems include hepatitis B virus (HBV), hepatitis
C virus (HCV), and hepatocellular carcinoma; human lymphotropic
virus-type 1 (HTLV-1) and adult T-cell leukemia/lymphoma; and human
papilloma virus (HPV) and cervical cancer. In general, these
malignancies occur relatively early in life, typically peaking in
middle-age or earlier.
Virus-Induced Hepatocellular Carcinoma
[0118] The causal relationship between both HBV and HCV and
hepatocellular carcinoma or liver cancer is established through
substantial epidemiologic evidence. Both appear to act via chronic
replication in the liver by causing cell death and subsequent
regeneration. Different types of treatments are available for
patients with liver cancer. These include surgery, immunotherapy,
radiation therapy, chemotherapy and percutaneous ethanol injection.
The types of surgery that may be used are cryosurgery, partial
hepatectomy, total hepatectomy and radiofrequency ablation.
Radiation therapy may be external beam radiation therapy,
brachytherapy, radiosensitizers or radiolabel antibodies. Other
types of treatment include hyperthermia therapy and
immunotherapy.
[0119] The methods provided by the invention can provide a
beneficial effect for virus induce hepatocellular carcinoma
patients, by administration of a nitrobenzamide metabolite compound
or a combination of administration of a nitrobenzamide metabolite
compound and surgery, radiation therapy, chemotherapy, percutaneous
ethanol injection, hyperthemia therapy and immunotherapy, or a
combination thereof.
Viral-Induced Adult T Cell Leukemia/Lymphoma
[0120] The association between lentiviruses (for example HTLV-1)
and Adult T cell leukemia (ATL) is established. Unlike the other
oncogenic viruses found throughout the world, HTLV-1 is highly
geographically restricted, being found primarily in southern Japan,
the Caribbean, west and central Africa, and the South Pacific
islands. Evidence for causality includes the monoclonal integration
of viral genome in almost all cases of ATL in carriers. The risk
factors for HTLV-1-associated malignancy appear to be preinatal
infection, high viral load, and being male sex.
[0121] Adult T cell leukemia is a cancer of the blood and bone
marrow. The standard treatments for adult T cell leukemia/lymphoma
are radiation therapy, immunotherapy, and chemotherapy. Radiation
therapy may be external beam radiation therapy or brachytherapy.
Other methods of treating adult T cell leukemia/lymphoma include
immunotherapy and high-dose chemotherapy with stem cell
transplantation.
[0122] The methods provided by the invention can provide a
beneficial effect for Adult T cell leukemia patients, by
administration of a nitrobenzamide metabolite compound or a
combination of administration of a nitrobenzamide metabolite
compound and radiation therapy, chemotherapy, immunotherapy and
high-dose chemotherapy with stem cell transplantation, or a
combination thereof.
Viral-Induced Cervical Cancer
[0123] Infection of the cervix with human papilloma virus (HPV) is
the most common cause of cervical cancer. Not all women with HPV
infection, however, will develop cervical cancer. Cervical cancer
usually develops slowly over time. Before cancer appears in the
cervix, the cells of the cervix go through changes known as
dysplasia, in which cells that are not normal begin to appear in
the cervical tissue. Later, cancer cells start to grow and spread
more deeply into the cervix and to surrounding areas. The standard
treatments for cervical cancers are surgery, immunotherapy,
radiation therapy and chemotherapy. The types of surgery that may
be used are conization, total hysterectomy, bilateral
salpingo-oophorectomy, radical hysterectomy, pelvic exenteration,
cryosurgery, laser surgery and loop electrosurgical excision
procedure. Radiation therapy may be external beam radiation therapy
or brachytherapy.
[0124] The methods provided by the invention can provide a
beneficial effect for adult cervical cancer, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and
radiation therapy, chemotherapy, or a combination thereof.
CNS Cancers
[0125] Brain and spinal cord tumors are abnormal growths of tissue
found inside the skull or the bony spinal column, which are the
primary components of the central nervous system (CNS). Benign
tumors are non-cancerous, and malignant tumors are cancerous. The
CNS is housed within rigid, bony quarters (i.e., the skull and
spinal column), so any abnormal growth, whether benign or
malignant, can place pressure on sensitive tissues and impair
function. Tumors that originate in the brain or spinal cord are
called primary tumors. Most primary tumors are caused by
out-of-control growth among cells that surround and support
neurons. In a small number of individuals, primary tumors may
result from specific genetic disease (e.g., neurofibromatosis,
tuberous sclerosis) or from exposure to radiation or cancer-causing
chemicals. The cause of most primary tumors remains a mystery.
[0126] The first test to diagnose brain and spinal column tumors is
a neurological examination. Special imaging techniques (computed
tomography, and magnetic resonance imaging, positron emission
tomography) are also employed. Laboratory tests include the EEG and
the spinal tap. A biopsy, a surgical procedure in which a sample of
tissue is taken from a suspected tumor, helps doctors diagnose the
type of tumor.
[0127] Tumors are classified according to the kind of cell from
which the tumor seems to originate. The most common brain tumor in
adults comes from cells in the brain called astrocytes that make up
the blood-brain barrier and contribute to the nutrition of the
central nervous system. These tumors are called gliomas
(astrocytoma, anaplastic astrocytoma, or glioblastoma multiforme)
and account for 65% of all primary central nervous system tumors.
Some of the tumors are, but not limited to, Oligodendroglioma,
Ependymoma, Meningioma, Lymphoma, Schwannoma, and
Medulloblastoma.
Neuroepithelial Tumors of the CNS
[0128] Astrocytic tumors, such as astrocytoma; anaplastic
(malignant) astrocytoma, such as hemispheric, diencephalic, optic,
brain stem, cerebellar; glioblastoma multiforme; pilocytic
astrocytoma, such as hemispheric, diencephalic, optic, brain stem,
cerebellar; subependymal giant cell astrocytoma; and pleomorphic
xanthoastrocytoma. Oligodendroglial tumors, such as
oligodendroglioma; and anaplastic (malignant) oligodendroglioma.
Ependymal cell tumors, such as ependymoma; anaplastic ependymoma;
myxopapillary ependymoma; and subependymoma. Mixed gliomas, such as
mixed oligoastrocytoma; anaplastic (malignant) oligoastrocytoma;
and others (e.g. ependymo-astrocytomas). Neuroepithelial tumors of
uncertain origin, such as polar spongioblastoma; astroblastoma; and
gliomatosis cerebri. Tumors of the choroid plexus, such as choroid
plexus papilloma; and choroid plexus carcinoma (anaplastic choroid
plexus papilloma). Neuronal and mixed neuronal-glial tumors, such
as gangliocytoma; dysplastic gangliocytoma of cerebellum
(Lhermitte-Duclos); ganglioglioma; anaplastic (malignant)
ganglioglioma; desmoplastic infantile ganglioglioma, such as
desmoplastic infantile astrocytoma; central neurocytoma;
dysembryoplastic neuroepithelial tumor; olfactory neuroblastoma
(esthesioneuroblastoma. Pineal Parenchyma Tumors, such as
pineocytoma; pineoblastoma; and mixed pineocytoma/pineoblastoma.
Tumors with neuroblastic or glioblastic elements (embryonal
tumors), such as medulloepithelioma; primitive neuroectodermal
tumors with multipotent differentiation, such as medulloblastoma;
cerebral primitive neuroectodermal tumor; neuroblastoma;
retinoblastoma; and ependymoblastoma.
Other CNS Neoplasms
[0129] Tumors of the Sellar Region, such as pituitary adenoma;
pituitary carcinoma; and craniopharyngioma. Hematopoietic tumors,
such as primary malignant lymphomas; plasmacytoma; and granulocytic
sarcoma. Germ Cell Tumors, such as germinoma; embryonal carcinoma;
yolk sac tumor (endodermal sinus tumor); choriocarcinoma; teratoma;
and mixed germ cell tumors. Tumors of the Meninges, such as
meningioma; atypical meningioma; and anaplastic (malignant)
meningioma. Non-menigothelial tumors of the meninges, such as
Benign Mesenchymal; Malignant Mesenchymal; Primary Melanocytic
Lesions; Hemopoietic Neoplasms; and Tumors of Uncertain
Histogenesis, such as hemangioblastoma (capillary
hemangioblastoma). Tumors of Cranial and Spinal Nerves, such as
schwannoma (neurinoma, neurilemoma); neurofibroma; malignant
peripheral nerve sheath tumor (malignant schwannoma), such as
epithelioid, divergent mesenchymal or epithelial differentiation,
and melanotic. Local Extensions from Regional Tumors; such as
paraganglioma (chemodectoma); chordoma; chodroma; chondrosarcoma;
and carcinoma. Metastatic tumours, Unclassified Tumors and Cysts
and Tumor-like Lesions, such as Rathke cleft cyst; Epidermoid;
dermoid; colloid cyst of the third ventricle; enterogenous cyst;
neuroglial cyst; granular cell tumor (choristoma, pituicytoma);
hypothalamic neuronal hamartoma; nasal glial herterotopia; and
plasma cell granuloma.
[0130] Chemotherapeutics available are, but not limited to,
alkylating agents such as, Cyclophosphamide, Ifosphamide,
Melphalan, Chlorambucil, BCNU, CCNU, Decarbazine, Procarbazine,
Busulfan, and Thiotepa; antimetabolites such as, Methotraxate,
5-Fluorouracil, Cytarabine, Gemcitabine (Gemzar.RTM.),
6-mercaptopurine, 6-thioguanine, Fludarabine, and Cladribine;
anthracyclins such as, daunorubicin. Doxorubicin, Idarubicin,
Epirubicin and Mitoxantrone; antibiotics such as, Bleomycin;
camptothecins such as, irinotecan and topotecan; taxanes such as,
paclitaxel and docetaxel; and platinums such as, Cisplatin,
carboplatin, and Oxaliplatin.
[0131] The treatments are surgery, radiation therapy,
immunotherapy, hyperthermia, gene therapy, chemotherapy, and
combination of radiation and chemotherapy. Doctors also may
prescribe steroids to reduce the swelling inside the CNS.
[0132] The methods provided by the invention can provide a
beneficial effect for adult cervical cancer, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and
radiation therapy, chemotherapy, or a combination thereof.
PNS Cancers
[0133] The peripheral nervous system consists of the nerves that
branch out from the brain and spinal cord. These nerves form the
communication network between the CNS and the body parts. The
peripheral nervous system is further subdivided into the somatic
nervous system and the autonomic nervous system. The somatic
nervous system consists of nerves that go to the skin and muscles
and is involved in conscious activities. The autonomic nervous
system consists of nerves that connect the CNS to the visceral
organs such as the heart, stomach, and intestines. It mediates
unconscious activities.
[0134] Acoustic neuromas are benign fibrous growths that arise from
the balance nerve, also called the eighth cranial nerve or
vestibulocochlear nerve. These tumors are non-malignant, meaning
that they do not spread or metastasize to other parts of the body.
The location of these tumors is deep inside the skull, adjacent to
vital brain centers in the brain stem. As the tumors enlarge, they
involve surrounding structures which have to do with vital
functions. In the majority of cases, these tumors grow slowly over
a period of years.
[0135] The malignant peripheral nerve sheath tumor (MPNST) is the
malignant counterpart to benign soft tissue tumors such as
neurofibromas and schwannomas. It is most common in the deep soft
tissue, usually in close proximity of a nerve trunk. The most
common sites include the sciatic nerve, brachial plexus, and sarcal
plexus. The most common symptom is pain which usually prompts a
biopsy. It is a rare, aggressive, and lethal orbital neoplasm that
usually arises from sensory branches of the trigeminal nerve in
adults. Malignant PNS tumor spreads along nerves to involve the
brain, and most patients die within 5 years of clinical diagnosis.
The MPNST may be classified into three major categories with
epithelioid, mesenchymal or glandular characteristics. Some of the
MPNST include but not limited to, Subcutaneous malignant
epithelioid schwannoma with cartilaginous differentiation,
Glandular malignant schwannoma, Malignant peripheral nerve sheath
tumor with perineurial differentiation, Cutaneous epithelioid
malignant nerve sheath tumor with rhabdoid features, Superficial
epithelioid MPNST, Triton Tumor (MPNST with rhabdomyoblastic
differentiation), Schwannoma with rhabdomyoblastic differentiation.
Rare MPNST cases contain multiple sarcomatous tissue types,
especially osteosarcoma, chondrosarcoma and angiosarcoma. These
have sometimes been indistinguishable from the malignant
mesenchymoma of soft tissue.
[0136] Other types of PNS cancers include but not limited to,
malignant fibrous cytoma, malignant fibrous histiocytoma, malignant
meningioma, malignant mesothelioma, and malignant mixed Mullerian
tumor.
[0137] The treatments are surgery, radiation therapy,
immunotherapy, chemotherapy, and combination of radiation and
chemotherapy.
[0138] The methods provided by the invention can provide a
beneficial effect for PNS cancers, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and
radiation therapy, chemotherapy, or a combination thereof.
Oral Cavity and Oropharyngeal Cancer
[0139] Management of patients with central nervous system (CNS)
cancers remains a formidable task. Cancers such as, hypopharyngeal
cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal
cancer, and the like, have been treated with surgery,
immunotherapy, chemotherapy, combination of chemotherapy and
radiation therapy. Etoposide and actinomycin D, two commonly used
oncology agents that inhibit topoisomerase II, fail to cross the
blood-brain barrier in useful amounts.
[0140] The methods provided by the invention can provide a
beneficial effect for Oral Cavity and Oropharyngeal cancer, by
administration of a nitrobenzamide metabolite compound or a
combination of administration of a nitrobenzamide metabolite
compound and radiation therapy, chemotherapy, or a combination
thereof.
Stomach Cancer
[0141] Stomach cancer is the result of cell changes in the lining
of the stomach. There are three main types of stomach cancers:
lymphomas, gastric stromal tumors, and carcinoid tumors. Lymphomas
are cancers of the immune system tissue that are sometimes found in
the wall of the stomach. Gastric stromal tumors develop from the
tissue of the stomach wall. Carcinoid tumors are tumors of
hormone-producing cells of the stomach.
[0142] The causes of stomach cancer continue to be debated. A
combination of heredity and environment (diet, smoking, etc) are
all thought to play a part. Common approaches to the treatment
include surgery, immunotherapy, chemotherapy, radiation therapy,
combination of chemotherapy and radiation therapy or biological
therapy.
[0143] The methods provided by the invention can provide a
beneficial effect for stomach cancer, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and
radiation therapy, chemotherapy, or a combination thereof.
Testicular Cancer
[0144] Testicular cancer is cancer that typically develops in one
or both testicles in young men. Cancers of the testicle develop in
certain cells known as germ cells. The 2 main types of germ cell
tumors (GCTs) that occur in men are seminomas (60%) and
nonseminomas (40%). Tumors can also arise in the supportive and
hormone-producing-tissues, or stroma, of the testicles. Such tumors
are known as gonadal stromal tumors. The 2 main types are Leydig
cell tumors and Sertoli cell tumors. Secondary testicular tumors
are those that start in another organ and then spread to the
testicle. Lymphoma is the most common secondary testicular
cancer.
[0145] Common approaches to the treatment include surgery,
immunotherapy, chemotherapy, radiation therapy, combination of
chemotherapy and radiation therapy or biological therapy. Several
drugs are typically used to treat testicular cancer: Platinol
(cisplatin), Vepesid or VP-16 (etoposide) and Blenoxane (bleomycin
sulfate). Additionally, Ifex (ifosamide), Velban (vinblastine
sulfate) and others may be used.
[0146] The methods provided by the invention can provide a
beneficial effect for stomach cancer, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and
radiation therapy, chemotherapy, or a combination thereof.
Thymus Cancer
[0147] The thymus is a small organ located in the upper/front
portion of your chest, extending from the base of the throat to the
front of the heart. The thymus contains 2 main types of cells,
thymic epithelial cells and lymphocytes. Thymic epithelial cells
can give origin to thymomas and thymic carcinomas. Lymphocytes,
whether in the thymus or in the lymph nodes, can become malignant
and develop into cancers called Hodgkin's disease and non-Hodgkin's
lymphomas. The thymus also contains another much less common type
of cells called Kulchitsky cells, or neuroendocrine cells, which
normally release certain hormones. These cells can give rise to
cancers, called carcinoids or carcinoid tumors that often release
the same type of hormones, and are similar to other tumors arising
from neuroendocrine cells elsewhere in the body.
[0148] Common approaches to the treatment include surgery,
immunotherapy, chemotherapy, radiation therapy, combination of
chemotherapy and radiation therapy or biological therapy.
Anticancer drugs that have been used in the treatment of thymomas
and thymic carcinomas are doxorubicin (Adriamycin), cisplatin,
ifosfamide, and corticosteroids (prednisone). Often, these drugs
are given in combination to increase their effectiveness.
Combinations used to treat thymic cancer include cisplatin,
doxorubicin, etoposide and cyclophosphamide, and the combination of
cisplatin, doxorubicin, cyclophosphamide, and vincristine.
[0149] The methods provided by the invention can provide a
beneficial effect for stomach cancer, by administration of a
nitrobenzamide metabolite compound or a combination of
administration of a nitrobenzamide metabolite compound and
radiation therapy, chemotherapy, or a combination thereof.
Combination Therapy
[0150] One aspect of the invention provides methods for treating
cancer using different combinations of treatment regimens. For
example, such combinations may include, but are not limited to, the
use of one or more of the nitrobenzamide compounds in conjunction
with one or more various antineoplastic chemotherapeutic agents,
chemopreventative agents, and/or side-effect limiting agents.
Antineoplastic Chemotherapeutic Agents
[0151] Suitable antineoplastic chemotherapeutic agents to be used
in the present invention include, but are not limited to,
alkylating agents, antimetabolites, natural antineoplastic agents,
hormonal antineoplastic agents, angiogenesis inhibitors,
differentiating reagents, RNA inhibitors, DNA inhibitors,
inhibitors of viral replication, antibodies or immunotherapeutic
agents, peptide agents, gene therapy agents, small molecule
enzymatic inhibitors, biological response modifiers, and
anti-metastatic agents.
Alkylating Agents
[0152] Alkylating agents are known to act through the alkylation of
macromolecules such as the DNA of cancer cells, and are usually
strong electrophiles. This activity can disrupt DNA synthesis and
cell division. Examples of alkylating reagents suitable for use
herein include nitrogen mustards and their analogues and
derivatives including, cyclophosphamide, ifosfamide, chlorambucil,
estramustine, mechlorethamine hydrochloride, melphalan, and uracil
mustard. Other examples of alkylating agents include alkyl
sulfonates (e.g. busulfan), nitrosoureas (e.g. carmustine,
lomustine, and streptozocin), triazenes (e.g. dacarbazine and
temozolomide), ethylenimines/methylmelamines (e.g. altretamine and
thiotepa), and methylhydrazine derivatives (e.g. procarbazine).
Included in the alkylating agent group are the alkylating-like
platinum-containing drugs comprising carboplatin, cisplatin, and
oxaliplatin.
Antimetabolites
[0153] Antimetabolic antineoplastic agents structurally resemble
natural metabolites, and are involved in normal metabolic processes
of cancer cells such as the synthesis of nucleic acids and
proteins. They differ enough from the natural metabolites so that
they interfere with the metabolic processes of cancer cells.
Suitable antimetabolic antineoplastic agents to be used in the
present invention can be classified according to the metabolic
process they affect, and can include, but are not limited to,
analogues and derivatives of folic acid, pyrimidines, purines, and
cytidine. Members of the folic acid group of agents suitable for
use herein include, but are not limited to, methotrexate
(amethopterin), pemetrexed and their analogues and derivatives.
Pyrimidine agents suitable for use herein include, but are not
limited to, cytarabine, floxuridine, fluorouracil (5-fluorouracil),
capecitabine, gemcitabine, and their analogues and derivatives.
Purine agents suitable for use herein include, but are not limited
to, mercaptopurine (6-mercaptopurine), pentostatin, thioguanine,
cladribine, and their analogues and derivatives. Cytidine agents
suitable for use herein include, but are not limited to, cytarabine
(cytosine arabinodside), azacitidine (5-azacytidine) and their
analogues and derivatives.
Natural Antineoplastic Agents
[0154] Natural antineoplastic agents comprise antimitotic agents,
antibiotic antineoplastic agents, camptothecin analogues, and
enzymes. Antimitotic agents suitable for use herein include, but
are not limited to, vinca alkaloids like vinblastine, vincristine,
vindesine, vinorelbine, and their analogues and derivatives. They
are derived from the Madagascar periwinkle plant and are usually
cell cycle-specific for the M phase, binding to tubulin in the
microtubules of cancer cells. Other antimitotic agents suitable for
use herein are the podophyllotoxins, which include, but are not
limited to etoposide, teniposide, and their analogues and
derivatives. These reagents predominantly target the G2 and late S
phase of the cell cycle.
[0155] Also included among the natural antineoplastic agents are
the antibiotic antineoplastic agents. Antibiotic antineoplastic
agents are antimicrobial drugs that have anti-tumor properties
usually through interacting with cancer cell DNA. Antibiotic
antineoplastic agents suitable for use herein include, but are not
limited to, belomycin, dactinomycin, doxorubicin, idarubicin,
epirubicin, mitomycin, mitoxantrone, pentostatin, plicamycin, and
their analogues and derivatives.
[0156] The natural antineoplastic agent classification also
includes camptothecin analogues and derivatives which are suitable
for use herein and include camptothecin, topotecan, and irinotecan.
These agents act primarily by targeting the nuclear enzyme
topoisomerase I. Another subclass under the natural antineoplastic
agents is the enzyme, L-asparaginase and its variants.
L-asparaginase acts by depriving some cancer cells of L-asparagine
by catalyzing the hydrolysis of circulating asparagine to aspartic
acid and ammonia.
Hormonal Antineoplastic Agents
[0157] Hormonal antineoplastic agents act predominantly on
hormone-dependent cancer cells associated with prostate tissue,
breast tissue, endometrial tissue, ovarian tissue, lymphoma, and
leukemia. Such tissues may be responsive to and dependent upon such
classes of agents as glucocorticoids, progestins, estrogens, and
androgens. Both analogues and derivatives that are agonists or
antagonists are suitable for use in the present invention to treat
tumors. Examples of glucocorticoid agonists/antagonists suitable
for use herein are dexamethasone, cortisol, corticosterone,
prednisone, mifepristone (RU486), their analogues and derivatives.
The progestin agonist/antagonist subclass of agents suitable for
use herein includes, but is not limited to, hydroxyprogesterone,
medroxyprogesterone, megestrol acetate, mifepristone (RU486),
ZK98299, their analogues and derivatives. Examples from the
estrogen agonist/antagonist subclass of agents suitable for use
herein include, but are not limited to, estrogen, tamoxifen,
toremifene, RU58668, SR16234, ZD164384, ZK191703, fulvestrant,
their analogues and derivatives. Examples of aromatase inhibitors
suitable for use herein, which inhibit estrogen production,
include, but are not limited to, androstenedione, formestane,
exemestane, aminoglutethimide, anastrozole, letrozole, their
analogues and derivatives. Examples from the androgen
agonist/antagonist subclass of agents suitable for use herein
include, but are not limited to, testosterone, dihydrotestosterone,
fluoxymesterone, testolactone, testosterone enanthate, testosterone
propionate, gonadotropin-releasing hormone agonists/antagonists
(e.g. leuprolide, goserelin, triptorelin, buserelin),
diethylstilbestrol, abarelix, cyproterone, flutamide, nilutamide,
bicalutamide, their analogues and derivatives.
Angiogenesis Inhibitors
[0158] Angiogenesis inhibitors work by inhibiting the
vascularization of tumors. Angiogenesis inhibitors encompass a wide
variety of agents including small molecule agents, antibody agents,
and agents that target RNA function. Examples of angiogenesis
inhibitors suitable for use herein include, but are not limited to,
ranibizumab, bevacizumab, SU11248, PTK787, ZK222584, CEP-7055,
angiozyme, dalteparin, thalidomide, suramin, CC-5013,
combretastatin A4 Phosphate, LY317615, soy isoflavones, AE-941,
interferon alpha, PTK787/ZK 222584, ZD6474, EMD 121974, ZD6474, BAY
543-9006, celecoxib, halofuginone hydrobromide, bevacizumab, their
analogues, variants, or derivatives.
Differentiating Reagents
[0159] Differentiating agents inhibit tumor growth through
mechanisms that induce cancer cells to differentiate. One such
subclass of these agents suitable for use herein includes, but is
not limited to, vitamin A analogues or retinoids, and peroxisome
proliferator-activated receptor agonists (PPARs). Retinoids
suitable for use herein include, but are not limited to, vitamin A,
vitamin A aldehyde (retinal), retinoic acid, fenretinide,
9-cis-retinoid acid, 13-cis-retinoid acid, all-trans-retinoic acid,
isotretinoin, tretinoin, retinyl palmitate, their analogues and
derivatives. Agonists of PPARs suitable for use herein include, but
are not limited to, troglitazone, ciglitazone, tesaglitazar, their
analogues and derivatives.
RNA Inhibitors
[0160] Certain RNA inhibiting agents may be utilized to inhibit the
expression or translation of messenger RNA ("mRNA") that is
associated with a cancer phenotype. Examples of such agents
suitable for use herein include, but are not limited to, short
interfering RNA ("siRNA"), micro RNA ("miRNA"), ribozymes, and
antisense oligonucleotides. Specific examples of RNA inhibiting
agents suitable for use herein include, but are not limited to,
Candy, Sirna-027, fomivirsen, and angiozyme.
Antibodies/Immunotherapeutic Agents
[0161] Antibody agents bind targets selectively expressed in cancer
cells and can either utilize a conjugate to kill the cell
associated with the target, or elicit the body's immune response to
destroy the cancer cells. Immunotherapeutic agents can either be
comprised of polyclonal or monoclonal antibodies. The antibodies
may be comprised of non-human animal (e.g. mouse) and human
components, or be comprised of entirely human components
("humanized antibodies"). Examples of monoclonal immunotherapeutic
agents suitable for use herein include, but are not limited to,
rituximab, tosibtumomab, ibritumomab which target the CD-20
protein. Other examples suitable for use herein include
trastuzumab, edrecolomab, bevacizumab, cetuximab, carcinoembryonic
antigen antibodies, gemtuzumab, alemtuzumab, mapatumumab,
panitumumab, EMD 72000, TheraCIM hR3, 2C4, HGS-TR2J, and
HGS-ETR2.
Gene Therapy Agents
[0162] Gene therapy agents insert copies of genes into a specific
set of a patient's cells, and can target both cancer and non-cancer
cells. The goal of gene therapy can be to replace altered genes
with functional genes, to stimulate a patient's immune response to
cancer, to make cancer cells more sensitive to chemotherapy, to
place "suicide" genes into cancer cells, or to inhibit
angiogenesis. Genes may be delivered to target cells using viruses,
liposomes, or other carriers or vectors. This may be done by
injecting the gene-carrier composition into the patient directly,
or ex vivo, with infected cells being introduced back into a
patient. Such compositions are suitable for use in the present
invention.
Small Molecule Enzymatic Inhibitors
[0163] Certain small molecule therapeutic agents are able to target
the tyrosine kinase enzymatic activity or downstream signal
transduction signals of certain cell receptors such as epidermal
growth factor receptor ("EGFR") or vascular endothelial growth
factor receptor ("VEGFR"). Such targeting by small molecule
therapeutics can result in anti-cancer effects. Examples of such
agents suitable for use herein include, but are not limited to,
imatinib, gefitinib, erlotinib, lapatinib, canertinib, ZD6474,
sorafenib (BAY 43-9006), ERB-569, and their analogues and
derivatives.
Biological Response Modifiers
[0164] Certain protein or small molecule agents can be used in
anti-cancer therapy through either direct anti-tumor effects or
through indirect effects. Examples of direct-acting agents suitable
for use herein include, but are not limited to, differentiating
reagents such as retinoids and retinoid derivatives.
Indirect-acting agents suitable for use herein include, but are not
limited to, agents that modify or enhance the immune or other
systems such as interferons, interleukins, hematopoietic growth
factors (e.g. erythropoietin), and antibodies (monoclonal and
polyclonal).
Anti-Metastatic Agents
[0165] The process whereby cancer cells spread from the site of the
original tumor to other locations around the body is termed cancer
metastasis. Certain agents have anti-metastatic properties,
designed to inhibit the spread of cancer cells. Examples of such
agents suitable for use herein include, but are not limited to,
marimastat, bevacizumab, trastuzumab, rituximab, erlotinib,
MMI-166, GRN163L, hunter-killer peptides, tissue inhibitors of
metalloproteinases (TIMPs), their analogues, derivatives and
variants.
Chemopreventative Agents
[0166] Certain pharmaceutical agents can be used to prevent initial
occurrences of cancer, or to prevent recurrence or metastasis.
Administration with such chemopreventative agents in combination
with one or more other anticancer agents including the
nitrobenzamide compounds can act to both treat and prevent the
recurrence of cancer. Examples of chemopreventative agents suitable
for use herein include, but are not limited to, tamoxifen,
raloxifene, tibolone, bisphosphonate, ibandronate, estrogen
receptor modulators, aromatase inhibitors (letrozole, anastrozole),
luteinizing hormone-releasing hormone agonists, goserelin, vitamin
A, retinal, retinoic acid, fenretinide, 9-cis-retinoid acid,
13-cis-retinoid acid, all-trans-retinoic acid, isotretinoin,
tretinoid, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin
E, cyclooxygenase inhibitors, non-steroidal anti-inflammatory drugs
(NSAIDs), aspirin, ibuprofen, celecoxib, polyphenols, polyphenol E,
green tea extract, folic acid, glucaric acid, interferon-alpha,
anethole dithiolethione, zinc, pyridoxine, finasteride, doxazosin,
selenium, indole-3-carbinal, alpha-difluoromethylomithine,
carotenoids, beta-carotene, lycopene, antioxidants, coenzyme Q10,
flavonoids, quercetin, curcumin, catechins, epigallocatechin
gallate, N-acetylcysteine, indole-3-carbinol, inositol
hexaphosphate, isoflavones, glucanic acid, rosemary, soy, saw
palmetto, and calcium. An additional example of chemopreventative
agents suitable for use in the present invention is cancer
vaccines. These can be created through immunizing a patient with
all or part of a cancer cell type that is targeted by the
vaccination process.
Side-Effect Limiting Agents
[0167] Treatment of cancer with nitrobenzamide compounds alone or
in combination with other antineoplastic compounds may be
accompanied by administration of pharmaceutical agents that can
alleviate the side effects produced by the antineoplastic agents.
Such agents suitable for use herein include, but are not limited
to, anti-emetics, anti-mucositis agents, pain management agents,
infection control agents, and anti-anemia/anti-thrombocytopenia
agents. Examples of anti-emetics suitable for use herein include,
but are not limited to, 5-hydroxytryptamine 3 receptor antagonists,
metoclopramide, steroids, lorazepam, ondansetron, cannabinoids,
their analogues and derivatives. Examples of anti-mucositis agents
suitable for use herein include, but are not limited to, palifermin
(keratinocyte growth factor), glucagon-like peptide-2, teduglutide,
L-glutamine, amifostin, and fibroblast growth factor 20. Examples
of pain management agents suitable for use herein include, but are
not limited to, opioids, opiates, and non-steroidal
anti-inflammatory compounds. Examples of agents used for control of
infection suitable for use herein include, but are not limited to,
antibacterials such as aminoglycosides, penicillins,
cephalosporins, tetracyclines, clindamycin, lincomycin, macrolides,
vancomycin, carbapenems, monobactams, fluoroquinolones,
sulfonamides, nitrofurantoin, their analogues and derivatives.
Examples of agents that can treat anemia or thrombocytopenia
associated with chemotherapy suitable for use herein include, but
are not limited to, erythropoietin, and thrombopoietin.
[0168] Several other suitable therapies for use in combination with
the nitrobenzamide compounds and other compounds described herein
are also available. For example, see Goodman & Gilman's The
Pharmacological Basis of Therapeutics 11th ed. Brunton L L, Lazo J
S, and Parker K L, ed. McGraw-Hill, New York, 2006. Formulations,
Routes of Administration, and Effective Doses
[0169] Another aspect of the present invention relates to
formulations and routes of administration for pharmaceutical
compositions comprising a nitrobenzamide metabolite compound. Such
pharmaceutical compositions can be used to treat cancer in the
methods described in detail above.
[0170] In some embodiments, the metabolite compounds depicted
herein above may be provided individually or in combination as a
prodrug and/or may be allowed to interconvert to a nitrosobenzamide
form in vivo after administration. Furthermore, said metabolite
compounds may be administered with the nitrobenzamide of formula Ia
which may be provided as a prodrug and/or may be allowed to
interconvert to a nitrosobenzamide form in vivo after
administration. That is, either the nitrobenzamide form and/or the
nitrosobenzamide form, or pharmaceutically acceptable salts may be
used in developing a formulation for use in the present invention.
Further, in some embodiments, the metabolite compound may be used
in combination with one or more other compounds or in one or more
other forms. For example a formulation may comprise both the
nitrobenzamide metabolite compound and acid forms in particular
proportions, depending on the relative potencies of each and the
intended indication. The two forms may be formulated together, in
the same dosage unit e.g. in one cream, suppository, tablet,
capsule, or packet of powder to be dissolved in a beverage; or each
form may be formulated in a separate unit, e.g., two creams, two
suppositories, two tablets, two capsules, a tablet and a liquid for
dissolving the tablet, a packet of powder and a liquid for
dissolving the powder, etc.
[0171] In compositions comprising combinations of a nitrobenzamide
metabolite compound and another active agent can be effective. The
two compounds and/or forms of a compound may be formulated
together, in the same dosage unit e.g. in one cream, suppository,
tablet, capsule, or packet of powder to be dissolved in a beverage;
or each form may be formulated in separate units, e.g., two creams,
suppositories, tablets, two capsules, a tablet and a liquid for
dissolving the tablet, a packet of powder and a liquid for
dissolving the powder, etc.
[0172] The term "pharmaceutically acceptable salt" means those
salts which retain the biological effectiveness and properties of
the compounds used in the present invention, and which are not
biologically or otherwise undesirable. For example, a
pharmaceutically acceptable salt does not interfere with the
beneficial effect of the compound of the invention in treating a
cancer.
[0173] Typical salts are those of the inorganic ions, such as, for
example, sodium, potassium, calcium and magnesium ions. Such salts
include salts with inorganic or organic acids, such as hydrochloric
acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric
acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid,
fumaric acid, succinic acid, lactic acid, mandelic acid, malic
acid, citric acid, tartaric acid or maleic acid. In addition, if
the compounds used in the present invention contain a carboxy group
or other acidic group, it may be converted into a pharmaceutically
acceptable addition salt with inorganic or organic bases. Examples
of suitable bases include sodium hydroxide, potassium hydroxide,
ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine,
diethanolamine and triethanolamine.
[0174] For oral administration, the metabolite compounds can be
formulated readily by combining the active compound(s) with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the compounds of the invention to be formulated as
tablets, including chewable tablets, pills, dragees, capsules,
lozenges, hard candy, liquids, gels, syrups, slurries, powders,
suspensions, elixirs, wafers, and the like, for oral ingestion by a
patient to be treated. Such formulations can comprise
pharmaceutically acceptable carriers including solid diluents or
fillers, sterile aqueous media and various non-toxic organic
solvents. Generally, the compounds of the invention will be
included at concentration levels ranging from about 0.5%, about 5%,
about 10%, about 20%, or about 30% to about 50%, about 60%, about
70%, about 80% or about 90% by weight of the total composition of
oral dosage forms, in an amount sufficient to provide a desired
unit of dosage.
[0175] Aqueous suspensions may contain a nitrobenzamide metabolite
compound with pharmaceutically acceptable excipients, such as a
suspending agent (e.g., methyl cellulose), a wetting agent (e.g.,
lecithin, lysolecithin and/or a long-chain fatty alcohol), as well
as coloring agents, preservatives, flavoring agents, and the
like.
[0176] In some embodiments, oils or non-aqueous solvents may be
required to bring the metabolite compounds into solution, due to,
for example, the presence of large lipophilic moieties.
Alternatively, emulsions, suspensions, or other preparations, for
example, liposomal preparations, may be used. With respect to
liposomal preparations, any known methods for preparing liposomes
for treatment of a condition may be used. See, for example, Bangham
et al., J. Mol. Biol, 23: 238-252 (1965) and Szoka et al., Proc.
Natl. Acad. Sci. 75: 4194-4198 (1978), incorporated herein by
reference. Ligands may also be attached to the liposomes to direct
these compositions to particular sites of action. Compounds of this
invention may also be integrated into foodstuffs, e.g, cream
cheese, butter, salad dressing, or ice cream to facilitate
solubilization, administration, and/or compliance in certain
patient populations.
[0177] Pharmaceutical preparations for oral use may be obtained as
a solid excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; flavoring
elements, cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinyl pyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. The compounds may also be
formulated as a sustained release preparation.
[0178] Dragee cores can be provided with suitable coatings. For
this purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0179] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for administration.
[0180] For injection, the inhibitors of the present invention may
be formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological saline buffer. Such compositions may also include one
or more excipients, for example, preservatives, solubilizers,
fillers, lubricants, stabilizers, albumin, and the like. Methods of
formulation are known in the art, for example, as disclosed in
Remington's Pharmaceutical Sciences, latest edition, Mack
Publishing Co., Easton P. These compounds may also be formulated
for transmucosal administration, buccal administration, for
administration by inhalation, for parental administration, for
transdermal administration, and rectal administration.
[0181] In addition to the formulations described previously, the
metabolite compounds may also be formulated as a depot preparation.
Such long acting formulations may be administered by implantation
or transcutaneous delivery (for example subcutaneously or
intramuscularly), intramuscular injection or use of a transdermal
patch. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0182] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
present in an effective amount, i.e., in an amount effective to
achieve therapeutic and/or prophylactic benefit in at least one of
the cancers described herein. The actual amount effective for a
particular application will depend on the condition or conditions
being treated, the condition of the subject, the formulation, and
the route of administration, as well as other factors known to
those of skill in the art. Determination of an effective amount of
a nitrobenzamide metabolite compound is well within the
capabilities of those skilled in the art, in light of the
disclosure herein, and will be determined using routine
optimization techniques.
EXAMPLES
Example 1
Identification of Benzamide Metabolites
[0183] Plasma samples were obtained from dog, rat and mouse
studies, in which animals were administered 4-iodo-3-nitrobenzamide
(BA). Plasma and tumor samples were prepared for HPLC injection by
precipitating plasma (50 n1) with 3.times. volumes (150 .mu.l) of
acetonitrile. Tissue samples were prepared for HPLC injection by
adding 1 .mu.l of acetonitrile per mg of tissue, then homogenizing
with an electric homogenizer. Following centrifugation, 150 .mu.l
of each supernatant was evaporated to dryness, reconstituted in 50
.mu.l of 0.2% formic acid in water and analyzed by chromatography
and mass spectrometry techniques (LC/MS/MS conditions). LC/MS/MS
conditions are: HPLC (Shimadzu VP System); Mobile Phase: 0.2%
formic acid in water (A) and 0.18% formic acid in methanol (B);
Column: 1.times.50 mm Thermo BetaBasic C18 column; Injection
volume: 25 .mu.L; Gradient: 0-60% B in 30 minutes; Flow Rate: 100
.mu.L/min; Mass Spectrometer: Applied Biosystems/MDS SCIEX Q-STAR;
Interface: IonSpray split at 1/10; Parent Ion Scan: TOF Positive
from 200-900 amu; Product Ion Scan: TOF Product Ion from 60-900 amu
of most intense Ion in Parent Ion Scan; TOF calibration: Externally
calibrated using Renin Substrate. Results of metabolite
identification from dog and mouse plasma are depicted in Tables
1-3, as follows:
TABLE-US-00001 TABLE 1 Summary of BA metabolite identification by
LC/MS/MS from canine plasma Compound or Retention Approximate Name
Metabolite Time (min) ng/ml .sup.a Parent (292.9 m/z) BSI-201 14.4
1.9 M1 (405 m/z) +112 amu 8.1 8.6 M2 (472 m/z) +179 amu 8.8 75 M3
(213 m/z) -80 amu 13.4 44 M4 (263 m/z) -29 amu 16.8 12 M5 (334 m/z)
+41 amu 19.4 5.0 M6 (569 m/z) +276 amu 19.7 2.5 M7 (413 m/z) +121
amu 21.0 20 .sup.a Approximate ng/ml equivalents were measured
using BSI-201 in the 1 .mu.g/ml spike sample as a reference
standard.
For clarity, the names in Tables 1 above correspond to compounds
disclosed above as follows (name: compound): M2:MS472, M3:MS213 and
M4:MS263.
TABLE-US-00002 TABLE 2 Summary of BA metabolite identification by
LC/MS/MS from murine plasma Compound or Retention Approximate Name
Metabolite Time (min) ng/ml .sup.a Parent (292.9 m/z) BSI-201 14.4
3.7 M1 (278 m/z) -14 amu 8.1 61 M2 (276 m/z) -16 amu 8.6 3.2 M3
(472 m/z) +179 amu 8.9 375 M4 (634 m/z) +341 amu 9.5 31 M5 (262
m/z) -30 amu 10.2 200 M6 (328 m/z) +35 amu 11.3 100 M7 (213 m/z)
-80 amu 13.5 44 M8 (263 m/z) -29 amu 16.8 56 .sup.a Approximate
ng/ml equivalents were measured using BSI-201 in the 1 .mu.g/ml
spike sample as a reference standard.
For clarity, the names in Tables 2 above correspond to compounds
disclosed herein as follows (name:compound):
M3:MS472, M6:MS328, M7:MS213 and M8:MS263.
TABLE-US-00003 [0184] TABLE 3 Summary of BA metabolite
identification by LC/MS/MS from canine plasma and red blood cells
Retention Approximate Approximate Compound or Time .mu.g/ml .sup.a
.mu.g/ml .sup.a Name Metabolite (min) Plasma RBC Parent BSI-201
14.2 2.1 1.6 (292.9 m/z) M1 (645 m/z) +352 amu 7.0 ND 0.8 M2 (472
m/z) +179 amu 8.8 2.3 0.02 M2 (601 m/z) +308 amu 8.9 0.8 0.4 .sup.a
Approximate .mu.g/ml equivalents were measured using the 270 nm UV
peak area of BSI-201 in the 5 .mu.g/ml spike sample as a reference
standard.
For clarity, the names in Tables 3 above correspond to compounds
disclosed herein as follows (name:compound): M2:MS472 and the
second M2:MS601.
Example 2
In Vitro Studies--Cytotoxicity Assays
[0185] Different types of cancer cell lines of different origin or
primary cells may be seeded (5.times.10.sup.4) on 48 wells plate,
or (2.times.10.sup.4) on 96 wells plate. The cells may be cultured
in the appropriate medium. Cultures can be maintained in a
37.degree. C. incubator in a humidified atmosphere of 95%
O.sub.2/5% CO.sub.2. After the cells are seeded (24 hours), medium
is removed and replaced with culture medium in the presence of
various concentrations of INO2BA or INH2BP, in the presence or not
of 200 .mu.M BSO. After 6 days of incubation at 37.degree. C., cell
viability is measured using the Cell Titer-Blue, Cell Viability
Assay (Promega) (See O'Brien, J. et al. (2000) Investigation of the
Alamar Blue (resazurin) fluorescent dye for the assessment of
mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421-26 and
Gonzalez, R. J. and Tarloff, J. B. (2001) Evaluation of hepatic
subcellular fractions for Alamar Blue and MTT reductase). This
assay incorporates a fluorometric/colorometric growth indicator
based on detection by vital dye reduction. Cytotoxicity is measured
by growth inhibition.
[0186] Cytotoxicity may also be assessed by counting the number of
viable cells. Cells were harvested by washing the monolayer with
PBS, followed by a brief incubation in 0.25% trypsin and 0.02%
EDTA. The cells are then collected, washed twice by centrifugation
and resuspended in PBS. Cell number and viability is determined by
staining a small volume of cell suspension with a 0.2% trypan blue
saline solution and examining the cells in a hemocytometer. See
Kerley-Hamilton et al. (2005) p53-dominant transcriptional response
to cisplatin in testicular germ cell tumor-derived human embryonal
carcinoma and Cheol et al. (2005) Induction of apoptosis and
inhibition of cyclooxygenase-2 expression by
N-methyl-N'-nitro-N-nitrosoguanidine in human leukemia cells.
Example 3
Cell Proliferation Measured with BrdU-ELISA
[0187] The cells may be incubated in the presence of various
concentrations of the test substance (drugs) in a black 96-well
MultiPlate (tissue culture grade; flat, clear bottom) at a final
volume of 100 .mu.l/well in a humidified atmosphere at 37.degree.
C. 10 .mu.l/well BrdU labeling solution was added if the cells were
cultured in 100 .mu.l/well (final concentration: 10 .mu.M BrdU) and
the cells are reincubated for additional 2 to 24 hours at
37.degree. C. (if the cells were cultured in 200 .mu.l/well, 20
.mu.l/well BrdU labeling solution was added). The MP is centrifuged
at 300.times.g for 10 min and the labeling medium was removed with
suction using a canulla. The cells are dried using a hair-dryer for
about 15 min or, alternatively, at 60.degree. C. for 1 h. 200
ul/well FixDenat is added to the cells and incubated for 30 min at
15-25.degree. C. FixDenat solution is removed thoroughly by
flicking off and tapping. 100 .mu.l/well Anti-BrdU-POD working
solution is added and incubated for approx. 90 min at 15-25.degree.
C. Alternatively, this incubation period can be varied between
30-120 min, depending on individual requirements. Antibody
conjugate is removed by flicking off and wells were rinsed three
times with 200-300 .mu.l/well washing solution. Washing solution is
removed by tapping. Then 100 .mu.l/well substrate solution is added
to each well. The light emission of the samples can be measured in
a microplate luminometer with photomultiplier.
Example 4
In Vivo Implantation and Tumor Growth
[0188] 100 female NU/NU-nuBR mice (Charles River, 5-6 wks) can be
implanted with 0.72 mg 17 .beta.-estradiol (human) pellets, ear
tagged using clips and weighed 24-48 hours prior to tumor cell
implantation. Tumor cells, BT474, (2.times.10.sup.7 cells/mouse)
are injected into the subscapular mammary fat pad (0.2 ml volume).
Caliper measurements begin on day 21 and three times weekly
thereafter (Mon, Wed, Fri). Animals are segregated according to the
presence and absence of tumor and then by tumor volume. Animals are
weighed twice weekly beginning the 3.sup.rd week post implantation
(Mon and Fri.). Drug treatment is started when tumor sizes were
150-250 mm.sup.3 (L*W*H). Drug and vehicle administration is BID by
gavage (BP+BSO) and SID by IP (BA) for five days. There is a two
day rest period before the next cycle begins. Animals may receive
three cycles (5 days each) unless there was unexpected toxicity.
Body weight loss that exceeded 15% of initial values or display of
certain symptoms may be used as criteria for animal euthanasia.
Drug is administered by gavage and IP in volume of 5 ml/kg. Drug
and vehicle are stored at 4.degree. C. in foil-covered bottle.
TABLE-US-00004 Study Design Implant # condi- Cells # Tumors Group
tions implanted Mice needed Treatment (BID) 1 sc 2 .times. 10.sup.7
20 10 none 2 sc 2 .times. 10.sup.7 20 10 Vehicle (10% DMSO in
saline) 3 sc 2 .times. 10.sup.7 20 10 BP + BSO (175 mg/kg + 220
mg/kg) P.O. 4 sc 2 .times. 10.sup.7 20 10 BA (5 g/kg) I.P. 5 sc 2
.times. 10.sup.7 20 10 Combo* (30 mg/kg) I.P. and P.O. *combination
of BP + BSO and BA
Example 4
Xenograft Studies
[0189] The effects of the metabolite compounds can be evaluated on
ovarian human cancer cells (OVCAR) xenografts in nude mice.
[0190] Female NU/NU 37-BU-04-BAC mice (Charles River, 5-6 weeks)
are ear tagged using clips and weighed 24-48 hours prior to tumor
cell implantation. Tumor cells Ovcar3 (5.times.10.sup.6
cells/mouse) are implanted subcutaneously into the subscapular
mammary fat pad of female nude mice hosts. Caliper measurement
begin on day 7 post tumor cells implantation and 2 times weekly
thereafter (Mon and Fri). Animals are segregated according to the
presence or absence of tumor and then tumor volume. Animals are
weighed once a week. Drug treatment starts when sizes were 0.4-0.5
cm in largest diameter. 4-Iodo-3-nitrobenzamide (BA) (in 50 .mu.L
of 100% DMSO/mouse and vehicle (50 .mu.L of 100% DMSO/mouse) are
injected by IP twice per day for five days. There is a two day rest
period before the next cycle begins.
TABLE-US-00005 STUDY DESIGN Implant Cells Group conditions
implanted TREATMENT 1 SC 5 .times. 10.sup.6 Vehicle .times. 2 (50
.mu.l of 100% DMSO/mouse) 2 SC 5 .times. 10.sup.6 BA 25 mg/kg
.times. 2/day (in 50 .mu.l of 100% DMSO/mouse) 3 SC 5 .times.
10.sup.6 BA 50 mg/kg .times. 2/day (in 50 .mu.l of 100% DMSO/mouse)
4 SC 5 .times. 10.sup.6 Nothing (Control)
Example 5
Evaluating the Efficacy of a Metabolite Compound
[0191] General Methods
[0192] MDA MB 231 human mammary cancer cells may be injected
subcutaneously into the right flank of female nude mice. For Task
1, BP may be administered for 5 consecutive days prior to tumor
cell implantation, and drug administration continued 5 days a week
for 4-8 weeks thereafter. For Task 2, cancer cells are injected
when the tumors reached a mean tumor volume of 50-60 mm.sup.3, and
mice are divided into groups of eight and treated with corn oil:PEG
400 (control), BP, or CTX (MDA MB 231 positive control). Tumor
volumes are monitored for 90 days (for MDA MB 231) after the
beginning of treatment.
Cell Lines
[0193] MDA MB 231 is a human mammary cancer cell line that was
established in 1973 from a pleural effusion of a patient who had
been treated with 5-FU, doxorubicin, methotrexate, and CTX in the 3
months before the cell line was initiated. This line is estrogen
receptor negative and has been used in screening anticancer drugs
that are not targeted as hormone antagonists. MDA MB 231 was grown
in Dulbecco's modified Eagle medium (DMEM) with 1.5 g
NaHCO.sub.3/L, 10% fetal bovine serum (FBS), and 2 mM L-glutamine
and was kept at 37.degree. C. in a humidified 5% CO.sub.2/air
incubator. Antibiotics were not added to the medium.
Animal Tumor Model
[0194] Mice
[0195] Female CB.17 SCID mice (Charles River) were 8-11 weeks old,
and had a body weight (BW) range of 12.6-23.0 g on D1 of the study.
The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl)
and NIH 31 Modified and Irradiated Lab Diet.RTM. consisting of
18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice
were housed on irradiated ALPHA-dri.RTM. Bed-O-Cobs.RTM. Laboratory
Animal Bedding in static microisolators on a 12-hour light cycle at
21-22.degree. C. (70-72.degree. F.) and 40-60% humidity in the
laboratory accredited by Association for Assessment and
Accreditation of Laboratory (AAALAC) International, which assures
compliance with accepted standards for the care and use of
laboratory animals.
[0196] Tumor Implantation
[0197] The human OVCAR-3 (NIH-OVCAR-3) ovarian adenocarcinoma
utilized in the study was maintained in athymic nude mice by serial
engraftment. A tumor fragment (1 mm.sup.3) was implanted
subcutaneously (s.c.) into the right flank of each test mouse.
Tumors were monitored twice weekly and then daily as their volumes
approached 80-120 mm.sup.3. On D1 of the study, animals were sorted
into treatment groups with tumor sizes of 63-221 mm.sup.3 and group
mean tumor sizes of .about.105 mm.sup.3. [0198] Tumor size, in
mm.sup.3, was calculated from:
[0198] Tumor Volume = w 2 .times. l 2 ##EQU00001##
[0199] Tumor weight may be estimated with the assumption that 1 mg
is equivalent to 1 mm.sup.3 of tumor volume.
[0200] Treatment
[0201] Mice were sorted into nine groups (n=10) and treated in
accordance with the protocol. Oral group received BA orally (p.o.)
twice daily from D1 p.m. until D68 a.m. (b.i.d. (twice daily) to
end). Alzet model osmotic pumps were implanted on Days 1, 15, and
29. The pumps were pre-warmed for .about.1 hour at 37.degree. C.,
and then implanted s.c. in the left flanks of isofluorane
anesthetized mice. Each pump delivered a total dose of 25
mg/kg/week of BA over 14 days.
[0202] Endpoint
[0203] Tumors were calipered twice weekly for the duration of the
study. Each animal was euthanized when its neoplasm reached the
predetermined endpoint size (1,000 mm.sup.3). The time to endpoint
(TTE) for each mouse was calculated by the following equation:
T T E = log 10 ( endpoint volume ) - b m ##EQU00002##
where TTE is expressed in days, endpoint volume is in mm3, b is the
intercept, and m is the slope of the line obtained by linear
regression of a log-transformed tumor growth data set.
[0204] The data set is comprised of the first observation that
exceeded the study endpoint volume and the three consecutive
observations that immediately preceded the attainment of the
endpoint volume. The calculated TTE is usually less than the day on
which an animal is euthanized for tumor size. Animals that do not
reach the endpoint are euthanized at the end of the study, and
assigned a TTE value equal to the last day (68 days). Treatment
efficacy was determined from tumor growth delay (TGD), which is
defined as the increase in the median TTE for a treatment group
compared to the control group: TGD=T-C, expressed in days, or as a
percentage of the median TTE of the control group:
% T G D = T - C C .times. 100 ##EQU00003##
where: T=median TTE for a treatment group, C=median TTE for control
Group 1.
[0205] The results of these studies are shown in FIG. 4.
[0206] Swiss NCr nude (nu/nu) female mice, age 4-5 weeks, are
commercially available from Taconic (Germantown, N.Y.). The animals
are housed three per cage in sterile filter-topped cages in a
barrier clean room purchased from Bio Bubble, Inc. (Fort Collins,
Colo.). Upon arrival, they are quarantined for four working days
before use. Temperature was maintained at 72.+-.5.degree. F. and
relative humidity at 35-70%, and a 12-hr light/dark cycle was used.
The mice are fed sterile, autoclavable, certified Purina rodent
chow ad libitum. Drinking water is acidified and autoclaved, and
the source water is recirculated, deionized, UV-treated, and 5
.mu.m filtered.
[0207] After the animals are released from quarantine, the mice are
injected subcutaneously in the right flank with 1 or
5.times.10.sup.6 MDA MB 231 cells (0.1-ml injection volume). The
mice for Task 1 received pretreatment for 5 days before cell
injection. Tumor dimensions and body weight are measured twice
weekly. Vernier calipers are used to measure tumors in three
planes, and tumor volume (V) is calculated as follows:
V=.pi.(x.times.y.times.z)/6, where x, y, and z are the tumor
measurements minus skin thickness. At the end of the experiment,
the mice are sacrificed by CO.sub.2 inhalation followed by cervical
dislocation.
Pharmaceuticals
[0208] MS472 can be made up in corn oil:PEG 400 (2:1, V/V) at
concentrations of 30 mg/ml and 100 mg/ml. The drug may be a
suspension at these concentrations. Positive control drugs are made
up on phosphate buffered saline (PBS) and CTX at 15 mg/ml. Both
drugs can be filter-sterilized (0.2-.mu.m filter) before use.
Treatment Protocol
[0209] For Task 1, mice to be implanted with MDA MB 231 tumor cells
can be pretreated for 5 days with MS472 (300 or 1000 mg/kg), and
following subcutaneous injection of the cell suspension, drug
treatment is continued 5 days a week (Monday through Friday) for a
minimum of 4 weeks.
[0210] For Task 2, after the tumor volumes reach a predetermined
size (mean tumor volume 50-60 mm3), mice are divided into treatment
groups of eight mice each. All treatments of BP are administered
five times per week (Monday through Friday) for at least 4 weeks.
CTX is administered intraperitoneally one time only at a dose of
150 mg/kg. All BP treatments are administered orally; the dosage
was 1000 or 2000 mg/kg for those implanted with MDA MB 231 cells.
For each task, all treatments would begin on the same day.
[0211] The tumors would be measured twice weekly for at least 9
weeks (MDA MB 231) after the first treatment. The mean tumor volume
for each group is calculated for each time point. Comparisons
between groups at specific times are made using an unpaired,
two-tailed t-test, and the results are analyzed using analysis of
variance (ANOVA). For Task 2, individual tumor volumes (V) are
expressed as a fraction of the tumor volume on Day 0, the first day
of treatment (V0). For each group, the mean of the ratio V/V0 is
plotted as a function of time after treatment. Response to
treatment is measured in two ways, depending on the tumor response
to treatment. The tumor volume doubling time (VDT) and volume
quadrupling time (VQT) is determined for each tumor by linear
regressions on the plot of time as a function of log(tumor volume)
in groups where there is a response to treatment. Tumor growth
delay for each treatment group is determined and comparisons
between groups are analyzed using ANOVA.
[0212] Systemic toxicity is assessed from reductions in body weight
after treatment. The mice are sacrificed at the end of the
follow-up period, or earlier in their tumor volumes reached 1600
mm.sup.3 or the tumors ulcerated.
Statistical Analysis
[0213] Statistical analysis can be performed using InStat (Graphpad
Software, San Diego, Calif.).
Tumor Growth
[0214] MDA MB 231 tumors may be measurable within 3 weeks of tumor
cell injection and usually grow more slowly, with a doubling time
of 7 days. Values may be calculated from the control group. Mean
tumor volumes and body weights at the start of treatment can be
shown under the formats depicted in Table 4 for Task 1 and Table 5
for Task 2.
TABLE-US-00006 TABLE 4 MOUSE PARAMETERS AT THE START OF TREATMENT -
TASK 1 Tumor Volume Mouse Weight Treatment Group (mm.sup.3 .+-.
SEM*) (g .+-. SEM*) PBS (control) 0 24.0 .+-. 0.8 MDA MB 231 300
mg/kg 0 24.6 .+-. 0.9 1000 mg/kg 0 23.6 .+-. 07 *SEM = Standard
error of the mean.
TABLE-US-00007 TABLE 5 MOUSE PARAMETERS AT THE START OF TREATMENT -
TASK 2 Treatment Group Tumor Volume Mouse Weight MDA MB 231
(mm.sup.3 .+-. SEM*) (g .+-. SEM*) Corn oil (control) 19.1 .+-. 5.1
24.4 .+-. 0.54 1000 mg/kg 24.4 .+-. 5.8 24.5 .+-. 0.7 2000 mg/kg
23.5 .+-. 5.8 23.0 .+-. 0.8 CTX, 150 mg/kg 24.0 .+-. 4.4 23.8 .+-.
0.4 *SEM = Standard error of the mean
[0215] The above examples are in no way intended to limit the scope
of the instant invention. Further, it can be appreciated to one of
ordinary skill in the art that many changes and modifications can
be made thereto without departing from the spirit or scope of the
appended claims, and such changes and modifications are
contemplated within the scope of the instant invention.
Example 6
Cell Cycle Analysis
[0216] HCT116 and Hela cells were obtained from ATCC. For DNA
content analysis, 2.times.10.sup.5 cells were washed twice with PBS
and fixed in 70% ethanol. Cells were treated with 100 units/mL
RNase A for 20 minutes at 37.degree. C., resuspended in cold PBS
containing Alexa Fluor.RTM. 405 fluorescent stain (Invitrogen)
according to the manufacturer's protocol. Cells were analyzed by
flow cytometry. FIG. 1 illustrates (FACS dot plots and histograms)
cell-cycle analysis in HTC116 cells treated with PARP-1 inhibitor
(4-iodo-3-nitrobenzamide or "BA") for 19 hours. BA treatment caused
an increase in the number of cells in the G1 phase (48.6%) as
compared to control (18.6%) with a concomitant decrease of S phase
cell cycle.
[0217] For DNA replication analysis, 2.times.105 cells were
incubated with 50 .mu.mol/L bromodeoxyuridine (BrdUrd) for 30
minutes. Cells were fixed in 70% ethanol and BrdUrd incorporation
was determined by flow cytometric analysis using an
anti-BrdUrd-FITC antibody (Becton Dickinson, Franklin Lakes, N.J.)
according to the manufacturer's protocol. To assess the degree of
G2/M checkpoint, mitotic cells were detected by flow cytometry
using the mitosis-specific antibody GF-7. Fixed cells were
incubated for 30 minutes with GF7-phycoerythrin (PE) antibody (BD
Biosciences Pharmingen), washed with PBS and analyzed by flow
cytometry. FIG. 2 compares the results obtained with BA, BNO
(4-iodo-3-nitrosobenzamide) and BNHOH
(4-iodo-3-hydroxyaminobenzamide) at 24 hr. incubation; FIG. 3 shows
the results for BA, BNO and BNHOH at 72 hr.
[0218] Image analysis and microscopy. Cells grown on coverslips
were fixed with 4% paraformaldehyde in PBS for 10 minutes and
permeabilized in 70% ethanol. After washing, the coverslips were
mounted on slides in 50% PBS/50% glycerol. Images were analyzed by
microscopy.
[0219] MDR1 Expression FACS Assay.
[0220] KB-V1 is a vinblastine resistant clone of KB-3-1 human
carcinoma cell line (Fojo, A. T., J. Whang-Peng, M. M. Gottesman,
and I. Pastan. 1985. Amplification of DNA sequences in human
multidrugresistant KB carcinoma cells. Proc. Natl. Acad. Sci. USA
82:7661-7665., Shen, D.-W, C. Cardarelli, J. Twang, M Cornwell, N.
Richert, S. Ishii, I. Pastan, and M. M. Gottesman. 1986. Multiple
drug-resistant human KB carcinoma cells independently selected for
high-level resistance to colchicine, Adriamycin, or vinblastine
show changes in expression of specific proteins. J. Biol. Chem.
261:7762-7770). Cells were propagated in Eagle minimal essential
medium with 10% fetal bovine serum.
[0221] For MDR1 staining cells were harvested by tripsinization,
washed in Iscove's Modified Dulbecco's Medium supplemented with 5%
FBS and then resuspended in of IMDM/5% FBS containing anti-Mdr1
antibody UIC2. After incubation with the primary antibody cells
were washed and then incubated with a secondary antibody conjugated
to phycoerythrin (PE) antibody (BD Biosciences Pharmingen). The
antibody-treated cells were washed and then analyzed on a
FACSCalibur.RTM. instrument (Becton Dickinson). Controls used in
the FACS analysis were cells that had not been incubated with
antibody, cells that had been incubated with an isotype control
antibody. Triplicate samples were assayed, and their histograms
were analyzed. The median fluorescence was plotted on a log scale.
FIGS. 6A and 6B show the level of MDR1 in KB 3-1 and KB V-1 cells,
respectively: Thus the FACS histograms demonstrate the
overexpression of MDR1 in KB V-1 cells. Overexpression of MDR1 is
associated with multidrug resistance.
[0222] Colony-Forming Assay
[0223] Cells were trypsinized, counted, and diluted to a final
concentration of 10.sup.6 cells/ml. Colony-forming assays were
performed by plating an appropriate number of cells into culture
dishes in triplicates and treated with 0-100 .mu.M of a compound.
After 14 days, cells were fixed and stained with 1% crystal violet,
and colonies were counted. FIG. 5C shows the results of this
experiment in KB 3-1 and KB V-1 cells. As can be seen in FIG. 5C,
BA reduces the number of cell colonies in a dose-dependant manner
for both MDR1 overexpressing and MDR1 normal cells. As MDR1
overexpression is associated with multidrug resistance, this result
suggests that BA will be effective against multidrug resistant
tumors.
[0224] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *