U.S. patent application number 12/114601 was filed with the patent office on 2008-11-06 for fulvene and fulvalene analogs and their use in treating cancers.
Invention is credited to Jack L. Arbiser.
Application Number | 20080275016 12/114601 |
Document ID | / |
Family ID | 39939979 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080275016 |
Kind Code |
A1 |
Arbiser; Jack L. |
November 6, 2008 |
FULVENE AND FULVALENE ANALOGS AND THEIR USE IN TREATING CANCERS
Abstract
Compounds, pharmaceutical compositions including the compounds,
and methods of preparation and use thereof are disclosed. The
compounds are fulvene and/or fulvalene analogs. The compounds and
compositions can be used to treat and/or prevent a wide variety of
cancers, including drug resistant cancers, as well as numerous
inflammatory, degenerative and vascular diseases, including various
ocular diseases. Representative fulvene and/or fulvalene analogs
include fulvene and fulvalene analogues of various dyes, hormones,
sugars, peptides, oligonucleotides, amino acids, nucleotides,
nucleosides, and polyols. The compounds are believed to function,
at least, by inhibiting Nox or ROS. In some embodiments, the Nox is
one that is selectively expressed in cancer cells over normal
cells, or one that is expressed in higher amounts in cancer cells
over normal cells. Thus, the compounds are novel therapeutic agents
for a variety of cancers and other diseases.
Inventors: |
Arbiser; Jack L.; (Atlanta,
GA) |
Correspondence
Address: |
INTELLECTUAL PROPERTY / TECHNOLOGY LAW
PO BOX 14329
RESEARCH TRIANGLE PARK
NC
27709
US
|
Family ID: |
39939979 |
Appl. No.: |
12/114601 |
Filed: |
May 2, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61047717 |
Apr 24, 2008 |
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60934381 |
Jun 13, 2007 |
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60927416 |
May 3, 2007 |
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Current U.S.
Class: |
514/182 ;
514/239.5; 514/334; 514/396; 514/411; 514/452; 514/474; 514/616;
514/730; 514/765; 548/335.1; 548/445; 549/475; 552/544;
564/153 |
Current CPC
Class: |
C07C 43/23 20130101;
C07C 2603/24 20170501; C07D 213/53 20130101; C07D 261/08 20130101;
A61K 31/444 20130101; C07C 237/26 20130101; C07C 2603/40 20170501;
C07D 407/04 20130101; A61K 31/403 20130101; A61P 25/00 20180101;
C07C 13/58 20130101; C07D 471/10 20130101; C07D 311/58 20130101;
C07D 233/58 20130101; C07D 295/03 20130101; A61K 31/045 20130101;
C07C 25/18 20130101; C07C 2603/46 20170501; C07C 39/23 20130101;
C07C 211/50 20130101; C07C 215/46 20130101; C07D 295/135 20130101;
A61P 19/10 20180101; A61P 27/04 20180101; C07C 251/28 20130101;
C07C 2601/10 20170501; A61K 31/167 20130101; A61P 39/06 20180101;
C07C 13/465 20130101; C07C 13/66 20130101; C07C 43/215 20130101;
C07D 233/68 20130101; A61P 43/00 20180101; C07C 13/40 20130101;
C07D 209/86 20130101; A61K 31/341 20130101; A61P 11/06 20180101;
C07C 275/64 20130101; A61P 27/06 20180101; C07D 473/34 20130101;
C07D 307/58 20130101; A61K 31/573 20130101; C07C 13/28 20130101;
C07C 35/32 20130101; C07D 311/60 20130101; A61P 17/06 20180101;
C07C 2602/08 20170501; C07D 277/40 20130101; A61K 31/015 20130101;
C07C 233/44 20130101; C07D 209/14 20130101; C07D 309/38 20130101;
A61P 19/02 20180101; A61P 27/14 20180101; C07D 209/82 20130101;
C07D 311/82 20130101; A61P 27/12 20180101; A61P 1/00 20180101; A61P
35/00 20180101; A61P 27/02 20180101; A61K 31/357 20130101; C07C
2602/42 20170501; A61K 31/4164 20130101; A61K 31/5375 20130101;
A61P 9/10 20180101; C07C 69/608 20130101; A61P 17/00 20180101 |
Class at
Publication: |
514/182 ;
514/730; 514/411; 514/396; 514/765; 514/474; 514/452; 514/334;
514/616; 514/239.5; 552/544; 548/335.1; 548/445; 549/475;
564/153 |
International
Class: |
A61K 31/573 20060101
A61K031/573; A61K 31/045 20060101 A61K031/045; A61K 31/403 20060101
A61K031/403; A61K 31/4164 20060101 A61K031/4164; A61K 31/015
20060101 A61K031/015; A61K 31/341 20060101 A61K031/341; A61K 31/357
20060101 A61K031/357; A61P 35/00 20060101 A61P035/00; A61K 31/444
20060101 A61K031/444; A61K 31/167 20060101 A61K031/167; A61K
31/5375 20060101 A61K031/5375; C07J 9/00 20060101 C07J009/00; C07D
233/58 20060101 C07D233/58; C07D 209/58 20060101 C07D209/58; C07D
307/58 20060101 C07D307/58; C07C 233/44 20060101 C07C233/44 |
Goverment Interests
[0002] This invention was made with government support under grant
number AR47901 awarded by National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method for treating a cancer in a mammal, comprising
administering to the mammal a therapeutically effective amount of a
composition comprising a fulvene or fulvalene-containing
therapeutic agent in an amount sufficient to inhibit a
therapeutically significant amount of NOX or ROS expression, such
that the growth of the tumor is at least partially inhibited.
2. The method of claim 1 wherein the mammal is a human.
3. The method of claim 1 wherein the cancer is selected from a
group consisting of hemangioma, melanoma, rectal carcinoma, colon
carcinoma, breast carcinoma, ovarian carcinoma, small cell lung
carcinoma, colon carcinoma, chronic lymphocytic carcinoma, hairy
cell leukemia, osophogeal carcinoma, prostate carcinoma, breast
cancer, myeloma, and lymphoma.
4. The method of claim 1, wherein the cancer is a metastasized
cancer.
5. The method of claim 1 wherein the human is immunosuppressed by
reason of having undergone anti-cancer therapy prior to
administration of the composition.
6. The method of claim 1, wherein the cancer is manifested in the
form of a solid tumor having cells that express one or more NOX
enzymes at concentrations higher than those expressed in a
non-cancer cell.
7. The method of claim 6, wherein the tumor is a tumor of
epithelial tissue, lymphoid tissue, connective tissue, bone, or the
central nervous system.
8. The method of claim 1, wherein the compounds are administered
parenterally, orally, or directly into the tumor.
9. The method of claim 1, wherein the compounds are administered
via an implanted device.
10. The method of claim 1, wherein the administration is provided
using a sustained release formulation.
11. The method of claim 1, wherein compound has one of the
following formulas: ##STR00027## wherein: X is O, S, NH or
CH.sub.2, x and y are integers of from 0 to 3, G is selected from
the group consisting of C.sub.1-6 alkyl, alkenyl, heterocyclyl,
aryl, heteroaryl, halo, --OR', --NR'R'', --CF.sub.3, --CN,
--NO.sub.2, --C.sub.2R', --SR', --N.sub.3, --C(.dbd.O)NR'R'',
--NR'C(.dbd.O) R'', --C(.dbd.O)R', --C(.dbd.O)OR', --OC(.dbd.O)R',
--OC(.dbd.O)NR'R'', --NR'C(.dbd.O)OR'', --SO.sub.2R',
--SO.sub.2NR'R'', and --NR'SO.sub.2R'', where R' and R'' are
individually hydrogen, C.sub.1-6 alkyl, cycloalkyl, heterocyclyl,
aryl, and arylalkyl.
12. The method of claim 1, wherein the compound has one of the
following formulas: ##STR00028## with the proviso that at least one
of R is other than H.
13. The method of claim 1, wherein the compound is selected from
the group consisting of: ##STR00029## ##STR00030## ##STR00031##
##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036##
##STR00037## ##STR00038##
14. A method for treating an ocular disorder, comprising
administering to a mammal a fulvene or fulvalene-containing
compound in an amount sufficient to treat the ocular disorder.
15. The method of claim 14, wherein the amount of the fulvene or
fulvalene-containing compound is sufficient to inhibit a
therapeutically significant amount of NADPH oxidase.
16. The method of claim 14, wherein the mammal is a human.
17. The method of claim 14, wherein the ocular disorder is selected
from the group consisting of wet or dry age-related macular
degeneration (AMD), diabetic retinopathy (DR), glaucoma,
neovascular glaucoma, retinal vasculitis, uveitis, such as
posterior uveitis, conjunctivitis, retinitis secondary to glaucoma,
episcleritis, scleritis, optic neuritis, retrobulbar neuritis,
cataract, ocular inflammation following ocular surgery, ocular
inflammation resulting from physical eye trauma, ocular allergy,
and dry eye.
18. The method of claim 14, wherein the compound is administered by
topical administration in the form of eye drops or other suitable
topical formulations for direct administration to the eye.
19. The method of claim 14, wherein the compound is administered by
subconjunctival injection, periocular injection, or intravitreal
injection.
20. The method of claim 14, wherein the compound is administered by
surgical implant.
21. The method of claim 14, wherein the compound is administered by
systemic administration or by iontophoresis.
22. The method of claim 14, wherein the administration is provided
using a sustained release formulation.
23. The method of claim 14, wherein compound has one of the
following formulae: ##STR00039## wherein: X is O, S, NH or
CH.sub.2, x and y are integers of from 0 to 3, G is selected from
the group consisting of C.sub.1-6 alkyl, alkenyl, heterocyclyl,
aryl, heteroaryl, halo, --OR', --NR'R'', --CF.sub.3, --CN,
--NO.sub.2, --C.sub.2R', --SR', --N.sub.3, --C(.dbd.O)NR'R'',
--NR'C(.dbd.O) R'', --C(.dbd.O)R', --C(.dbd.O)OR', --OC(.dbd.O)R',
--OC(.dbd.O)NR'R'', --NR'C(.dbd.O)OR'', --SO.sub.2R',
--SO.sub.2NR'R'', and --NR'SO.sub.2R'', where R' and R'' are
individually hydrogen, C.sub.1-6 alkyl, cycloalkyl, heterocyclyl,
aryl, and arylalkyl.
24. The method of claim 14, wherein the compound has one of the
following formulae: ##STR00040## with the proviso that at least one
of R is other than H.
25. The method of claim 14, wherein the compound is selected from
the group consisting of: ##STR00041## ##STR00042## ##STR00043##
##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048##
##STR00049## ##STR00050##
26. A compound of one of the following formulae: ##STR00051##
wherein: X is O, S, NH or CH.sub.2, x and y are integers of from 0
to 3, G is selected from the group consisting of C.sub.1-6 alkyl,
alkenyl, heterocyclyl, aryl, heteroaryl, halo, --OR', --NR'R'',
--CF.sub.3, --CN, --NO.sub.2, --C.sub.2R', --SR', --N.sub.3,
--C(.dbd.O)NR'R'', --NR'C(.dbd.O) R'', --C(.dbd.O)R',
--C(.dbd.O)OR', --OC(.dbd.O)R', --OC(.dbd.O)NR'R'',
--NR'C(.dbd.O)OR'', --SO.sub.2R', --SO.sub.2NR'R'', and
--NR'SO.sub.2R'', where R' and R'' are individually hydrogen,
C.sub.1-6 alkyl, cycloalkyl, heterocyclyl, aryl, and arylalkyl.
27. A compound of any of the following formulae: ##STR00052## with
the proviso that at least one of R is other than H.
28. A compound selected from the group consisting of: ##STR00053##
##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##
##STR00059## ##STR00060## ##STR00061## ##STR00062##
29. A compound of the formulae: ##STR00063## wherein R' is selected
from the group consisting of H, C.sub.1-6 straight chain, branched,
or cyclic alkyl, aryl, arylalkyl, or alkylaryl.
Description
STATEMENT OF RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional Patent
Application Nos. (A) 60/927,416 filed on May 3, 2007, (B)
60/934,381 filed on Jun. 13, 2007, and (C) 61/047,717 filed on Apr.
24, 2008.
FIELD OF THE INVENTION
[0003] The present invention relates to novel methods and
compositions for the treatment of primary and metastatic cancers.
These methods and compositions use fulvenes and/or fulvalenes.
These compounds, and pharmaceutical compositions including the
compounds, are particularly useful for treating primary and
metastatic cancers in humans. The invention also encompasses the
varying modes of administration of the therapeutic compounds or
compositions.
BACKGROUND OF THE INVENTION
[0004] Cancer is characterized primarily by an increase in the
number of abnormal cells derived from a given normal tissue,
invasion of adjacent tissues by these abnormal cells, and lymphatic
or blood-borne spread of malignant cells to regional lymph nodes
and to distant sites (metastasis). Cancer is a multistep process,
beginning with minor preneoplastic changes, which may under certain
conditions progress to neoplasia. Malignant endothelial tumors
arise in the setting of autocrine loops involving vascular
endothelial growth factor (VEGF) and its major mitogenic receptor
vascular endothelial growth factor receptor 2.
[0005] Reactive oxygen species (ROS) are believed to be mediators
of growth and angiogenesis in cancer. Increased ROS often
correlates with cell growth, e.g., Ras-transformed cells and cells
treated with growth factors. While non-transformed cells respond to
growth factors/cytokines with the regulated production of ROS,
tumor cells in culture frequently overproduce H.sub.2O.sub.2.
[0006] NAD(P)H oxidase (Nox) is a cell surface protein with
hydroquinone (NADH) oxidase and protein disulfide-thiol interchange
activities. In general, most forms of the enzyme can utilize either
NADH or NADPH equally efficiently. There are many forms of Nox,
including Nox 1-5, Dual oxidase 1 and 2 (Duox 1 and 2), as well as
p22(phox), p47(phox) and the small G-protein Rac1.
[0007] Nox are believed to account for increased levels of ROS in
certain cancers. Reactive oxygen-generating Nox enzymes are
implicated in the angiogenic switch, and Nox inhibitors have an
effect on ang-2 production in vitro and on bEnd.3 tumor growth in
vivo. ang-2 production can be inhibited pharmacologically using Nox
enzyme inhibitors, which nearly abolishes bEnd.3 hemangioma growth
in vivo. Signal-transduction blockade targeting ang-2 production
may therefore be useful for treating human hemangiomas in vivo.
Journal of Investigative Dermatology advance online publication, 1
Jun. 2006; doi:10.1038/sj.jid.5700413.
[0008] With respect to specific Nox enzymes, it has been shown that
transfection of Nox1 into a prostate cancer cell line dramatically
enhanced tumor growth (Arbiser et al.: PNAS 99:715-720, 2001), and
prostate tumors show increased H.sub.2O.sub.2 levels. Further,
prostate tumors were recently found to show increased levels of
Nox1 and hydrogen peroxide (Lim et al., Prostate. Feb. 1,
2005;62(2):200-7). Nox1-dependent superoxide production has also
been shown to control colon adenocarcinoma cell migration (Sadok et
al., Biochim. Biophys. Acta. 1783(1):23-33 (January 2008). Sadok
showed that Nox1 inhibition or down-regulation led to a decrease of
superoxide production and alpha 2 beta 1 integrin membrane
availability. Thus, there is a correlation between Nox protein
levels and ROS in prostate cancer, and increased
Nox1/H.sub.2O.sub.2 correlates with increased tumorigenicity.
[0009] Nox4 is believed to be implicated in inhibition of apoptosis
in cancer cells, such as pancreatic cancer cells (Vaquero et al., J
Biol Chem. Aug. 13, 2004;279(33):34643-54). Vaquero suggested that
growth factor-induced ROS produced by NAD(P)H oxidase (probably
Nox4) protects pancreatic cancer cells from apoptosis, and that
transfection with a Nox4 antisense oligonucleotide inhibited
NAD(P)H oxidase activity and ROS production in certain pancreatic
cells (i.e., MIA PaCa-2 and PANC-1 cells), and stimulated apoptosis
in these cells.
[0010] Akt, a signaling molecule downstream of PI3K, is known to
induce expression of the ROS-generating enzyme Nox4. One study
introduced Akt into a radial growth WM35 melanoma in order to test
whether Akt overexpression was sufficient to transform the cells
from radial growth to vertical growth. Overexpression of Akt led to
upregulation of VEGF, increased production of superoxide ROS, and
the switch to a more pronounced glycolytic metabolism. Subcutaneous
implantation of WM35 cells overexpressing Akt led to rapidly
growing tumors in vivo, while vector control cells did not form
tumors. Arbiser et al., J. Clini. Invest. 117(10): 2762-2765
(2007). This data supports the premise that inhibition of Akt can
inhibit downstream production of Nox 4, which then would inhibit
superoxide generation, and therefore treat melanoma.
[0011] Duox 1 and 2 are the major Nox species in airway endothelia,
and are believed to be one of the main sources for reactive oxygen
species production in the airway (Luxen et al., Cancer Res. Feb.
15, 2008;68(4):1037-45). Accordingly, inhibition of these enzymes
may be useful in treating human lung cancer.
[0012] Some authors have characterized Nox as falling into two
categories. One is hormone-insensitive and drug-responsive (i.e.,
by quinine-site inhibitors such as capsaicin or the antitumor
sulfonylurea, LY181984), designated "tNox," which is specific to
cancer cells. The other is a drug-indifferent constituted form
associated with the plasma membrane of non-transformed cells,
designated "CNox" (Bruno et al., 1992, Biochem. J. 284:625-628 and
Morre and Morre, 1995, Protoplasma 184:188-195).
[0013] Cancer cells exhibit both drug-responsive and hormone and
growth factor-indifferent (tNox), and drug inhibited and hormone
and growth factor dependent (CNox) activities, whereas
non-transformed cells exhibit only the drug inhibited hormone- and
drug-responsive CNox. Like the tNox of cancer cells, CNox is
capable of oxidizing NADH, but has an activity which is modulated
by hormones and growth factors. Thus, some authors have theorized
that inhibitors of tNox (which are believed to include one or more
of the Nox enzymes listed above, such as Nox4) will be useful for
treating cancer.
[0014] In addition to treating cancer, Nox inhibitors are also
expected to have provide therapeutic effects for numerous other
inflammatory, degenerative and vascular diseases in which reactive
oxygen species have been implicated.
[0015] For example, Nox has been reported to have a role in retinal
vascular inflammation, as well as ischemia-induced increases in
vascular endothelial growth factor (VEGF) and retinal
neovascularization (Al-Shabrawey et al., Invest, Ophthalmol, Vis,
Sci. (2008)). Studies performed using wild type mice, mice lacking
Nox2 and mice treated with the NADPH oxidase inhibitor apocynin in
models of endotoxemia and streptozotocin-induced diabetes showed
that both endotoxemia- and diabetes-induced increases in ICAM-1
expression and leukostasis were significantly inhibited by deletion
of Nox2. Apocynin treatment was as effective as deletion of Nox2 in
preventing diabetes-induced increases in ICAM-1, leukostasis, and
breakdown of the blood-retinal barrier, suggesting that Nox2 is
primarily responsible for these early signs of diabetic
retinopathy.
[0016] Elevated ROS initiate and anti-oxidants inhibit the
apoptotic cell loss in the retinal pigment epithelium (Glotkin et
al, 2006 IOVS, 47: 4614-4623). This is thought to play a role in
the development of dry age-related macular degeneration. Likewise,
the use of antioxidants had been shown to reduce the progression to
neovascularization in patients with large drusen in AMD (Coleman
and Chew, 2007, Curr. Opin. Ophthalmol. 18(3): 220-223).
[0017] NADP+ reductases lower the concentration of retinaldehyde
and retinoic acid, which in turn protect cells from
retinaldehyde-induced cell death (Lee et al., J. Biol. Chem.,
282(49)35621-8 (2007). By extension, inhibition of NADPH oxidase
can have the same effect as increasing the rate of a NADP+
reductase, and have a beneficial effect on retinal degeneration
mediated by retinaldehyde or retinoic acid.
[0018] Specific inhibition of NADPH oxidase has been shown to
reduce angiogenesis in models of retinopathy of prematurity
(Al-Shabraway et al, 2005, Am. J. Pathol. 167(2): 599-607 and Saito
et al, 2007, Mol. Vision, 13: 840-853). In addition elevated ROS
have been observed in diabetic animals and the elevation correlates
with increase VEGF activity. Similarly, oxidative stress is thought
to be a significant factor in the development of diabetic
retinopathy (Kowluru and Chan, 2007, Expt. Diabetes Res. Article ID
43603).
[0019] ROS may have two separate effects in the development of
glaucoma. First, increased ROS led to increased cellularity of the
trabecular meshwork (and thereby increased intraocular pressure,
Sacca et al, 2007, Exp. Eye Res. 84(3): 389-399). Over time
increased reactive oxygen species are also thought to stimulate
apoptosis of retinal ganglion cells (Tezel, 2006, Prog. Retin. Eye
Res. 25(5): 490-513), the anatomic basis of visual field loss.
[0020] In non-ocular cutaneous tissues, NADPH oxidase from pollen
has been shown to perpetuate the allergic response. Inhibition of
NADPH oxidase reduces mast cell degranulation and may be useful in
allergic eye disease (Nishikawa et al, 2007, BBRC, 362(2):
504-509).
[0021] Although direct experimental evidence that inhibition of
NADPH oxidase will provide a therapeutic effect in the some of the
eye diseases mentioned is lacking, NADPH oxidase inhibition can be
expected to alter the cellular redox balance and thus may be
therapeutic in the various condition by indirect means.
[0022] NADPH oxidase inhibitors may also be effective for the
treatment of dry eye based on the observation that NADPH oxidase is
constituitively expressed in corneal epithelial and stromal cells
(O'Brien et al, 2006, IOVS, 47: 853-863). The authors suggest that
the production of superoxide anion may play a role in inflammation
of the cornea.
[0023] With respect to the role of specific Nox enzymes in
inflammatory disorders, Nox2-containing NADPH oxidase and Akt
activation are believed to play a key role in angiotensin
II-induced cardiomyocyte hypertrophy (Physiol. Genomics 26:
180-191, 2006).
[0024] Accordingly, Nox are believed to be responsible for
increased levels of ROS in some cancers and inflammatory disorders,
and treatment with appropriate inhibitors may be useful in treating
such cancers and inflammatory disorders.
[0025] There remains a need for treatment of cancer that does not
have the adverse effects generally caused by the non-selectivity of
conventional chemotherapeutic agents. There further remains a need
to have additional treatments for inflammatory, degenerative and
vascular diseases in which a reactive oxygen species has been
implicated. The present invention provides such compounds,
compositions and methods.
SUMMARY OF THE INVENTION
[0026] Compounds, pharmaceutical compositions including the
compounds, and methods of preparation and use thereof are
disclosed. In one embodiment, the compounds are fulvene and/or
fulvalene analogs, which can be formed by reacting a
cyclopentadienyl anion with one or more ketone or aldehyde groups
on a suitable intermediate. In another embodiment, the compounds
are fulvene and/or fulvene analogues which can be formed by
reacting a fulvene and/or fulvalene-containing carboxylic acid (or
acid halide or anhydride thereof) with a hydroxyl, thiol, or amine
group on a sugar, nucleoside, nucleotide, or amino acid, or
oligonucleotides and peptides including the nucleotides or amino
acids.
[0027] Representative compounds include fulvene and/or fulvalene
analogues of steroids and steroid precursors, such as cholesterol,
progesterone, testosterone, or estrogen; dyes such as indigo and
benzophenones; curcumin and aldehyde and ketone-containing
curcumenes.
[0028] The synthesis, characterization and an evaluation of the
anti-tumor potential of these fulvene and/or fulvalene-containing
compounds is also disclosed.
[0029] While not wishing to be bound by a particular theory, it is
believed that the compounds function by one or more of the
following mechanisms:
[0030] a) inhibiting all forms of Nox,
[0031] b) specifically inhibiting Nox 1-5,
[0032] c) specifically inhibiting Nox 2 and/or Nox 4 (the latter of
which is more prevalent in cancer cells than normal cells),
[0033] d) inhibiting a Nox enzyme that is more prevelant in cancer
cells than normal cells, hereinafter referred to as tNox,
[0034] e) inhibiting ROS, and
[0035] f) stimulating superoxide scavengers, such as scavenger
enzyme systems catalase, superoxide dismutase I (Zn2+/Cu2+ SOD) and
II (MN-SOD), and glutathione peroxidase.
[0036] Evidence that the compounds can inhibit ROS is demonstrated
herein in the working examples, which show that electron spin
resonance spectra show that when the compounds are added to
superoxide dismutase, they alter the spectra of the superoxide
dismutase, and appear to be converted to a free radical.
[0037] Treatment with one or more of these compounds selectively
kills cancer cells, without killing healthy cells, thus providing a
selective anti-cancer therapy. Most importantly, these compounds
are potent against cancer cells that have become metastacized. As
discussed above, the mechanism for killing the cancer cells may
involve inhibition of tNOX, without significantly affecting CNox,
thereby effectively inhibiting cell proliferation, particularly in
metastacized tumors, or the inhibition of any of the Nox enzymes,
such as Nox4, which is prevalent in cancer cells. That is, in some
embodiments, the Nox is one that is selectively expressed in cancer
cells over normal cells, and in other embodiments, the Nox is one
that is expressed in higher concentrations in cancer cells than in
normal cells.
[0038] The pharmaceutical compositions include an effective amount
of the compounds described herein, along with a pharmaceutically
acceptable carrier or excipient. When employed in effective
amounts, the compounds can act as a therapeutic agent to prevent
and/or treat a wide variety of cancers, particularly metasticized
cancers, and are believed to be both safe and effective in this
role. Representative cancers that can be treated and/or prevented
include melanoma, leukemia, non-small cell lung, colon, central
nervous system (CNS), renal, ovarian, breast and prostate cancer.
Additional pharmaceutical compositions may be useful for the
treatment of ocular diseases.
[0039] The foregoing and other aspects of the present invention are
explained in detail in the detailed description and examples set
forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a graphic representation of inhibition of Nox2
activity by various test compounds as determined by H.sub.2O.sub.2
production in PMA-stimulated Cos-phox cells treated with different
concentrations of a vehicle control or the various test
compounds.
[0041] FIG. 2 is a chart showing the effect of curcumin fulvene
against tumor cells in vivo (average tumor volume).
[0042] FIG. 3 is a chart showing the mean ERG b-wave amplitude
(.mu.m) for mice treated with either vehicle or Fulvene-5
(4-(cyclopenta-2,4-dienylidine methyl)-5-methyl-1H-imidazole), and
exposed to either dim light or bright light.
[0043] FIG. 4 is an electron spin resonance ("ESR") spectra of
superoxide dismutase and Fulvene 5 ("Indigo fulvene").
DETAILED DESCRIPTION OF THE INVENTION
[0044] Compounds, pharmaceutical compositions including the
compounds, and methods of preparation and use thereof are
disclosed.
[0045] The following definitions will be useful in understanding
the metes and bounds of the invention as described herein.
[0046] As used herein, "alkyl" refers to straight chain or branched
alkyl radicals including C.sub.1-C.sub.8, preferably
C.sub.1-C.sub.5, such as methyl, ethyl, or isopropyl; "substituted
alkyl" refers to alkyl radicals further bearing one or more
substituent groups such as hydroxy, alkoxy, aryloxy, mercapto,
aryl, heterocyclo, halo, amino, carboxyl, carbamyl, cyano, and the
like; "alkenyl" refers to straight chain or branched hydrocarbon
radicals including C.sub.1-C.sub.8, preferably C.sub.1-C.sub.5 and
having at least one carbon-carbon double bond; "substituted
alkenyl" refers to alkenyl radicals further bearing one or more
substituent groups as defined above; "cycloalkyl" refers to
saturated or unsaturated, non-aromatic, cyclic ring-containing
radicals containing three to eight carbon atoms, preferably three
to six carbon atoms; "substituted cycloalkyl" refers to cycloalkyl
radicals further bearing one or more substituent groups as defined
above; "aryl" refers to aromatic radicals having six to ten carbon
atoms; "substituted aryl" refers to aryl radicals further bearing
one or more substituent groups as defined above; "alkylaryl" refers
to alkyl-substituted aryl radicals; "substituted alkylaryl" refers
to alkylaryl radicals further bearing one or more substituent
groups as defined above; "arylalkyl" refers to aryl-substituted
alkyl radicals; "substituted arylalkyl" refers to arylalkyl
radicals further bearing one or more substituent groups as defined
above; "heterocyclyl" refers to saturated or unsaturated cyclic
radicals containing one or more heteroatoms (e.g., O, N, S) as part
of the ring structure and having two to seven carbon atoms in the
ring; "substituted heterocyclyl" refers to heterocyclyl radicals
further bearing one or more substituent groups as defined
above.
I. Compounds
[0047] The compounds are fulvene and/or fulvalene analogs, prodrugs
or metabolites of these compounds, and pharmaceutically acceptable
salts thereof. In one embodiment, the compounds generally fall
within one of the formulas provided below:
##STR00001##
[0048] wherein:
[0049] X is O, S, CH.sub.2, or NR', where each R' is, individually,
hydrogen, C.sub.1-6 alkyl, cycloalkyl, heterocyclyl, aryl, or
arylalkyl (such as benzyl); and
[0050] the aryl or heteroaryl rings can be substituted at any free
position with H or a substituent, G, as described herein, and x and
y are integers between 0 and 3.
[0051] In other embodiments, the compounds are ether, thioether, or
amine derivatives of compounds which originally included a
hydroxyl, thiol, or amine group, where this group has been reacted
with a compound that includes a fulvene or fulvalene moiety, and a
carboxylic acid or an activated carboxylic acid moiety, as
described herein. One fulvene-containing carboxylic acid is shown
below:
##STR00002##
where the carbonyl group is attached to a hydroxyl, thiol, or amine
group on an intermediate to form an ester, thiolester, or amide
linkage. Analogous compounds can be prepared, for example, by using
different keto- or aldehyde-containing carboxylic acids, by
analogous reaction with cyclopentadienyl anion.
[0052] Representative hydroxyl, thiol, and amine-containing
moieties that can be used to prepare the compounds described
herein, by reaction with a fulvene- or fulvalene-containing
carboxylic acid, acid halide, or anhydride, include natural or
synthetic sugars, polyols, polyalkylene glycols, such as
polyethylene glycol, nucleosides and nucleotides (for example, by
reaction with the 3' and/or 5'-hydroxy groups on these compounds),
short (i.e., 25 mer or less) oligonucleotides including these
nucleosides, hydroxyl, thiol, and/or amine-containing amino acids,
peptides and proteins including these amino acids, and compounds of
the following formulas:
##STR00003##
or another fulvene- or fulvalene-containing carboxylic acid moiety
or activated carboxylic acid moiety as described above, with the
proviso that at least one of R is other than H.
[0053] Representative substituents, G, include C.sub.1-6 alkyl
(including cycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl,
halo (e.g., F, Cl, Br, or I), --OR', --NR'R'', --CF.sub.3, --CN,
--NO.sub.2, --C.sub.2R', --SR', --N.sub.3, --C(.dbd.O)NR'R'',
--NR'C(.dbd.O) R'', --C(.dbd.O)R', --C(.dbd.O)OR', --OC(.dbd.O)R',
--OC(.dbd.O)NR'R'', --NR'C(.dbd.O)O R'', --SO.sub.2R',
--SO.sub.2NR'R'', and --NR'SO.sub.2R'', where R' and R'' are
individually hydrogen, C.sub.1-6 alkyl, cycloalkyl, heterocyclyl,
aryl, or arylalkyl (such as benzyl);
[0054] The compounds can occur in varying degrees of enantiomeric
excess, and racemic mixtures can be purified using known chiral
separation techniques.
[0055] The compounds can be in a free base form or in a salt form
(e.g., as pharmaceutically acceptable salts). Examples of suitable
pharmaceutically acceptable salts include inorganic acid addition
salts such as sulfate, phosphate, and nitrate; organic acid
addition salts such as acetate, dichloroacetate, galactarate,
propionate, succinate, lactate, glycolate, malate, tartrate,
citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate,
and ascorbate; salts with an acidic amino acid such as aspartate
and glutamate; alkali metal salts such as sodium and potassium;
alkaline earth metal salts such as magnesium and calcium; ammonium
salt; organic basic salts such as trimethylamine, triethylamine,
pyridine, picoline, dicyclohexylamine, and
N,N'-dibenzylethylenediamine; and salts with a basic amino acid
such as lysine and arginine. The salts can be in some cases
hydrates or ethanol solvates. The stoichiometry of the salt will
vary with the nature of the components.
[0056] Representative compounds include the following:
##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008##
##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014## ##STR00015##
[0057] The compound identified above as indigo fulvene is also
referred to herein as "Fulvene-5."
[0058] If desired, certain of these compounds can be rendered more
hydrophobic by substituting a C.sub.1-6 alkyl, cycloalkyl,
heterocyclyl, aryl, or arylalkyl moiety for hydrogen, or a
cycloalkyl, heterocyclyl, aryl, or arylalkyl moiety for an alkyl
moiety, on a nitrogen atom in the structure. Examples include the
following:
##STR00016##
II. Methods of Preparing the Compounds
[0059] In some embodiments, the compounds can be prepared by
reacting sodium cyclopentadienide with any aldehyde or ketone.
Using this approach, numerous fulvenes can be made from readily
available ketone- or aldehyde-containing starting materials.
[0060] Representative aldehydes and ketones are provided below:
##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023## ##STR00024##
[0061] wherein any of the aryl/heteroaryl rings can be substituted
with one or more substituents as described herein, and amines
(i.e., --NH groups) can be substituted with R' groups as described
herein.
[0062] In other embodiments, the compounds are prepared by reacting
a hydroxyl, thiol, or amine group with a compound that includes a
fulvene or fulvalene moiety, and a carboxylic acid or an activated
carboxylic acid moiety.
[0063] Generally, the hydroxyl, thiol, or amine group is reacted
with either a fulvene- or fulvalene-containing carboxylic acid or
an activated derivative thereof (e.g., an acid chloride or
anhydride), in the presence of dehydrating agents and/or bases. A
variety of conditions are possible.
[0064] A carboxylic acid can be coupled to a hydroxyl or ester
group directly, with an acid catalyst and subsequent formation of
water (typically removed by azeotropic distillation), or by
reaction with an acid halide or anhydride, typically in the
presence of a tertiary amine such as triethylamine. The resulting
compound has an ester or thiolester linkage, and the fulvene and/or
fulvalene moiety is attached via this linkage.
[0065] Intermediates with a free amine functionality can be coupled
to a carboxylic acid-containing, fulvene or fulvalene-containing
moiety using any one of various agents used for forming amide bonds
(for instance, those used in peptide synthesis). Such reagents
include N,N'-dicyclohexylcarbodiimide (DCC),
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP),
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP), O-(benzotriazol-1-yl)-N,N,N',N'-bis(tetramethylene)uronium
hexafluorophosphate (HBPyU),
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU),
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU), and
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDCI) with
1-hydroxybenzotriazole (HOBt). In some cases these reagents are
commercially available as polymer supported modifications, which
greatly facilitate isolation of coupling products. An example of
such a reagent is polystyrene bound N,N'-dicyclohexylcarbodiimide
(PS-DCC).
[0066] Acid halides can be prepared, for example, by reacting the
carboxylic acid-containing moeity with any of various reagents,
such as thionyl chloride or oxalyl chloride. The reaction between
the acid chloride and the carboxylic acid is typically performed in
the presence of a tertiary amine, usually a hindered one.
[0067] Typically, after ester, thiolester, or amide bond formation,
any protecting groups (e.g., a tert-butoxycarbonyl group or a
benzyl group) are removed to generate the desired compounds.
Protecting groups, and methods for their removal, are well known to
those of skill in the art, and are described for example, in T. W.
Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,
3.sup.rd Edition, John Wiley & Sons, New York (1999).
[0068] The fulvene-containing and/or fulvalene-containing
carboxylic acids used to make compounds described herein are either
commercially available, or can be prepared from commercially
available starting materials. Those that are not commercially
available can be made by a variety of synthetic methodologies,
related to the particular moieties and the particular substitution
desired. The variation in synthetic methodology will be readily
apparent to those of skill in the art of organic synthesis.
[0069] For example, one fulvene-containing and/or
fulvalene-containing carboxylic acid is shown below:
##STR00025##
where the carbonyl group is attached to a hydroxyl, thiol, or amine
group on an intermediate to form an ester, thiolester, or amide
linkage. This intermediate can be prepared, for example, by
reacting a suitably protected 3-keto butyric acid (or the
corresponding butyrate salt) with cyclopentadienyl anion to form
the fulvene ring. The carboxylate salt can be acidified to reform
the carboxylic acid moiety, which can be further reacted to form an
anhydride or acid halide, if desired. This carboxylic acid, acid
halide, or acid anhydride intermediate can be used to form a
fulvene analogue of virtually any hydroxyl, thiol, or
amine-containing compounds, using the esterification,
thiolesterification, or amidation chemistry described above.
[0070] The above intermediate is just one of a number of compounds
that can be used to incorporate a fulvene or fulvalene moiety onto
a compound. Analogous compounds can be prepared, for example, by
using different keto- or aldehyde-containing carboxylic acids, by
analogous reaction with cyclopentadienyl anion.
[0071] Representative hydroxyl, thiol, and amine-containing
moieties that can be used to prepare the compounds described
herein, by reaction with a fulvene- or fulvalene-containing
carboxylic acid, acid halide, or anhydride, are described
below.
[0072] Natural or synthetic sugars, polyols, polyalkylene glycols,
such as polyethylene glycol, nucleosides and nucleotides (for
example, by reaction with the 3' and/or 5'-hydroxy groups on these
compounds), short (i.e., 25 mer or less) oligonucleotides including
these nucleosides, hydroxyl, thiol, and/or amine-containing amino
acids, peptides and proteins including these amino acids, and
compounds of the following formulas:
##STR00026##
or another fulvene- or fulvalene-containing carboxylic acid moiety
or activated carboxylic acid moiety as described above, with the
proviso that at least one of R is other than H.
[0073] Those skilled in the art will readily understand that
incorporation of other substituents onto the cyclopentadiene ring
used as a starting material to prepare the fulvenes/fulvalenes, and
other positions in the fulvene/fulvalene framework, can be readily
realized. Such substituents can provide useful properties in and of
themselves or serve as a handle for further synthetic
elaboration.
[0074] Substituents typically can be added to a cyclopentadiene
before forming the sodium cyclopentadienide (i.e., by addition of
base) that is reacted with a suitable ketone or aldehyde to form
the compounds described herein, or to form the fulvene/fulvalene
containing carboxylic acid/acid halide/acid anhydride reacted with
hydroxyl, thiol, or amine groups to form the compounds described
herein.
[0075] For example, diazocyclopentadiene can be prepared using the
techniques in Cram and Partos, Electrophilic Substitution and Other
Reactions of Diazocyclopentadiene, J.A.C.S. p. 1273-1277
(1962).
[0076] Diazocyclopentadiene can be halogenated using various known
procedures, which vary depending on the particular halogen.
Examples of suitable reagents include bromine/water in concentrated
HBr, thionyl chloride, pyr-ICl, fluorine and Amberlyst-A
[0077] A number of other analogs, bearing substituents in the
diazotized position of the diazocyclopentadiene, can be synthesized
from the corresponding amino compounds, via the
diazocyclopentadiene intermediate. The diazocyclopentadiene can be
prepared using known chemistry, for example, as described
above.
[0078] Nitration of the diazocyclopentadiene results in two
isomers, the 2-nitro and 3-nitro cyclopentadiene compounds.
Benzenediazonium tetrafluoroborate leads to 2-substitution
products, whereas bromination provides
tetrabromodiazocyclopentadiene. Mercuration with mercury iodide can
provide 2,5-di-iododiazocyclopentadiene.
[0079] The nitro derivatives can be reduced to the amine compound
by reaction with a nitrite salt, typically in the presence of an
acid. Other substituted analogs can be produced from diazonium salt
intermediates, including, but are not limited to, hydroxy, alkoxy,
fluoro, chloro, iodo, cyano, and mercapto, using general techniques
known to those of skill in the art. For example, hydroxy-fulvene
analogues can be prepared by reacting the diazonium salt
intermediate with water, protecting the resulting hydroxyl group,
forming the cyclopentadienyl anion, and reacting it with a suitable
aldehyde or ketone. Likewise, alkoxy fulvene analogues can be made
by reacting the diazocyclopentadiene with alcohols. The
diazocyclopentadiene can also be used to synthesize cyano or halo
compounds, as will be known to those skilled in the art. Mercapto
substitutions can be obtained using techniques described in Hoffman
et al., J. Med. Chem. 36: 953 (1993). The mercaptan so generated
can, in turn, be converted to an alkylthio substitutuent by
reaction with sodium hydride and an appropriate alkyl bromide.
Subsequent oxidation would then provide a sulfone. Acylamido
analogs of the aforementioned compounds can be prepared by reacting
the corresponding amino compounds with an appropriate acid
anhydride or acid chloride using techniques known to those skilled
in the art of organic synthesis.
[0080] Hydroxy-substituted analogs can be used to prepare
corresponding alkanoyloxy-substituted compounds by reaction with
the appropriate acid, acid chloride, or acid anhydride. Likewise,
the hydroxy compounds are precursors of both the aryloxy and
heteroaryloxy via nucleophilic aromatic substitution at electron
deficient aromatic rings. Such chemistry is well known to those
skilled in the art of organic synthesis. Ether derivatives can also
be prepared from the hydroxy compounds by alkylation with alkyl
halides and a suitable base or via Mitsunobu chemistry, in which a
trialkyl- or triarylphosphine and diethyl azodicarboxylate are
typically used. See Hughes, Org. React. (N.Y.) 42: 335 (1992) and
Hughes, Org. Prep. Proced. Int. 28: 127 (1996) for typical
Mitsunobu conditions.
[0081] Cyano-substituted analogs can be hydrolyzed to afford the
corresponding carboxamido-substituted compounds. Further hydrolysis
results in formation of the corresponding carboxylic
acid-substituted analogs. Reduction of the cyano-substituted
analogs with lithium aluminum hydride yields the corresponding
aminomethyl analogs. Acyl-substituted analogs can be prepared from
corresponding carboxylic acid-substituted analogs by reaction with
an appropriate alkyllithium using techniques known to those skilled
in the art of organic synthesis.
[0082] Carboxylic acid-substituted analogs can be converted to the
corresponding esters by reaction with an appropriate alcohol and
acid catalyst. Compounds with an ester group can be reduced with
sodium borohydride or lithium aluminum hydride to produce the
corresponding hydroxymethyl-substituted analogs. These analogs in
turn can be converted to compounds bearing an ether moiety by
reaction with sodium hydride and an appropriate alkyl halide, using
conventional techniques. Alternatively, the
hydroxymethyl-substituted analogs can be reacted with tosyl
chloride to provide the corresponding tosyloxymethyl analogs, which
can be converted to the corresponding alkylaminoacyl analogs by
sequential treatment with thionyl chloride and an appropriate
alkylamine. Certain of these amides are known to readily undergo
nucleophilic acyl substitution to produce ketones.
[0083] Hydroxy-substituted analogs can be used to prepare N-alkyl-
or N-arylcarbamoyloxy-substituted compounds by reaction with
N-alkyl- or N-arylisocyanates. Amino-substituted analogs can be
used to prepare alkoxycarboxamido-substituted compounds and urea
derivatives by reaction with alkyl chloroformate esters and
N-alkyl- or N-arylisocyanates, respectively, using techniques known
to those skilled in the art of organic synthesis.
[0084] Similarly, benzene rings (and pyridine, pyrimidine,
pyrazine, and other heteroaryl rings) can be substituted using
known chemistry, including the reactions discussed above. For
example, the nitro group on nitrobenzene can be reacted with sodium
nitrite to form the diazonium salt, and the diazonium salt
manipulated as discussed above to form the various substituents on
a benzene ring.
III. Pharmaceutical Compositions
[0085] The compounds described herein can be incorporated into
pharmaceutical compositions and used to prevent a condition or
disorder in a subject susceptible to such a condition or disorder,
and/or to treat a subject suffering from the condition or disorder.
The pharmaceutical compositions described herein include one or
more of the fulvene and/or fulvalene analogues described herein,
and/or pharmaceutically acceptable salts thereof. Optically active
compounds can be employed as racemic mixtures, as pure enantiomers,
or as compounds of varying enantiomeric purity.
[0086] The manner in which the compounds are administered can vary.
The compositions are preferably administered orally (e.g., in
liquid form within a solvent such as an aqueous or non-aqueous
liquid, or within a solid carrier). Preferred compositions for oral
administration include pills, tablets, capsules, caplets, syrups,
and solutions, including hard gelatin capsules and time-release
capsules. Compositions may be formulated in unit dose form, or in
multiple or subunit doses. Preferred compositions are in liquid or
semisolid form. Compositions including a liquid pharmaceutically
inert carrier such as water or other pharmaceutically compatible
liquids or semisolids may be used. The use of such liquids and
semisolids is well known to those of skill in the art.
[0087] The compositions can also be administered via injection,
i.e., intraveneously, intramuscularly, subcutaneously,
intraperitoneally, intraarterially, intrathecally; and
intracerebroventricularly. Intravenous administration is a
preferred method of injection. Suitable carriers for injection are
well known to those of skill in the art, and include 5% dextrose
solutions, saline, and phosphate buffered saline. The compounds can
also be administered as an infusion or injection (e.g., as a
suspension or as an emulsion in a pharmaceutically acceptable
liquid or mixture of liquids).
[0088] The formulations may also be administered using other means,
for example, rectal administration. Formulations useful for rectal
administration, such as suppositories, are well known to those of
skill in the art. The compounds can also be administered by
inhalation (e.g., in the form of an aerosol either nasally or using
delivery articles of the type set forth in U.S. Pat. No. 4,922,901
to Brooks et al., the disclosure of which is incorporated herein in
its entirety); topically (e.g., in lotion form); or transdermally
(e.g., using a transdermal patch, using technology that is
commercially available from Novartis and Alza Corporation).
Although it is possible to administer the compounds in the form of
a bulk active chemical, it is preferred to present each compound in
the form of a pharmaceutical composition or formulation for
efficient and effective administration.
[0089] The compounds can be incorporated into drug delivery devices
such as nanoparticles, microparticles, microcapsules, and the like.
Representative microparticles/nanoparticles include those prepared
with cyclodextrins, such as pegylated cyclodextrins, liposomes,
including small unilamellar vesicles, and liposomes of a size
designed to lodge in capillary beds around growing tumors. Suitable
drug delivery devices are described, for example, in Heidel J D, et
al., Administration in non-human primates of escalating intravenous
doses of targeted nanoparticles containing ribonucleotide reductase
subunit M2 siRNA, Proc Natl Acad Sci USA. Apr. 3,
2007;104(14):5715-21; Wongmekiat et al., Preparation of drug
nanoparticles by co-grinding with cyclodextrin: formation mechanism
and factors affecting nanoparticle formation, Chem Pharm Bull
(Tokyo). March 2007;55(3):359-63; Bartlett and Davis,
Physicochemical and biological characterization of targeted,
nucleic acid-containing nanoparticles, Bioconjug Chem. March-April
2007;18(2):456-68; Villalonga et al., Amperometric biosensor for
xanthine with supramolecular architecture, Chem Commun (Camb). Mar.
7, 2007;(9):942-4; Defaye et al., Pharmaceutical use of
cyclodextrines: perspectives for drug targeting and control of
membrane interactions, Ann Pharm Fr. January 2007;65(1):33-49; Wang
et al., Synthesis of Oligo(ethylenediamino)-beta-Cyclodextrin
Modified Gold Nanoparticle as a DNA Concentrator; Mol Pharm.
March-April 2007;4(2):189-98; Xia et al., Controlled synthesis of
Y-junction polyaniline nanorods and nanotubes using in situ
self-assembly of magnetic nanoparticles, J Nanosci Nanotechnol.,
December 2006;6(12):3950-4; and Nijhuis et al., Room-temperature
single-electron tunneling in dendrimer-stabilized gold
nanoparticles anchored at a molecular printboard, Small. December
2006;2(12): 1422-6.
[0090] Exemplary methods for administering such compounds will be
apparent to the skilled artisan. The usefulness of these
formulations may depend on the particular composition used and the
particular subject receiving the treatment. These formulations may
contain a liquid carrier that may be oily, aqueous, emulsified or
contain certain solvents suitable to the mode of
administration.
[0091] The compositions can be administered intermittently or at a
gradual, continuous, constant or controlled rate to a warm-blooded
animal (e.g., a mammal such as a mouse, rat, cat, rabbit, dog, pig,
cow, or monkey), but advantageously are administered to a human
being. In addition, the time of day and the number of times per day
that the pharmaceutical formulation is administered can vary.
[0092] Preferably, the compositions are administered such that
active ingredients interact with regions where cancer cells are
located. The compounds described herein are very potent at treating
these cancers.
[0093] In certain circumstances, the compounds described herein can
be employed as part of a pharmaceutical composition with other
compounds intended to prevent or treat a particular cancer, i.e.,
combination therapy. In addition to effective amounts of the
compounds described herein, the pharmaceutical compositions can
also include various other components as additives or adjuncts.
[0094] Complexation with Proteins
[0095] The fulvene and fulvalene analogues described herein can be
complexed with peptides and proteins, including albumin,
transferrin, VEGF, bFGF, and the like. These complexes are easy to
make and tend to have lower toxicity than the un-complexed
compounds.
[0096] Those of skill in the art can readily appreciate how to
complex the compounds described herein with a protein or peptide.
The complexes can be administered in any manner in which the
un-complexed compounds can be administered.
[0097] Combination Therapy
[0098] The combination therapy may be administered as (a) a single
pharmaceutical composition which comprises a fulvene and/or
fulvalene analogue as described herein, at least one additional
pharmaceutical agent described herein, and a pharmaceutically
acceptable excipient, diluent, or carrier; or (b) two separate
pharmaceutical compositions comprising (i) a first composition
comprising a fulvene and/or fulvalene analogue as described herein
and a pharmaceutically acceptable excipient, diluent, or carrier,
and (ii) a second composition comprising at least one additional
pharmaceutical agent described herein and a pharmaceutically
acceptable excipient, diluent, or carrier. The pharmaceutical
compositions can be administered simultaneously or sequentially and
in any order.
[0099] In use in treating or preventing cancer, the fulvene and/or
fulvalene analogues described herein can be administered together
with at least one other chemotherapeutic agent as part of a unitary
pharmaceutical composition. Alternatively, the fulvene and/or
fulvalene analogues can be administered apart from the other
anticancer chemotherapeutic agent. In this embodiment, the fulvene
and/or fulvalene analogues and the at least one other anticancer
chemotherapeutic agent are administered substantially
simultaneously, i.e. the compounds are administered at the same
time or one after the other, so long as the compounds reach
therapeutic levels for a period of time in the blood.
[0100] Combination therapy involves administering a fulvene and/or
fulvalene analogue, as described herein, or a pharmaceutically
acceptable salt or prodrug of a compound described herein, in
combination with at least one anti-cancer chemotherapeutic agent,
ideally one which functions by a different mechanism (i.e., VEGF
inhibitors, alkylating agents, and the like).
[0101] Examples of known anticancer agents which can be used for
combination therapy include, but are not limited to alkylating
agents, such as busulfan, cis-platin, mitomycin C, and carboplatin;
antimitotic agents, such as colchicine, vinblastine, paclitaxel,
and docetaxel; topo I inhibitors, such as camptothecin and
topotecan; topo II inhibitors, such as doxorubicin and etoposide;
RNA/DNA antimetabolites, such as 5-azacytidine, 5-fluorouracil and
methotrexate; DNA antimetabolites, such as
5-fluoro-2'-deoxy-uridine, ara-C, hydroxyurea and thioguanine; and
antibodies, such as Herceptin.RTM. and Rituxan.RTM.. Other known
anti-cancer agents, which can be used for combination therapy,
include arsenic trioxide, gamcitabine, melphalan, chlorambucil,
cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin,
aclarubicin, bleomycin, mitoxantrone, elliptinium, fludarabine,
octreotide, retinoic acid, tamoxifen and alanosine. Other classes
of anti-cancer compounds that can be used in combination with the
fulvene and/or fulvalene analogues are described below.
[0102] The fulvene and/or fulvalene analogues can be combined with
alpha-1-adrenoceptor antagonists, such as doxazosin, terazosin, and
tamsulosin, which can inhibit the growth of prostate cancer cell
via induction of apoptosis (Kyprianou, N., et al., Cancer Res
60:4550 4555, (2000)).
[0103] Sigma-2 receptors are expressed in high densities in a
variety of tumor cell types (Vilner, B. J., et al., Cancer Res. 55:
408 413 (1995)) and sigma-2 receptor agonists, such as CB-64D,
CB-184 and haloperidol, activate a novel apoptotic pathway and
potentiate antineoplastic drugs in breast tumor cell lines.
(Kyprianou, N., et al., Cancer Res. 62:313 322 (2002)).
Accordingly, the fulvene and/or fulvalene analogues can be combined
with at least one known sigma-2 receptor agonists, or a
pharmaceutically acceptable salt of said agent.
[0104] The fulvene and/or fulvalene analogues can be combined with
lovastatin, a HMG-CoA reductase inhibitor, and butyrate, an inducer
of apoptosis in the Lewis lung carcinoma model in mice, can
potentiate antitumor effects (Giermasz, A., et al., Int. J. Cancer
97:746 750 (2002)). Examples of known HMG-CoA reductase inhibitors,
which can be used for combination therapy include, but are not
limited to, lovastatin, simvastatin, pravastatin, fluvastatin,
atorvastatin and cerivastatin, and pharmaceutically acceptable
salts thereof.
[0105] Certain HIV protease inhibitors, such as indinavir or
saquinavir, have potent anti-angiogenic activities and promote
regression of Kaposi sarcoma (Sgadari, C., et al., Nat. Med. 8:225
232 (2002)). Accordingly (in addition to forming fulvene and/or
fulvalene analogues of these compounds), the fulvene and/or
fulvalene analogues can be combined with HIV protease inhibitors,
or a pharmaceutically acceptable salt of said agent. Representative
HIV protease inhibitors include, but are not limited to,
amprenavir, abacavir, CGP-73547, CGP-61755, DMP-450, indinavir,
nelfinavir, tipranavir, ritonavir, saquinavir, ABT-378, AG 1776,
and BMS-232,632.
[0106] Synthetic retinoids, such as fenretinide
(N-(4-hydroxyphenyl)retinamide, 4HPR), can have good activity in
combination with other chemotherapeutic agents, such as cisplatin,
etoposide or paclitaxel in small-cell lung cancer cell lines
(Kalemkerian, G. P., et al., Cancer Chemother. Pharmacol. 43:145
150 (1999)). 4HPR also was reported to have good activity in
combination with gamma-radiation on bladder cancer cell lines (Zou,
C., et al., Int. J. Oncol. 13:1037 1041 (1998)). Representative
retinoids and synthetic retinoids include, but are not limited to,
bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,
.alpha.-difluoromethylornithine, ILX23-7553, fenretinide, and
N-4-carboxyphenyl retinamide.
[0107] Proteasome inhibitors, such as lactacystin, exert anti-tumor
activity in vivo and in tumor cells in vitro, including those
resistant to conventional chemotherapeutic agents. By inhibiting
NF-kappaB transcriptional activity, proteasome inhibitors may also
prevent angiogenesis and metastasis in vivo and further increase
the sensitivity of cancer cells to apoptosis (Almond, J. B., et
al., Leukemia 16:433 443 (2002)). Representative proteasome
inhibitors include, but are not limited to, lactacystin, MG-132,
and PS-341.
[0108] Tyrosine kinase inhibitors, such as STI571 (Imatinib
mesilate, Gleevec.RTM.), have potent synergetic effects in
combination with other anti-leukemic agents, such as etoposide
(Liu, W. M., et al. Br. J. Cancer 86:1472 1478 (2002)).
Representative tyrosine kinase inhibitors include, but are not
limited to, Gleevec.RTM., ZD1839 (Iressa.RTM.), SH268, genistein,
CEP2563, SU6668, SU11248, and EMD121974.
[0109] Prenyl-protein transferase inhibitors, such as farnesyl
protein transferase inhibitor R115777, possess antitumor activity
against human breast cancer (Kelland, L. R., et. al., Clin. Cancer
Res. 7:3544 3550 (2001)). Synergy of the protein
farnesyltransferase inhibitor SCH66336 and cisplatin in human
cancer cell lines also has been reported (Adjei, A. A., et al.,
Clin. Cancer Res. 7:1438 1445 (2001)). Prenyl-protein transferase
inhibitors, including farnesyl protein transferase inhibitor,
inhibitors of geranylgeranyl-protein transferase type I (GGPTase-I)
and geranylgeranyl-protein transferase type-II, or a
pharmaceutically acceptable salt of said agent, can be used in
combination with the fulvene and/or fulvalene analogues described
herein. Examples of known prenylprotein transferase inhibitors
include, but are not limited to, R115777, SCH66336, L-778,123,
BAL9611 and TAN-1813.
[0110] Cyclin-dependent kinase (CDK) inhibitors, such as
flavopiridol, have potent, often synergetic, effects in combination
with other anticancer agents, such as CPT-11, a DNA topoisomerase I
inhibitor in human colon cancer cells (Motwani, M., et al., Clin.
Cancer Res. 7:4209 4219, (2001)). Representative cyclin-dependent
kinase inhibitors include, but are not limited to, flavopiridol,
UCN-01, roscovitine and olomoucine.
[0111] Certain COX-2 inhibitors are known to block angiogenesis,
suppress solid tumor metastases, and slow the growth of implanted
gastrointestinal cancer cells (Blanke, C. D., Oncology (Hunting)
16(No. 4 Suppl. 3):17 21 (2002)). Representative COX-2 inhibitors
include, but are not limited to, celecoxib, valecoxib, and
rofecoxib.
[0112] Any of the above-mentioned compounds can be used in
combination therapy with the fulvene and/or fulvalene analogues.
Additionally, many of these compounds can be converted to fulvene
and/or fulvalene analogues by reaction of ketone, aldehyde,
hydroxyl, thiol, and/or amine functional groups on the compounds
using the chemistry described herein. The fulvene and/or fulvalene
analogues of these compounds are within the scope of this
invention.
[0113] Further, the fulvene and/or fulvalene analogues can be
targeted to a tumor site by conjugation with therapeutically useful
antibodies, such as Herceptin.RTM. or Rituxan.RTM., growth factors,
such as DGF, NGF; cytokines, such as IL-2, IL-4, or any molecule
that binds to the cell surface. The antibodies and other molecules
will deliver a compound described herein to its targets and make it
an effective anticancer agent. The bioconjugates can also enhance
the anticancer effect of therapeutically useful antibodies, such as
Herceptin.RTM. or Rituxan.RTM..
[0114] The compounds can also be used in conjunction with surgical
tumor removal, by administering the compounds before and/or after
surgery, and in conjunction with radiation therapy, by
administering the compounds before, during, and/or after radiation
therapy.
[0115] The appropriate dose of the compound is that amount
effective to prevent occurrence of the symptoms of the disorder or
to treat some symptoms of the disorder from which the patient
suffers. By "effective amount", "therapeutic amount" or "effective
dose" is meant that amount sufficient to elicit the desired
pharmacological or therapeutic effects, thus resulting in effective
prevention or treatment of the disorder.
[0116] When treating cancers, an effective amount of the fulvene
and/or fulvalene analogue is an amount sufficient to suppress the
growth of the tumor(s), and, ideally, is a sufficient amount to
shrink the tumor, and, more ideally, to destroy the tumor. Cancer
can be prevented, either initially, or from re-occurring, by
administering the compounds described herein in a prophylactic
manner. Preferably, the effective amount is sufficient to obtain
the desired result, but insufficient to cause appreciable side
effects.
[0117] The effective dose can vary, depending upon factors such as
the condition of the patient, the severity of the cancer, and the
manner in which the pharmaceutical composition is administered. The
effective dose of compounds will of course differ from patient to
patient, but in general includes amounts starting where desired
therapeutic effects occur but below the amount where significant
side effects are observed.
[0118] The compounds, when employed in effective amounts in
accordance with the method described herein, are selective to
certain cancer cells, but do not significantly affect normal
cells.
[0119] For human patients, the effective dose of typical compounds
generally requires administering the compound in an amount of at
least about 1, often at least about 10, and frequently at least
about 25 .mu.g/24 hr/patient. The effective dose generally does not
exceed about 500, often does not exceed about 400, and frequently
does not exceed about 300 .mu.g/24 hr/patient. In addition,
administration of the effective dose is such that the concentration
of the compound within the plasma of the patient normally does not
exceed 500 ng/mL and frequently does not exceed 100 ng/mL.
IV. Methods of Using the Compounds and/or Pharmaceutical
Compositions
[0120] The compounds described herein, and pharmaceutical
compositions including the compounds, can be used to treat cancers.
Representative disorders that can be treated include neoplasms,
such as hemangiomas, and malignant tumors, for example, those which
arise in the setting of autocrine loops involving vascular
endothelial growth factor (VEGF) and its major mitogenic receptor
vascular endothelial growth factor receptor 2.
[0121] The cancers include those in which one of the Nox enzymes is
present in elevated concentrations (i.e., Nox 1, Nox 4, and the
like), or those in which cancer growth is mediated by ROS.
[0122] Representative malignant tumors include malignant
endothelial tumors such as melanoma. Additional cancers that can be
treated include, but not limited to human sarcomas and carcinomas,
e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and
acute myelocytic leukemia (myeloblastic, promyelocytic,
myelomonocytic, monocytic and erythroleukemia); chronic leukemia
(chronic myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease, and malignant forms of
these cancers.
[0123] In one embodiment, the cancer is melanoma, rectal carcinoma,
colon carcinoma, breast carcinoma, ovarian carcinoma, small cell
lung carcinoma, colon carcinoma, chronic lymphocytic carcinoma,
hairy cell leukemia, esophogeal carcinoma, prostate carcinoma,
breast cancer, myeloma, or lymphoma. It is believed that these
cancers have circulating levels of tNOX (which may include Nox4 or
other Nox enzymes) present in the sera of patients suffering from
the cancer (see, for example, U.S. Pat. No. 5,605,810, which is
hereby incorporated by reference in its entirety).
[0124] In some embodiments, the patient already has cancer and is
undergoing treatment for the cancer, and may or may not have tumor
metastasis (i.e., secondary cancer).
[0125] The cancer may be manifested in the form of a tumor, such as
a tumor of epithelial tissue, lymphoid tissue, connective tissue,
bone, or central nervous system.
[0126] The compounds can also be used as adjunct therapy in
combination with existing therapies in the management of the
aforementioned types of cancers. In such situations, it is
preferably to administer the active ingredients to in a manner that
optimizes effects upon cancer cells, including drug resistant
cancer cells, while minimizing effects upon normal cell types.
While this is primarily accomplished by virtue of the behavior of
the compounds themselves, this can also be accomplished by targeted
drug delivery and/or by adjusting the dosage such that a desired
effect is obtained without meeting the threshold dosage required to
achieve significant side effects.
[0127] Treatment of Osteoporosis
[0128] The compounds described herein can also be used to treat
osteoporosis. The cytokine RANKL (receptor activator of NF-.kappa.B
ligand) causes osteoporosis by activating osteoclasts. The
compounds inhibit RANKL activity by potentiating apoptosis,
suppresses osteoclastogenesis, and inhibits invasion through
modulation of nuclear factor-kappaB activation pathway (see, for
example, Mol Cancer Res. September 2006;4(9):621-33).
[0129] Treatment of Inflammatory Disorders
[0130] The compounds described herein are useful for treating or
preventing inflammatory disorders. Reactive oxygen drives NFkB in
inflammatory disorders such as rheumatoid arthitis, asthma,
psoriasis, excema, lupus, scleroderma, certain heart diseases such
atherosclerosis and coronary artery disease, and the like. Because
the compounds are effective at inhibiting production of reactive
oxygen species, they are active against inflammatory disorders.
[0131] The compounds also inhibit certain inflammatory signals, and
can alleviate inflammatory disorders such as inflammatory arthritis
by inhibiting these signals.
[0132] Rheumatoid arthritis (RA) is considered the most common
systemic autoimmune disease, but other disorders, such as
hypothyroidism, systemic lupus erythematosus (SLE), and the like
can also be treated using the compounds described herein. A number
of conditions are associated with chronic inflammation and elevated
levels of TNF-.alpha. and IL-6, including rheumatoid arthritis,
heart disease, and cancer. Numerous gastrointestinal disorders are
caused by inflammation, including, but not limited to, Chrohn's
disease, irritable bowel syndrome, and inflammatory bowel syndrome,
and these disorders can also be treated and/or prevented using the
compounds described herein.
[0133] There is a suggested link between rheumatoid arthritis and
chronic inflammation due to the re-activation of Epstein-Barr virus
(EBV), which latently infects a proportion of memory B cells in
>90% of the world's population. Among the EBV-encoded proteins
implicated in viral pathogenesis, considerable attention has
focused upon latent membrane protein 1 (LMP1). Of the nine EBV
genes expressed as proteins in EBV-transformed cells, LMP1 is the
best characterized, and is the only EBV-encoded gene product
capable of transforming cells in vitro and in vivo, resulting in
the potential for lymphoproliferative changes and malignancy. In
addition to its established role in the pathogenesis of B cell
lymphoma and other malignancies, EBV infection may be linked to
exacerbation of various human autoimmune diseases, including RA and
SLE.
[0134] The mouse collagen-induced arthritis (CIA) model (Myers, et
al., Life Science 61: 1861-1878 (1997)) has many pathologic and
immunologic parallels to rheumatoid arthritis, and provides a
stable, predictable model for evaluating the therapeutic potential
of compounds for treating chronic inflammatory conditions. This
model can be used, for example, to evaluate the ability of the
compounds described herein to treat and/or prevent these
disorders.
[0135] Treatment of mouse B cell lines with compounds described
herein in vitro can be shown to recapitulate the cytokine profile
seen in primary mouse B cells with a concomitant dose-dependent
decrease in CD40 and LMP1-mediated NFkB and AP-1 activation. Those
compounds which decrease CD40 and LMP1-mediated NFkB and AP-1
activation in a dose-dependent manner will be expected to have
anti-inflammatory properties, potentially in both the cognitive
phase of the immune response, as well as the effector phase, by
inhibiting cytokines that lead to chronic inflammation and
additional pathology.
[0136] Treatment of Ocular Disorders
[0137] The compounds are also suitable for use in treating ocular
disorders with an inflammatory component, such as wet and dry
age-related macular degeneration (AMD), diabetic retinopathy (DR),
glaucoma, neovascular glaucoma, retinal vasculitis, uveitis, such
as posterior uveitis, conjunctivitis, retinitis secondary to
glaucoma, episcleritis, scleritis, optic neuritis, retrobulbar
neuritis, ocular inflammation following ocular surgery, ocular
inflammation resulting from physical eye trauma, cataract, ocular
allergy and dry eye.
[0138] Current methods for ocular delivery include topical
administration (eye drops or other suitable topical formulations
for direct administration to the eye), subconjunctival injections,
periocular injections, intravitreal injections, surgical implants,
and systemic routes.
[0139] Particularly where systemic toxicity is of concern when the
oral and intravenous routes of administration are used,
intravitreal injections, periocular injections, and
sustained-release implants can be used to achieve therapeutic
levels of drugs in ocular tissues. Eye drops are useful in treating
conditions affecting either the exterior surface of the eye or
tissues in the front of the eye, and some formulations can
penetrate to the back of the eye for treatment of retinal
diseases.
[0140] Certain disorders affect tissues at the back of the eye,
where treatment is difficult to deliver. In these embodiments,
iontophoresis can be used to deliver the compounds described herein
to the back of the eye. For example, the ocular iontophoresis
system, OcuPhor.TM., can deliver drugs safely and noninvasively to
the back of the eye (Iomed). Iontophoresis uses a small electrical
current to transport ionized drugs into and through body tissues.
Care must be taken not to use too high of a current density, which
can damage eye tissues.
[0141] Iontophoresis typically involves using a drug applicator, a
dispersive electrode, and an electronic iontophoresis dose
controller. The drug applicator can be a small silicone shell that
contains a conductive element, such as silver-silver chloride. A
hydrogel pad can absorb the drug formulation. A small, flexible
wire can connect the conductive element to the dose controller. The
drug pad can be hydrated with a drug solution immediately before
use, with the applicator is placed on the sclera of the eye under
the lower eyelid. The eyelid holds the applicator in place during
treatment. The drug dose and rate of administration can be
controlled by programming and setting the electronic
controller.
[0142] Treatment of Neurodegenerative Disorders and/or Providing
Neuroprotection
[0143] Reactive oxygen species also induce inflammation and
neurodegeneration. Inhibition of these species can also result in
neuroprotection, including protection from further damage following
an ischemic brain injury such as a stroke, or that caused from
blunt trauma, and treatment or prevention of neurodegenerative
disorders such as retinal degenerations, Alzheimer's disease,
senile dementia, pre-senile dementia, Parkinsons disease,
Huntington's Chorea, multiple sclerosis, and the like.
[0144] Reactive oxygen species also drive seizures, and the
compounds described herein have GABAergic activity which may
ameliorate seizures as well.
[0145] Treatment of Vascular Disorders
[0146] Vascular diseases such as erectile dysfunction and migraines
in which ROS have been implicated may also respond to NADPH oxidase
inhibitors.
[0147] In all of these treatments, the compounds are believed to
function by inhibiting one or more Nox enzymes, such as Nox1-5, or
by stimulating superoxide scavengers (and thus inhibiting ROS
production), or directly reacting with and inactivating ROS.
[0148] Nox2-containing NADPH oxidase and Akt activation are
believed to play a key role in angiotensin II-induced cardiomyocyte
hypertrophy (Physiol. Genomics 26: 180-191, 2006). Inhibition of
this Nox enzyme can therefore be used to treat or prevent
angiotensin II-induced cardiomyocyte hypertrophy.
[0149] The following examples are provided to illustrate the
present invention, and should not be construed as limiting thereof.
In these examples, all parts and percentages are by weight, unless
otherwise noted. Reaction yields are reported in mole
percentages.
EXAMPLES
[0150] The following examples are provided to illustrate the
present invention and should not be construed as limiting the scope
thereof. In these examples, all parts and percentages are by
weight, unless otherwise noted. Reaction yields are reported in
mole percentage.
Example 1
Spectrophotometric Assay of NADH Oxidase
[0151] NADH oxidase activity can be determined as the disappearance
of NADH measured at 340 nm in a reaction mixture containing 25 mM
Tris-Mes buffer (pH 7.2), 1 mM KCN, and 150 .mu.M NADH at
37.degree. C. Activity can be measured, for example, using a
Hitachi U3210 spectrophotometer with stirring and continuous
recording over two intervals of 5 min each. A millimolar extinction
coefficient of 6.22 can be used to determine specific activity.
Example 2
Measuring Cell Growth
[0152] A mouse mammary tumor subpopulation line 4T1 arising from a
BALB/cf C3H mouse can be grown in DME-10, Dulbecco's modified
Eagle's medium supplemented with 5% fetal calf serum, 5% newborn
calf serum, 1 mM mixed non-essential amino acids, 2 mM L-glutamine,
penicillin (100 units/ml), and streptomycin (100 .mu.g/ml) (Miller
et al., 1987, Brit. J. Can. 56:561-569 and Miller et al., 1990,
Invasion Metastasis 10:101-112).
Example 3
Inhibition of Nox2 Enzyme by Various Test Compounds
[0153] Various test compounds were examined for activity against
Nox2 enzyme by determining hydrogen peroxide (H.sub.2O.sub.2)
production in phorbol 12-myristate 13-acetate (PMA)-stimulated
Cos-phox cells treated with different concentrations of vehicle
control or a test compound listed in Table 1.
TABLE-US-00001 TABLE 1 Test Compounds No. Name 1 BWL-63-11 2
BWL-90-3C 3 BWL-304-1 4 BWL-115-7 5 BWL-325-3F 6 BWL-42-2 7
cyclopentadiene hydrazine 8 6-dimethylamino fulvene 9 BWL-42-2 10
Indigo fulvene 11 Dihydroxy-tert-butyl-fulvene 12
Phosphorous-oxy-fulvene 13 Carbazole blue 14 Ethylcarbazole blue 15
Impramine blue 16 Curcumin fulvene 17 Ninhydrine fulvene 18
Dodecane fulvene
[0154] Cos-phox cells have been described previously in Price et
al., Blood, 99: 2653-61 (2002), which is incorporated herein by
reference.
[0155] H.sub.2O.sub.2 release was measured using the homovanillic
acid assay as described previously in Martyn et al., Cellular
Signalling, 18:69-82 (2006) and Perry et al., J. Invest. Dermatol.,
126:2316-22 (2006), which are incorporated herein by reference.
Briefly, 1.5-1.75.times.10.sup.5 cells/well of a 12-well plate were
seeded with Cos-phox cells. The following day, cells were washed
once with Hank's balanced salt solution, stimulated with 0.4 mg/ml
phorbol 12-myristate 13-acetate (PMA), and then preincubated for 15
minutes with either vehicle control or different concentrations
(i.e., 1 .mu.M, 5 .mu.M, or 20 .mu.M) of test compound no. 8, 10,
15, 16, 17, or 18 in 1 ml of media. The cells were then washed once
with Hank's balanced salt solution. Vehicle control or different
concentrations of test compound no. 8, 10, 15, 16, 17, or 18 were
added at the same concentrations as in pretreatment to 0.5 ml of
homovanillic acid assay solution (100 mM homovanillic acid assay, 4
U/ml horseradish peroxidase in Hank's balanced salt solution with
Ca.sup.2+ and Mg.sup.2+) and incubated with the cells for 1 hour at
37.degree. C. The reaction was stopped by adding 75 ml of
homovanillic acid assay stop buffer (0.1 M glycine/0.1 M NaOH (pH
12) and 25 mM EDTA in phosphatebuffered saline). Fluorescence was
read on a BioTek Synergy HT (BioTek Instruments Inc., Winooski,
Vermont, Calif.) with an excitation of 320 nm and emission of 440
nm.
[0156] Cox-phox cells did not produce H.sub.2O.sub.2 without PMA
stimulation with (data not shown) or without the addition of the
test compounds, therefore, in this particular system, detection of
Nox2 activity required PMA. The ability of test compound no. 8, 10,
15, 16, 17, or 18 to inhibit production of H.sub.2O.sub.2 in
Cox-phox cells is shown in FIG. 1 as a percentage relative to the
untreated control (100%).
[0157] The results showed that test compound nos. 8, 10, 15, 16,
17, and 18 inhibited Nox2 enzyme in a dose-dependent manner.
Example 4
In vitro Testing of Various Test Compounds
[0158] Nude mice were injected subcutaneously with approximately
one million tumor cells. Once tumors became visible, they were
treated with 40 mg/kg daily of circumin fulvene. The compound was
reconstituted in 100 microliters of ethanol and diluted with 900
microliters of 20% Intralipid, and 0.3 ml of this mixture was
injected intraperitoneally daily. Tumors were measured with vernier
calipers, and tumor volume was calculated using the formula
(width.sup.2.times.length) 0.52, where width is the smallest
dimension, 2 represents squared, and 1 represents the length.
[0159] The results are shown in Table 1, below, and in FIG. 2.
TABLE-US-00002 TABLE 1 Treatment with Curcumin Fulvene Tumor Group
L W Volume Average Control 12.42 10.18 669.2994922 21.82 21.24
5118.787665 Circumin Control Fulvene 22.58 11.98 1685.159129
2491.082 2491.082 1767.914 Curcumin 14.52 12.09 1103.627622 Fulvene
13.76 8.89 565.4904819 25.83 16.45 3634.624539 1767.914
Example 5
The NADPH Oxidase Inhibitor Fulvene-5 Diminishes Light-Induced
Retinal Function Loss in Albino Mice
[0160] Exposing albino mice to bright light causes loss of retinal
function, an effect partially mediated by damage caused by reactive
oxygen species (ROS). Activation of NADPH oxidase by various
stressors increases ROS production. The purpose of these
experiments was to test whether light-induced retinal function loss
is mediated by NADPH oxidase activity.
[0161] Methods:
[0162] Balb-C mice were exposed to dim (20 lux) or bright (10,000
lux) white light for 6 hours. Mice were injected with Fulvene-5, a
triphenylmethane that inhibits NADPH oxidase, dissolved in vehicle
(intralipid-DMSO) or vehicle alone. Intraperitoneal injections were
given daily for two weeks. Electroretinograms (ERGs) were taken 0,
7, and 14 days following light exposure.
[0163] Results:
[0164] Mice injected with vehicle and exposed to bright light
exhibited significantly diminished ERG a-wave and b-wave amplitudes
compared to mice exposed to bright light but treated with Fulvene-5
or compared to mice exposed to dim light. The results are shown in
FIG. 3.
[0165] Conclusions:
[0166] Treatment with the NADPH oxidase inhibitor Fulvene-5
precluded the damaging effects of bright light exposure on retinal
function as measured by ERG. It may be that bright light exposure
results in activation of NADPH oxidase resulting in increased ROS
production causing retinal cell damage. Retinal morphology,
apoptosis, NADPH oxidase enzyme activity, redox status, and ROS
content are currently being analyzed.
[0167] Mice were exposed to either dim light (control) or bright
light of an intensity that causes retinal degeneration (Light
Induced Retinal Degeneration; LIRD). This is a classic rodent model
of retinal degeneration. For each lighting condition, half the
animals were injected with vehicle and the other half were injected
with Fulvene 5. Electroretinograms (ERGs) of the treated mice were
measured at one week post-exposure. An ERG is a measure of the
change in electrical potential across the eyeball in response to a
flash of light, and is used as an indication of retinal
function.
[0168] The data showed that bright light exposure induced about a
50% suppression of ERG b-wave amplitude at one week. However, rats
injected daily with Fulvene 5 showed no suppression of ERG
amplitude, suggesting that Fulvene 5 prevented visual function loss
at one week. The data is summarized in FIG. 3.
Example 6
ESR Spectrum of a Representative Fulvene and Superoxide
Dismutase
[0169] Li used ESR to confirm the production of NADPH-dependent
.O2- by isolated endosomes (Li et al., Molecular and Cellular
Biology, January 2006, p. 140-154, 26(1):140-154 (2006)). ESR
assays were conducted at room temperature using a Bruker model EMX
ESR spectrometer (Bruker). Vesicular fractions from each sample
were mixed with the spin trap, 50 mM 5,5-dimethyl-1-pyrroline
N-oxide (DMPO), in a total volume of 500 .mu.l of PBS, pH 7.4. This
solution contained iminodiacetic acid-chelating resin (10 ml/liter;
Sigma-Aldrich). The reaction was initiated by adding NADPH to 100
.mu.M and was immediately placed into the ESR spectrometer.
DMPO-hydroxyl radical adduct formation was assayed for 10 min.
Instrument settings were as follows: receiver gain,
1.times.10.sup.6; modulation frequency, 100 kHz; microwave power,
40.14 mW; modulation amplitude, 1.0 G; and sweep rate, 1 G/s.
[0170] In the instant application, the ESR spectrum of Fulvene 5
and of superoxide dismutase were taken using conditions
substantially as described in Li et al. The ESR spectra (FIG. 4)
shows that Fulvene 5 appears to form a radical by reacting with
superoxide, thus inhibiting the ability of superoxide dismutase to
generate ROS.
[0171] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described will
become apparent to those skilled in the art from the foregoing
description and accompanying figures. Such modifications are
intended to fall within the scope of the appended claims.
[0172] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
* * * * *