U.S. patent application number 14/789928 was filed with the patent office on 2016-01-07 for method for treating fibrosis and cancer with imidazolium and imidazolinium compounds.
This patent application is currently assigned to AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH. The applicant listed for this patent is AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH. Invention is credited to Zhaobing Ding, Began Gopalan, Zhiyuan Ke, Jackie Y. Ying, Chunyan Zhang, Yugen Zhang, Lang Zhuo.
Application Number | 20160000757 14/789928 |
Document ID | / |
Family ID | 40913057 |
Filed Date | 2016-01-07 |
United States Patent
Application |
20160000757 |
Kind Code |
A1 |
Zhuo; Lang ; et al. |
January 7, 2016 |
METHOD FOR TREATING FIBROSIS AND CANCER WITH IMIDAZOLIUM AND
IMIDAZOLINIUM COMPOUNDS
Abstract
There is presently provided methods for delivering an
anti-fibrotic or anti-cancer agent to a cell. The methods comprise
contacting a cell with an effective amount of imidazolium and
imidazolinium compounds as described herein, including imidazolium
and imidazolinium salts.
Inventors: |
Zhuo; Lang; (Singapore,
SG) ; Zhang; Chunyan; (Singapore, SG) ; Zhang;
Yugen; (Singapore, SG) ; Ying; Jackie Y.;
(Singapore, SG) ; Gopalan; Began; (Singapore,
SG) ; Ke; Zhiyuan; (Singapore, SG) ; Ding;
Zhaobing; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH |
Singapore |
|
SG |
|
|
Assignee: |
AGENCY FOR SCIENCE, TECHNOLOGY AND
RESEARCH
Singapore
SG
|
Family ID: |
40913057 |
Appl. No.: |
14/789928 |
Filed: |
July 1, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12865668 |
Feb 28, 2011 |
9072729 |
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PCT/SG2009/000037 |
Jan 30, 2009 |
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14789928 |
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61006769 |
Jan 30, 2008 |
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Current U.S.
Class: |
514/397 ;
435/375 |
Current CPC
Class: |
A61K 31/4184 20130101;
A61K 31/4178 20130101; A61K 31/4439 20130101; C07D 233/61 20130101;
C07D 401/04 20130101; C07D 403/04 20130101; C07D 401/14 20130101;
A61P 1/16 20180101; A61K 31/439 20130101; A61P 35/00 20180101; A61K
31/4188 20130101; C07D 233/58 20130101; C07D 235/06 20130101; C07D
471/04 20130101; C07D 487/22 20130101; C07D 487/04 20130101; A61P
43/00 20180101; C07D 233/60 20130101; A61P 19/04 20180101; A61P
39/06 20180101 |
International
Class: |
A61K 31/4178 20060101
A61K031/4178 |
Claims
1. A method for treating cancer selected from the group consisting
of brain cancer, bone cancer, skin cancer, gallbladder cancer,
laryngeal cancer, oral cancer, pleural mesothelioma, testicular
cancer, uterine cancer, thyroid cancer, hepatocellular carcinoma
and glioma in a subject in need of such cancer treatment, the
method comprising administering an effective amount of an
anti-cancer agent to the subject, the anti-cancer agent being: a)
an oligomer or polymer comprising three or more compounds of
general formula I connected together, general formula I being
defined as: ##STR00005## wherein: R.sup.1: (i) is H, straight or
branched C.sub.1-C.sub.6 alkyl, straight or branched
C.sub.2-C.sub.6 alkenyl, straight or branched C.sub.2-C.sub.6
alkynyl, C.sub.6-C.sub.10 aryl; (ii) together with R.sup.2 and
their ring atoms form a 6- to 10-membered fused saturated,
unsaturated or aromatic ring system; (iii) together with R.sup.5
and their ring atoms form a 5- to 10-membered fused saturated,
unsaturated or aromatic ring system when R.sup.2 is as defined
above in (i); or (iv) together with R.sup.5 and their ring atoms
form a 5- to 10-membered fused saturated, unsaturated or aromatic
ring system, when R.sup.2 and R.sup.6 together with their ring
atoms also form a 5- to 10-membered fused saturated, unsaturated or
aromatic ring system; R.sup.2: (i) is H, straight or branched
C.sub.1-C.sub.6 alkyl, straight or branched C.sub.2-C.sub.6
alkenyl, straight or branched C.sub.2-C.sub.6 alkynyl,
C.sub.6-C.sub.10 aryl; (ii) together with R.sup.1 and their ring
atoms form a 6- to 10-membered fused saturated, unsaturated or
aromatic ring system; (iii) together with R.sup.6 and their ring
atoms form a 5- to 10-membered fused saturated, unsaturated or
aromatic ring system when R.sup.1 is as defined above in (i); or
(iv) together with R.sup.6 and their ring atoms form a 5- to
10-membered fused saturated, unsaturated or aromatic ring system,
when R.sup.1 and R.sup.5 together with their ring atoms also form a
5- to 10-membered fused saturated, unsaturated or aromatic ring
system; R.sup.3 is H, or, when R.sup.1 and R.sup.2 together with
their ring atoms form a 6- to 10-membered fused aromatic ring
system, R.sup.3 is absent; R.sup.4 is H, or, when R.sup.1 and
R.sup.2 together with their ring atoms form a 6- to 10-membered
fused aromatic ring system, R.sup.4 is absent; R.sup.5 is: (i) as
defined above for R.sup.1; or (ii) straight or branched
C.sub.1-C.sub.6 alkyl, straight or branched C.sub.2-C.sub.6
alkenyl, straight or branched C.sub.2-C.sub.6 alkynyl,
C.sub.3-C.sub.18 cycloalkyl including fused cycloalkyl ring
systems, C.sub.6-C.sub.10 aryl, C.sub.6-C.sub.10
aryl-C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl-C.sub.2-C.sub.6
alkenyl, or C.sub.6-C.sub.10 aryl-C.sub.2-C.sub.6 alkynyl,
C.sub.1-C.sub.6 alkyl-C.sub.6-C.sub.10 aryl, C.sub.2-C.sub.6
alkenyl-C.sub.6-C.sub.10 aryl, or C.sub.2-C.sub.6
alkynyl-C.sub.6-C.sub.10 aryl; R.sup.6 is: (i) as defined above for
R.sup.2; or (ii) straight or branched C.sub.1-C.sub.6 alkyl,
straight or branched C.sub.2-C.sub.6 alkenyl, straight or branched
C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.18 cycloalkyl including
fused cycloalkyl ring systems, C.sub.6-C.sub.10 aryl,
C.sub.6-C.sub.10 aryl-C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10
aryl-C.sub.2-C.sub.6 alkenyl, or C.sub.6-C.sub.10
aryl-C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6
alkyl-C.sub.6-C.sub.10 aryl, C.sub.2-C.sub.6
alkenyl-C.sub.6-C.sub.10 aryl, or C.sub.2-C.sub.6
alkynyl-C.sub.6-C.sub.10 aryl; R.sup.7 is H, C.sub.1-C.sub.6 alkyl,
phenyl, substituted C.sub.1-C.sub.6 alkyl or halo; in which any of
R.sup.1 to R.sup.7, where applicable, optionally has one or more
carbon atoms replaced with a heteroatom selected from N, O, S and P
and is optionally substituted with one or more of straight or
branched C.sub.1-C.sub.6 alkyl, straight or branched
C.sub.2-C.sub.6 alkenyl, straight or branched C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.18 cycloalkyl including fused cycloalkyl
ring systems, C.sub.6-C.sub.10 aryl, fluoro, tri-fluoro-methyl,
cyanato, isocyanato, carboxyl, C.sub.1-C.sub.6 acyloxy,
C.sub.1-C.sub.6 acyl, carbonyl, amino, acetyl, acetoxy, oxo, nitro,
hydroxyl, C.sub.1-C.sub.6 alkylcarboxy, C.sub.1-C.sub.6 alkoxy,
C.sub.2-C.sub.6 alkenoxy, C.sub.2-C.sub.6 alkynoxy; and in which
one of the ring carbon atom to which R.sup.1 and R.sup.3 are
attached and the ring carbon to which R.sup.2 and R.sup.4 are
attached is optionally replaced with a nitrogen atom; or any
pharmaceutically acceptable salt of the compound or of the oligomer
or polymer of the compound; or b) selected from the group
consisting of 1,3-di-tert-butylimidazolinium,
1,3-bis(1-adamantyl)imidazolium,
1,3-bis(2,4,6-trimethylphenyl)-imidazolinium,
1,3-bis(2,6-diisopropyl-phenyl)-imidazolinium,
1-(1-adamantyl)-3-(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium,
2-benzylimidazo[1,5-a]quinolinium,
1,3-bis(1-adamantyl)-benzimidazolium, 1,3-diisopropylimidazolinium,
2-(2,6-diisopropylphenyl)-5-methylimidazo[1,5-a]pyridinium,
diisopropylphenyl)-3-(2,4,6-trimethylphenyl)-imidazolinium,
2-mesityl-5-methylimidazo[1,5-a]pyridinium,
2-mesityl-2,5,6,7-tetrahydropyrrolo[2,1-c][1,2,4]triazol-4-ium,
1,3-bis(1-adamantyl)imidazolinium,
6,7-dihydro-2-pentafluorophneyl-5H-pyrrolo[2,1-c]-1,2,4-trizolium,
1-methyl-3-(2-hydroxylethyl)-imidazolium,
1-methyl-3-(4-isocynatobenzyl)-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxylethyl)-imidazolium,
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium,
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium,
1-benzyl-3-(2,2-dimethoxylethyl)-2-methyl-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-methylbenzyl)-5-phenylimidazolium,
1-benzyl-3-(3-hydroxyl-propyl)-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-imidazolium,
1-(4-cyanatobenzyl)-3-methyl-imidazolium,
1-(4-carboxybenzyl)-3-methyl-imidazolium,
1,3-Bis(2,6-diisopropylphenyl)imidazolium and
1,3-Di-tert-butylimidazolium, or being any dimer thereof; or any
pharmaceutically acceptable salt thereof; or c) selected from the
group consisting of 2,6-di-(3-benzyl-imidazolium)-pyridine,
2,2'-di-(3-benzyl-imidazolium)-1,1'-binaphthalene,
(1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-benzene,
1,3,5-tris-(4-methyl-imiazolium)-linked cyclophane and
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole; or any pharmaceutically acceptable salt thereof.
2. The method of claim 1 wherein the anti-cancer agent is an
oligomer or polymer comprising three or more compounds of general
formula I connected together and wherein R.sup.5 is the same as
R.sup.6.
3. The method of claim 1 wherein the anti-cancer agent is an
oligomer or polymer comprising three or more compounds of general
formula I connected together and wherein R.sup.5 and R.sup.6 are
hydrocarbons.
4. The method of claim 1 wherein the anti-cancer agent is a trimer
of a compound having a structure of general formula I or any
pharmaceutically acceptable salt thereof.
5. The method of claim 1 wherein the anti-cancer agent is
1,3-di-tert-butylimidazolinium, 1,3-bis(1-adamantyl)imidazolium,
1,3-bis(2,4,6-trimethylphenyl)-imidazolinium,
1,3-bis(2,6-diisopropyl-phenyl)-imidazolinium,
1-(1-adamantyl)-3-(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium,
2-benzylimidazo[1,5-a]quinolinium,
1,3-bis(1-adamantyl)-benzimidazolium, 1,3-diisopropylimidazolinium,
2-(2,6-diisopropylphenyl)-5-methylimidazo[1,5-a]pyridinium,
1-(2,6-diisopropylphenyl)-3-(2,4,6-trimethylphenyl)-imidazolinium,
2-mesityl-5-methylimidazo[1,5-a]pyridinium,
2-mesityl-2,5,6,7-tetrahydropyrrolo[2,1-c][1,2,4]triazol-4-ium,
1,3-bis(1-adamantyl)imidazolinium,
6,7-dihydro-2-pentafluorophneyl-5H-pyrrolo[2,1-c]-1,2,4-trizolium,
1-methyl-3-(2-hydroxylethyl)-imidazolium,
1-methyl-3-(4-isocynatobenzyl)-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxylethyl)-imidazolium,
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium,
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium,
1-benzyl-3-(2,2-dimethoxylethyl)-2-methyl-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-methylbenzyl)-5-phenylimidazolium,
1-benzyl-3-(3-hydroxyl-propyl)-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-imidazolium,
1-(4-cyanatobenzyl)-3-methyl-imidazolium,
1-(4-carboxybenzyl)-3-methyl-imidazolium,
1,3-Bis(2,6-diisopropylphenyl)imidazolium or
1,3-Di-tert-butylimidazolium, or any dimer thereof or any
pharmaceutically acceptable salt thereof.
6. The method of claim 1 wherein the anti-cancer agent is
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole, (1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-benzene,
2,6-di-(3-benzyl-imidazolium)-pyridine or
2,2'-di-(3-benzyl-imidazolium)-1,1'-binaphthalene, or any
pharmaceutically acceptable salt thereof.
7. The method of claim 1 wherein the anti-cancer agent is
1,3,5-tris(4-methyl-imidazolium)-linked cyclophane or any
pharmaceutically acceptable salt thereof.
8. The method of claim 1 in wherein the pharmaceutically acceptable
salt is a chloride, bromide, tetrafluoroborate or
hexafluorophosphate salt.
9-13. (canceled)
14. The method of claim 1 wherein the cancer is hepatocellular
carcinoma or glioma.
15. The method of claim 1 wherein the cancer is brain cancer, bone
cancer, skin cancer, gallbladder cancer, laryngeal cancer, oral
cancer, pleural mesothelioma, testicular cancer, uterine cancer or
thyroid cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of application Ser. No. 12/865,668,
filed Feb. 28, 2011, now U.S. Pat. No. 9,072,729, which was the
National Stage of International Application No. PCT/SG2009/000037,
filed Jan. 30, 2009, which claims benefit of U.S. Provisional
Patent Application No. 61/006,769, filed Jan. 30, 2008, the
contents of all of which are fully incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for delivering an
anti-fibrotic or an anti-cancer agent to a cell, including methods
for treating fibrotic disease or cancer.
BACKGROUND OF THE INVENTION
[0003] Hepatic stellate cells (HSCs) play a pivotal role in hepatic
fibrogenesis and are considered a major cellular target for
therapeutic intervention. However, no approved anti-fibrotic drug
is currently on the market.
[0004] Hepatic fibrogenesis may be incurred as a result of insults
arising from oxidative stress, chemical toxicity or viral
infection. Thus fibrotic disease, including cell and tissue
fibrosis, is among the physiological disorders that have been
associated with oxidative stress. For example, oxidative stress
resulting from the metabolic generation of ROS has been linked to
HSC activation and liver fibrosis (Britton et al. 1994, Tsukamoto
et al. 1995, Galli et al. 2005). It has been shown that products of
lipid peroxidation lead to increased collagen synthesis by HSCs
(Casini et al. 1997 and Parola et al. 1996).
[0005] Reactive oxygen species (ROS) are oxygen derivatives that
include free radicals and non-radical reactive molecules such as
peroxides and oxygen derivatives. Oxidative stress occurs when
there is an excess amount of ROS due to an imbalance between the
generation and the elimination or neutralization of these
molecules. Excess amounts of ROS may damage cell lipids, proteins
and DNA resulting in the inhibition of normal cell function or the
development of abnormal cellular behaviour which can in turn lead
to a variety of diseases and conditions, depending on the cells
affected. ROS have been implicated in many physiological disorders
and in the onset and development of a number of diseases.
[0006] Some anti-oxidants have been explored as potential
inhibitors of hepatic fibrosis (Kawada et al. 1998).
(-)-Epigallocatechin gallate (EGCG), a major active component of
tea catechin, has been shown to inhibit HSC activation in vitro via
transforming growth factor-beta (TGF-.beta.) signalling (Chen et
al. 2002, Nakanuta et al. 2005, Fu et al. 2006). In clinical
studies, a combination of vitamins E and C was shown to decrease
the fibrosis score in non-alcoholic steatohepatitis patients, but
did not affect hepatic inflammation (Harrison et al. 2003).
N-acetyl-L-cysteine (NAC), a synthetic precursor of glutathione
(GSH) that has been clinically used as an antioxidant, showed
anti-fibrogenic properties through the suppression of TGF-.beta.
signalling transduction as well (Meurer et al. 2005).
[0007] Oxidative stress has also been associated with the onset and
progression of cancer. It has been reported that oxidative stress
as indicated by reduced anti-oxidant enzyme activities is
associated with the development of primary carcinogenesis and
metastasis in clinical patients (Vali et al. 2008). ROS have been
found to cause DNA damage increasing the risk of DNA mutation and
thus the development of cancer (Hussain et al. 2005). Tea
polyphenols are anti-oxidants and have been shown to inhibit
carcinogen-induced DNA damage in animal models of skin, lung,
colon, liver and pancreatic cancers (Frei et al. 2003). In
addition, anti-oxidants PBN and NXY-059 have demonstrated
anti-cancer activity in hepatocellular carcinoma. (Floyd 2006).
However, the effectiveness of the anti-oxidants investigated
remains unclear (Valko et al, 2004).
[0008] The inflammatory response is the immune system's response to
infection or injury and involves the activation of cells of the
immune system which produce mediators, such as cytokines, that
further activate other cells, leading to a cascade of immune
reactions that fight off the infection or repair the injury Like
oxidative stress, inflammatory stresses have been associated with
fibrotic disease and cancer (Rakoff-Nahoum 2006; Tsukamoto et al.
1999; Bachem et al. 1992; Vasiliou et al. 2000). For example, in
the development of liver fibrosis, HSCs play a critical role.
Responding to liver injury, HSCs undergo a process called
"activation" and trans-differentiate to myofibroblast-like cells.
This process is characterized by phenotypic changes including cell
proliferation, over-expression of smooth muscle actin-.alpha.
(SMAA), and deposition of extracellular matrix (ECM) proteins,
including collagen type .alpha.I (I) (col1a1) and fibronectin.
[0009] Inflammatory cytokines represent major mediators for HSC
activation.
[0010] Among them, transforming growth factor-beta I (TGF-.beta.1)
and interleukin 6 (IL-6) have been categorized as profibrogenic
cytokines mainly responsible for the induction of ECM proteins
(Tsukamoto 1999). HSCs respond to TGF-.beta.1 secreted from Kupffer
cells and endothelial cells during liver injury, and themselves via
autocrine action, resulting in HSC activation and liver fibrosis
(Bachem et al. 1992). In addition, the activation of HSCs can also
be attributed to chronic hepatic inflammation through the secretion
of proinflammatory cytokine IL-6, leading to cirrhosis (Vasiliou et
al. 2000) and hepatocellular carcinoma (Naugler et al, 2007).
Transcription factor nuclear factor kappa B (NF-.kappa.B) is an
important regulator for the secretion of inflammatory cytokines,
and its subunit NF-.kappa.B p65 was reported to mediate liver
fibrosis (Vasiliou et al. 2000).
[0011] Beyond sharing a common association with fibrotic diseases
and cancer, the production and regulation of oxidative and
inflammatory stress appear to be interconnected, as an inflammatory
response can trigger oxidative stress and vice versa.
[0012] For example, in hepatic fibrogenesis, both ROS and
pro-inflammatory cytokines have been shown to be involved in the
activation of HSCs, the central event in the development of liver
fibrogenesis. TGF-.beta. is a major mediator for HSC activation and
TGF-.beta. signalling is affected by both oxidative stress and the
induction of an inflammatory response (Tsukamoto et al. 1999,
Bachem et al. 1992, Chen et al. 2002, Nakanuta et al. 2005, Fu et
al. 2006, Meurer et al. 2005). Another key molecule in the
development of fibrosis, is NF-.kappa.B which is an important
regulator of the secretion of inflammatory cytokines but is also
known to be sensitive to oxidative stress. Most agents activating
NF-.kappa.B are either modulated by ROS or oxidant themselves. It
has been reported that treatment with anti-oxidant resveratrol
(Chavez et al. 2007) or vitamin E (Liu et al. 1995) attenuated
NF-.kappa.B elevation induced in carbon tetrachloride experimental
fibrotic rodents.
[0013] Oxidative stress and inflammatory response have also been
found to be interrelated in the pathogenesis of cancer. Oxidative
stress can cause DNA mutations, some of which will result in the
formation of cancer cells. However other mutations will result in
cell death, stimulating an inflammatory response. In turn, an
inflammatory response will not only provide survival and
proliferative signals to cancer cells but may also induce the
production of ROS (Rakoff-Nahoum 2006).
[0014] Dietary anti-oxidants have been widely used as a general
approach to ameliorate excessive oxidative stress both in animal
models and humans. For example, resveratrol has been shown to
extend the lifespan of various species, and to be effective at
improving the health and survival of mice on a high-calorie diet
(Baur et al. 2006).
[0015] To date, the development of effective treatments for
oxidative stress and certain diseases associated with oxidative
stress using natural or synthetic anti-oxidants has been
problematic. Stringent scientific proof for the efficacy of natural
anti-oxidants has not been established (Droge et al. 2001). Some of
the notable limitations for using natural anti-oxidants as
therapeutics include low potency and fast turnover during
metabolism. In contrast, the development of synthetic anti-oxidants
has been inhibited by safety concerns. Nevertheless, some progress
has been made in this direction. Modification of a natural
anti-oxidant has been performed to enhance its potency (Keum et al.
2007). In addition, synthetic mimics of superoxide dismutase (SOD)
and catalase have been shown to be effective in rodent models of
ischemia and Parkinson's disease (Peng et al. 2005). Even more
encouragingly, a class of nitron-free radical trap agents,
alpha-phenyl-N-tert-butyl-nitron (PBN) and disodium
2,4-disulfophenyl-N-tert-butylnitrone (NXY-059), have been shown to
be potent neuroprotective agent (Maples et al. 2004), and have
demonstrated anti-cancer activity in hepatocellular carcinoma
through its anti-inflammatory property (Floyd 2006).
SUMMARY OF THE INVENTION
[0016] There is presently provided methods for delivering an
anti-fibrotic or anti-cancer agent to a cell, the method comprising
contacting a cell with an effective amount of imidazolium and
imidazolinium compounds, including imidazolium and imidazolinium
salts, as described herein.
[0017] In one aspect, there is provided a method for delivering an
anti-fibrotic or anti-cancer agent to a cell, the method comprising
contacting the cell with an effective amount of a compound of
general formula I or an oligomer or polymer thereof:
##STR00001##
wherein: the dashed line is absent or is present as a bond to form
a second bond between the carbon to which R.sup.1 and R.sup.3 are
attached and the carbon to which R.sup.2 and R.sup.4 are
attached;
R.sup.1 and R.sup.2:
[0018] (i) are each independently H, straight or branched
C.sub.1-C.sub.6 alkyl, straight or branched C.sub.2-C.sub.6
alkenyl, straight or branched C.sub.2-C.sub.6 alkynyl,
C.sub.6-C.sub.10 aryl; [0019] (ii) together with their ring atoms
form a 6- to 10-membered fused saturated, unsaturated or aromatic
ring system; [0020] (iii) R.sup.1 and R.sup.5 together with their
ring atoms, or R.sup.2 and R.sup.6 together with their ring atoms,
form a 5- to 10-membered fused saturated, unsaturated or aromatic
ring system and the other of R.sup.1 and R.sup.2 is as defined
above in (i); or [0021] (iv) R.sup.1 and R.sup.5 together with
their ring atoms and R.sup.2 and R.sup.6 together with their ring
atoms each form a 5- to 10-membered fused saturated, unsaturated or
aromatic ring system; R.sup.3 and R.sup.4 are both H, or, when
R.sup.1 and R.sup.2 together with their ring atoms form a 6- to
10-membered fused aromatic ring system or when the dashed line is
present as a bond, R.sup.3 and R.sup.4 are absent;
R.sup.5 or R.sup.6:
[0021] [0022] (i) are as defined above for R.sup.1 and R.sup.2; or
[0023] (ii) are each independently straight or branched
C.sub.1-C.sub.6 alkyl, straight or branched C.sub.2-C.sub.6
alkenyl, straight or branched C.sub.2-C.sub.6 alkynyl, C.sub.3-Cis
cycloalkyl including fused cycloalkyl ring systems,
C.sub.6-C.sub.10 aryl, C.sub.6-C.sub.10 aryl-C.sub.1-C.sub.6 alkyl,
C.sub.6-C.sub.10 aryl-C.sub.2-C.sub.6 alkenyl, or C.sub.6-C.sub.10
aryl-C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6
alkyl-C.sub.6-C.sub.10 aryl, C.sub.2-C.sub.6
alkenyl-C.sub.6-C.sub.10 aryl, or C.sub.2-C.sub.6
alkynyl-C.sub.6-C.sub.10 aryl; R.sup.7 is H, C.sub.1-C.sub.6 alkyl,
phenyl, substituted C.sub.1-C.sub.6 alkyl or halo; in which any of
R.sup.1 to R.sup.7, where applicable, optionally has one or more
carbon atoms replaced with a heteroatom selected from N, O, S and P
and is optionally substituted with one or more of straight or
branched C.sub.1-C.sub.6 alkyl, straight or branched
C.sub.2-C.sub.6 alkenyl, straight or branched C.sub.2-C.sub.6
alkynyl, C.sub.3-Cis cycloalkyl including fused cycloalkyl ring
systems, C.sub.6-C.sub.10 aryl, fluoro, tri-fluoro-methyl, cyanato,
isocyanato, carboxyl, C.sub.1-C.sub.6 acyloxy, C.sub.1-C.sub.6
acyl, carbonyl, amino, acetyl, acetoxy, oxo, nitro, hydroxyl,
C.sub.1-C.sub.6 alkylcarboxy, C.sub.1-C.sub.6 alkoxy,
C.sub.2-C.sub.6 alkenoxy, C.sub.2-C.sub.6 alkynoxy; and and in
which one of the ring carbon atom to which R.sup.1 and R.sup.5 are
attached and the ring carbon to R.sup.2 and R.sup.6 are attached is
optionally replaced with a nitrogen atom; or a pharmaceutically
acceptable salt thereof.
[0024] In one embodiment, the compound is an imidazolium or an
imidazolium. In another embodiment, the compound is an
imidazolinium or an imidazolinium salt.
[0025] In one embodiment, R.sup.5 is the same as R.sup.6. In
another embodiment R.sup.5 and R.sup.6 are hydrocarbons.
[0026] In certain embodiments, the pharmaceutically acceptable salt
of general formula I may be a chloride, bromide, tetrafluoroborate
or hexafluorophosphate salt.
[0027] In various embodiments, the compound may be
1-ethyl-3-methylimidazolium, 1,3-bisbenzylimidazolium,
1,3-diisopropylimidazolium, 1,3-di-tert-butylimidazolinium,
1,3-bis(1-adamantyl)imidazolium,
1,3-bis(2,4,6-trimethylphenyl)-imidazolinium,
1,3-bis(2,6-diisopropyl-phenyl)-imidazolinium,
1,3-diallylimidazolium, 1-benzyl-3-methylimidazolium,
1-butyl-3-methylimidazolium,
1-(1-adamantyl)-3-(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium,
2-benzylimidazo[1,5-a]quinolinium,
1,3-bis(1-adamantyl)-benzimidazolium,
1,3-dicyclohexylbenzimidazolium, 1,3-diisopropylimidazolinium
tetrafluoroborate, 1,3-diisopropylimidazolium,
2-(2,6-diisopropylphenyl)-5-methylimidazo[1,5-a]pyridinium,
1-(2,6-diisopropylphenyl)-3-(2,4,6-trimethylphenyl)-imidazolinium,
2-mesityl-5-methylimidazo[1,5-a]pyridinium,
2-mesityl-2,5,6,7-tetrahydropyrrolo[2,1-c][1,2,4]triazol-4-ium,
1,3-bis(1-adamantyl)imidazolinium,
1-butyl-3-(2-pyridinylmethyl)-1H-imidazolium,
6,7-dihydro-2-pentafluorophneyl-5H-pyrrolo[2,1-c]-1,2,4-trizolium,
1-methyl-3-(2-hydroxylethyl)-imidazolium,
1-methyl-3-(4-isocynatobenzyl)-imidazolium,
1-methyl-3-(4-carboxylbenzyl)-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxylethyl)-imidazolium,
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium,
1,3-Dibenzyl-5-phenylimidazolium,
1-benzyl-3-(4-carboxylbenzyl)-2-methylimidazolium,
1-benzyl-3-(3,4,5-trimethoxylbenzyl)-2-methylimidazolium,
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium,
1-benzyl-3-(4-methylcarboxylatebenzyl)-2-methyl-imidazolium,
1-benzyl-3-(2,2-dimethoxylethyl)-2-methyl-imidazolium,
2,6-di-(3-benzyl-imidazolium)-pyridine,
2,2'-di-(3-benzyl-imidazolium)-1,1'-binaphthalene,
1-benzyl-3-(4-methylbenzyl)-imidazolium,
1-benzyl-3-(2-trifluoromethylbenzyl)-2-methylimidazolium,
1-benzyl-3-(4-methylcarboxylatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-methylbenzyl)-5-phenylimidazolium,
(1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-benzene,
1-benzyl-3-(2-propyn-1-yl)-imidazolium,
1-benzyl-3-(3-hydroxyl-propyl)-imidazolium,
1,3-di(2-phenylethyl)-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-imidazolium,
1-benzyl-3-(pyridin-2-yl)-imidazolium,
1,3,5-tris-(4-methyl-imiazolium)-linked cyclophane,
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole, 1-benzyl-3-methyl-imidazolium,
1-(4-cyanatobenzyl)-3-methyl-imidazolium,
1-(4-carboxybenzyl)-3-methyl-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxyethyl)-imidazolium, or
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium, or a
pharmaceutically acceptable salt thereof.
[0028] In one embodiment, the compound is a dimer of a compound
having a structure of general formula I. For example, the compound
may be
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole, (1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-benzene,
2,6-di-(3-benzyl-imidazolium)-pyridine or
2,2'-di-(3-benzyl-imidazolium)-1,1'-binaphthalene, or a
pharmaceutically acceptable salt thereof.
[0029] In another embodiment, the compound is a trimer of a
compound having a structure of general formula I. For example, the
compound is 1,3,5-tris(4-methyl-imidazolium)-linked cyclophane or a
pharmaceutically acceptable salt thereof.
[0030] In one embodiment of the present methods, the cell may be in
vitro.
[0031] In another embodiment, the cell may be in vivo. For example,
the method may comprise administering the agent to a subject for
the treatment of fibrotic disease or the treatment of cancer. In
one particular embodiment, the fibrotic disease may be hepatic
fibrosis amd the compound may be for example,
3-diisopropylimidazolium or a pharmaceutically acceptable salt
thereof. In other embodiments, the cancer may be hepatocellular
carcinoma, lung cancer, breast cancer, stomach cancer or glioma or
the compound may be for example, 3-Bisbenzylimidazolium,
3-dibenzyl-2-methylimidazolium,
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium,
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium,
1-benzyl-3-(4-methylbenzyl)-imidazolium,
1-benzyl-3-(2-trifluoromethylbenzyl)-2-methylimidazolium or
1,3-di(2-phenylethyl)-imidazolium, or a pharmaceutically acceptable
salt thereof.
[0032] In another aspect, there is provided use of a compound
having a structure of general formula I, or an oligomer or polymer
thereof, for delivering an anti-fibrotic or anti-cancer agent to a
cell in vivo:
[0033] In another aspect, there is provided use of a compound
having a structure of general formula I, or an oligomer or polymer
thereof, in the manufacture of a medicament for delivering an
anti-fibrotic or anti-cancer agent to a cell in vivo.
[0034] In one embodiment of the uses described herein, the compound
is an imidazolium or an imidazolium salt. In another embodiment,
the compound is an imidazolinium or an imidazolinium salt.
[0035] In other embodiments, R.sup.5 is the same as R.sup.6. In
another embodiment R.sup.5 and R.sup.6 are hydrocarbons.
[0036] In certain embodiments, the pharmaceutically acceptable salt
of general formula I may be a chloride, bromide, tetrafluoroborate
or hexafluorophosphate salt.
[0037] In various embodiments of the present uses, the compound may
be 1-ethyl-3-methylimidazolium, 1,3-bisbenzylimidazolium,
1,3-diisopropylimidazolium, 1,3-di-tert-butylimidazolinium,
1,3-bis(1-adamantyl)imidazolium,
1,3-bis(2,4,6-trimethylphenyl)-imidazolinium,
1,3-bis(2,6-diisopropyl-phenyl)-imidazolinium,
1,3-diallylimidazolium, 1-benzyl-3-methylimidazolium,
1-butyl-3-methylimidazolium,
1-(1-adamantyl)-3-(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium,
2-benzylimidazo[1,5-a]quinolinium,
1,3-bis(1-adamantyl)-benzimidazolium,
1,3-dicyclohexylbenzimidazolium, 1,3-diisopropylimidazolinium
tetrafluoroborate, 1,3-diisopropylimidazolium,
2-(2,6-diisopropylphenyl)-5-methylimidazo[1,5-a]pyridinium,
1-(2,6-diisopropylphenyl)-3-(2,4,6-trimethylphenyl)-imidazolinium,
2-mesityl-5-methylimidazo[1,5-a]pyridinium,
2-mesityl-2,5,6,7-tetrahydropyrrolo[2,1-c][1,2,4]triazol-4-ium,
1,3-bis(1-adamantyl)imidazolinium,
1-butyl-3-(2-pyridinylmethyl)-1H-imidazolium,
6,7-dihydro-2-pentafluorophneyl-5H-pyrrolo[2,1-c]-1,2,4-trizolium,
1-methyl-3-(2-hydroxylethyl)-imidazolium,
1-methyl-3-(4-isocynatobenzyl)-imidazolium,
1-methyl-3-(4-carboxylbenzyl)-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxylethyl)-imidazolium,
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium,
1,3-Dibenzyl-5-phenylimidazolium,
1-benzyl-3-(4-carboxylbenzyl)-2-methylimidazolium,
1-benzyl-3-(3,4,5-trimethoxylbenzyl)-2-methylimidazolium,
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium,
1-benzyl-3-(4-methylcarboxylatebenzyl)-2-methyl-imidazolium,
1-benzyl-3-(2,2-dimethoxylethyl)-2-methyl-imidazolium,
2,6-di-(3-benzyl-imidazolium)-pyridine,
2,2'-di-(3-benzyl-imidazolium)-1,1'-binaphthalene,
1-benzyl-3-(4-methylbenzyl)-imidazolium,
1-benzyl-3-(2-trifluoromethylbenzyl)-2-methylimidazolium,
1-benzyl-3-(4-methylcarboxylatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-methylbenzyl)-5-phenylimidazolium,
(1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-benzene,
1-benzyl-3-(2-propyn-1-yl)-imidazolium,
1-benzyl-3-(3-hydroxyl-propyl)-imidazolium,
1,3-di(2-phenylethyl)-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-imidazolium,
1-benzyl-3-(pyridin-2-yl)-imidazolium,
1,3,5-tris-(4-methyl-imiazolium)-linked cyclophane,
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole, 1-benzyl-3-methyl-imidazolium,
1-(4-cyanatobenzyl)-3-methyl-imidazolium,
1-(4-carboxybenzyl)-3-methyl-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxyethyl)-imidazolium, or
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium, or a
pharmaceutically acceptable salt thereof.
[0038] In one embodiment, the compound is a dimer of a compound
having a structure of general formula I. For example, the compound
may be
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole, (1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-benzene,
2,6-di-(3-benzyl-imidazolium)-pyridine or
2,2'-di-(3-benzyl-imidazolium)-1,1'-binaphthalene, or a
pharmaceutically acceptable salt thereof.
[0039] In another embodiment, the compound is a trimer of a
compound having a structure of general formula I. For example, the
compound is 1,3,5-tris(4-methyl-imidazolium)-linked cyclophane or a
pharmaceutically acceptable salt thereof.
[0040] In one embodiment of the present uses, the anti-fibrotic
agent is delivered for the treatment of fibrotic disease. In a
particular embodiment, the fibrotic disease may be hepatic fibrosis
amd the compound may be for example, 3-diisopropylimidazolium or a
pharmaceutically acceptable salt thereof.
[0041] In another embodiment of the present uses, the anti-cancer
agent is delivered for the treatment of cancer. In various
embodiments the cancer s hepatocellular carcinoma, lung cancer,
breast cancer, stomach cancer or glioma and the compounds may be,
for example, 3-Bisbenzylimidazolium,
3-Dibenzyl-2-methylimidazolium,
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium,
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium,
1-benzyl-3-(4-methylbenzyl)-imidazolium,
1-benzyl-3-(2-trifluoromethylbenzyl)-2-methylimidazolium or
1,3-di(2-phenylethyl)-imidazolium, or a pharmaceutically acceptable
salt thereof.
[0042] In another aspect there is provided, a compound that is an
imidazolium or imidazolinium that is 1-ethyl-3-methylimidazolium,
1-methyl-3-(2-hydroxylethyl)-imidazolium,
1-methyl-3-(4-isocynatobenzyl)-imidazolium,
1-methyl-3-(4-carboxylbenzyl)-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxylethyl)-imidazolium,
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium,
1,3-Dibenzyl-5-phenylimidazolium,
1-benzyl-3-(4-carboxylbenzyl)-2-methylimidazolium,
1-benzyl-3-(3,4,5-trimethoxylbenzyl)-2-methylimidazolium,
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium,
1-benzyl-3-(4-methylcarboxylatebenzyl)-2-methyl-imidazolium,
1-benzyl-3-(2,2-dimethoxylethyl)-2-methyl-imidazolium,
2,6-di-(3-benzyl-imidazolium)-pyridine,
2,2'-di-(3-benzyl-imidazolium)-1,1'-binaphthalene,
1-benzyl-3-(4-methylbenzyl)-imidazolium,
1-benzyl-3-(2-trifluoromethylbenzyl)-2-methylimidazolium,
1-benzyl-3-(4-methylcarboxylatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-methylbenzyl)-5-phenylimidazolium,
(1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-benzene,
1-benzyl-3-(2-propyn-1-yl)-imidazolium,
1-benzyl-3-(3-hydroxyl-propyl)-imidazolium,
1,3-di(2-phenylethyl)-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-imidazolium,
1-benzyl-3-(pyridin-2-yl)-imidazolium,
1,3,5-tris-(4-methyl-imiazolium)-linked cyclophane,
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole, 1-benzyl-3-methyl-imidazolium,
1-(4-cyanatobenzyl)-3-methyl-imidazolium,
1-(4-carboxybenzyl)-3-methyl-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxyethyl)-imidazolium, or
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium, or a
pharmaceutically acceptable salt thereof.
[0043] Other aspects and features of the present invention will
become apparent to those of ordinary skill in the art upon review
of the following description of specific embodiments of the
invention in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] In the figures, which illustrate, by way of example only,
embodiments of the present invention:
[0045] FIG. 1. Cytotoxicity of (a) DBZIM and (b) TDBZIM. HSC T6
cells were seeded at a density of 5000 cells/well in 96-well plate,
and cultured for 18-24 h in 10% FBS DMEM before the addition of
various compounds (0-400 .mu.M) in 10% FBS DMEM for 48 h of
proliferation before assaying. The signal was normalized against a
vehicle control treatment (0 .mu.M of compound). The data were
obtained from four independent experiments, and presented as mean
and standard error or mean (SEM), *.sup.a P<0.05, *.sup.b
P<0.01 and *.sup.c P<0.005.
[0046] FIG. 2. (a) DBZIM and (b) TDBZIM attenuated cellular ROS
level. HSC T6 cells were incubated with DBZIM (10, 50, 100 and 300
.mu.M), TDBZIM (10, 50 and 100 .mu.M), NAC (1 mM) and EGCG (25
.mu.M) for 48 h before assaying for cellular ROS levels. The data
were presented as relative values after normalizing against a
vehicle control. The data were presented as mean and SEM, N=6,
*.sup.b P<0.01, *.sup.c P<0.005, and *.sup.d P<0.0005 when
compared to the vehicle control.
[0047] FIG. 3. Effect of DBZIM and TDBZIM on amount of GSH (a)
DBZIM attenuated total cellular GSH amount, and (b) TDBZIM did not
change the total GSH. (c) DBZIM and (d) TDBZIM both suppressed
cellular GSSG (the oxidation product of GSH). (e) DBZIM and (f)
TDBZIM both enhanced the GSH/GSSG ratio. HSC-T6 cells were
incubated with compounds of various concentrations for 48 h, and
assayed for the total GSH and GSSG amounts. The GSH/GSSG ratio was
calculated as (GSH-GSSG)/GSSG. The GSH and GSSG amounts were
normalized against the total protein. Note that DBZIM was dissolved
in DMSO, and TDBZIM was dissolved in H.sub.2O. The data were
presented as mean and SEM, N=6, *.sup.aP<0.05, *.sup.bp<0.01,
*.sup.c P<0.005, and *.sup.d P<0.0005, when compared to the
vehicle control.
[0048] FIG. 4. Effect of DBZIM and TDBZIM on GPx, CAT, SOD and GST.
DBZIM affected (a) GPx activity and (c) CAT activity in a
dosage-dependent manner. TDBZIM suppressed (b) GPx activity and (d)
CAT activity. (e) DBZIM and (f) TDBZIM had little effect on SOD
activity. (g) DBZIM induced GST activity. HSC T6 cells were treated
with compounds of various concentrations for 48 h, and homogenized
by sonication (60% frequency for 30 s) in PBS (pH 7.4, 1 mM of
EDTA) for GPx, GST and CAT assays, or in 20 mM of HEPES buffer
containing 1 mM of EGTA, 210 mM of mannitol and 70 mM of sucrose
(pH 7.2) for SOD assay. Enzyme activity was normalized against
total protein. DBZIM was dissolved in DMSO, and TDBZIM was
dissolved in H.sub.2O. The data were presented as mean and SEM,
N=6, *.sup.a P<0.05, *.sup.b P<0.01, *.sup.c P<0.005, and
*.sup.d P<0.0005, when compared to the vehicle control.
[0049] FIG. 5. DBZIM and TDBZIM protected primary HSC cells against
oxidative stress induced by 0.2% (v/v) of DMSO. (a) Optical images
of cells treated with IMSs for 26 and 93 h. Scale bar=60 .mu.m.
0.2% (v/v) of DMSO induced oxidative stress by depleting the total
GSH level (b), inducing the GSSG level (c), reducing the GSH/GSSG
ratio (d), and inducing GPX (e), CAT (f) and SOD (g) activity
levels. The data were presented as mean and SEM, N=6, *.sup.c
P<0.005, and *.sup.d P<0.0005, when compared to the untreated
sample.
[0050] FIG. 6. (a) DBZIM and (b) TDBZIM suppressed the mRNA
expression of HSC activation markers (GFAP and SMAA) and fibrotic
endpoints (col1a1 and fibronectin) in a dosage-dependent manner.
HSC T6 cells were treated with DBZIM (1, 10, 100 and 300 .mu.M) and
TDBZIM (1, 10 and 100 .mu.M) for 48 h. .beta.-actin was used as the
normalization gene; its expression level was constant under the
experimental conditions. The .DELTA..DELTA.Ct method was used for
relative quantification with vehicle control sample being the
calibrator. The data were presented as mean and SEM, N=6, *.sup.a
P<0.05, *.sup.b P<0.01, *.sup.c P<0.005, and *.sup.d
P<0.0005, when compared to the vehicle control.
[0051] FIG. 7. DBZIM (100 .mu.M) and TDBZIM (100 .mu.M) suppressed
(a) GFAP, (b) SMAA, (c) col1a1 and (d) fibronectin mRNA expression
in a time-dependent manner. HSC T6 cells were treated with DBZIM
and TDBZIM, and assayed after 8, 24 and 48 h. A transient elevation
of SMAA expression was observed for TDBZIM at 24 h. The mRNA
expression was calculated using the .DELTA..DELTA.Ct method by
normalizing to .beta.-actin house-keeping gene and vehicle control
sample, and presented as relative quantification data with mean and
SEM, N=6, *.sup.a P<0.05, *.sup.b P<0.01, *.sup.c P<0.005,
and *.sup.d P<0.0005, when compared to the vehicle control.
[0052] FIG. 8. Western blotting of col1a1, fibronectin, GFAP and
SMAA protein expression in HSC T6 cells treated with DBZIM (100
.mu.M) and TDBZIM (50 .mu.M) for 48 h. 15 .mu.g of total proteins
were resolved in 3-8% of tris-acetate gel for col1a1 and
fibronectin, or 4-12% gradient PAGE-SDS gel for GFAP and SMAA. The
target proteins were recognized by the respective antibodies, and
visualized by the ECL method. The membrane was stripped and
re-probed for .alpha.-tubulin as the loading control.
[0053] FIG. 9. DBZIM and TDBZIM suppressed TGF-.beta.1 mRNA. HSC T6
cells were incubated with (a) DBZIM (1, 10, 100 and 300 .mu.M) and
(b) TDBZIM (1, 10 and 100 .mu.M) for 48 h for the dosage-dependent
study, and (c) with DBZIM (100 .mu.M) and TDBZIM (100 .mu.M) for 8,
24 and 48 h for the time-dependent study. The expression level of
TGF-.beta.1 mRNA was quantified with real-time RT-PCR. The data
were presented as mean and SEM, N=6, *.sup.a P<0.05, *.sup.b
P<0.01, *.sup.c P<0.005, when compared to vehicle
control.
[0054] FIG. 10. DBZIM and TDBZIM suppressed IL-6 mRNA. HSC T6 cells
were incubated with (a) DBZIM (1, 10, 100 and 300 .mu.M) and (b)
TDBZIM (1, 10 and 100 .mu.M) for 48 h for the dosage-dependent
study, and (c) with DBZIM (100 .mu.M) and TDBZIM (100 .mu.M) for 8,
24 and 48 h for the time-dependent study. The expression level of
IL-6 mRNA was quantified with real-time RT-PCR. The data were
presented as mean and SEM, N=6, *.sup.b P<0.01, *.sup.c
P<0.005, and *.sup.d P<0.0005, when compared to the vehicle
control.
[0055] FIG. 11. Western blotting of NF-.kappa.B and AP-1 (c-Fos,
JunD and Fra-1) protein expressions in HSC T6 cells treated with
DBZIM (100 .mu.M) and TDBZIM (50 .mu.M) for 48 h. 15 .mu.g of
nuclear protein was resolved in 4-12% gradient PAGE-SDS gel. The
target proteins were recognized by the respective antibodies, and
visualized by the ECL method. The membrane was stripped and probed
for the Tata binding protein TBP used as the loading control.
[0056] FIG. 12. DBZIM (top panel) and TDBZIM (bottom panel)
attenuated GFAP-LacZ transgene as reported by .beta.-galactosidase
activity. Stable transfected T6 GFAP-LacZ cells were treated with
compounds of 1-300 .mu.M concentrations, and assayed after 24 h and
48 h. The .beta.-galactosidase activity was normalized to the total
protein, and presented as relative value after comparing to the
vehicle control. The data were presented as mean and SEM, N=6,
*.sup.c P<0.005, and *.sup.d P<0.0005, when compared to the
vehicle control.
[0057] FIG. 13. Western blotting of SMAA, col1a1, fibronectin,
TGF-.beta.1, TGF.beta. RI protein expressions in HSC T6 cells
treated with various IMSs for 48 h. (A) DBIM (1 mM), AMIM (50
.mu.M), TMPHIM (50 .mu.M), DPPHIM (10 .mu.M) and DBZBIM (100 .mu.M)
were dissolved in DMSO with a final DMSO concentration of 0.2%
(v/v). DPIM (2 mM), EGCG (25 .mu.M) and DBZIM (100 .mu.M) were
dissolved in H.sub.2O. Alpha-tubulin was used as the loading
control. Densitometric measurement of protein band intensity was
normalized against the respective vehicle control. (B) Relative
densitometric quantification of protein band intensity. The vehicle
treated sample was normalized to 1.
[0058] FIG. 14. Real-time PCR quantification of (a) GFAP, (b) SMAA,
(c) fibronectin and (d) col1a1 mRNA expressions in HSC T6 cells
treated with various IMSs for 48 h. DBIM (1 mM), AMIM (50 .mu.M),
TMPHIM (50 .mu.M), DPPHIM (10 .mu.M) and DBZBIM (100 .mu.M) were
dissolved in DMSO with a final DMSO concentration of 0.2% (v/v).
DPIM (2 mM), EGCG (25 .mu.M) and DBZIM (100 .mu.M) were dissolved
in H.sub.2O. .beta.-actin was used as the normalization gene; its
expression was constant under the experimental conditions. Relative
quantification was normalized against the respective vehicle
control. The data were presented as mean and SEM, N=6, *.sup.a
P<0.05, *.sup.c P<0.005, and *.sup.d P<0.0005, when
compared to the vehicle control.
[0059] FIG. 15 Effect of DBZIM and DPIM on liver fibrosis in mice.
DBZIM was tested on mice induced by (a) liver toxin thioacetamide
(TAA) or by (b) bile duct ligation (BDL). DPIM was also tested on
mice induced by TAA (data not shown)) or by (c) bile duct ligation
(BDL). DBZIM attenuated fibrosis in the TAA model (at 500 mg/L, 12
weeks) and the BDL model (at 10 mg/L, 4 weeks), whereas the DPIM
displayed its effect in the BDL model at 1 g/L (4 wks).
[0060] FIG. 16. Representative of images of Sirius red staining in
liver sections from different DPIM compound treated group mice.
Each treated group had 6-8 mice, except for 500 mg/l treated group,
which had only one mouse in the group. Red staining areas represent
collagen deposits. (A) sham operated, (B) sham operated+DPIM 1 g/l
for 4 weeks, (C) BDL for 4 weeks, (D) BDL 4 weeks+500 mg/l DPIM
treatment, (E) BDL 4 weeks+750 mg/l DPIM treatment, and (F) BDL 4
weeks+1 g/l DPIM treatment.
[0061] FIG. 17. Percentage of Sirius red staining areas. Sirius red
staining areas from different treatment group mice were quantified
with Image J software, at least 6 different areas from left and
middle lobes of liver of each mouse were covered. * P<0.05, BDL
vs. DPIM treated groups, # P<0.05, 750 mg/l vs. 1 g/l treated
groups.
[0062] FIG. 18. Liver weights of different treatment groups. Each
group consisted of 6-8 mice which were 10-12 weeks old at the start
of surgery procedure. *P<0.05, sham operated control group vs.
BDL 4 weeks, ** P<0.05, DPIM compound treated groups vs. BDL
weeks group, # P<0.05, 750 mg/l DPIM treated group vs. 1 g/l
DPIM treated group.
[0063] FIG. 19. Representative images of H&E staining of liver
sections. (A) control group; (B) BDL 4 weeks group; and (C) BDL 4
weeks+DPIM1 g/l treatment group.
[0064] FIG. 20. Quantitative real-time PCR for collagen 1a1 mRNA.
DPIM reduced collagen 1a1 mRNA.
[0065] FIG. 21. DBZIM inhibited cell proliferation and disrupted
cell cycle in HLE cells (A). DBZIM inhibited HCC cell
proliferation. HLE cells were seeded at a density of 5000
cells/well in 96-well plate, and cultured for 18-24 h in 10% FBS
DMEM before the addition of various compounds (0-1.25 mM) in 10%
FBS DMEM for 48 h of proliferation before assaying. Cell count was
calculated based on Hoechst dye staining and BrdU incorporation was
based on the total fluorescence intensity of antibody staining.
(B). DBZIM arrested HCC cells at G0/G1 phase. HLE cells were
incubated with DBZIM (0, 1, 3 mM) for 24 hr before fixation and
DAPI staining for DNA content (2N vs. 4N) determination.
[0066] FIG. 22. DBZIM induced caspase 3/7 activity in HLE cells (A)
and HepG2 (C) and did not cause LDH releasing in HLE (B) and HepG2
(D). HLE or HepG2 cells were incubated with DBZIM of various
concentrations for 6 hr, and assayed for caspase 3/7 activity and
LDH releasing (expressed as % toxicity). DBZIM induced apoptosis as
characterized by annexin V staining using flow cytometer in HLE (E)
and HepG2 (F). Scatter plot and histogram gave the representative
information on the cell population distribution while bar graphs
quantified the percentage of apoptotic cells from the histogram.
HLE and HepG2 cells were treated by DBZIM of various concentrations
for 24 hr before harvesting for annexin V staining and flow
cytometric analysis.
[0067] FIG. 23. (A). DBZIM did not change caspase 8 activity and
induced caspase 9 and 3 activity in HLE cells. HLE cells were grown
in a T75 flask and treated with DBZIM for 24 hr. The cells were
then lysed and cytoplasmic protein was collected for assaying
caspase 8, 9 and 3 using kits from Biovision. The data was
normalized against the vehicle control. (B). DBZIM did not cause
cytochrome c release from mitochondria to cytosol. HLE cells were
treated by DBZIM of 1.5 mM for 24 hr. Cytosolic and mitochondria
protein fractions were collected using a kit from Biovision. 15
.mu.g of each protein was resolved in 4-12% gradient PAGE-SDS gel.
The target proteins were recognized by antibody of cytochrome C,
and visualized by the ECL method. The membrane was stripped and
probed for the .alpha.-tubulin as the loading control for cytosolic
fraction and Cox4 for mitochondria fraction.
[0068] FIG. 24. DBZIM affected Bcl-2 and IAP protein expression.
HepG2 cells were treated with DBZIM (0, 0.5, 1.0, 1.5 and 2.0 mM)
for 24 hr. Total protein was collected using RIPA buffer. 15 .mu.g
of total protein was resolved in 4-12% gradient PAGE-SDS gel. The
target proteins were recognized by respective antibodies, and
visualized by the ECL method. The membrane was stripped and probed
for the .alpha.-tubulin as the loading control.
[0069] FIG. 25. DBZIM induced nucleus to cytoplasm translocation of
survivin protein. (A) Representative immunocytostaining images of
HLE cells treated by DBZIM (0 and 1.0 mM) for 24 hr. Arrows
indicate the accumulation and the disappearance of survivin in the
nucleus of the control and the DBZIM-treated cells, respectively.
The images were acquired and analyzed using ArrayScan VTI HCS
reader (Thermal Scientific, PA, USA) and Target Activation
BioApplication software. (B) Quantitation of translocation event.
Cells were grown in 96-well plate and treated by DBZIM of various
concentrations for 24 hr before fixation and immunocytostaining for
survivin. Lower value in "Cytoplasmic-Nuclear Difference" indicated
a lower amount of cytoplasmic survivin.
[0070] FIG. 26. DBZIM induced cytoplasm-to-nucleus translocation of
AIF. (A). DBZIM translocated AIF from cytoplasm to nucleus. HepG2
cells were treated by DBZIM for 24 hr. Cytoplasmic and nuclear
proteins were fractionated using a kit from Pierce. 15 .mu.g of
proteins were resolved in 4-12% gradient PAGE-SDS gel. The target
proteins were recognized by the antibody of AIF, and visualized by
the ECL method. The membrane was stripped and re-probed for
.alpha.-tubulin as the loading control for cytoplasmic fraction and
Tata binding protein TBP for nuclear protein. (B). HLE cells were
grown in 96-well plate and treated by DBZIM for 24 hr. The cells
were then fixed and stained by antibody of AIF. The images were
acquired and analyzed using ArrayScan VTI HCS reader (Thermal
Scientific, PA, USA) and Target Activation BioApplication software.
The arrows indicate the cytoplasmic and the nuclear location of the
AIF staining in the control and the DBZIM, respectively. (C).
Quantification of AIF staining in different subcellular
compartments. Higher value in "Cytoplasmic-Nuclear
[0071] Difference" indicated higher nuclear AIF.
[0072] FIG. 27. High dose DBZIM induced ROS production in HLE
cells. HLE cells were grown in 96-well plate and treated by DBZIM
for 24 hr. The cells were then fixed and stained by DHE for
quantification of ROS generation. The images were acquired and
analyzed using ArrayScan VTI HCS reader (Thermal Scientific, PA,
USA) and Target Activation BioApplication software.
[0073] FIG. 28. DBZIM induced up-regulation of AP-1 expression in
HepG2 cells. HepG2 cells were treated with DBZIM for 24 hr.
Cytoplasmic and nuclear proteins were fractionated using a kit from
Pierce. 15 .mu.g of nuclear proteins were resolved in 4-12%
gradient PAGE-SDS gel. The target proteins were recognized by their
respective antibodies, and visualized by the ECL method. The
membrane was stripped and re-probed for Tata binding protein TBP as
loading control.
[0074] FIG. 29. DMZIM induced apoptosis. Pictures from
phase-contrast microscopy show the apoptotic morphology of gastric
cancer cell AGS (A) and lung cancer cell H1299 (B) after exposure
to DBZIM for 72 h, respectively. Extending the length of exposure
caused increased loss of confluence with apoptotic cells in cell
cultures.
[0075] FIG. 30. DBZIM inhibited cell proliferation in glioma cell
line C6 (A) and U87 MG (C) and induced caspase 3/7 activity in C6
(B) and U87 MG (D). C6 or U87 cells were incubated with DBZIM of
various concentrations for 48 hr for proliferation assay and 6 hr
for caspase 3/7 activation assay. Proliferation was measured using
MTS kit from Promega and expressed as relative cell proliferation
with control group was normalized to 1. Caspase 3/7 activity was
quantified using a kit from Promega and also expressed as relative
caspase 3/7 activity of treated groups over control group.
[0076] FIG. 31. DBZIM induces cleavage of caspase-3, caspase-9 and
PARP as indication of apoptosis. Western blot analysis for the
effect of DBZIM, on the cleavage of caspase-3, Caspase-9 and PARP
A) in p53 wild type gastric cancer cell AGS and B) in p53 wild type
breast cancer cell line MCF-7. Western blot analysis conducted on
cells after 72 h exposure to 50 .mu.M of DBZIM.
[0077] FIG. 32. DBZIM inhibits phosphorylation of Akt. When AGS,
MKN28, H1299 and MCF-7 cells were treated with DBZIM at their
corresponding IC50 concentration, phosphorylation at serine 473 of
Akt was downregulated in p53 wild type cancer cell line AGS (A), in
p53 mutant gastric cancer cell MKN 28 (B), in p53 null Lung cancer
cell H1299 (C), and in p53 wild type breast cancer cell MCF-7 (D).
Time course inhibition of phosphorylated-AKT was shown by Western
blot. .beta.-actin was used as an internal loading control.
[0078] FIG. 33. DBZIM reduced HCC tumor in vivo. Tumors in the
HCC-xenografted mice in the control group and in the treatment
group, 3 weeks post inoculation, are shown in (A). The tumor growth
curve and body weight chart are depicted in (B) and (C),
respectively. * P<0.01.
[0079] FIG. 34. Endogenous expression of p53. Cell lysates from
Hep3B (p53 null), Hep G2 (wild type), Hle and Plc (p53 mutant) were
subjected to Western blotting using p53 (DO-1) and actin
antibodies.
[0080] FIG. 35. Phase-contrast microscopy of cells exposed to IMSs.
Pictures from phase-contrast microscopy show cell cultures after
exposure to IMSs (IBN 15, IBn 19, IBN 24, IBN 25 and IBN 32) for 72
h, respectively. Extending the length of exposure caused increased
loss of confluency in cell cultures.
[0081] FIG. 36. IMSs induce apoptosis in Hepatocarcinoma cells. A)
Cells were treated with vehicle and IBN 15, IBN 19, IBN 24, IBN 25
and IBN 32 for 72 h, and apoptosis was quantified using Annexin
V-FITC/PI staining followed by flow cytometric analysis. 10,000
cells were detected for every sample. Living cells are in the
bottom left quadrants (FITC- and PI-), early apoptotic cells in the
bottom right quadrants (FITC+ and PI-), and late apoptotic cells in
the upper right quadrants (apoptotic FITC+ and PI+). B) The
percentage of apoptotic cells with respect to different IMSs.
[0082] FIG. 37. IMSs arrest Hepatocarcinoma cells. Cells were
treated with vehicle and IBN 15, IBN 19, IBN 24, IBN 25 and IBN 32
for 72 h, and cell cycle distribution was assessed by flow
cytometry. A) Example FACS profiles of propidium iodide-stained
control cells, and cells treated with IMSs respectively. B) % of
cells in cell cycle phase C) % of cells in subG0 population. Each
value is the mean.+-.S.D. of three determinations.
[0083] FIG. 38. IMSs induce cleavage of caspase-3, caspase-9 and
PARP. Western blot analysis for the effect of IBN 15, IBN 19, IBN
24, IBN 25, IBN 32 on the cleavage of caspase-3, Caspase-9 and PARP
in HLE cells after 72 h exposure to indicated doses of IMSs.
[0084] FIG. 39. IMSs-induced apoptosis was accompanied by
accumulation of p53 in the nucleus. HLE cells were treated with the
indicated dose of IBN 19 and IBN 24 for 48 h. The treated cells
were fixed with 4% formaldehyde, immunostained with anti-p53 (green
fluorescence) and DAPI (blue fluorescence), and analysed by
confocal microscopy.
[0085] FIG. 40. IMSs-induced accumulation of p53 and p53
phosphorylation on serines 15, 20, 46, and 392 in p53mutant HLE
cells. (A) Hepatocarcinoma cancer cell HLE were treated with
indicated concentration of IBN 15, IBN 19, IBN 24, IBN 25 and IBN
32 for 72 h. The levels of phosphorylation of p53 protein and
Ser-15, Ser-20, Ser-46 and Ser-392 were determined by Western
blotting. .beta.-actin was used as a loading control.
[0086] FIG. 41. IMSs-induced apoptosis through the initiation of
the mitochondrial pathway. Cells were treated with various IMSs and
the levels of Bcl-2 and BAX were assessed by Western blot
assay.
[0087] FIG. 42. Real-Time PCR array analysis of p53 signaling
pathway-associated gene expression. (A) Anti-apoptotic genes. (B)
Growth inhibition gene SESN2. (C) Cell cycle check point genes. (D)
Cell proliferation genes. (E) DNA repair gene ATM. The relative
level of gene expression was normalized with housekeeping gene
GAPDH. Values are expressed as mean.+-.standard deviation.
[0088] FIG. 43. Effect of Compound C (DBZMIM) and compound 9
(MABZMIM) on lung cancer and gastric cancer cells. Compound C
(DBZMIM) and compound 9 (MABZMIM) induced morphological change and
apoptosis in lung cancer cells MDAMB 231 (A) and in gastric cancer
cells MKN 28 (B), 120 h after the treatment.
[0089] FIG. 44. Effect of compound 9on HCC-xenografted mice. Tumors
in the HCC-xenografted mice in the control group and in the
treatment group, 3 weeks post inoculation, were shown in (A). The
tumor growth curve and body weight chart was depicted in (B) and
(C), respectively. * P<0.01.
[0090] FIG. 45 (Table 1). Names and Structures of IMSs.
[0091] FIG. 46 (Table 2). IC50 values of IMSs.
[0092] FIG. 47 (Table 3). IC50 values of DBZIM in various cancer
cell lines.
[0093] FIG. 48 (Table 4). IC50 values of IBN-15, 19, 24, 25 and 32
in HLE cells.
[0094] FIG. 49 (Table 5). IC50 values of DBZMIM and Compound 9 in
gastric cancer cells.
[0095] FIG. 50 (Table 6). IC50 values of DBZMIM and Compound 9 in
breast cancer cells.
[0096] FIG. 51 (Table 7). IC50 values of DBZMIM and Compound 9 in
normal breast cells.
DETAILED DESCRIPTION
[0097] The methods described herein relate to the discovery that
the imidazolium and imidazolinium compounds (collectively "IMSs")
as described herein, including in the form of imidazolium and
imidazolinium salts, may be used as anti-fibrotic and anti-cancer
agents or to treat fibrotic disease or cancer. The inventors have
synthesized novel IMSs that may be used in the methods described
herein.
[0098] Both imidazolium and imidazoliniums are based on an
imidazole ring and both are N,N'-substituted; imidazoliums are
N,N'-substituted imidazoles, while imidazoliniums are
N,N'-substituted imidazolines and do not have the carbon-carbon
double bond between positions C4 and C5 that is present in
imidazole. Imidazole itself is incorporated in many biological
molecules, and synthetic C-substituted imidazoles have become an
important part of many pharmaceuticals (Olmos et al. 1999)
(Casanovas et al. 2000).
[0099] One attractive feature of using IMSs in synthetic chemistry
is the structural versatility they provide. The electronic
structure and stability, and thus the therapeutic safety and
efficacy of IMSs, can be fine-tuned by varying the N-substituents
(substituents on the nitrogen atoms of the central ring) and the
central ring of the molecule. It will be understood that the
"central ring" of IMSs refers to the five membered ring containing
2 nitrogen atoms (either the imidazole or imidazoline ring) to
which various substituents may be bound to create different IMS
molecules. IMSs provide inexpensive and chemically tunable building
blocks for the development of novel therapeutics.
[0100] Some IMSs are precursors of N-heterocyclic carbenes (NHCs).
NHCs can be easily generated from IMSs having a hydrogen atom at
the C.sub.2 position of the central ring and substituents on both
nitrogen atoms of the central ring i.e. two N-substituents. NHCs
are generated from these IMSs by the deprotonation of the IMS under
the appropriate conditions. Deprotonation of IMSs may be carried
out under basic conditions or in a diluted solution under neutral
conditions.
[0101] It is difficult to generate NHCs from IMSs with substituents
other than a hydrogen atom at the C.sub.2 position of the central
ring. However radical species may be formed from these IMSs by
cleaving the N-substituents.
[0102] The inventors have discovered that the IMSs as described
herein may be used as anti-fibrotic or anti-cancer agents or to
treat fibrotic disease or cancer. Without being limited to any
particular theory, the IMSs as described herein may exhibit
anti-oxidative and anti-inflammatory properties. Both fibrotic
disease and cancer have been linked to oxidative stress response.
In turn, in both fibrotic disease and cancer, oxidative stress has
been shown to be closely linked with inflammatory response.
[0103] IMSs that may be used as anti-fibrotic agents or as
anti-cancer agents are compounds having a structure of the general
formula I:
##STR00002##
[0104] In formula I, the dashed line is absent or is present as a
bond to form a second bond between the carbon to which R.sup.1 and
R.sup.3 are attached and the carbon to which R.sup.2 and R.sup.4
are attached. Thus, the carbon to which R.sup.1 and R.sup.3 are
attached and the carbon to which R.sup.2 and R.sup.4 are attached
may be connected by either a single bond or a double bond.
[0105] R.sup.1 and R.sup.2 (i) are each independently H, straight
or branched C.sub.1-C.sub.6 alkyl, straight or branched
C.sub.2-C.sub.6 alkenyl, straight or branched C.sub.2-C.sub.6
alkynyl, C.sub.6-C.sub.10 aryl; or (ii) together with their ring
atoms form a 6- to 10-membered fused saturated, unsaturated or
aromatic ring system; or (iii) R.sup.1 and R.sup.5 together with
their ring atoms or R.sup.2 and R.sup.6 together with their ring
atoms, form a 5- to 10-membered fused saturated, unsaturated or
aromatic ring system and the other of R.sup.1 and R.sup.2 is as
defined above in (i); or (iv) R.sup.1 and R.sup.5 together with
their ring atoms and R.sup.2 and R.sup.6 together with their ring
atoms form 5- to 10-membered fused saturated, unsaturated or
aromatic ring system.
[0106] R.sup.3 and R.sup.4 are both H, or, when R.sup.1 and R.sup.2
together with their ring atoms form a 6- to 10-membered fused
aromatic ring system or when the dashed line is present as a bond,
R.sup.3 and R.sup.4 are absent.
[0107] When R.sup.5 or R.sup.6 is not fused together with R.sup.1
or R.sup.2, respectively, as set out above, R.sup.5 and R.sup.6 are
each independently straight or branched C.sub.1-C.sub.6 alkyl,
straight or branched C.sub.2-C.sub.6 alkenyl, straight or branched
C.sub.2-C.sub.6 alkynyl, C.sub.3-Cis cycloalkyl including fused
cycloalkyl ring systems, C.sub.6-C.sub.10 aryl, C.sub.6-C.sub.10
aryl-C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl-C.sub.2-C.sub.6
alkenyl, or C.sub.6-C.sub.10 aryl-C.sub.2-C.sub.6 alkynyl,
C.sub.1-C.sub.6 alkyl-C.sub.6-C.sub.10 aryl, C.sub.2-C.sub.6
alkenyl-C.sub.6-C.sub.10 aryl, or C.sub.2-C.sub.6
alkynyl-C.sub.6-C.sub.10 aryl.
[0108] R.sup.7 may be H, C.sub.1-C.sub.6 alkyl, phenyl, substituted
C.sub.1-C.sub.6 alkyl or halo.
[0109] Any of the above substituents R.sup.1 to R.sup.7, where
applicable, may optionally have one or more carbon atoms replaced
with a heteroatom selected from N, O, S and P. As well, any of the
above substituents R.sup.1 to R.sup.7, where applicable, may
optionally be substituted with one or more of straight or branched
C.sub.1-C.sub.6 alkyl, straight or branched C.sub.2-C.sub.6
alkenyl, straight or branched C.sub.2-C.sub.6 alkynyl,
C.sub.3-C.sub.18 cycloalkyl including fused cycloalkyl ring
systems, C.sub.6-C.sub.10 aryl, fluoro, tri-fluoro-methyl, cyanato,
isocyanato, carboxyl, C.sub.1-C.sub.6 acyloxy, C.sub.1-C.sub.6
acyl, carbonyl, amino, acetyl, acetoxy, oxo, nitro, hydroxyl,
C.sub.1-C.sub.6 alkylcarboxy, C.sub.1-C.sub.6 alkoxy,
C.sub.2-C.sub.6 alkenoxy, C.sub.2-C.sub.6 alkynoxy.
[0110] One of the ring carbon atom to which R.sup.1 and R.sup.5 are
attached and the ring carbon to which R.sup.2 and R.sup.6 are
attached may be optionally replaced with a nitrogen atom.
[0111] The above compounds may be present in salt form. Thus, IMSs
that may be used in the present methods include pharmaceutically
acceptable salts of compounds of formula I and oligomers and
polymers of such salts. Such salts have a structure of the general
formula II:
##STR00003##
[0112] General formula II is as defined above for general formula
I, with the further feature of counterion X. X is a
pharmaceutically acceptable anion, including but not limited to
chloride, bromide, tetrafluoroborate, hexafluorophosphate.
[0113] The term "fused" as used herein in reference to ring
structures refers to the sharing of at least two atoms between ring
structures. When two ring atoms (either of which may be C or N) are
included in a fused ring system, the ring system is fused to the
central imidazolium or imidazolinium ring.
[0114] In particular embodiments, the dashed line may be absent or
is present as a bond to form a second bond between the carbon to
which R.sup.1 and R.sup.3 are attached and the carbon to which
R.sup.2 and R.sup.4 are attached; R.sup.1 and R.sup.2 are both H or
together with their ring atoms form a 6- to 10-membered fused
aromatic ring system; R.sup.3 and R.sup.4 are both H or nothing;
R.sup.5 and R.sup.6 are each independently straight or branched
C.sub.1-C.sub.6 alkyl, straight or branched C.sub.2-C.sub.6
alkenyl, straight or branched C.sub.2-C.sub.6 alkynyl,
C.sub.3-C.sub.18 cycloalkyl including fused cycloalkyl ring
systems, C.sub.6-C.sub.10 aryl, C.sub.6-C.sub.10
aryl-C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl-C.sub.2-C.sub.6
alkenyl or C.sub.6-C.sub.10 aryl-C.sub.2-C.sub.6 alkynyl; R.sup.7
is H; any of which substituents R.sup.1 to R.sup.7, where
applicable may have one or more carbon atoms replaced with a
heteroatom selected from N, O, S and P and any of which may
optionally be substituted with one or more of straight or branched
C.sub.1-C.sub.6 alkyl, straight or branched C.sub.2-C.sub.6
alkenyl, straight or branched C.sub.2-C.sub.6 alkynyl,
C.sub.3-C.sub.18 cycloalkyl including fused cycloalkyl ring
systems, C.sub.6-C.sub.10 aryl, fluoro, tri-fluoro-methyl, cyanato,
carboxyl, carbonyl, amino, acetyl, oxo, nitro, C.sub.1-C.sub.6
alkoxy, C.sub.2-C.sub.6 alkenoxy, C.sub.2-C.sub.6 alkynoxy.
[0115] For example, in various embodiments, the compound may be an
imidazolium salt, with a double bond between the two ring atoms to
which R.sup.1 and R.sup.2 are attached. In various other
embodiments, the compound may be an imidazolinium salt, wherein
R.sup.3 and R.sup.4 are hydrogens and there is only a single bond
between their ring carbons. In yet other embodiments, R.sup.3 and
R.sup.4 are nothing and R.sup.1 and R.sup.2 together with their
ring atoms form a 6- to 10-membered fused aromatic ring system.
[0116] The substituents comprising R.sup.5 and R.sup.6 may also
vary. For example, in some embodiments, the IMS of the present
method may be a compound wherein R.sup.5 and R.sup.6 are the same.
In different embodiments, R.sup.5 or R.sup.6 may be aralkyls,
branched alkyls or cycloalkyls including fused ring systems. In
other embodiments R.sup.5 or R.sup.6 may comprise a phenalkyl
group. In other embodiments, one or both of R.sup.5 and R.sup.6 may
comprise an adamantanyl group.
[0117] IMSs that may be used in the present method also include
oligomers or polymers formed from compounds of general formula I.
Thus, "oligomer" as used herein, refers to, for example, 2 or more,
3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or
more, 25 or more, 50 or more, 100 or more, 150 or more compounds of
general formula I connected together in the manner described below.
Thus, oligomer includes dimers and trimers. "Polymers" as used
herein refers to, for example, 100 or more, 150 or more, 200 or
more, 250 or more, 500 or more, 1000 or more compounds of general
formula I connected together in the manner described below.
[0118] In oligomers or polymers as used herein, compounds of
general formula I are connected to each other to form a
macromolecular structure, which may be cyclised.
[0119] For example, trimers as described herein include
1,3,5-tris(4-methyl-imidazolium)-linked cyclophane or a
pharmaceutically acceptable salt thereof (TDBZIM).
[0120] In the macromolecular structure, two compounds of general
formula I may be connected together so that one or more R.sup.1,
R.sup.2, R.sup.5, R.sup.6 or R.sup.7 substituent is bivalent and is
thus shared between the two compounds, rather than have a relevant
substituent present in the oligomer or polymer for every compound
of general formula I present. For example, in such a dimer, two
compounds may be connected by a shared bivalent R.sup.1
substituent, and thus in the complete dimer, there is only one
occurrence of an R.sup.1 substituent for the two compounds of
general formula I.
[0121] Thus, the compounds of general formula I may be joined to
each other via a bivalent substituent such as R.sup.1, R.sup.2,
R.sup.5, R.sup.6, or R.sup.7. For example, an R.sup.5 substituent
on one compound of general formula I may also be R.sup.5 on a
second compound of general formula I such that the two compounds
are linked via a shared R.sup.5 substituent. Similarly, oligmers or
polymers may be formed by linking compounds via shared R.sup.1,
R.sup.2, R.sup.5, R.sup.6, or R.sup.7 substituents. For example,
dimers of this description include 2,6-di-(3-benzyl-imidazolium
bromide)-pyridine or a pharmaceutically acceptable salt thereof
(IBN-22).
[0122] Sharing of substituents as described above can include
sharing of two substituents that together with their ring carbons
form a fused ring system. Thus, for example, if R.sup.1 and R.sup.2
together with their ring carbons form a ring system on a first
compound of formula I, additional carbons within the ring will be
ring carbons from a second compound of general formula I. For
example, dimers of this description include
benzo(1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-di-bromide
or a pharmaceutically acceptable salt thereof (IBN-29).
[0123] Alternatively, a substituent such as one or more of R.sup.1,
R.sup.2, R.sup.5, R.sup.6 or R.sup.7 may be bonded to another such
substituent on a second compound in order to connect two compounds
together to form an oligomer or polymer, and thus there will be one
occurrence of the relevant linking substituent for each compound of
general formula I in the oligomer or polymer. Multiple compounds
falling within general formula I may be joined together
(oligomerised) in this way, by one or more shared bivalent R.sup.1,
R.sup.2, R.sup.5, R.sup.6 or R.sup.7 substituents. For example,
such dimers include 2,2'-di-(3-benzyl-imidazolium
bromide)-1,1'-binaphthalene or a pharmaceutically acceptable salt
thereof (IBN-23).
[0124] Additionally, two compounds of general formula I may be
attached by a single or double bond between respective carbon atoms
to which R.sup.7 is attached in general formula I to form a dimer.
These molecules include for example,
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole or a pharmaceutically acceptable salt thereof (compound
H).
[0125] The oligomerised or polymerised compounds may be the same
compound, such that all substituents in general formula I are the
same in each oligomerised or polymerised compound, or may be
different, with one or more substituents differing between
compounds, with the exception that shared bivalent substituent or
substituents will obviously be the same in the compounds in which
the bivalent substituent is shared.
[0126] Table 1 contains the names and structures of particular
examples of the IMSs described herein.
[0127] In particular embodiments, the compounds of the present
invention may be 1-ethyl-3-methylimidazolium,
1,3-bisbenzylimidazolium, 1,3-diisopropylimidazolium,
1,3-di-tert-butylimidazolinium, 1,3-bis(1-adamantyl)imidazolium,
1,3-bis(2,4,6-trimethylphenyl)-imidazolinium,
1,3-bis(2,6-diisopropyl-phenyl)-imidazolinium,
1,3-diallylimidazolium, 1-benzyl-3-methylimidazolium,
1-butyl-3-methylimidazolium,
1-(1-adamantyl)-3-(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium,
2-benzylimidazo[1,5-a]quinolinium,
1,3-bis(1-adamantyl)-benzimidazolium,
1,3-dicyclohexylbenzimidazolium, 1,3-diisopropylimidazolinium,
1,3-diisopropylimidazolium,
2-(2,6-diisopropylphenyl)-5-methylimidazo[1,5-a]pyridinium,
1-(2,6-diisopropylphenyl)-3-(2,4,6-trimethylphenyl)-imidazolinium,
2-mesityl-5-methylimidazo[1,5-a]pyridinium,
2-mesityl-2,5,6,7-tetrahydropyrrolo[2,1-c][1,2,4]triazol-4-ium,
1,3-bis(1-adamantyl)imidazolinium,
1-butyl-3-(2-pyridinylmethyl)-1H-imidazolium,
6,7-dihydro-2-pentafluorophneyl-5H-pyrrolo[2,1-c]-1,2,4-trizolium,
1,3-dibenzylbenzimidazolium, 1,3-dibenzyl-2-methylimidazolium,
1,3-Bis(2,6-diisopropylphenyl)imidazolium,
1,3-Di-tert-butylimidazolium or any pharmaceutically acceptable
salt thereof.
[0128] In particular embodiments, the compounds of the present
invention may be 1-ethyl-3-methylimidazolium bromide,
1,3-bisbenzylimidazolium bromide (DBZIM),
1,3-diisopropylimidazolium tetrafluoroborate (DPIM),
1,3-di-tert-butylimidazoliniumtetrafluoroborate (DBIM),
1,3-bis(1-adamantyl)imidazolium tetrafluoroborate (AMIM),
1,3-bis(2,4,6-trimethylphenyl)-imidazolinium chloride (TMPHIM),
1,3-bis(2,6-diisopropyl-phenyl)-imidazolinium chloride (DPPHIM),
1,3-diallylimidazolium bromide (Compound D),
1-benzyl-3-methylimidazolium bromide (Compound E),
1-butyl-3-methylimidazolium chloride (Compound G),
1-(1-adamantyl)-3-(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium
chloride (Compound S1), 2-benzylimidazo[1,5-a]quinolinium chloride
(Compound S2), 1,3-bis(1-adamantyl)-benzimidazolium chloride
(Compound S3), 1,3-dicyclohexylbenzimidazolium chloride (Compound
S6), 1,3-diisopropylimidazolinium tetrafluoroborate (Compound S7),
1,3-diisopropylimidazolium chloride (Compound S8),
2-(2,6-diisopropylpheneyl)-5-methylimidazo[1,5-a]pyridinium
hexafluorophosphate (Compound S9),
1-(2,6-diisopropylphenyl)-3-(2,4,6-trimethylphenyl)-imidazolinium
chloride (Compound S10), 2-mesityl-5-methylimidazo[1,5-a]pyridinium
chloride (Compound S11),
2-mesityl-2,5,6,7-tetrahydropyrrolo[2,1-c][1,2,4]triazol-4-ium
chloride (Compound S12), 1,3-bis(1-adamantyl)imidazolinium
tetrafluoroborate (Compound S13),
1-butyl-3-(2-pyridinylmethyl)-1H-imidazolium hexafluorophosphate
(Compound S14),
6,7-dihydro-2-pentafluorophneyl-5H-pyrrolo[2,1-c]-1,2,4-trizolium
tetrafluroborate (Compound S15) 1,3-dibenzylbenzimidazolium
bromide, 1,3-dibenzyl-2-methylimidazolium bromide,
1,3-Bis(2,6-diisopropylphenyl)imidazolium chloride,
1,3-Di-tert-butylimidazolium tetrafluoroborate.
[0129] The compounds of general formula I include novel compounds,
including 1-ethyl-3-methylimidazolium,
1-methyl-3-(2-hydroxylethyl)-imidazolium,
1-methyl-3-(4-isocynatobenzyl)-imidazolium,
1-methyl-3-(4-carboxylbenzyl)-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxylethyl)-imidazolium,
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium,
1,3-Dibenzyl-5-phenylimidazolium,
1-benzyl-3-(4-carboxylbenzyl)-2-methylimidazolium,
1-benzyl-3-(3,4,5-trimethoxylbenzyl)-2-methylimidazolium,
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium,
1-benzyl-3-(4-methylcarboxylatebenzyl)-2-methyl-imidazolium,
1-benzyl-3-(2,2-dimethoxylethyl)-2-methyl-imidazolium,
2,6-di-(3-benzyl-imidazolium)-pyridine,
2,2'-di-(3-benzyl-imidazolium)-1,1'-binaphthalene,
1-benzyl-3-(4-methylbenzyl)-imidazolium,
1-benzyl-3-(2-trifluoromethylbenzyl)-2-methylimidazolium,
1-benzyl-3-(4-methylcarboxylatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-5-phenyl-imidazolium,
1-benzyl-3-(4-methylbenzyl)-5-phenylimidazolium,
(1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-benzene,
1-benzyl-3-(2-propyn-1-yl)-imidazolium,
1-benzyl-3-(3-hydroxyl-propyl)-imidazolium,
1,3-di(2-phenylethyl)-imidazolium,
1-benzyl-3-(4-acetatebenzyl)-imidazolium,
1-benzyl-3-(pyridin-2-yl)-imidazolium,
1,3,5-tris-(4-methyl-imiazolium)-linked cyclophane,
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole, 1-benzyl-3-methyl-imidazolium,
1-(4-cyanatobenzyl)-3-methyl-imidazolium,
1-(4-carboxybenzyl)-3-methyl-imidazolium,
1-methyl-3-(4-acetate-benzyl)-imidazolium,
1-methyl-3-(2,2-dimethoxyethyl)-imidazolium, and
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium, or any
pharmaceutically acceptable salt thereof.
[0130] The compounds of general formula I include novel compounds,
including 1-ethyl-3-methylimidazolium bromide,
1-methyl-3-(2-hydroxylethyl)-imidazolium bromide (IBN-2),
1-methyl-3-(4-isocynatobenzyl)-imidazolium chloride (IBN-3),
1-methyl-3-(4-carboxylbenzyl)-imidazolium bromide (IBN-4),
1-methyl-3-(4-acetate-benzyl)-imidazolium chloride (IBN-6),
1-methyl-3-(2,2-dimethoxylethyl)-imidazolium bromide (IBN-8),
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium chloride
(IBN-9), 1,3-Dibenzyl-5-phenylimidazolium bromide (IBN-15),
1-benzyl-3-(4-carboxylbenzyl)-2-methylimidazolium chloride
(IBN-17), 1-benzyl-3-(3,4,5-trimethoxylbenzyl)-2-methylimidazolium
chloride (IBN-18),
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium chloride
(IBN-19),
1-benzyl-3-(4-methylcarboxylatebenzyl)-2-methyl-imidazolium
chloride (IBN-20),
1-benzyl-3-(2,2-dimethoxylethyl)-2-methyl-imidazolium bromide
(IBN-21), 2,6-di-(3-benzyl-imidazolium bromide)-pyridine (IBN-22),
2,2'-di-(3-benzyl-imidazolium bromide)-1,1'-binaphthalene (IBN-23),
1-benzyl-3-(4-methylbenzyl)-imidazolium chloride (IBN-24),
1-benzyl-3-(2-trifluoromethylbenzyl)-2-methylimidazolium chloride
(IBN-25),
1-benzyl-3-(4-methylcarboxylatebenzyl)-5-phenyl-imidazolium bromide
(IBN-26), 1-benzyl-3-(4-acetatebenzyl)-5-phenyl-imidazolium bromide
(IBN-27), 1-benzyl-3-(4-methylbenzyl)-5-phenylimidazolium chloride
(IBN-28), Benzo(1,2-4,5-diimidazolium)-N,N',N'',N'''-tetrabenzyl-,
di-bromide (IBN-29), 1-benzyl-3-(2-propyn-1-yl)-imidazolium bromide
(IBN-30), 1-benzyl-3-(3-hydroxyl-propyl)-imidazolium bromide
(IBN-31), 1,3-di(2-phenylethyl)-imidazolium bromide (IBN-32),
1-benzyl-3-(4-acetatebenzyl)-imidazolium chloride (IBN-33),
1-benzyl-3-(pyridin-2-yl)-imidazolium bromide (IBN-34),
1,3,5-tris-(4-methyl-imiazolium)-linked cyclophane.3Br (TDBZIM),
1,3-dibenzyl-2-(1,3-dibenzyl-1H-imidazol-2(3H)-ylidene)-2,3-dihydro-1H-im-
idazole (compound H), 1-benzyl-3-methyl-imidazolium bromide
(compound 12), 1-(4-cyanatobenzyl)-3-methyl-imidazolium chloride,
1-(4-carboxybenzyl)-3-methyl-imidazolium bromide,
1-methyl-3-(4-acetate-benzyl)-imidazolium chloride,
1-methyl-3-(2,2-dimethoxyethyl)-imidazolium bromide, and
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium
chloride.
[0131] The compounds of general formula I and oligomers and
polymers thereof are commercially available or may be synthesized
using routine chemistry, as described in the Examples set out
below. Methods of synthesis have also been described in Harlow et
al. 1996, Zhang et al. 2007; Chianese and Cratree 2005 and Boydston
et al. 2005.
[0132] Thus, there is presently provided a method for delivering an
anti-fibrotic or anti-cancer agent to a cell, the method comprising
contacting the cell with an effective amount of a compound of
general formula I, a pharmaceutically acceptable salt thereof or an
oligomer or polymer thereof.
[0133] As used herein, "delivering" an anti-fibrotic or anti-cancer
agent to a cell refers to providing the agent in sufficiently close
proximity to the cell such that the agent can exert its
anti-fibrotic or anti-cancer effects on the cell. In vitro, for
example, the agent may be delivered to the cell by adding the agent
to the cell culture media. In vivo, for example, the agent may be
delivered by administering the agent to a subject as a
pharmaceutical composition.
[0134] As used herein, "contacting" a cell refers to direct and
indirect contact with the cell. Direct contact refers to a direct
interaction between the agent and the cell. In contrast, indirect
contact involves "contacting the cell" via interactions with other
molecules or compounds. For example, the agent may interact with a
molecule, affecting a change in that molecule that causes it to
interact with the cell or interact with another molecule which will
then interact with the cell to exert an effect. Indirect contact
may involve a series of molecular interactions resulting in an
effect on the cell.
[0135] An anti-fibrotic agent as used herein refers to a compound
that alters, reduces or inhibits molecules, mechanisms or effects
associated with fibrotic disease. For example, the agent may be an
anti-inflammatory or an anti-oxidative, induce apoptosis, alter,
reduce or inhibit the activity of fibrogenic mediators such as
TGF-.beta., NF-.kappa.B or IL-6 or neutralize proliferative,
fibrogenic, contractile or pro-inflammatory responses of cells.
Without being limited to any particular theory, the anti-fibrotic
agent may reduce or inhibit fibrotic disease by: (a) reducing
inflammation to avoid stimulating activation of cells involved in
fibrotic disease such as stellate cells, (b) directly
down-regulating activation of cells involved in fibrotic disease,
(c) neutralizing proliferative, fibrogenic, contractile or
pro-inflammatory responses of cells involved in fibrotic disease,
(d) inducing apoptosis such as, for example, Bcl-xL or Fas; or (e)
inducing ECM degradation.
[0136] An anti-cancer agent as used herein refers to a compound
that alters, reduces or inhibits molecules, mechanisms or effects
associated with cancer. For example, the agent may be an
anti-inflammatory or an anti-oxidative, induce apoptosis, inhibit
abnormal cell growth, inhibit cell proliferation, inhibit DNA
mutation or modify the activity or level of activity of molecules
associated with cancer such as survivin, caspase 9, caspase 3,
Bcl-XL, Bak, BAX, ATM, p53, Cdc25A, Cdc2, SESN2 and AP-1.
[0137] The cell to which the anti-fibrotic or anti-cancer agent is
to be provided may be any cell, including an in vitro cell, a cell
in culture, or an in vivo cell within a subject. The term "cell" as
used herein refers to and includes a single cell, a plurality of
cells or a population of cells where context permits, unless
otherwise specified. The cell may be an in vitro cell including a
cell explanted from a subject or it may be an in vivo cell in a
subject. Similarly, reference to "cells" also includes reference to
a single cell where context permits, unless otherwise
specified.
[0138] The cell may be derived from any organism, for example an
insect, a microorganism including a bacterium, or an animal
including a mammal including a human.
[0139] The cell of the present method may be within a subject
having fibrotic disease or cancer, a subject requiring treatment
for fibrotic disease or cancer or a subject in which prevention of
fibrotic disease or cancer is desired. In some embodiments, the
subject is a human subject.
[0140] Fibrotic disease will be understood by those skilled in the
art to refer to a condition which may be characterized or caused by
the development of excess fibrous tissue or over production of
extracellular matrix in an organ or tissue and may include for
example liver fibrosis, kidney fibrosis, lung fibrosis, bone
fibrosis., systemic sclerosis, mixed connective tissue. Futher
examples of fibrotic disease are provided in US 2007/0043016 which
is herein incorporated by reference.
[0141] In one embodiment of the present methods, an anti-fibrotic
agent may be delivered to a cell in a subject having hepatic
fibrosis. The compound may be for example
1,3-diisopropylimidazolium or a pharmaceutically acceptable salt
thereof.
[0142] A skilled person will understand cancer to encompass a class
of diseases in which cells exhibit abnormal cell growth and the
potential to invade nearby tissues. In some forms of cancer, the
abnormal cells may also spread to other locations in the body.
Different types of cancer include for example, breast cancer,
colorectal cancer, brain cancer, prostate cancer, cervical cancer,
ovarian cancer, bone cancer, skin cancer, lung cancer, pancreatic
cancer, bladder cancer, gallbladder cancer, kidney cancer,
esophageal cancer, Hodgkin lymphoma, Non-Hodgkin lymphoma,
laryngeal cancer, leukemia, multiple myeloma, oral cancer, pleural
mesothelioma, small intestine cancer, testicular cancer, uterine
cancer, thyroid cancer and stomach cancer.
[0143] In one embodiment of the present methods, an anti-cancer
agent may be deliverd to a cell in a subject having hepatocellular
carcinoma, lung cancer, breast cancer, stomach cancer or glioma.
The compound may be for example 1,3-Bisbenzylimidazolium, 1,
3,-Dibenzyl-2-methylimidazolium,
1-(2,4,6-trimethylphenyl)-3-(4-acetate-benzyl)-imidazolium,
1,3-Dibenzyl-5-phenylimidazolium,
1-benzyl-3-(4-acetate-benzyl)-2-methyl-imidazolium,
1-benzyl-3-(4-methylbenzyl)-imidazolium,
1-benzyl-3-(2-trifluoromethylbenzyl)-2-methylimidazolium or
1,3-di(2-phenylethyl)-imidazolium, or a pharmaceutically acceptable
salt thereof. [0144] The term "effective amount" as used herein
means an amount effective, at dosages and periods of time necessary
to achieve the desired result, for example to treat fibrotic
disease or cancer. The total amount of IMS to be administered will
vary, depending on several factors, including the severity and type
of the disorder, the mode of administration, and the age and health
of the subject. Methods for determining an effective amount of a
particular IMS for treating fibrotic disease or cancer will be
readily apparent to a person skilled in the art.
[0145] "Treating" fibrotic disease or cancer refers to an approach
for obtaining beneficial or desired results, including clinical
results. Beneficial or desired clinical results can include, but
are not limited to, alleviation or amelioration of one or more
symptoms or conditions, diminishment of extent of disorder or
disease, stabilization of the state of disease, prevention of
development of disorder or disease, prevention of spread of
disorder or disease, delay or slowing of disorder or disease
progression, delay or slowing of disorder or disease onset,
amelioration or palliation of the disorder or disease state, and
remission (whether partial or total). "Treating" can also mean
prolonging survival of a subject beyond that expected in the
absence of treatment. "Treating" can also mean inhibiting the
progression of disorder or disease, slowing the progression of
disorder or disease temporarily, although more preferably, it
involves halting the progression of the disorder or disease
permanently.
[0146] To aid in administration, the IMS may be formulated as an
ingredient in a pharmaceutical composition. The compositions may
contain pharmaceutically acceptable concentrations of salt,
buffering agents, preservatives and various compatible carriers or
diluents.
[0147] The proportion and identity of the pharmaceutically
acceptable carrier is dependant on a variety of factors including
the chosen route of administration, compatibility with the IMS
molecule and standard pharmaceutical practice. Generally, the
pharmaceutical composition will be formulated with components that
will not significantly impair the biological properties of the
IMS.
[0148] Suitable vehicles and diluents are described, for example,
in Remington's Pharmaceutical Sciences (Remington, The Science and
Practice of Pharmacy, 21.sup.st edition, Lippincott Williams &
Wilkins, Philadelphia, Pa., 2006). It would be known to a person
skilled in the art how to prepare a suitable pharmaceutical
composition.
[0149] The pharmaceutical composition may be administered to a
subject in a variety of forms depending on the selected route of
administration, as will be understood by those skilled in the art.
The composition of the invention may be administered for example,
by oral administration, surgically or by injection to the desired
site.
[0150] In different embodiments, the composition is administered by
injection (subcutaneously, intravenously, intramuscularly, etc.)
directly at a desired site, for example in the vicinity of fibrotic
disease or cancer that is to be treated.
[0151] The dose of the pharmaceutical composition that is to be
used depends on the particular fibrotic disease or cancer disorder
being treated, the severity of the condition, individual patient
parameters including age, physical condition, size and weight, the
duration of the treatment, the nature of concurrent therapy (if
any), the specific route of administration and other similar
factors that are within the knowledge and expertise of the health
practitioner. These factors are known to those of skill in the art
and can be addressed with minimal routine experimentation.
[0152] It will be understood that pharmaceutical compositions may
be provided in a variety of dosage forms and thus, in different
embodiments, IMSs may be administered in different dosage forms
including for example pills, tablets, capsules, solutions,
suspensions, powder and injections. Conventional procedures and
ingredients for preparing and administering the different dosage
forms would be known to a skilled person and are described for
example, in Remington's Pharmaceutical Sciences (Remington, The
Science and Practice of Pharmacy, 21.sup.st edition, Lippincott
Williams & Wilkins, Philadelphia, Pa., 2006).
[0153] Uses of the IMSs as described herein, including compounds
having a structure of general formula I, a pharmaceutically
acceptable salt thereof or an oligomer or polymer thereof for
delivering an anti-fibrotic or anti-cancer agent to a cell in vivo
and in the preparation of a medicament for delivering an
anti-fibrotic or anti-cancer agent to a cell in vivo are also
contemplated.
[0154] The IMSs of the present method may exert their anti-fibrotic
and anti-cancer effect as a result of anti-oxidative properties,
which may function via a multitude of molecular mechanisms.
Anti-oxidants generally exert their effect mainly through three
different pathways: (1) neutralization of cellular free radical ROS
generated during metabolism and immune response, (2) induction of
endogenous anti-oxidative enzymatic activity, and (3) chelation of
iron or copper ions that catalyze the generation of hydroxyl
radical. Without being limited to any particular theory, it appears
that the anti-oxidative properties of the IMSs may result, at least
in part, due to their activity as a radical scavenger. It appears
that the neutralization of free radicals by IMSs may occur after a
series of chemical reactions including (1) the spontaneous
conversion of IMSs to NHCs preferably under a basic condition, (2)
the interaction of the NHCs at the carbon 2-position with free
radicals such as ROS resulting in the formation of intermediate
active radicals and the neutralization of the free radicals.
[0155] The present inventors have discovered that the IMSs
described herein also exhibit anti-inflammatory properties. Without
being limited to any particular theory, the dual anti-oxidative and
anti-inflammatory properties of the IMSs may be attributable, at
least in part, to the interplay between oxidative stress and
inflammation. For example, it is hypothesized that in the treatment
of liver fibrosis, the anti-inflammatory effects of the IMSs
compounds defined herein may be attributable to the IMSs'
anti-oxidative properties that inhibit HSC activation and thus the
resulting production of pro-inflammatory cytokines. Similarly,
without being limited to any particular theory, the inhibition of
inflammatory mediator IL-6 by an IMS may be partially attributable
to the anti-oxidative properties of the IMS which suppress the
activation of transcription factor NF-.kappa.B, an important
regulator of the secretion of inflammatory cytokines.
[0156] In addition to their anti-oxidative and anti-inflammatory
properties, the IMSs may exhibit other effects that are useful in
treating fibrotic disease and cancer. Anti-oxidants have been found
to also have apoptosis-inducing properties. For example, EGCG, a
green tea extract has shown to induce apoptosis of human
hepatocellular carcinoma in cultured cell lines and a xenograft
model (Nishikawa et al. 2006) while garlic extract, a natural
anti-oxidant, has also been shown to induce apoptosis in human
glioblastoma cells (Das et al. 2007) and colon carcinoma cells
(Jakubilova et al. 2006) by inducing oxidative stress.
[0157] Without being limited to any particular theory, it appears
that the IMSs may be effective in treating cancer due to inducement
of apoptosis and inhibition of cell proliferation through cell
cycle arrest. For example, IMSs may trigger apoptosis and cell
cycle arrest by increasing or decreasing the activity and
expression of molecules involved in apoptosis and cell cycling
including for example survivin, caspase 9, caspase 3, Bcl-XL, Bak,
BAX, ATM, p53, Cdc25A, Cdc2, SESN2 and AP-1.
[0158] The present methods and compounds are further exemplified by
way of the following non-limited examples.
EXAMPLES
Materials and Methods
[0159] Anti-Oxidative, Anti-Inflammatory and Anti-Fibrotic
Properties of IMSs
[0160] Synthesis and Characterization of IMSs
[0161] DBZIM, TDBZIM and DBZBIM were synthesized in-house. DPIM,
DBIM, AMIM, TMPHIM and DPPHIM were purchased from Sigma Chemicals
(USA). The chemical structures of DBZIM (1,3-bisbenzylimidazolium
bromide), DBZBIM (1,3-bisbenzyl-benzimidazolium bromide) and TDBZIM
(1,3,5-tris(4-methyl-imidazolium)-linked cyclophane.3Br) are
illustrated in Table 1. DBZIM and DBZBIM were synthesized using a
method disclosed in the literature (Harlow et al. 1996). TDBZIM was
synthesized by mixing 4-methylimidazole (123 mg, 1.5 mmol) and
2,4,6-tris(bromomethyl)mesitylene (400 mg, 1 mmol) in 200 ml of
N,N'-dimethylformamide (DMF) in a reaction vial. The reaction
mixture was heated to 100.degree. C. for 2 days. Colorless crystals
of TDBZIM were precipitated, and collected in 40% yield (160 mg).
Nuclear magnetic resonance was conducted with the following
results: .sup.1H nuclear magnetic resonance (NMR) (400 MHz,
CD.sub.3OD): .delta. 7.77 (s, 1H), 5.53 (s, 2H), 5.41 (s, 2H), 4.60
(s, 1H), 2.55 (s, 3H), 2.19 (s, 6H); .sup.13C NMR (100 MHz,
CD.sub.3OD): .delta. 143.49, 136.27, 131.73, 131.54, 123.45, 67.06,
16.35, 9.57. Elemental analysis of TDBZIM
(C.sub.36H.sub.45Br.sub.3N.sub.6) provided the following results:
C, 53.21; H, 5.88; N, 10.12 (calc. C, 53.95; H, 5.66; N,
10.49).
[0162] Primary HSC Isolation and HSC-T6 Line
[0163] The isolation of primary HSCs from Wistar rat liver was
performed primarily following the method provided in a previous
report (Weiskirchen et al. 2005). Briefly, the supernatant of cell
suspension after hepatocyte removal was washed and resuspended in
9.5 ml of Gey's balance salt solution (GBSS), and mixed with 8 ml
of 28.7% (w/v) Histodenz in GBSS. The gradient was prepared by
laying the cell suspension underneath 6 ml of GBSS, and centrifuged
in Histodenz gradient at 1400.times.g for 20 min. The stellate
cells were separated into a fuzzy band just above the interface of
the Histodenz solution and the aqueous buffer. The stellate cell
band was harvested, washed, and 1.times.10.sup.6 cells/well were
seeded in a 6-well culture plate in Dulbecco modified Eagle's
medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The
viability of the HSCs was >95% as determined by trypan blue
exclusion staining, and the purity of the HSCs was >90% as
assessed by glial fibrillary acidic protein (GFAP) positive
staining in >200 cells counted. Primary HSCs were routinely
cultured in DMEM with 10% FBS in a humidified CO.sub.2 incubator at
37.degree. C., and split at 1:4 ratio by trypsinization (0.05%
trypsin/0.53 mM ethylenediaminetetraacetic acid (EDTA)) when they
grew to confluency. The HSC-T6 cell line was kindly provided by Dr.
Scott Friedman of Mount Sinai School of Medicine, New York. A
stable T6/GFAP-LacZ clonal cell line was established as previously
reported (Maubach et al. 2006). HSC-T6 and T6/GFAP-LacZ cells were
cultured in the same conditions as the primary HSCs.
[0164] Cell Treatment with IMSs
[0165] Stock solutions of IMSs were prepared either in
dimethylsulfoxide (DMSO) or H.sub.2O depending on their solubility.
The final DMSO concentration in the culture medium was kept below
0.2% (v/v). NAC was obtained from Merck KGaA (Germany), and EGCG
was purchased from Sigma and used directly without further
purification. Cells were initially seeded in cell culture plates or
flasks in DMEM supplemented with 10% FBS for 18-24 h before the
addition of compounds of various concentrations for different time
periods. Details of the each treatment can be found in the figure
legends.
[0166] Cellular ROS Determination
[0167] The ROS level of treated HSC-T6 cells was determined using
the dichlorofluorescein (DCF) labeling method (Molecular Probes
Inc., OR, USA). Cultured cells were harvested by trypsinization,
and resuspended in phenol red-free DMEM (Invitrogen). Cells were
then incubated with 10 .mu.g/ml of 2',7'-dichloro-fluorescein
diacetate (DCFH-DA) in DMEM for 15 min before resuspension in DMEM
for assaying. Cell solution was dispensed into a black-wall 96-well
pate in triplicate for fluorescence readout at Ex=485 nm and Em=530
nm with a Tecan Safire II plate reader. The fluorescence unit was
normalized against viable cell numbers, which were determined using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT
assay, Promega, WI, USA). Cellular ROS level was expressed relative
to the control sample.
[0168] Glutathione (GSH) and GSH/Disulfide Dimer of Glutathione
(GSSG) Assays
[0169] Total cellular GSH and GSSG levels were determined by using
assay kits from Cayman Chemical (Ann Arbor, Mich., USA) following
the manufacturer's instructions. Briefly, HSC-T6 cells treated with
DBZIM, TDBZIM, NAC, or EGCG were scalped and homogenized in
phosphate buffered saline (PBS) by sonication at 60% frequency for
30 s, and the supernatant was collected after centrifugation at
16,000.times.g for 10 min at 4.degree. C. Protein content of the
samples was determined by bicinchonicic acid (BCA) assay.
[0170] For GSH assays, the protein sample was deproteinated by
metaphosphoric acid (Aldrich, Catalog No. 23927-5), and the pH was
adjusted by triethanolamine (Aldrich, Catalog No. T5830-0)
according to the manual. The sample was ready for assaying total
GSH including both the reduced and oxidized forms. Quantification
of GSSG, exclusive of GSH, was performed by first derivatizing GSH
with 2-vinylpyridine and assaying separately. Colorimetric signal
was recorded at 405 nm with a Tecan S afire II plate reader. The
levels of GSH or GSSG were determined as nmol/.mu.g protein.
[0171] Glutathione Peroxidase (GPx), Catalase (CAT) and Superoxide
Dimutase (SOD) Assays
[0172] Activity levels of three anti-oxidant enzymes, GPx, CAT and
SOD, were determined using assay kits from Cayman Chemical. To
assay GPx and CAT activities, protein samples were harvested in
ice-cold PBS containing 1 mM of EDTA. To assay SOD activity,
protein samples were prepared in 20 mM of
N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES)
buffer containing 1 mM of ethylene
glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA),
210 mM of mannitol and 70 mM of sucrose. Protein content was
determined by BCA assay.
[0173] GPx activity was indirectly measured by a coupled reaction
with GSH reductase. Oxidized GSH, produced upon reduction of
H.sub.2O.sub.2 by GPx, is recycled to its reduced state by
glutathione reductase (GR) and nicotinamide adenine dinucleotide
phosphate (NADPH). The oxidation of NAPDH is proportional to the
GPx activity, and can be measured as a decrease in absorbance at
340 nm. GPx activity was expressed as nmol/min/mg protein.
[0174] CAT activity was determined based on reaction of the enzyme
with methanol in the presence of H.sub.2O.sub.2. The formaldehyde
produced was measured spectrophotometrically at 540 nm using
Purpald as the chromogen. CAT activity was expressed as nmol/min/mg
protein.
[0175] Total SOD, including all three types of SOD (Cu/Zn-, Mn- and
Fe-SOD), was quantified using a tetrazolium salt to detect
superoxide radicals generated by xanthine oxidase and hyposanthine.
One unit of SOD was defined as the amount of enzyme needed to
exhibit 50% dismutation of the superoxide radical. SOD activity was
expressed as U/mg protein.
[0176] Glutathione S-Transferase (GST) Assay
[0177] The activity of phase II anti-oxidant enzyme, GST, was
assayed using a kit from Cayman Chemical. Protein samples were
harvested in PBS buffer containing 1 mM of EDTA, and the protein
concentration was determined by BCA assay. Total GST (cytosolic and
microsomal) activity was assayed by measuring the conjugation of
1-chloro-2,4-dinitrobenzene (CDNB) with reduced GSH, and monitored
by an increase in absorbance at 340 nm. GST activity was expressed
as nmol/min/mg protein.
[0178] Total RNA Isolation and Real-Time RT-PCR
[0179] Total RNA was isolated using NucleoSpin RNAII isolation kit
(Nacherey & Nagel, Germany), and quantified by ND-100
spectrophotometer (Nanodrop Technologies, DE, USA). The details of
real-time RT-PCR were reported elsewhere (Zhang et al. 2006).
Briefly, it was a two-step real-time RT-PCR using Taqman chemistry.
Total RNA was first reverse transcribed to cDNA, and real-time PCR
was run and detected in ABI 7500 Fast Real-Time PCR System (Applied
Biosystems, CA, USA). Primers and probes for rat GFAP, SMAA,
collagen Ia1, fibronectin, TGF-.beta.1, TGF-.beta. receptor I
(TGF.beta. RI) and IL-6 were ordered from Taqman's assay-on-demand
database. Rat .beta.-actin was used as normalization gene, and its
expression level was constant (CT difference was <.+-.0.5)
throughout the experimental settings in this study. Relative
quantification of target mRNA was calculated using comparative
threshold cycle method (.DELTA..DELTA.C.sub.T) as described in User
Bulletin #2 (ABI Prism 7700 Sequence Detection System). Relative
quantification was given by 2.sup.-.DELTA..DELTA.CT to express the
up-regulation or down regulation of the target gene under the
treatments compared to the control.
[0180] Western Blotting
[0181] Protein extracts were prepared by lysing a cell pellet in 10
volumes of boiled 1% sodium dodecyl sulfate (SDS) (90-95.degree.
C.) for 10 min for GFAP, or using NE-PER nuclear and cytoplasmic
extraction reagents from Pierce (IL, USA) for nuclear proteins, or
RIPA (Pierce, IL, USA) for SMAA, fibronectin, collagen .alpha.I(I),
TGF-.beta.1 and TGF.beta. RI. Protein content was quantified using
a BCA kit (Pierce, IL, USA). 15 .mu.g of total protein were
resolved in 4-12% gradient SDS/polyacrylamide gel electrophoresis
(PAGE) gel, and transferred to a nitrocellular membrane. The
membrane was then probed with primary antibodies against GFAP
(Dako, Denmark), SMAA (Sigma, USA), procollagen fibronectin, c-Jun,
c-Fos, JunB, JunD, Fra-1, Fra-2 (Santa Cruz Biotechnology, CA,
USA), NF-.kappa.B P65, TGF-.beta.1 and TGF.beta. RI (Abcam, UK),
and detected by horseradish peroxidase-conjugated secondary
antibody (Santa Cruz Biotechnology, CA, USA). For fibronectin and
col1a1 blotting, the protein extract was separated in 3-8%
tris-acetate gel. Protein bands were recorded on X-ray film by
reacting with ECL chemiluminescence reagents (Amersham Biosciences,
NJ, USA). Alpha-tubulin (Abcam, UK) was used as the loading control
for total protein, and Tata binding protein (TBP) (Abcam, UK) was
used as the loading control for nuclear protein.
[0182] Quantification of .beta.-Galactosidase Activity
[0183] Beta-galactosidase activity in extracts from T6/GFAP-lacZ
cells treated by DBZIM or TDBZIM was measured using a
chemiluminescent assay kit (Roche, Mannheim, Germany) in a 96-well
plate format, according to the manufacturer's instructions.
Briefly, cells were washed twice with ice-cold 1.times.PBS (pH
7.4), and lysed for 30 min at room temperature in the lysis buffer.
The supernatant was collected for assays with a Tecan Safire II
plate reader. Protein concentration was determined with a BCA
protein assay kit (Pierce, IL, USA). Specific .beta.-galactosidase
activity was obtained by normalizing against total cellular protein
content.
[0184] General Toxicity of IMSs in Mice
[0185] IMSs (DPIM and DBZIM) were dissolved in saline, and
administered to Friend virus B-Type (FVB) mice (weighing
.about.25-30 g) intraperitoneally every other day for 2 weeks. The
dosages were 0, 200, 300, 350, 400 and 500 mg/kg for DPIM, and 0,
10, 30, 35, 40 and 55 mg/kg for DBZIM. The injection volume was
maintained at 100-160 .mu.l. The mortality rate of mice was
assessed after the treatment. The animal experimental protocol was
approved by the Institutional Animal Care and Use Committee (IACUC)
of Biological Resource Center (BRC) at Biopolis, Singapore.
[0186] Statistical Analysis
[0187] All quantitative results were presented as mean and standard
error of mean (SEM). Experimental data were analyzed using
two-tailed Student's t-test assuming unequal variances. A P-value
of .ltoreq.0.05 was considered statistically significant.
[0188] Effect IMSs on Liver Fibrosis in Mice
[0189] For the liver toxin thioacetamide (TAA) model, TAA was given
by i.p. injection at 200 mg/kg body weight, 3 times per week, for
12 weeks, to induce hepatic fibrosis in mice. For the bile duct
ligation model (BDL), adult mice were anesthetized with Ketamine
(150 mg/kg)/Xylazine (10 mg/kg) via i.p. injection. An abdominal
incision at the midline is made to expose the common bile conduct,
which was then ligated using 5-0 silk suture. In the control
animal, the bile duct is exposed, but not ligated. DBZIM or DPIM
was provided to the mice in drinking water at concentrations from 1
to 1000 mg/liter. The collagen content in the fibrotic liver was
assessed by the Sirius Red staining of the liver tissue
sections.
[0190] Additional studies were carried out on the effect of DPIM on
liver fibrosis inducted by BDL. DPIM was purchased from Sigma
Chemicals (St. Louis, Mo., USA).
[0191] Mice and Compound Dosing
[0192] Male FVB/N mice (8-10 weeks old) housed in a specific
pathogen-free (SFP) facility on a 12 hour dark-light cycle and with
free access to water and diet were used in the study. The animal
experiment protocol was approved by the institutional animal care
and use committee (IACUC), the Biomedical Research Council (BMRC)
of Singapore.
[0193] Cholestatic fibrosis was induced by BDL, briefly, mice were
anesthetized with intraperitoneal injection of ketamine (150 mg/kg)
and xylazine (10 mg/kg), after midline laparotomy, the common bile
duct was ligated twice with 5-0 silk suture. The sham operation was
performed similarly except that bile duct was not double ligated.
The experimental mice were divided into four groups: 1) control
only (sham operated, but no BDL); 2) control+DPIM; 3) BDL only; 4)
BDL+DPIM. Each group consists of 6-8 mice. DPIM compound for
treatment was supplied in the drinking water at three
concentrations, 500 mg/1, 750 mg/1, 1 g/l, respectively. The
drinking water containing DPIM was changed weekly. It was observed
that on average each mouse (normal or operated) drank approximately
3-4 ml plain or DPIM-containing water every day. Testing by NMR
analysis was also conducted to confirm that DPIM is stable in water
at room temperature condition for at least two weeks. The compound
treatment was started on the third day after animals recovered from
the BDL procedure. After 4 weeks, livers were removed from mice for
fixation 10% formalin, subsequently embedded in paraffin.
[0194] Measurement of Hepatic Enzymes
[0195] Serum was collected by retro-orbital method and serum
alanine transaminase (ALT) activity was measured by automated
procedures with Cobase c 111 (Roche diagnostic).
[0196] Collagen Staining by Sirius Red
[0197] For consistency, paraffin-sections prepared from the left
and median lobes were stained with Sirius-Red to visualize the
collagen content. Briefly, after de-wax, re-hydration, air-drying,
the sections were stained in 0.1% Sirius Red solution for one hour,
washed twice with 0.5% acetic acid, then dehydrated in three
changes of ethanol, the sections were finally cleared in xylene and
mounted with Histomount (National diagnostics, Georgia, USA). The
collagen stained areas were measured in at least 6 independent
fields under low magnification (4.times.) for each left and median
lobe, respectively, and quantified with Image J software.
[0198] Statistical Analysis
[0199] All images displayed are representative of 6-8 mice, unless
stated otherwise. All quantitative data were expressed as
mean.+-.SE, differences among different treatment groups were
analysed by two-tailed unpaired Student t-test, P value less than
0.05 were considered statistically significant.
[0200] Effect of DBZIM on Hepatocellular Carcinoma and Other Tumour
Cells
[0201] DBZIM (1,3-bisbenzylimidazolium bromide) was synthesized
based on a published method (Harlow et al. 1996).
[0202] Cell Culture and Compound Treatment
[0203] Human heptocellular carcinoma cell lines: HLE (an
undifferentiated cell line) and HepG2 (differentiated cell line)
were purchased from JCRB (Japanese Collection of Research
Bioresources). Human gastric cancer cell lines AGS and MKN28, lung
cancer cell line H1299, breast cancer cell line MCF-7, glioma cell
line U87 MG, and C6 were all purchase from ATCC. Cell lines were
routinely cultured in DMEM with 10% FBS in a humidified CO.sub.2
incubator at 37.degree. C., and split at 1:4 ratio by
trypsinization (0.05% trypsin/0.53 mM ethylenediaminetetraacetic
acid (EDTA) when they grew to confluence.
[0204] Stock solution of DBZIM was prepared in water. Cells were
initially seeded in cell culture plates or flasks in DMEM
supplemented with 10% FBS for 18-24 h before the addition of
compound of various concentrations for different time periods.
Details of the each treatment can be found in the figure
legends.
[0205] DNA Content Analysis
[0206] HLE cells grown in a 96-well plate were treated with
compounds in 6 replicates for 24 hr. Cells were then fixed with 4%
paraformaldehyde and stained with DAPI. Cells were analyzed and
imaged on ArrayScan VTI HCS reader (Thermal Scientific, PA, USA)
and DNA content (2N vs. 4N) was determined based on DAPI
staining.
[0207] Cell Proliferation Assay
[0208] HLE cells were grown, treated by compounds in 6 replicates
for 48 hr in a 96-well plate. Cells were then fixed by using 4%
paraformaldehyde and stained by DAPI and BrdU antibody using Hitkit
(Thermo Scientific, IL, USA). Cells were imaged and analyzed on
ArrayScan VTI HCS reader (Thermal Scientific, PA, USA). Cell
numbers were counted based on nuclear staining and BrdU
incorporation was determined and expressed as percentage of
positively stained cells.
[0209] Proliferation of Glioma cell lines: U87 MG and C6 under the
treatment of DBZIM was examined using
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS) based proliferation assay kit (Promega, WI,
USA). MTS reagent was added to the control and treated cells (after
48 hr) directly and the absorbance signal was read at 490 nm using
a Tecan Safire II plate reader. Proliferation was similarly
performed for gastric cancer cell line AGS, lung cancer cell line
H1299, and breast cancer cell line MCF-7, but the IC50 value was
determined at 72 h, instead of 48 h.
[0210] Cell Based Caspase 3/7 and LDH Assays
[0211] Capspase 3/7 activity and lactate dehydrogenase (LDH)
release were measured by Fluorometric assays (Promega Corp., WI,
USA). The HCC cells were cultured in 96-well plate and treated with
DBZIM of various concentrations for 6 hr before assays. For caspase
3/7 assay, medium was removed and substrate buffer was added to
lyse the cell and react with caspase 3/7 to give rise to
fluorescence signal which was read at Ex=485 nm and Em=530 nm on a
Tecan Safire II plate reader after 1 hr incubation. The caspase 3/7
activity of sample cells with the compound treatment was expressed
as relative fold change after normalizing to the control cells. The
LDH assay (indicative of membrane integrity) was performed using a
Kit from Promega Corp according to manufactor's instructions.
[0212] Protein Extract Based Caspase 3/8/9 Colorimetric Assays
[0213] Capspase 3/8/9 activities were determined using total
protein extract as starting material (Biovision Inc., CA, USA). HLE
cells were cultured in T75 flask and treated with DBZIM of various
concentrations. Cytosolic protein extract was collected by
pelleting and lysing the cells and centrifugation to remove the
debris. Capspase 3, 8 and 9 activities were assayed using the same
protein extract but adding the respective substrate. Absorbance
readings were taken at 405 nm using Tecan Safire II plate reader.
Results were expressed as relative caspase activity by normalizing
to control cells without compound treatment.
[0214] Annexin V Staining and Analysis by Cytometry
[0215] HLE and HepG2 cells were treated with various concentrations
of DBZIM or TRAIL (100 ng/ml) for 24 hr, harvested and labeled with
Annexin V-FITC (BioVision, CA, USA). The labeled cells were
analysed by flow cytometry FACSCalibur (Beckman-Dickson, NJ, USA)
and CellQuest software. Cells showing green staining in the plasma
membrane were considered as apoptotic cells.
[0216] AIF/Survivin Translocation Assay
[0217] HLE cells cultured in 96-well plate were treated with DBZIM
for 24 hr before staining and imaging. Cells were then fixed and
stained with antibodies against apoptosis-inducing protein (AIF)
and Survivin (Lab Vision Corp. CA, USA) and buffer reagent from
Hitkit (Thermo Scientific, IL, USA). The cells were imaged and
analyzed on ArrayScan VTI HCS reader (Thermal Scientific, PA, USA).
Fluorescence intensity in the cytoplasmic and nuclear area was
plotted for indication of cytoplasm to nucleus translocation.
[0218] SDS-PAGE and Western Blot
[0219] Protein extracts were prepared by lysing cell pellet in RIPA
buffer (Pierce, IL, USA) for total protein extract, or in NE-PER
reagent from Pierce (Pierce, IL, USA) for nuclear and cytoplasmic
fractions, or in a Kit from BioVision (CA, USA) for mitochondria
and cytosol fractions. Protein content was quantified using BCA Kit
(Pierce, IL, USA). Fifteen micrograms of protein were resolved in
4-12% gradient SDS/polyacrylamide gel electrophoresis (PAGE) gel,
and transferred to nitrocellular membrane. The membrane was then
probed with primary antibodies against Bcl-XL, Bak, Bcl-2.alpha.,
Bax (Lab Vision Corp., CA, USA), survivin, xIAP, cIAP (R&D
systems Inc., MN, USA), c-Jun, c-Fos, JunB, JunD, Fra-1, Fra-2
(Santa Cruz Biotechnology, CA, USA) respectively, and detected by
horseradish peroxidase-conjugated secondary antibody (Santa Cruz
Biotechnology, CA, USA). Protein bands were recorded on X-ray film
by reacting with ECL chemiluminescence reagents (Amersham
Biosciences, NJ, USA). Alpha-tubulin (Abcam, UK) was used as the
loading control for total protein, Tata binding protein (TBP)
(Abcam, UK) was used as the loading control for nuclear protein,
and Cox4 (Abcam, UK) was used as loading control for mitochondria
protein.
[0220] ROS Determination
[0221] Oxidative stress was measured using Cellomic HitKit (Thermo
Scientific, IL, USA). HLE cells were treated with DBZIM for 24 hr,
fixed and labeled by Dihydroethidium probe for quantitation of ROS
generation and Hoechst dye for nuclear staining. The result was
analyzed by Target Activation BioApplication software. The
percentage of positive responding cells was plotted as the
indication of ROS amount.
[0222] High-Content Analysis for Cytotoxicity and Apoptosis
[0223] Hitkit: Multiparameter Cytotoxicity 1 and Multiparameter
Apoptosis 1 assay kits (Thermal Scientific, PA, USA) were used to
exam the cytotoxicity and apoptosis profile of additional IMSs.
Cells were cultured and treated in 96-well plate for 24 hr before
assaying. Cells were fixed and stained essentially following the
manufacturer's instruction. The images were taken by ArrayScan VTI
HCS reader (Thermal Scientific, PA, USA) and analyzed using Cell
Viability and Cell Health Profiling BioApplication software
respectively.
[0224] HCC Xenograft Model and Dosing
[0225] Balb/c Nude mice, 5-6 weeks old, were inoculated with
1.times.10.sup.7 Huh7 cells in 0.2 ml volume of Matrigel/DMEM mix.
Since not every mouse developed a tumor after the inoculation of
HCC cells, only those with visible tumors (about 8 weeks after
inoculation) were used for subsequent experiment. The tumor-bearing
mice were randomly divided to the control and the treatment group
(n=3), respectively. Mice in the treatment group had free access to
drinking water containing DBZIM (2 grams/liter) only, while mice in
the control group had free access to water only. The compound
treatment lasted for 3 weeks. The tumor size was measured weekly,
and the tumor volume was calculated using the formula:
0.52.times.width.sup.2.times.length.
[0226] Anticancer Activities of Additional IMSs
[0227] Synthesis and Characterization of IMSs
[0228] All solvents and chemicals were used as obtained from
commercial suppliers, unless otherwise indicated. Centrifugation
was performed on Eppendorf Centrifuge 5810R (4000 rpm, 10 min).
.sup.1H and .sup.13C NMR spectra were recorded on Bruker AV-400
(400 MHz) instrument. Data for .sup.1H and .sup.13C NMR were
reported in chemical shift (.delta. ppm) and multiplicity
(s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet). GC-MS
was performed on Shimadzu GCMS QP2010. Gas liquid chromatography
(GLC) was performed on Agilent 6890N gas chromatographs equipped
with split-mode capillary injection system and flame ionization
detector.
[0229] To prepare substituted imidazoles, NaH (60% in oil, 420 mg,
10.5 mmol) was added to a DMF solution of imidazole (A.sub.1) (680
mg, 10 mmol) at 0.degree. C., and the resulting suspension was
stirred at room temperature for 2 h. Benzylbromide (1.71 g, 10
mmol) was added to the residue. The resulting solution was stirred
at room temperature for another 4 h. The solvent was removed under
vacuum. The product was extracted with dichloromethane (DCM), and
B.sub.1 was obtained in quantitative yield after removing the
solvent. B.sub.2 to B.sub.4 were synthesized in the same protocol
with A.sub.2 to A.sub.4 as imidazole starting materials. The
products were confirmed by MS and NMR.
##STR00004##
[0230] DBZIM, DBZBIM, 1,3,-Dibenzyl-2-methylimidazolium bromide
(DBZMIM, Compound C), IBN-2, 3, 4, 6, 8, 9 (Compound), 12, 13, 15,
17, 18, 19, 20, 21, 24, 25, 26, 27, 28, 30, 31, 32 33, 34 were
synthesized by mixing 1 mmol of substituted imidazole (like
B.sub.1-B.sub.4) and 1 mmol of substituted benzyl bromide(chloride)
or alkyl bromide(chloride) in 1 ml of DMF. The mixture was stirred
at 100.degree. C. for 16 hours. The reaction mixture was cooled
down to room temperature and ether (10 ml) was added to precipitate
product. The powder or gel precipitate was collected as imidazolium
product and purified by re-crystalline or chromatograph method and
characterized by NMR.
[0231] IBN-22, 23, 29 were made based on literature methods (Zhang
et al. 2007; Chianese and Cratree 2005, Boydston et al. 2005).
[0232] The remaining IMSs listed in Table 2 were purchased from
Sigma.
[0233] Typically 100 mM of stock solution of IMSs was prepared in
DMSO. Aliquots of stock solution were stored until use at -20
C.
[0234] Cell Lines and Cell Culture
[0235] Human Hepatocellular cancer cell lines HLE (p53 Mutant) and
human gastric cancer cell line MKN 28 were obtained from Japan
Health Sciences Foundation (Osaka, Japan). The human breast cancer
cell line MDAMB231 (mutant p53) and breast epithelial cells MCF-10A
(wild type p53) were obtained from ATCC. These cell lines were
maintained in DMEM medium (RPMI 1640 for MDAMB231) supplemented
with 10% fetal bovine serum and 1% of mixture Penicillin and
Stretomycin incubated at 37.degree. C. in an atmosphere of 5%
CO.sub.2.
[0236] Growth Inhibition Assay
[0237] The percentage of growth inhibition was determined by using
a MTT assay to measure viable cells. For the MTT assay,
1.times.10.sup.3 cells per well were cultured in 96-well plates and
treated with 0 to 100 .mu.M of various types of IMSs for 72 h.
After incubation for specified times at 37.degree. C. in a
humidified incubator, culture medium replaced with 10 .mu.L of MTT
reagent (5 mg/mL) contains 100 .mu.L medium was added to each well
and further incubated for 4 h. The reaction was stopped by adding
100 .mu.L of DMSO. Absorbance was measured at 570 nm on a micro
plate reader (SpectroMax 190, Molecular Devices). Data were
presented from three separate experiments, and the percentage of
IMSs induced cell growth inhibition was determined where
DMSO-treated cells (control) were taken as 100%.
[0238] Cell Cycle Analysis
[0239] To perform cell cycle distribution analysis,
4.times.10.sup.5 cells were plated in 10 cm dish. The cells (70%
confluence) were serum-starved for 24 h to synchronize them in the
G.sub.0 phase of the cell cycle. After 24 h incubation, the cells
were replaced with fresh 10% DMEM medium and then treated 60 .mu.M
of IBN-15, 90 .mu.M of IBN-19, 90 .mu.M of IBN-24, 30 .mu.M of
IBN-24 and 20 .mu.M of IBN-32) and 0.045% DMSO as control for 72 h.
The floating and trypsinized adherent cells were collected and
washed with PBS. The cell pellets were resuspended in 1 ml of PBS
and fixed by the addition of 70% ice-cold ethanol and stored at
-20.degree. C. After incubation for 24 hr, the cells were
re-pelleted by centrifugation, the cells were washed once with PBS
and resuspended in 0.1 mL of PBS containing 100 .mu.g/ml of RNase
and then incubated at 37.degree. C. for 15 min. Finally, the cells
were stained with 0.5 ml of PI solution (100 .mu.g/mL in PBS) for
30 min. Cell cycle distribution was detected by a BD FACS array
flow cytometer (Becton Dickinson). Cells with sub-G.sub.0 DNA were
classified as apoptotic cells.
[0240] Apoptosis Assay
[0241] To determine apoptosis analysis, 4.times.10.sup.5 cells were
plated in 10 cm dish. The cells were then treated with 60 .mu.M of
IBN-15, 90 .mu.M of IBN-19, 90 .mu.M of IBN-24, 30 .mu.M of IBN-25
and 20 .mu.M of IBN-32) and 0.045% DMSO as control for 72 h. The
floating and trypsinized adherent cells were collected and the
apoptosis was quantified using Annexin V-FITC reagent (BD
Biosciences Pharmingen) and PI (Invitrogen) following the
manufacturer's protocol. Briefly, the cells were washed twice with
cold PBS and then resuspend cell in 100 .mu.l of.times.binding
buffer containing a 10 of Annexin V-FITC (BD-Pharminagen) and 2
.mu.l of PI from the stock of 100 .mu.g/ml. The cells were gently
vortexed and incubated for 15 min at room temperature in the dark.
Subsequently 400 .mu.l of 1.times. binding buffer were added to
each tube, and the samples were immediately analyzed by FACScan
flow cytometer (Becton Dickinson). The fluorescent signals of the
Annexin-V conjugate and PI were detected at channels of
fluorescence intensity FL1 and FL2 (BD LSR II with software
BD).
[0242] SDS-PAGE and Western Blotting
[0243] The HLE cells were seeded at a density of 4.times.10.sup.5
in 10 cm dish. After 24 h incubation, the cells were replaced with
fresh 10% DMEM medium and then treated with 60 .mu.M of IBN-15, 90
.mu.M of IBN-19, 90 .mu.M of IBN-24, 30 .mu.M of IBN-25 and 20
.mu.M of IBN 32 and 0.045% DMSO as control for 72 h. Whole cell
lysates were prepared on day 3 with lysis buffer [125 mM Tris (pH
7.4), 2% Sodium dodecyl sulfate, 10% glycerol, 6M urea and a
protease inhibitor cocktail (Sigma) and 0.02% Bromophenol blue].
The lysates were sonicated for 30 seconds and then heated for 5
mins at boiling temperature. The lysates were centrifuged at 13000
rpm for 10 mins and the protein concentration of each sample was
estimated using the BCA protein assay. The lysates was then mixed
with 5% 2-mercaptoethanol and stored at -80 C. For Western
blotting, samples containing 50 .mu.g of total cell lysate were
loaded onto a SDS-PAGE and subjected to electrophoresis. Proteins
were transferred to a nitrocellulose membrane and then blocked with
5% non-fat dry milk in Tris buffer saline containing 0.1% Tween 20
for 60 min at 37 C. The membranes were probed with a primary
antibody overnight at 4 C in TBST containing 5% Bovine serum
albumin. Then the membrane was incubated with horseradish
peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary
antibodies (GE Health care, Chalfont St Giles, UK). Detection was
performed with enhanced chemiluminescence (ECL) reagent (Amersham
Arlington Heights, Ill.) according to the manufacturer's protocol.
Rabbit polyclonal Phospho-p53 (Ser15, 20, 46, 392), anti-caspase-9,
anti-caspase-3, anti-PARP antibodies were purchased from Cell
Signaling Technology, Inc. (Beverly, Mass., USA). Antibodies
against p53 and p21 were purchased from Santa Cruz Biotechnology
(Santa Cruz, Calif., USA). Equal loading of protein was
demonstrated by probing the membranes with a mouse
anti-.beta.-actin monoclonal antibody (Sigma Chemical Co. (St.
Louis, Mo.).
[0244] Immunostaining and Confocal Microscopy
[0245] For immunostaining, HLE cells were seeded at a density of
1.times.10.sup.4 in 8 well chamber slide. After 24 h incubation,
the cells were replaced with 200 .mu.l fresh 10% DMEM medium and
then treated with 60 .mu.M of IBN-15, 90 .mu.M of IBN-19, 90 .mu.M
of IBN-24, 30 .mu.M of IBN-25 and 20 .mu.M of IBN-32 and 0.045%
DMSO as control for 24 and 48 h. After washing, cells were fixed in
4% formaldehyde for 15 mins and permeabilized with 0.5% Triton
X-100 in PBS for 10 mins. The cells were then blocked for 60 min in
PBS containing 3% bovine serum albumin and then incubated for 1 h
with a mouse antibody against nucleolin (MS-3, Santa Cruz; 1:100 in
blocking buffer). After washing extensively with PBS, the cells
were then incubated for 1 h at room temperature in dark with a
FITC-conjugated antibody against mouse IgG (1:20 ilution). The
slides were mounted with a VectaShield medium containing DAPI
(Vector Laboratories, Burlingame, Calif.). Microscopy was performed
with a Zeiss LSM 510 Meta (upright stand) confocal microscope.
[0246] Real-Time Quantitative RT-PCR Analysis
[0247] Cells were seeded at a density of 4.times.10.sup.5 cells per
dish in 10 cm dishes. Next day, the cells were exposed to 90 uM of
IBN-19, 90 uM of IBN-24, 40 uM of IBN-25 and 0.045% of DMSO
(Dimethyl Sulphoxide) as the control. After 48 hrs, total RNAs were
extracted from the cells using Nucleospin RNA II kit as per the
manufacturer's istructions. RNA quality and yield were evaluated
after spectrophotometer measurements.
[0248] The human apoptosis PCR array (RT.sup.2 Profiler) was used
to analyze mRNA levels of 84 key genes involved in apoptosis, in a
96-well format, according to the manufacturer's instructions (Super
Array Bioscience). First-strand cDNA was synthesized with 1 .mu.g
of RNA by using a PCR array first strand-synthesis kit (C-03; Super
array Bioscience). This kit uses reverse transcriptase (Power
Script; Super array Bioscience) and a combination of random primers
and oligo dT primers. The total volume of the reaction was 20 .mu.K
diluted to 100 .mu.L. PCR reactions were performed using real-time
PCR (79s00HT 96-well block with RT.sup.2 Real-Time SYBR Green PCR
master mix PA-012; Applied Biosystems Instruments 7500). The total
volume of the PCR reaction was 25 .mu.l. An equivalent of 1 .mu.g
of RNA was applied to the PCR reaction. The thermocycler parameters
were 95.degree. C. for 10 minutes, followed by 40 cycles of
95.degree. C. for 15 seconds and 60.degree. C. for 1 minute.
Relative changes in gene expression were calculated using the
.DELTA..DELTA.C.sub.t (cycle threshold) method. An average of the
number of cycles of the five housekeeping genes, GAPDH,
Actin-.beta., .beta.2m, Hprt1, and Rp113d, was used to normalize
the expression between samples. The expression data are presented
as fold regulation. GAPDH gene expression levels were utilized as
an internal control to normalize the data. Reported fold changes in
expression are the ratios of treatment over control values.
[0249] Treatment of HCC Mice with Compound 9
[0250] Balb/c Nude mice, 5-6 weeks old, were inoculated with
1.times.10.sup.7 Huh7 cells in 0.2 ml volume of Matrigel/DMEM mix.
Since not every mouse developed a tumor after the inoculation of
HCC cells, only those with visible tumors (about 8 weeks after
inoculation) were used for subsequent experiment. The tumor-bearing
mice were randomly divided to the control and the treatment group
(n=3), respectively. Mice in the treatment group had free access to
drinking water containing compound 9 (IBN-9) at 0.6 g/l or 1.5 g/l
only, while mice in the control group had free access to water
only. The compound treatment lasted for 3 weeks. The tumor size was
measured weekly, and the tumor volume was calculated using the
formula: 0.52.times.width.sup.2.times.length.
[0251] Statistical Analysis
[0252] All data are presented as mean.+-.SD. Significant
differences between the groups were determined using the unpaired
Student's t-test. Levels of significance were defined as follows:
p<0.05 (*), p<0.01 (**) and p<0.001 (***).
[0253] Results
[0254] Anti-Oxidative, Anti-Inflammatory and Anti-Fibrotic
Properties of IMSs
[0255] Determination of Toxicity of IMSs on Cultured HSC T6 Cells
and FVB Mice
[0256] Firstly we assessed the cytotoxicity of DBZIM and TDBZIM on
cultured HSC-T6 cells using a
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS) based proliferation assay kit (Promega, WI,
USA). FIGS. 1a and 1b show the effect of DBZIM (0-400 .mu.M) and
TDBZIM (0-200 .mu.M) on the proliferation of T6 cells cultured in
DMEM containing 10% of FBS. At high concentrations of 250-400
.mu.M, DBZIM inhibited cell proliferation by 3.5-8.9% with
statistical significance. At high concentrations of 125-200 .mu.M,
TDBZIM demonstrated a stronger inhibition of 13-19% as compared to
the control.
[0257] In the preliminary toxicity studies, two forms of IMSs were
selected for in vivo testing with FVB inbred mice. For DBZIM, the
lethal dosage was 50 mg/kg and LD50 was estimated to be 30-40
mg/kg. For DPIM, the lethal dosage was 500 mg/kg and LD50 was
.about.300-400 mg/kg.
[0258] DBZIM and TDBZIM Attenuated Cellular ROS Level and Enhanced
GSH/GSSG Ratio
[0259] To measure the cellular oxidative stress level of HSC T6
cells treated with IMSs, cells cultured with DBZIM (10, 50, 100 and
300 .mu.M) or TDBZIM (10, 50 and 100 .mu.M) for 48 h in full serum
medium were assayed for ROS, GSH and GSSG. Cells treated with NAC
(1 or 5 mM), and EGCG (25 .mu.M) were included as references. IMSs
treatment led to significantly attenuated cellular ROS level. As
shown in FIG. 2a, ROS was suppressed by DBZIM in a dosage-dependent
manner (by 25% at 50 .mu.M (P<0.005) and by 34% at 300 .mu.M
(P<0.0005)). TDBZIM, which is comprised of a trimer of DBZIM,
substantially suppressed cellular ROS level by 19% at 10 .mu.M
(P<0.01) and by 36% at 100 .mu.M (P<0.00001) (FIG. 2b). In
comparison, EGCG was able to attenuate ROS by 14% at 25 .mu.M
(P<0.005), whilst NAC did not show apparent inhibition on ROS
level even at 1 mM.
[0260] The effect of the compounds on the level of an important
endogenous antioxidant, GSH, was also investigated. As indicated in
FIGS. 3a and 3b, the total cellular GSH amount was quantified
including the reduced glutathione (GSH) and oxidized glutathione
(GSSG). As shown in FIG. 3a, DBZIM attenuated the total GSH level
by 7%, 12% (P<0.005) and 20% (P<0.0001) at 10, 50 and 100
.mu.M, respectively. At a high dosage of 300 .mu.M of DBZIM, slight
enhancement by 8% was observed, but without statistical
significance. NAC (5 mM) did not show any effect on the total GSH,
while EGCG (25 .mu.M) induced the total GSH synthesis by 19%
(P<0.01), which was in agreement with an earlier report (Fu et
al. 2006). In contrast to its monomer, TDBZIM did not alter the
total cellular GSH level at 10-100 .mu.M (FIG. 3b). GSH was
oxidized to GSSG in the reaction to reduce H.sub.2O.sub.2 to
H.sub.2O. The amount of GSSG was also quantified as shown in FIGS.
3c and 3d. DBZIM depleted GSSG by 13% at 10 .mu.M, 23% at 50 .mu.M
(P<0.0005), 35% at 100 .mu.M (P<0.0001) and 85% at 300 .mu.M
(P<0.0001). In contrast, EGCG reduced GSSG by 42% at 25 .mu.M
(P<0.05) and NAC had no effect on GSSG at 5 mM. TDBZIM also
suppressed GSSG production by 12% at 10 .mu.M (P<0.05), by 35%
at 50 .mu.M (P<0.05), and by 50% at 100 .mu.M (P<0.0001). The
GSH/GSSG ratio was enhanced by 8%, 15% (P<0.001) and 23%
(P<0.0001) under the influence of DBZIM at 10, 50 and 100 .mu.M,
respectively (see FIG. 3e). At a high dosage of DBZIM (300 .mu.M),
the increase in GSH/GSSG ratio was more dramatic, reaching 7.4 fold
of that of the control group. There was no change in the GSH/GSSG
ratio for NAC at 5 mM, whereas a 2-fold increase in the GSH/GSSG
ratio was observed for EGCG at 25 .mu.M (P<0.0001). For TDBZIM,
the GSH/GSSG ratio was enhanced by 14% at 10 .mu.M, by 65% at 50
.mu.M (P<0.05) and by 106% at 100 .mu.M (P<0.001) (FIG. 3f).
These results suggested that these two forms of IMSs significantly
enhanced the GSH/GSSG ratio mainly through attenuating the
production of GSSG from the oxidation of GSH, resulting in improved
anti-oxidation.
[0261] DBZIM and TDBZIM Attenuated GPx and CAT Activities, and
DBZIM Enhanced GST Activity.
[0262] To further investigate the anti-oxidant properties of the
IMSs, the activities of anti-oxidant enzymes: GPx, CAT, SOD and GST
were assayed. FIG. 4a shows that DBZIM attenuated the GPx activity
by 10% at 10 .mu.M (P<0.005) and by 10% at 50 .mu.M (P<0.01),
but enhanced the GPx activity by 15% at a high concentration of 300
.mu.M (P<0.005), with a slight biphasic pattern. For TDBZIM, GPx
activity was suppressed by 18% at 10 .mu.M (P<0.05), by 25% at
50 .mu.M (P<0.01) and by 34% at 100 .mu.M (P<0.005) (FIG.
4b). In comparison, EGCG attenuated the GPx activity by 53%
(P<0.005) at 25 .mu.M, while NAC did not show much effect at 5
mM (FIG. 4a).
[0263] A similar pattern was observed for DBZIM's effect on CAT
activity. DBZIM was able to attenuate CAT activity by 19%, 16% and
12%, respectively at 10 .mu.M, 50 .mu.M and 100 .mu.M, respectively
(P<0.05) (FIG. 4c). When the concentration was increased to 300
.mu.M, the attenuating effect decreased to 8% without statistical
significance. TDBZIM inhibited the CAT activity by 25% at 10 .mu.M
(P<0.005), 31% at 50 .mu.M (P<0.0005) and 20% at 100 .mu.M
(P<0.005), with a less potent effect above 50 .mu.M (FIG. 4d).
EGCG showed 13% suppression on CAT activity, but the data were not
significant, whereas NAC demonstrated 35% inhibition (P<0.01)
(FIG. 4c).
[0264] As shown in FIGS. 4e and 4f, DBZIM, TDBZIM, EGCG and NAC all
did not show much effect on the SOD activity, suggesting that these
compounds did not act on superoxides. FIG. 4g illustrates the GST
activity of cells under the treatment of DBZIM (10-300 .mu.M). GST
activity was enhanced by 22% at 100 .mu.M (P<0.005) and by 32%
at 300 .mu.M (P<0.0001). In contrast, EGCG and NAC did not have
much affect on the GST activity.
[0265] DBZIM and TDBZIM Protected Primary HSCs from DMSO
Cytotoxicity
[0266] DMSO is a common solvent used in cell-based assays (at
concentrations of .ltoreq.0.5% (v/v)) and cell cryopreservation (at
a concentration of .about.10% (v/v). For the primary HSCs, however,
DMSO imposed significant toxicity at a concentration of 0.2% (v/v)
(FIG. 5a). Control cells incubated with 0.2% (v/v) of DMSO died
after 26 h. In contrast, the cells survived in the co-presence of
100 .mu.M of DBZIM or 50 .mu.M of TDBZIM and 0.2% (v/v) of DMSO.
When they were cultivated for over 93 h, the cells remained viable
and proliferated to confluency in the presence of TDBZIM. However,
most cells died in the presence of DBZIM after 93 h. This suggested
that TDBZIM provided a more potent protective effect against DMSO's
toxicity on primary HSCs.
[0267] DMSO-induced oxidative stress in cultured HSC T6 cells was
quantitatively characterized by the depleted total cellular GSH
level (74% of the control, P<0.0001), the dramatically increased
GSSG amount (4.4 fold of the control, P<0.0001), the decreased
GSH/GSSG ratio (17% of the control, P<0.001), the induced GPx
(2.62 times of the control, P<0.0001) and CAT (1.7 fold of the
control, P<0.0001) anti-oxidant enzymes (see FIGS. 5b,c,d,e,'f).
DMSO also enhanced SOD activity slightly (10% increase), but the
data were not statistically significant (FIG. 5g). This finding
suggested that the protective effect of IMSs against DMSO
cytotoxicty on primary HSCs was through the attenuation of
oxidative stress. A similar observation was reported in another
study that found that quercetin, a flavonoid (0.1 .mu.M), can
effectively protect human lens epithelial cells from dying from 1%
(v/v) of DMSO, presumably through the attenuation of oxidative
stress (Cao et al. 2007).
[0268] IMSs Suppressed HSC Activation Markers and Fibrogenic
Endpoints in a Time- and Dosage-Dependent Manner
[0269] It has earlier been shown that GFAP can be used as a
biomarker along with SMAA for HSC activation in vitro (Mang et. al
2006). Deposition of ECM (including collagen and fibronectin) was
the endpoint used to assess the degree of fibrosis. Two IMSs (DBZIM
and TDBZIM) suppressed the mRNA level of both HSC activation
markers (GFAP and SMAA) and fibrotic endpoint (col1a1 and
fibronectin) in a dosage-dependent (FIGS. 6a and 6b) and
time-dependent (FIGS. 7a-d) manner. As shown in FIG. 6a, DBZIM
attenuated GFAP mRNA expression levels by 18% (P<0.05) and 86%
(P<0.001) at 10 .mu.M and 300 respectively. It attenuated SMAA
by 21% at 100 .mu.M (P<0.05) and by 58% at 300 .mu.M
(P<0.001), col1a1 by 21% at 100 .mu.M (P<0.05) and by 44% at
300 .mu.M (P<0.001), and fibronectin by 19% (P<0.005) to 40%
(P<0.0001) with dosages of 1 .mu.M to 300
[0270] The dosage dependency of TDBZIM was less prominent compared
to its monomer counterpart (FIG. 6b). TDBZIM decreased GFAP mRNA by
32% at 1 .mu.M (P<0.01), by 20% at 10 .mu.M (P<0.01) and by
60% at 100 .mu.M (P<0.0001), SMAA by 27% only at a low dosage of
1 .mu.M (P<0.01), col1a1 by 39% at 1 .mu.M (P<0.005), by 36%
at 10 .mu.M (P<0.005) and by 53% at 100 .mu.M (P<0.001), and
fibronectin by 32% at 1 .mu.M (P<0.0010), by 28% at 10 .mu.M
(P<0.0005) and by 43% at 100 .mu.M (P<0.0001).
[0271] Time course study showed that 100 .mu.M of DBZIM and TDBZIM,
respectively, suppressed GFAP by 28% (P<0.05) and 30%
(P<0.05) at 8 h, by 40% (P<0.005) and 36% (P<0.005) at 24
h, and by 56% (P<0.0001) and 60% (P<0.0001) at 48 h. They
suppressed col1a1 by 22% and 22% (P<0.01) at 8 h, by 29%
(P<0.005) and 40% (P<0.0001) at 24 h, and by 21% (P<0.05)
and 53% (P<0.001) at 48 h. They also suppressed fibronectin by
15% (P<0.05) and 25% (P<0.05) at 8 h, by 17% (P<0.05) and
29% (P<0.05) at 24 h, and by 25% (P<0.0005) and 43%
(P<0.0001) at 48 h. It was generally true that the IMSs
suppressed almost all HSC activation markers and endpoint molecules
in a time- and dosage-dependent manner, except that SMAA was
transiently induced by up to 79% by 100 .mu.M of TDBZIM at 24 h,
while no change was observed with both compounds at 8 h, and 21%
suppression was noted for DBZIM (P<0.05) at 48 h. SMAA
expression was particularly induced by TDBZIM at a high dosage.
Western blotting of GFAP, SMAA, col1a1 and fibronectin was also
performed to evaluate the influence of compounds on the protein
expression level. As shown in FIG. 8, both DBZIM (100 .mu.M) and
TDBZIM (50 .mu.M) attenuated procollagen .alpha.I(I) and
fibronectin significantly, and GFAP expression to a less degree,
when the cells were treated for 48 h. DBZIM suppressed SMAA
protein, but TDBZIM enhanced SMAA, which agreed with the real-time
PCR data. Other time points (8 h and 24 h) were also examined, but
no significant change was observed (data were not shown).
[0272] IMSs Suppressed Transcription of TGF-.beta.1 and TGF.beta.
RI
[0273] TGF-.beta.1 is the most important pro-fibrogenic cytokine.
When the liver is insulted, quiescent HSCs respond to TGF-.beta.1
secreted by Kupffer cells and endothelial cells, and start to
produce TGF-.beta.1 themselves via autocrine loops. TGF-.beta.1 is
over-expressed during HSC activation and liver fibrogenesis (Bachem
et al. 1992). The total TGF-.beta.1 synthesis under the treatment
of IMSs was measured. As shown in FIG. 9a, DBZIM suppressed the
TGF-.beta.1 mRNA by 28% (P<0.05) and 33% (P<0.005) at high
concentrations of 100 .mu.M and 300 .mu.M, respectively. TDBZIM
inhibited TGF-.beta.1 by 23% regardless of dosage (1-100 .mu.M)
with statistical significance (P<0.05) (FIG. 9b). For 8-24 h
treatment, both DBZIM and TDBZIM suppressed TGF-.beta.1
transcription by 40-50% (P<0.05) (FIG. 9c). The less apparent
dosage- or time-dependency might be due to the auto-looping
characteristic of TGF-.beta.1.
[0274] DBZIM and TDBZIM Inhibited Pro-Inflammatory and
Pro-Fibrogenic Cytokine IL-6
[0275] IL-6 is regulated by NF-.kappa.B and involved in
inflammatory responses. Being a pro-contractile and
pro-inflammatory cytokine, IL-6 plays an important role in liver
fibrogenesis. To study the influence of IMSs on IL-6, IL-6 mRNA
expression was quantified using real-time PCR. As shown in FIG.
10a, DBZIM inhibited IL-6 transcription in a dosage-dependent
manner by 37% at 1 .mu.M (P<0.0005), by 46% at 10 .mu.M
(P<0.0005), by 53% at 100 .mu.M (P<0.0001), and by 59% at 300
.mu.M (P<0.0001). TDBZIM demonstrated more potent inhibition of
37% at 1 .mu.M (P<0.01), 55% at 10 (P<0.0001) and 80% at 100
.mu.M (P<0.0001) (FIG. 10b). Time course study showed that 100
.mu.M of DBZIM significantly suppressed IL-6 mRNA expression by 53%
at 48 h (P<0.005). Shorter treatment of DBZIM did not show
statistically significant suppression on IL-6. 100 .mu.M of TDBZIM
demonstrated an inhibitory effect of 52% at 24 h (P<0.005), and
80% at 48 h (P<0.0001) (FIG. 10c).
[0276] DBZIM and TDBZIM Mediated Through NF-.kappa.B and AP-1
Transcription Factors
[0277] Transcription factors NF-.kappa.B and AP-1 are known to be
sensitive to cellular redox states. NF-.kappa.B also plays an
essential role in physiological conditions such as inflammation and
tissue repair. Protein expression of the NF-.kappa.B P65 and
activation protein 1 (AP-1) family members (c-Fos, c-Jun, Jun B/D,
Fra-1/2) were examined when the cells were treated with IMSs. As
shown in FIG. 11, nuclear NF-.kappa.B P65 in an active form was
significantly attenuated by both DBZIM and TDBZIM. The amount of
latent NF-.kappa.B in cytosol was not affected (data not shown).
AP-1, c-Fos, Jun D and Fra-1 were suppressed, while c-Jun, Jun B
and Fra-2 were less affected.
[0278] IMSs Regulated the Expression of a HSC-Specific
GFAP-Reporter Surrogate
[0279] It has been shown earlier that the GFAP-lacZ transgene
reporter could be used to screen anti-fibrotic compounds (Maubach
et. al 2006, Zhang et. al 2006). In the current experiment, the
.beta.-galactosidase activity under the treatment of DBZIM and
TDBZIM at different dosages (0-300 .mu.M) and time points (24 h and
48 h) was assayed. Significant suppression on .beta.-galactosidase
activity was observed for DBZIM: by 25% at 100 .mu.M (P<0.001)
and by 34% at 300 .mu.M (P<0.001) (FIG. 12 (top panel)). For
TDBZIM, a smaller and transient suppression (20%) was observed at
100 .mu.M at 24 h (FIG. 12 (bottom panel)).
[0280] Safety and Efficacy of Additional IMSs with Different
Substituents
[0281] IMSs of different structures were also tested in a
preliminary structure-activity-relationship (SAR) study. Testing
was performed on 3-Bisbenzylimidazolium bromide (DBZIM),
1,3,5-tris(4-methyl-imidazolium)-linked cyclophane.3Br (TDBZIM),
1,3-Diisopropylimidazolium tetrafluoroborate (DPIM),
1,3-Di-tert-butylimidazoliniumtetrafluoroborate (DBIM),
1,3-Bis(1-adamantyl)imidazolium tetrafluoroborate (AMIM),
1,3-Bis(2,4,6-trimethylphenyl)-imidazolinium chloride (TMPHIM),
1,3-Bis(2,6-diisopropyl-phenyl)-imidazolinium chloride (DPPHIM) and
1,3-Bisbenzyl-benzimida-zolium bromide (DBZBIM). In general, those
with smaller aliphatic groups, such as 1,3-diisopropylimidazolinium
tetrafluoroborate (DPIM) and 1,3-di-tert-butylimidazolium
tetrafluoroborate (DBIM), showed less toxicity on T6 cells,
compared to those with bulky aromatic groups, such as
1,3-bis(2,4,6-trimethylphenyl)imidazolium chloride (TMPHIM) and
1,3-bis(2,6-diisopropylphenyl)imidazolium chloride (DPPHIM). Those
with a methyl spacer between the imidazolium and aromatic ring,
such as 1,3-bisbenzylimidazolium bromide (DBZIM) and
1,3-bisbenzylbenzimidazolium bromide (DBZBIM), showed moderate
toxicity. The toxicity of compounds was ranked based on IC50 as
follows: DPIM (IC50=3.1 mM)<DBIM (IC50 was undetermined due to
limited solubility in DMSO, but estimated to be 2-3 mM)<DBZIM
(IC50=1.7 mM)<TDBZIM (IC50.about.500 .mu.M)<DBZBIM (IC50=310
.mu.M)<AMIM (IC50=166 .mu.M)<TMPHIM (IC50=110
.mu.M)<DPPHIM (IC50=34 .mu.M).
[0282] Efficacy of compounds in suppressing fibrosis was also
evaluated. HSC T6 cells were treated with IMSs at concentrations
that were below their respective IC50. Compounds were either
dissolved in DMSO or H.sub.2O depending on their solubility, and
the intensities of the bands were compared to the respective
control. As shown in FIG. 13A, proteins of SMAA, Col1a1,
fibronectin, TGF-.beta.1 and TGF.beta. RI were all suppressed with
the treatment of IMSs. Semiquantitative data from measuring the
gray scale of the band pixels is summarized in FIG. 13B. If all 5
parameters are assigned with equal importance, the efficacy of IMSs
could be roughly ranked as
DPIM>TMPHIM>AMIM>DBIM>DBZBIM>DBZIM>DPPHIM>TDBZIM.
[0283] GFAP, SMAA, fibronectin and col1a1 transcripts were also
quantified by real-time PCR. As shown in FIGS. 14a,b,c,d, GFAP mRNA
was suppressed by 2 mM of DPIM by 78% (P<0.0001), by 50 .mu.M of
AMIM by 69% (P<0.0005), by 50 .mu.M of TMPHIM by 66%
(P<0.001), and by 100 .mu.M of DBZBIM by 50% (P<0.005). SMAA
mRNA was suppressed by 1 mM of DBIM by 49% (P<0.05), by 2 mM of
DPIM by 78% (P<0.0001), by 50 .mu.M of AMIM by 23% (P<0.05),
and by 50 .mu.M of TMPHIM by 49% (P<0.05). Fibronectin mRNA was
suppressed by DPIM by 48% (P<0.05), by TMPHIM by 29%
(P<0.005), and by DBZIM by 26% (P<0.005). Col1a1 mRNA was
suppressed by DBIM by 58% (P<0.0005), by DPIM by 61%
(P<0.0001), by TMPHIM by 62% (P<0.0001), and by DPPHIM by 47%
(P<0.05). EGCG did not show significant inhibition on HSC
activation and fibrosis. This might due to the chemical instability
of EGCG under the prolonged incubation of 48 h. It was reported
that the half life of EGCG was only 30 min in the culture medium
for human esophageal cancer cells (Hou et al. 2005) and .about.72
min in rat blood at a dosage of 30 mg/kg (Lin et al. 2004). Among
all the IMSs, DPIM and TMPHIM showed significant suppression on all
four genes, and DPIM was the most potent agent under the test
conditions.
[0284] Effect IMSs on Liver Fibrosis in Mice
[0285] DBZIM and DPIM were tested in two liver fibrosis models in
mice induced by liver toxin thioacetamide (TAA) or by a surgical
procedure called bile duct ligation (BDL), respectively. As shown
in FIG. 15, the two IMSs, when administered in drinking water,
showed anti-fibrotic properties in the two mouse models of liver
fibrosis. Particularly DBZIM attenuated fibrosis in the TAA model
(at 500 mg/L, 12 weeks) (FIG. 15a) and the BDL model (at 10 mg/L, 4
weeks) (FIG. 15b), whereas the DPIM displayed its effect in the BDL
model at 1 g/L (4 wks) (FIG. 15c). This data suggests that IMSs
have potential as anti-liver fibrosis therapeutic compounds.
[0286] DPIM Attenuated Liver Fibrosis
[0287] DPIM was chosen for further in vivo study, To ensure
proficiency in BDL procedure, a number of exercises and tests were
performed to eventually achieve over 90% success rate on the BDL
surgery and over 4 weeks of survival time for the operated mice.
The extent of fibrosis was assessed by the Sirius-Red collagen
staining at 4 weeks post-BDL. An extensive collagen accumulation
around the sinusoidal areas was observed in the BDL group (FIG.
16C), whereas only minimal collagen staining was seen in the sham
operated group (FIG. 16A). Treatment of sham mice with 1 g/l of
DPIM for 4 weeks did not induce any significant build-up of
collagen (FIG. 16B). Four weeks after compound treatment, the
collagen contents were dramatically reduced by 750 mg/l (FIG. 16E)
and 1 g/l (FIG. 16F) of DPIM. The effect by the 500 mg/l of DPIM
was not conclusive, since only one mouse was treated with this dose
at the time of data collection (FIG. 16D). Quantitatively the
Sirius-Red positive areas were reduced from 8.69 to 4.33 by the
dose of 750 mg/l, and even more (and dose-dependently) down to 3.42
by the dose of 1 g/l (FIG. 17).
[0288] DPIM Reduced Fibrotic Cellular Proliferation
[0289] Liver weight increases in BDL-mice mainly due to 1) the
build-up of bile, and 2) fibrotic cellular proliferation. The
livers of different treated groups were weighed, 4 weeks after BDL,
liver weight was increased by 83% compared with sham operated mice
(1.50.+-.0.19 g for sham operated, vs. 2.74.+-.0.51 g for BDL 4
weeks, P<0.05). However, DPIM compound treatments showed a
significant liver weight reduction (P<0.05, DPIM compound
treatment vs. BDL 4 weeks): 1.97.+-.0.44 g for the 750 mg/l
treatment group, 1.65.+-.0.20 g for the 1 g/l treatment group (FIG.
18). This dose-dependant effect was consistent with Sirius red
staining areas. Compared with sham operated mice, DPIM treatments
in sham operated mice for 4 weeks did not result in significant
liver weight change (1.56.+-.0.08 g for DPIM treated vs.
1.50.+-.0.19 g for sham operated, P=0.46)(FIG. 18).
[0290] H&E staining of the liver sections (FIG. 19) showed that
DPIM treatment (FIG. 19C) reduced the number of proliferative cells
(identified by the nuclear staining of Hamehexlin) around the
sinusoidal area, when compared to the DBL group (FIG. 19B) and
control sham group (FIG. 19A). At the messenger level, DPIM also
reduced the collagen 1a1 mRNA (FIG. 20).
[0291] Effect of DBZIM on Hepatocellular Carcinoma and Other Tumour
Cells
[0292] DBZIM Inhibited Cell Proliferation and Disrupted Cell Cycle
in HLE Cells
[0293] The effect of DBZIM on the HCC cell proliferation and cell
cycle was characterized on a high-content screening (HCS) platform.
Under each concentration, at lease 1000 cells were analyzed.
5-bromo-2'-deoxyuridine (BrdU), which incorporates into replicating
DNA, is used to identify cells which have progressed through S
phase of cell cycle. DAPI staining was used for nuclear
identification and DNA content determination. DBZIM inhibited the
HLE cell proliferation as indicated by less total cell number and
less BrdU positive cells in a dose-dependant manner (FIG. 21A), and
arrested HLE cells in the G0/G1 phase (FIG. 21B).
[0294] DBZIM Induced Cell Death Through Apoptosis in HCC Cells
[0295] Cell-based caspase 3/7 activity and lactate dehydrogenase
(LDH) release assay allow the differentiation of apoptosis and
necrosis of cells under the influence of DBZIM. As shown in FIG.
22A-D, caspase 3/7 activity (indicative of apoptosis) in both HLE
and HepG2 cells was enhanced by DBZIM, while LDH release
(indicative of cytotoxicity) was least affected under the same
treatment regimen. It suggested that DBZIM used at the current
treatment condition caused the HCC cell death mainly by apoptosis,
rather than necrosis.
[0296] Apoptosis was further confirmed by annexin V staining. In
the initial phase of apoptosis, cells translocated the membrane
phosphatidylserine (PS) from the inner face of the plasma membrane
to the cell surface. The surface PS is readily detected by
fluorescent conjugate of annexin V, which has a high affinity for
PS and often used as an apoptosis marker. As shown in the scatter
plots in FIG. 22E-F, DBZIM dose-dependently induced apoptosis in
both HepG2 and HLE cells. It was noted that as a positive control,
the TRAIL (TNF-Related Apoptosis Inducing Ligand) only induced
apoptosis in HepG2, but not in HLE cells.
[0297] DBZIM Triggered Mitochondria-Mediated Apoptosis Pathway
[0298] In order to understand the apoptosis pathway involved by
DBZIM, caspase 3, 8, 9 activities were assayed using colorimetric
assays. As shown in FIG. 23A, caspase 8 activity was not affected
by DBZIM, whilst caspase 9 and 3 activities were dose-dependently
enhanced by DBZIM, pointing a mitochondria-mediated apoptosis
pathway. However, DBZIM was found to cause a minimal release of
cytochrome C from the mitochondria to the cytosol (FIG. 23B).
[0299] The mitochondria-mediated intrinsic apoptosis pathway is
mainly regulated by the Bcl-2 and the IAP (Inhibitor of Apoptosis
Protein) families. As shown with Western blotting in FIG. 24, the
anti-apoptotic protein Bcl-XL and the pro-apoptotic protein Bak
were slightly down-regulated and up-regulated by DBZIM
respectively, whereas the other two members of the Bcl-2 family,
Bcl-2a and Bax, were not affected by the treatment. Notably in the
IAP family, survivin was significantly suppressed by DBZIM, whereas
xIAP and cIAP were not affected (FIG. 24). In addition, HCS
analysis revealed that survivin was translocated from the nucleus
to the cytoplasm under the influence of DBZIM (FIG. 25).
[0300] DBZIM Induced Cytoplasm-to-Nucleus Translocation of AIF
[0301] Apoptosis-inducing factor (AIF) is normally localized in the
mitochondria, and translocated to the nucleus upon induction of
apoptosis. It induces mitochondria to release caspase 9. In
nucleus, AIF also induces chromatin condensation and DNA
fragmentation, a process known as caspase independent apoptosis
pathway. DBZIM was found to induce the translocation of AIF to the
nucleus by Western blotting (FIG. 26A). Immunocytostaining showed
that AIF was translocated from the mitochondria/cytoplasm to the
nucleus (FIG. 26B). It is worth noting that under DBZIM treatment
the nuclear AIF (arrows) tends to be associated with the smaller
condensed nuclei which are presumably going through the apoptosis.
Quantitative analysis with HCS suggested that the DBZIM-induced
translocation is dose-dependent (FIG. 26C).
[0302] DBZIM Induced Apoptosis Through Inducing ROS and AP-1
[0303] Reactive oxygen species (ROS) not only directly modifies
cellular macromolecules, it also actively participates in various
cellular signaling. An elevated level of ROS has been reported to
trigger the programmed cell death by apoptosis. When treated with
DBZIM at the millimolar range, HLE cells generated significant
amount of ROS and coincidently underwent mass cell death (FIG.
27).
[0304] A sustained and elevated level of transcription factor
activation protein 1 (AP-1) complex is known to participate in
apoptosis. Western blotting (FIG. 28) of nuclear extracts from HLE
cells showed that DBZIM significantly induced several key members
of the AP-1 complex, including C-Jun, Jun B, Jun D, and Fra-1, but
not Fra-2.
[0305] DBZIM Induced Apoptosis and Reduced pAKT in Several Other
Tumor Cell Lines
[0306] To investigate whether the effect of DBZIM is liver cell
specific, tumor cells of lung, breast and gastric cancer cells were
treated with DBZIM, and it was found that the same agent caused
significant cell death in all the tumor cell lines tested. The IC50
value (determined at day 3) was 50 .mu.M for H1299 (lung cancer),
50 .mu.M for MCF-7 (breast cancer), and 40 .mu.M for AGS gastric
cancer at day 3 (Table 3). Interestingly, DBZIM showed much less
cytotoxicity on the normal (non-cancerous) lung cells (IMR90).
Further, treatment of AGS and H1299 with DBZIM resulted in
morphological (apoptotic look) changes and significant reduction in
the number of cells (FIGS. 29A and 29B). It was also found that
DBZIM significantly reduced the growth of two glioma cell lines,
U87MG (human) and C6 (rat) (FIGS. 30A and 30C), with IC50
(determined at day 2) for U87 MG and C6 being 2.1 mM and 1.0 mM,
respectively.
[0307] To determine whether cells undergo apoptosis, Western
blotting was first performed for a caspase-3/9 and PARP activation
with proteins from MCF-7 and AGS treated with DBZIM. Results showed
that DBZIM time-dependently increased caspase-3/9 and PARP
activity, suggesting that the DBZIM-treated cells undergo apoptosis
in breast cancer cell line MCF-7 (FIG. 31A) and in wild-type p53
gastric cancer cell AGS (FIG. 31B). Similarly we observed a
DBZIM-induced elevation of caspase 3/7 in C.sub.6 (by 3 folds at
2.0 mM) and in U87 MG (by 17 folds at 2 mM) (FIGS. 30B and
30D).
[0308] The Akt (also known as protein kinase B) kinase signaling
pathway is activated in a variety of carcinomas of lung, breast,
colon, and pancreatic and other tumor types through over-expressing
the Akt protein itself and/or increasing phosphorylation of Akt.
This study provided evidence that DBZIM is particularly efficacious
in inducing apoptosis in AGS, H1299 and MCF-7 cells through a
mechanism involving the reduction in the levels of the,
phospho-AKT. Next it was investigated whether DBZIM modulated the
Akt pathway in H1299, MCF-7, AGS and NKN28, DBZIM treatment
resulted in an appreciable down-regulation of the active form of
Akt (phospho-Ser473-Akt) from 24 h to 72 h, without any changes in
.beta.-actin in all four cancer cell lines tested (FIG. 32). Taken
together, these data suggest that DBZIM is a potent suppressor of
AKT-mediated signaling and is independent of p53 status.
[0309] DBZIM Reduced Tumor Growth in a Xenograft HCC Mouse
Model
[0310] Several HCC cell lines (HepG2, Hep3B, Huh7, PLC, and HLE)
were tested for their ability to efficiently induce HCC in nude
mice, and it was found that Huh7 line was among the most efficient.
Therefore a murine HCC model was established using the Huh7 cells
for testing the anti-tumor efficacy of DBZIM in vivo. Three weeks
into the treatment, difference in tumor size was apparent between
mice in the control and the treatment group (FIG. 33A). Measurement
showed that the tumor volume in the DBZIM-treated mice was
significantly reduced by 40% (P<0.01) when compared to the
control mice (FIG. 33B). It was noted that the body weight
(including a reduction in the tumor weight) of the DBZIM-treated
mice decreased by 17% (P<0.01) after three weeks of treatment
(FIG. 33C).
[0311] Anticancer Activities of Additional IMSs
[0312] Determination of IC50 for 46 IMS in HLE Cells
[0313] A total of 46 IMSs were cultured in the HLE cell line for 48
h to determine rough IC50 values for individual compounds. (Table
2) After the initial screening, seven of the IMSs, compound C,
compound 9, IBN-15, IBN-19, IBN-24, IBN-25 and IBN-32, were used
for subsequent experiments.
[0314] Endogenous Expression of p53
[0315] The expression level of p53 in Hep 3B (p53null), HepG2 (p53
wild type), and HLE and PLC (p53 Mutant) hepatocarcinoma cells was
determined by Western Blot. (FIG. 34). The expression of p53 was
high in cells bearing mutant p53, as compared to cells with the
wild type p53.
[0316] IMSs Inhibited Proliferation of HLE Cells
[0317] The effect of IMSs (IBN-15, IBN-19, IBN-24, IBN-25 and
IBN-32) on the proliferative properties of p53 mutant cell line HLE
was examined. The cytotoxicity activity and proliferation
inhibition of IMSs in tumor cells were measured by MTT. The
treatment of HLE liver cancer cells with IMSs resulted in a dose
dependent inhibition of cell proliferation. The control experiments
with DMSO alone (0.05% v/v) had no effect on cell proliferation.
The IC50 value for IBN-15, IBN-19, IBN-24, IBN-25, and IBN-32 in
HLE cells was estimated to be 60 .mu.m, 90 .mu.m, 90 .mu.m, 30
.mu.m and 20 .mu.m at 72 h. While the IC50 value for IBN-15,
IBN-19, IBN-24, IBN-25 and IBN-32 in HLE cells at 120 h was found
to be 20 .mu.m, 28 .mu.m, 30 .mu.m, 35 .mu.m, and 20 .mu.m (Table
4) Microscopic observation on the cell cultures visually confirmed
the inhibition on cell proliferation by IMSs. (FIG. 35)
[0318] IMSs Induced Apoptosis
[0319] As the data indicated that IMSs significantly inhibited cell
growth in HLE cells, Annexin V-PI dual-staining assay was performed
in the same cell type under the same conditions to investigate
whether the growth inhibition resulted from cell apoptosis. Cells
undergoing apoptosis would stain positive for Annexin V-FITC and
negative for PI (quadrant 4, Q4). Cells which stained positive for
both Annexin V-FITC and PI (Q2) are either at the end stage of
apoptosis or undergoing necrosis, and those which stained negative
for both Annexin V-FITC and PI (Q3) were alive or undergoing
undetectable apoptosis. In contrast, cell debris stained only for
PI (Q1). As shown in FIGS. 36 A and B, IMSs IBN-15, IBN-19, IBN-24,
IBN-25, and IBN-32 induced 33%, 29%, 29%, 21%, and 18% (Q2) cell
apoptosis, respectively, whereas fewer apoptotic cells (7.2%) were
observed for DMSO treated cells, confirming that the IMS-induced
cell growth inhibition has a significant apoptotic component
[0320] IMSs Changed Cell Cycle Distribution
[0321] To analyze whether the IMSs-induced inhibition of cell
growth in HLE cells was accompanied by alterations in cell cycle
distribution, the percentage of cells in the different phases of
cell cycle and apoptotic index were analysed by flow cytometry.
When cells were treated with IBN-15, IBN-19, IBN-24, IBN-25, and
IBN-32 at a concentration corresponding to their respective IC-50
value (i.e., 60 .mu.m, 90 .mu.m, 90 .mu.m, 30 .mu.m, and 20 .mu.m)
for 72 h, a significant accumulation of cells in the sub-G0 phase
and a corresponding reduction in the number of cells in the G2/M
phase was found. The representative results are shown in FIGS. 37A,
37B and 37C). Two percent of the cells in the control culture (with
DMSO) were in sub-G0 phase, whereas the numbers dramatically
climbed up to 20% in the IMS-treated cultures. Similarly in control
culture, 50% cells were in the G1 phase, 24% were in the S phase,
and 30% were in the G2/M phase at 72 h, whereas in the IMS-treated
cultures (with IBN-15, IBN-19, IBN-24, IBN-25, and IBN-32), 42%,
29%, 28.6%, 34.5%, and 32.3% of the cells were in the G1 phase; and
19.7%, 19.8%, 11.34%, 14.3%, and 14.6% in the S phase; and 21.%,
23.7%, 16.3%, 19.4%, and 18.9% in the G2/M phase, respectively.
[0322] IMSs Induced Apoptosis Via Caspase Activation
[0323] To further determine whether the apoptotic activity of IMSs
is due to caspase activation, Western blot analysis was performed
with total protein lysates from HLE cells. It was observed that IBN
19, 24 and 25 induced significant activation and cleavage of
caspase 3 and caspase 9 and PARP (FIG. 38), suggesting that the
compounds induce cells to undergo apoptosis.
[0324] IMSs Increased the Expression of p53 and Phosphorylated p53
(Ser15, 20, 46 and Ser 392)
[0325] In order to determine whether the activation of the p53
pathway was involved in IMS-induced apoptosis, analyses of p53 and
its down stream effectors were performed in HLE cells, which carry
the mutant p53. Immunocytochemical analyses revealed that IMSs
(IBN-19 and IBN-24) induced p53 expression and accumulation in the
nucleus (FIG. 39).
[0326] Because phosphorylation at the Ser 15 site of p53 by ATM is
often observed when cells receive DNA damage signals. It was
speculated that ATM may also respond to IMSs in the HLE cells. Tt
has been reported that chemical agents that damage DNA act through
posttranslational modifications of p53 and activate its downstream
targets in various human cancer cells (Banin et al. 1998; Canman et
al. 1998). The present results also demonstrated that these IMSs
induced p53 modifications, such as phosphorylation at Ser-15 (FIG.
40) due to up-regulation of ataxia telangiectasia-mutated (ATM)
kinase gene. Furthermore, IMSs induced phosphorylation at position
at Ser-20, -46 and -392. These posttranslational modifications of
p53 appear to be responsible for the cell cycle arrest (FIG.
40).
[0327] IMSs Induced the Execution of Apoptosis Through Activation
of the Mitochondrial Pathway
[0328] To investigate the mitochondrial events involved in
IMSs-induced apoptosis, the changes in the levels of pro-apoptotic
protein Bax, and anti-apoptotic protein Bcl-2 were analyzed.
Immunoblot analysis showed that treatment of HLE cells with various
IMSs increased Bax protein level and simultaneously decreased Bcl-2
level (FIG. 41).
[0329] Real-Time RT-PCR Profiling of Gene Expression in IMS-Treated
Cells
[0330] To obtain more information on how the IMSs influence other
gene expression in the HLE cells, 84 genes related to p53 signal
transduction were profiled by using the RT.sup.2 Profiler array
(Super Array Bioscience) (FIG. 42A, B, C, D, E). The array includes
keys genes relevant to p53, apoptosis, cell cycle, cell growth,
proliferation, differentiation, and DNA repair. It was found that
ATM gene expression at 48 h after IBN-19, IBN24 and IBN-25
treatment was significantly increased by 36-, 4.6- and 9-folds
respectively, when compared to that of the control (FIG. 42E).
These data show that IMSs induce transcriptional up-regulation of
ATM gene, which in term elicits a specific p53 phosphorylation at
Ser-15 in the HLE cells.
[0331] The results demonstrated that IBN-24 and IBN-25
significantly down regulated anti-apoptotic genes BCl-2 (FIG. 42A).
The results from the quantitative RT-PCR analysis also indicated
that SESN2 gene was markedly induced by IBN-19, as compared to
untreated (FIG. 42B). Further, the results also demonstrated that
cell proliferation genes Mcl1, Egr1, Foxo3, Jun, Mdm4, Nf1, Prm1D,
Sesn1, E2f11, Prkca and, Brca1 were significantly down regulated in
the IBN-24 and IBN-25 treated cells as compared to control cells.
(FIG. 42 D) The important cell cycle regulatory genes cdk4, cdc25A,
cdc2, e2f1, and Hk2 were significantly downregulated. (FIG.
42C)
[0332] Compound C (DBZMIM) and Compound 9 (MABZIM) Inhibit
Proliferation of Gastric and Breast Cancer Cells
[0333] In this study, the anti-cancer effects of compound C and
compound 9 on p53 mutant gastric cancer cell line MKN 28 and p53
mutant breast cancer cell line MDAMB231, as well as on wild type
p53 breast epithelial cells (MCF-10A) was preliminarily explored.
First, the cytotoxic effect of these drugs on these cells was
investigated using the MTT assay. Cells were seeded into 96 well
plates and treated with increasing concentrations of these
synthetic organic molecules for 72 h, and then the cytotoxic effect
was measured. In response to these synthetic organic molecules, the
normal MCF-10A cells showed resistance to both organic molecules
with no IC.sub.50 (concentration of the drug that leads to 50%
lethality) (Table 7). Both the cancer cell lines MKN 28 and
MDAMB231 were the most sensitive towards compound 9, with IC-50
being 10 and 20 .mu.M (Tables 5 and 6). On the other hand, compound
C killed both MKN 28 and MDAMB231 with the same IC50 of 35 .mu.M
(Tables 5 and 6). These IC50 values were similar to those obtained
with a commonly used cancer drug 5-Flurouracil (5-FU). Morphology
of the cells treated with compound C and compound 9 respectively
was shown in FIGS. 43A and 43B.
[0334] Compound 9 Inhibited Proliferation of HCC Huh7 Cells in
Mice
[0335] Several HCC cell lines (HepG2, Hep3B, Huh7, PLC, and HLE)
were tested for their ability to efficiently establish HCC in nude
mice, and found that Huh7 line was among the most efficient.
Therefore a murine HCC model was established using the Huh7 line
and one of the IMSs (compound 9) was tested to assess its
anti-tumor efficacy. Three weeks into the treatment, significant
difference in tumor size was apparent between mice in the control
and the treatment group (FIG. 44A). Measurement showed that the
tumor volume in the C9-treated mice was significantly reduced by
43% and 62% (P<0.01) at 0.6 g/l and 1.5 g/l, respectively, when
compared to the control mice (FIG. 44B). No change in body weight
or gross pathology was noted for the C9-treated mice, when compared
to the control mice (FIG. 44C), implying lack of significant
general toxicity associated with the C9 under the current dosing
scheme.
Discussion
[0336] Anti-Oxidative, Anti-Inflammatory and Anti-Fibrotic
Properties of IMSs
[0337] Toxicity of IMSs
[0338] The IC50 of the most frequently tested compound in this
study, DBZIM, was 1.7 mM, and the IC50 values for DPIM and DBIM
with aliphatic substitutions were 2-4 mM. The in vivo toxicity for
DPIM and DBIM was roughly one-tenth of that for DBZIM. In contrast,
the IC50 values for natural antioxidants EGCG and genistein in HSCs
were much lower (25-75 .mu.M) (Kang et al. 2001, Chen et. al 2002,
Chen et. al 2003, Higashi et al. 2005, Zhang et al. 2006).
Intriguingly, preliminary in vitro and in vivo data seemed to
suggest, in contrast to common belief, that the synthetic IMSs
might have a better safety profile than the natural anti-oxidants.
The mild toxicity of this class of compounds certainly helped to
put forth the prospect of using IMSs as the basic units to
synthesize additional candidates with novel properties and
functionalities tailored
to various specific therapeutic needs.
[0339] IMSs Demonstrated Anti-Oxidative Property Through
Neutralization of ROS and Induction of Phase II Anti-Oxidative
Enzyme
[0340] Cells or tissues maintain a redox hemeostasis when the rate
of ROS production and scavenging capacity are essentially in
balance. Changes in oxidant/anti-oxidant balance will trigger
responsive redox signaling. It has been reported that oxidative
stimuli can induce expression of anti-oxidative defense enzymes
such as SODs, CAT and GPx, etc., to restore the original redox
homeostasis or to reach a new equilibrium (Droge 2002). Redox
regulation is also partly mediated through the cellular GSH and the
GSH/GSSG redox state. GSH plays a key role in cellular
anti-oxidative processes by serving as an electron donor to GPx in
the reduction of hydrogen peroxide to water, and as a nucleophilic
co-substrate to GST in the detoxification of xenobiotics.
Anti-oxidants exert their effect mainly through three different
pathways: (1) neutralization of cellular ROS generated during
metabolism and immune response, (2) induction of endogenous
anti-oxidative enzymatic activity, and (3) chelation of iron or
copper ions that catalyze the generation of hydroxyl radical. GPx,
CAT and SOD, which constitute the first line of cellular
anti-oxidative defense, are directly involved in the neutralization
of ROS. GPx enzyme reduces H.sub.2O.sub.2 to H.sub.2O, while GSH
functions as a cofactor and is consequently oxidized to GSSG. CAT
is a peroxisomal enzyme and converts H.sub.2O.sub.2 to H.sub.2O.
SOD catalyzes the dismutation of superoxide. There are three forms
of SOD: extracellular and intracellular copper/zinc (Cu/Zn) SODs,
and a mitochondrial manganese (Mn) SOD. GST is a phase II
anti-oxidant enzyme. It has been reported that activation of HSCs
is associated with the loss of GST activity in culture (Whalen et.
al 1990).
[0341] In this study, IMSs effectively attenuated ROS (in
particular H.sub.2O.sub.2 and lipid peroxides, which react to
2',7'-dichloro-fluorescein diacetate (DCF-DA) probe (Halliwell et
al. 2004) in a dosage-dependent manner. While the level of GSH
remained relatively constant, the amount of GSSG was dramatically
decreased, resulting in a significant increase in the GSH/GSSG
ratio. Moreover, the enzymatic activities of GPx and CAT, which are
responsible for reducing hydrogen peroxide to water, were slightly
attenuated. Taking all the data together, it is proposed that IMSs
might have mainly exerted their anti-oxidant effect through the
direct neutralization of ROS, such as hydrogen peroxide. Therefore,
the burden on cellular anti-oxidative defense was alleviated by the
exogenous synthetic anti-oxidative IMSs, resulting in a decrease in
the expression of first-tier anti-oxidative enzymes. In the
meantime, the effect of IMSs on GSH synthesis and anti-oxidative
enzymes showed a general pattern of attenuation at low dosages,
less potent attenuation and even enhancement at high dosages. This
suggested that DBZIM worked through different mechanisms at low and
high dosages. At a low dosage, the anti-oxidative pathway
predominated by direct reaction to remove H.sub.2O.sub.2 so that
GPx and CAT activities were reduced. In contrast, there might be a
synergistic effect between direct neutralization of H.sub.2O.sub.2
and induction of anti-oxidant enzymes at a high dosage. It was
reported that a reduction in the battery of anti-oxidative enzymes
GPx, CAT and SOD as observed in the experimental cholestasis can be
reversed by the endogenous anti-oxidant melatonin (Padillo et al.
2004). Interferon-alpha has been shown to increase GPx activity in
activated HSCs (Lu et al. 2002). On the other hand, it has been
shown that oxidative stimuli could induce anti-oxidative enzymatic
activities (Droge 2002). One recent paper also showed that a high
dosage of NAC (30 mM) shifted cellular redox state towards
oxidative stress, as indicated by enhanced reduced glutathione,
oxidized glutathione, GPx activity and ROS production in
endothelial cells. In this study, high-dosage IMSs showed a trend
of enhanced GPx and CAT activities. This result, along with other
findings that high-dosage IMSs imposed greater effect in
attenuating ROS production (FIGS. 2a and 2b) and GSSG (FIGS. 3c and
3d), and inducing GSH/GSSG ratio (FIGS. 3e and 3f), suggested that
IMSs at high dosage enhanced cellular defense activity against
oxidative stress by synergizing neutralization of ROS and induction
of endogenous anti-oxidant enzymes. In addition, IMSs had little
effect on the total SOD (Gu/Zn-SOD and Mn-SOD) activity, suggesting
that the compounds did not react with superoxide and have little
influence on the cellular response to remove superoxide.
[0342] Meanwhile, phase II enzymes such as total GST were enhanced
in activity under the treatment of IMSs. It was known that GSTs
protect the cells against oxidative toxicants by conjugating the
xenobiotics to GSH, thereby neutralizing their electrophilic sites
and rendering the product water-soluble. The enhancement of GST
activity indicated that the cells have better capability of getting
rid of oxidants such as lipid peroxidation products, thereby
minimizing the cellular oxidative injuries.
[0343] The anti-oxidative property of IMSs was also demonstrated in
primary HSC cells. DMSO induced cellular oxidative stress, and
imposed lethal effect on primary HSC cells. The presence of IMSs
effectively protected the primary HSC cells from dying in the
culture medium containing 0.2% (v/v) of DMSO. This finding implies
a potential application of this group of compounds in cell-based
drug screening assays and tissue engineering.
[0344] IMSs Showed Anti-Inflammatory Effect Through Suppression of
NF-.kappa.B and IL-6
[0345] Transcription factor NF-.kappa.B is found in the cytosol
bound to inhibitory protein known as I.kappa.B. When the cells are
under various stress conditions, such as infection, inflammation
and tissue repair, NF-.kappa.B is activated and translocated into
the nucleus where it binds to DNA and regulates the transcription
of gene-encoding proteins involved in immune or inflammatory
responses (Vasiliou et al. 2000, Ali et al. 2004). The activation
of NF-.kappa.B is associated with phosphorylation and subsequent
degradation of I.kappa.B. The NF-.kappa.B-responsive site has been
identified and characterized in the promoters and enhancers of many
genes, including IL-6 and ICAM-1 (Ali et al. 2004). NF-.kappa.B is
also known to be sensitive to oxidative stress. Most agents
activating NF-.kappa.B are either modulated by ROS or oxidant
themselves. It has been reported that the treatment of anti-oxidant
resveratrol (Chavez et al. 2007) or vitamin E (Liu et al. 1995)
attenuated NF-.kappa.B elevation induced in carbon tetrachloride
experimental fibrotic rodents.
[0346] To test whether IMSs are able to suppress NF-.kappa.B
activation and in turn regulate immune and inflammatory responses,
the protein expression of active NF-.kappa.B was assayed by Western
blotting and NF-.kappa.B-responsive IL-6 gene transcription. Our
data showed that IMSs could effectively interrupt the activation of
NF-.kappa.B, and as a result, down-regulate the gene expression of
IL-6 (FIGS. 10a-c and 11). These data indicated that IMSs have
anti-inflammatory effect by down-regulating IL-6 transcription
through NF-.kappa.B signaling.
[0347] IMSs Showed Anti-Fibrotic Properties Through Suppression of
HSC Activation
[0348] HSCs are well-recognized cellular regulators in the
development of hepatic fibrosis. HSC activation, associated with
enhanced secretion of pro-fibrogenic, pro-inflammatory cytokines
(such as TGF-.beta.1 and IL-6) is characterized by the
over-expression of SMAA, and results in the over-deposition of ECM
proteins including col1a1 and fibronectin. The anti-fibrotic
effects of IMSs were evident by the suppression of HSC activation
markers (GFAP and SMAA) and fibrotic endpoints (col1a1 and
fibronectin) in a dosage- and time-dependent manner. A transiently
induced SMAA expression was observed under the treatment of
high-dosage TDBZIM (FIG. 7b). The underlying mechanism was not
clear, but might be related to the radical-like nature of NHC with
bulky N-substituents (Bourissou et al. 2000). SMAA might have a
transient over-response to excess radicals. Nevertheless, IMSs of
moderate concentrations were generally able to attenuate SMAA
expression (FIGS. 6a, 6b and 14b). IMSs also consistently
suppressed the mRNA and protein expression of col1a1 and
fibronectin (FIGS. 6a-b, 7c-d, 13 and 14c-d), which as a net effect
prevented or slowed down the progression of fibrosis.
[0349] TGF-.beta.1 is the most potent pro-fibrogenic cytokine,
which is over-expressed in activated HSCs. TGF-.beta.1 signaling is
initiated by the binding of the active form of TGF-.beta.1 to type
II receptor, and subsequently leading to the phosphorylation of
type I receptor (TGF.beta. RI). Activated TGF.beta. RI recruits
Smads protein, and propagates the downstream signal to regulate the
gene expression of matrix proteins (Fu et al. 2006). To understand
IMSs' effect on treating liver fibrosis, TGF-.beta.1 and TGF.beta.
RI in T6 cells under IMS treatment were measured. The data showed
that IMSs could effectively interfere with TGF-.beta.1 signaling by
suppressing the gene and protein expressions of total TGF-.beta.1
and TGF.beta. RI (FIGS. 9a-c and FIG. 13). It was recently reported
that EGCG could decrease the active form of TGF-.beta.1 in primary
HSC cells (Fu et al. 2006). In the present study it was also shown
that EGCG could suppress the production of total TGF-.beta.1
protein in T6 cells (FIG. 13). Some IMSs, particularly DPIM, showed
greater suppression of TGF-.beta.1 and TGF.beta. RI than EGCG under
the test conditions. Resveratrol, another anti-oxidant from grape
skin, has been reported to effectively attenuate TGF-.beta.1
protein elevation induced in an experimental fibrotic rat model
(Chavez et al. 2007).
[0350] Activation of HSC is also associated with elevated levels of
NF-.kappa.B and NF-.kappa.B-responsive genes, including IL-6 (Mann
et al. 2006). The role of NF-.kappa.B is to maintain HSCs in the
activated state and promote a chronic wound healing response. In
fact, NF-.kappa.B has been implicated in various aspects of liver
disorders, including hepatic inflammation, fibrosis, and the
development of hepatocellular carcinoma (Elsharkawy et al. 2007).
In this study, IMSs greatly suppressed active NF-.kappa.B p65
protein (FIG. 11), while not affecting the cytosolic NF-.kappa.B
P65 level (data not shown). AP-1 is a homodimer or heterodimer
composed of at least one Jun family protein (c-Jun, JunB and JunD)
and another member from the Fos family (c-Fos, Fra1 and Fra2). It
has been reported that JunD is functionally the most important AP-1
factor in activated HSCs required for the induction of IL-6 and the
tissue inhibitor of metalloproteinase I (TIMP-1) gene transcription
(Smart el al. 2001). It is known that HSC T6 cells express all
members of Jun and Fos proteins (Zhang et al., 2006). Under the
influence of IMSs, c-Fos, JunD and Fra-1 levels were attenuated
(FIG. 11). In line with the down-regulation of JunD, IL-6 gene
transcription was suppressed (FIGS. 10a-c). In short, IMSs might
prevent hepatic fibrosis through the suppression of HSC activation,
and by decreasing the deposition of ECM proteins (col1a1 and
fibronectin) and the production of pro-fibrogenic cytokines
(TGF-.beta.1, TGF.beta. RI and IL-6) mediated through NF-.kappa.B
and AP-1 signaling.
[0351] NHC Precursors could be Drug-Like Compounds
[0352] ROS has been implicated in many physiological conditions,
including neurodegenerative diseases, cardiovascular diseases,
stroke, heart attack, cancer, aging and fibrosis, etc. Therefore,
anti-oxidation has been pursued as a therapeutic strategy for a
number of diseases. Dietary anti-oxidants have been widely used to
ameliorate excessive oxidative stress both in animal models and
humans. For example, resveratrol has been shown to extend the
lifespan of various species, and to be effective at improving the
health and survival of mice on a high-calorie diet (Baur et al.
2006). Tea polyphenols (EGCG being the most abundant catechin) have
been shown to inhibit carcinogen-induced DNA damage in animal
models of skin, lung, colon, liver and pancreatic cancers (Frei et
al. 2003). However, stringent scientific proof for the efficacy of
natural anti-oxidants has not been established (Droge et al. 2001)
due to various factors. Some of the notable limitations for using
natural anti-oxidants as therapeutics include low potency and fast
turnover during metabolism. By comparison, less effort has been
devoted towards studying synthetic anti-oxidants due to safety
concerns. Nevertheless, some progress has been made in this
direction. For example, natural anti-oxidant has been modified to
enhance its potency (Keum et al. 2007). Synthetic mimics of SOD and
catalase have been shown to be effective in rodent models of
ischemia and Parkinson's disease (Peng et al. 2005). Even more
encouragingly, a class of nitron-free radical trap agents,
alpha-phenyl-N-tert-butyl-nitron (PBN) and disodium
2,4-disulfophenyl-N-tert-butylnitrone (NXY-059), has been shown to
be potent neuroprotective agent (Maples et al. 2004), and has
demonstrated anti-cancer activity in hepatocellular carcinoma
through its anti-inflammatory properties (Floyd 2006). These
examples suggest that it is possible to develop therapeutic drug
candidates based on synthetic anti-oxidants. It is believed that
this report represents the first effort towards exploring the
potential therapeutic effect of IMSs. IMSs are precursors of NHCs,
in which the electronic structure and stability, and thus,
therapeutic safety and efficacy can be tuned by varying the
N-substituents. NHCs with bulky substituents favor a triplet ground
state and exhibit radical-like reactivity, while those with smaller
substituents adopt a singlet ground state and show both
nucleophilic and electrophilic behavior (Bourissou et al.
2000).
[0353] In this study, IMSs demonstrated potent anti-oxidative,
anti-inflammatory and anti-fibrotic properties in cultured HSCs. At
the same time, IMSs showed relatively low and tunable toxicity with
IC50 in the range of 35 .mu.M to 3.1 mM. It is hypothesized that
the anti-fibrogenic and anti-inflammatory effects of the IMS
compounds are derived through the anti-oxidative property targeted
at inhibiting HSC activation. The radical scavenging property of
the IMSs is presumably originated from the product after a series
of chemical reactions including: (1) the spontaneous conversion of
IMSs to NHCs preferably under a basic condition, (2) the
interaction of the NHCs at the carbon 2-position with free radicals
such as ROS resulting in the formation of intermediate active
radicals and the neutralization of the free radicals.
[0354] The findings of this study support this hypothesis. Although
the underlying molecular mechanism is not yet well understood, the
effects of IMSs are shown to be mediated through NF-.kappa.B and
AP-1 transcription factors, and TGF-.beta.1 and IL-6 signaling
pathways. Large-scale screening of lead compounds for the treatment
of liver fibrosis can be performed using the surrogate GFAP
biomarker driving a fluorescent reporter. The radical scavenging
and the anti-inflammatory properties are central to IMSs'
therapeutic effect in treating liver fibrosis in this study.
Further exploration may reveal therapeutic potential for this group
of compounds in treating other degenerative and aging-related
diseases.
[0355] Effect of DBZIM on Hepatocellular Carcinoma and Other Tumour
Cells
[0356] Hepatocellular carcinoma (HCC) is known to develop over
liver insults, which leads to hepatocyte inflammation and
regeneration, matrix protein remodeling, fibrosis/cirrhosis and
eventually HCC. HCC is inherently chemotherapy-resistant and is
know to express the drug-resistant gene MDR-1. (Huang et al. 1999,
Kato et al. 2001) Advanced HCC also shows poor prognosis even after
a successful surgical resection (Ariizumi et al. 2004). The data
showed that 1 IMS compound DBZIM can induce apoptosis an in both
differentiated (HepG2) and undifferentiated (HLE) HCC cell line
without significant cytotoxicity (FIG. 22). DBZIM can also inhibit
the proliferation of HLE (FIG. 21A) via cell cycle arrest at G0/G1
phase (FIG. 21B).
[0357] Apoptosis can be triggered through either the extrinsic
pathway or the intrinsic pathway. The extrinsic pathway is
initiated through the simulation of transmembrane death receptors
such as Fas receptors and activation of caspase 8 which leads to
caspase 3/7 cleavage. The intrinsic pathway is initiated through
the release of apoptosis activators from mitochondria and
activation of caspase 9 is involved to cleave caspase 3/7 which
leads to apoptosis. In this study, enzyme activity assays showed
that DBZIM induced the activities of caspase 9 and 3 but not
caspase 8 (FIG. 23). Further experiment by Western blotting showed
that DBZIM also regulated the expression of Bcl-XL and Bak to
induce the apoptosis (FIG. 24). This evidence suggests that DBZIM
mainly caused the mitochondria-mediated apoptosis through the
regulation of Bcl-2 favoring the occurrence of apoptosis.
Meanwhile, it was found that AIF but not cytochrome C (FIG. 23d and
FIG. 26) was released from mitochondria to activate caspases and/or
cause chromatin condensation/DNA fragmentation. AIF has been
generally implicated in the caspase-independent mode of cell
apoptosis and been proposed as a new drug target. (Mignotte et al.
1998, Lorenzo et al. 2007). However it has also been shown to act
in a caspase-dependant manner. (Amoult et al. 2003) DBZIM was also
shown to down-regulate survivin (FIG. 24) and induce cytoplasm
location of survivin (FIG. 25). Survivin is one member of IAPs
protein family, expressed during human foetal development and in
several neoplasms, but not in normal tissue except in thymus and
placenta. It has been shown that nuclear translocation of surviving
in HCC promotes the cancer cell growth. (Moon et al. 2003) Nuclear
and cytoplasmic expression of survivin has also been related to
prognosis of pancreatic cancer (Tonini et al. 2005). DBZIM
perturbated the regulatory mechanism by translocation of survivin
and suggesting a possible new angle for searching for HCC
chemotherapy.
[0358] The alteration of mitochondria function is associated with
the early phases of the cell death. Meanwhile, observed inhibition
of the mitochondrial respiratory chain causes the over production
of ROS, which mediates the death signaling pathway. (Mignotte et
al. 1998) The mode of cell death, either by apoptosis or necrosis
is largely dependant on the level of intracellular ROS. Also it is
difficult to differentiate whether ROS is the result or the cause
of the killing process or both. In this study, it was interesting
that IMSs of different concentration had different physiological
effect, in terms of ROS production. In HCC cells, when DBZIM was in
the micromolar range, it attenuated the ROS level slightly, which
agreed with the findings in HSC-T6 cells. When DBZIM was at much
higher concentration in the milimolar range, it promoted the
accumulation of ROS (FIG. 27). Meanwhile, accompanying the ROS
production, some members of the transcription factor AP-1 complex
was found to be up-regulated by DBZIM (FIG. 28). It was reported
that the integration of oxidative stress and the up-regulation of
JNK/c-Jun/AP-1 cascade is necessary to promote the cell death
(Singh et al. 2007). Taking these results together, it is suggested
that the cell killing caused by DBZIM at high dose may be mostly
due to apoptosis and ROS induction.
[0359] The observations that DBZIM not only inhibited cell
proliferation in HCC lines, but also several other tumor cell lines
commonly used in various cancer studies, through multiple
mechanisms (cell cycle arrest, apoptosis, ROS, AP-1, inhibition of
pAKT, etc.) provided further support for the speculation that DBZIM
indeed exerts a number of beneficial anti-tumor properties in
vitro. Its potential in vivo efficacy will need to be tested in
various specific tumor models for confirmation.
[0360] In the initial in vivo trial, DBZIM was administered to a
HCC xenograft mouse model (Huh7cells in nude mice) via drinking
water, after the tumor growth was evident and at the log phase
about 8 weeks after cell inoculation. Several observations were
made. First, 3 weeks of treatment with DBZIM resulted in 40%
reduction in tumor volume, which is a preliminary and significant
demonstration for using DBZIM as an effective anti-tumor agent.
Second, the effective dose observed for in vivo study (2 g/l) is
much lower that those for in vitro cell culture study (generally in
1-2 mM). This disparity could suggest that DBZIM may have a very
different PK/PD profile in vivo than in vitro. Third, the
effectiveness achieved by dosing the agent in drinking water (with
weekly replacement) indicates that the DBZIM is rather stable in
water and can be absorbed through gastrointestinal (GI) tract
effectively to enter the general circulation. Finally, we also
noticed a significant loss in body weight (by 17%) in the 3-week
treatment with DBZIM, which may suggest some type of general
toxicity in the mice, though no gross pathological abnormality
could be identified in all major organs. Perhaps an alternative
dosing scheme using this agent can be further explored to achieve
maximal therapeutic effect, yet with minimal side effect.
[0361] In conclusion, by using DBZIM as a model compound for
research in IMSs, it has been demonstrated that this agent has
anti-tumor activity in vitro and in vivo, which warrants further
efforts in chemically synthesizing and biologically characterizing
additional IMS compounds with different substituents on the
imidazolium ring, in order to identify candidates with more potent
efficacy with reduced side effect.
[0362] Anticancer Activities of Additional IMSs
[0363] The ability of "DBZIM-like" candidate compounds to induce
cancer cell death was investigated and molecular mechanisms
(apoptosis and cell cycle arrest) underlying this ability were
dissected. It was discovered that the basic anti-tumor properties
(apoptosis and cell cycle arrest) embedded in the model DBZIM were
preserved in the some of additional IMSs with different subgroups,
mainly including IBN-15, -19, -24, -25, -32, compound C (DBZMIM)
and compound 9 (MABZIM).
[0364] It was found that IMSs effectively inhibit tumor cell growth
concomitant with induction of cell cycle arrest and apoptosis. IMSs
activate ATM system and subsequently the phosphorylation and
stabilization of p53. A correlation was drawn between the level of
p53, phospho-p53 (Ser-15) and ATM in HLE liver cancer cells. It is
well known that ATM and ATR are activated by DNA damage. Activation
of the ATM pathways has long been associated with the cell cycle
checkpoint induced by several DNA damage/genotoxic stress. Once
activated, ATM phosphorylates various downstream molecules such as
p53, MDM2, Chk1, Chk2, resulting in cell cycle arrest or cell
death. Tumor suppressor gene p53 is a key element in the induction
of cell cycle arrest and apoptosis following DNA damage or cellular
stress in human cells and cell cycle arrest. (Saito et al. 2002,
Hofseth et al, 2003) In this study, it has been shown that
treatment of HLE cells with IMSs resulted in the accumulation of
p53 and phospho-p53 (Ser15). It has also been found that the
induction of ATM gene expression causes DNA damage and p53
activity, suggesting that p53 induced by DNA damage play an
important role in IMSs-mediated cell cycle arrest. Indeed, this
result is consistent with the report of Canman et al., (1998) who
showed that phosphorylation of p53 at serine 15 was in response to
DNA damage. The in vitro studies also showed that phosphorylation
of this specific site is critical for the p53 response and
subsequent IMS-induced anticancer activity. It was also found that
IMSs decrease the expression of a number of genes important for
cell cycling, including Cdc25A, Cdc2, and SESN2.
[0365] Up-regulated p53 protein can activate the pro-apoptotic
protein BAX and inhibits Bcl-2 to cause mitochondrial dysfunction
and cytochrome c release, stimulating the mitochondrial apoptotic
pathway (Melino et al., 2004). In this study, it was demonstrated
that IMSs inhibited Bcl-2 and activated Bax in the HLE cancer
cells. It was found that IMSs effectively inhibit tumor cell
growth, concomitant with induction of cell cycle arrest and
apoptosis. Furthermore, the fact that IMSs do not exhibit any
significant toxicity in normal lung and breast cells suggests that
IMSs possesses selectivity between normal and cancer cells. The
data on caspase-3 activation strongly suggest that the mechanism of
IMS induced apoptosis in these liver cancer-derived cell lines
might be mediated by caspase-dependent signaling leading to
caspase-3 activation. In the present study, it was shown that IMSs
have cytotoxic activity toward cancer cells harboring mutant p53
and can activate mutant p53. In the present study, the inhibitory
effects of IMSs to malignant tumor cell lines with mutant p53
status was investigated. The mitochondrial apoptotic pathway has
been described as important in signalling apoptotic cell death for
mammalian cells (Pilch et al 2003; Kluck et al., 1997). Following
the treatment of HLE cells with IMSs, it was observed that IMSs
treatment resulted in a significant increase of Bax expression, and
a decrease of Bcl-2 suggesting that changes in the ratio of
pro-apoptotic and anti-apoptotic Bcl-2 proteins might contribute to
the apoptosis-promotion activity of IMSs. The activation of
caspase-3 and caspase-9 was also observed after HLE cells were
treated with IMSs. These results confirm that IMS-induced apoptosis
is associated with regulation of BAX and Bcl-2 proteins.
[0366] Here it is demonstrated that, in addition to DBZIM, compound
9 or C.sub.9 displays a potent anti-tumor activity in the HCC
xenograft model, and yet shows no apparent general toxicity. This
preliminary evidence provides proof of the concept that chemical
modification based on a core DBZIM structure can significantly
improve the anti-tumor efficacy and reduce side effects. For
example, C9 at concentration (1.5 g/l) can have much higher potency
than DBZIM at a higher concentration (2 g/l), when administered via
same route for the same period of time.
[0367] Thus the present study demonstrated that: (1) human liver
cancer HLE cells are highly sensitive to growth inhibition by IMSs.
(2) IMSs reduced survival of HLE cells via cell cycle arrest and
apoptosis induction in a p53-dependent manner, and by decreasing
the expression of Cdc2, Cdc25A. (3) IMSs inhibited cell growth in
the HLE cells via activation of ATM, which stabilizes p53 by
phosphorylation of p53 at Ser15 and decreasing the interaction of
p53 and MDM2. (4) IMSs triggered mitochondrial apoptotic pathway by
regulation of Bcl-2 and BAX proteins expression. These findings
suggest that IMSs are a promising class of low toxic
chemopreventive agents against human liver cancer cells, and other
tumor cell types.
[0368] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
[0369] Concentrations given in this specification, when given in
terms of percentages, include weight/weight (w/w), weight/volume
(w/v) and volume/volume (v/v) percentages.
[0370] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural reference unless
the context clearly dictates otherwise. As used in this
specification and the appended claims, the terms "comprise",
"comprising", "comprises" and other forms of these terms are
intended in the non-limiting inclusive sense, that is, to include
particular recited elements or components without excluding any
other element or component. Unless defined otherwise all technical
and scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this
invention belongs.
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