U.S. patent application number 16/059679 was filed with the patent office on 2020-02-13 for platinum (ii) complexes containing n-heterocyclic carbene ligand and pincer ligands, synthesis, and their applications in cancer.
The applicant listed for this patent is THE UNIVERSITY OF HONG KONG. Invention is credited to Chi Ming CHE, Tian Feng CHEN, Sin Ki FUNG.
Application Number | 20200048291 16/059679 |
Document ID | / |
Family ID | 69405529 |
Filed Date | 2020-02-13 |
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United States Patent
Application |
20200048291 |
Kind Code |
A1 |
CHE; Chi Ming ; et
al. |
February 13, 2020 |
PLATINUM (II) COMPLEXES CONTAINING N-HETEROCYCLIC CARBENE LIGAND
AND PINCER LIGANDS, SYNTHESIS, AND THEIR APPLICATIONS IN CANCER
TREATMENT
Abstract
Provided herein is a method of synthesis of Pt(II) complexes, a
pharmaceutical composition comprises thereof. Also provided herein
are the methods for the treatment and prevention of cancer/tumor in
patients in need thereof by the administration of the Pt(II)
complexes. Also provided is a method of detecting the Pt(II)
complex in a biological system. Also provided is a method of making
the Pt(II) complex The Pt(II) complexes possess anticancer activity
such as the induction of cell death, inhibition of cellular
proliferation, and inhibition of tumor growth in vivo.
Inventors: |
CHE; Chi Ming; (Mid-Level,
HK) ; FUNG; Sin Ki; (New Territories, HK) ;
CHEN; Tian Feng; (Guangzhou, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF HONG KONG |
Hong Kong |
|
CN |
|
|
Family ID: |
69405529 |
Appl. No.: |
16/059679 |
Filed: |
August 9, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/57415 20130101;
C07F 15/0093 20130101; C07F 15/0086 20130101; G01N 21/6486
20130101; A61P 35/00 20180101; G01N 21/6458 20130101 |
International
Class: |
C07F 15/00 20060101
C07F015/00; A61P 35/00 20060101 A61P035/00; G01N 21/64 20060101
G01N021/64; G01N 33/574 20060101 G01N033/574 |
Claims
1. A Pt(II) complex consisting of a Pt(II)-N-heterocyclic carbene
("NHC") ligand and 1, 3-bis(2-pyridylimino) isoindoline (BPI)
ligand, and wherein the Pt(II) complex has formula I, ##STR00020##
wherein R.sup.1 is --CH.sub.3, --C.sub.4H.sub.9, -nC.sub.6H.sub.13,
--CH.sub.3, or --CH.sub.2Ph, and wherein R.sup.2 is --CH.sub.3,
--C.sub.4H.sub.9, -nC.sub.6H.sub.13, --C.sub.8H.sub.17,
--C.sub.16H.sub.33, or --CH.sub.2Ph.
2. The Pt(II) complex of claim 1 wherein the NHC ligand is about
90.degree. to the BPI ligand.
3. The Pt(II) complex of claim 1 wherein the Pt(II) complex has
anti-tumor or anti-angiogenic properties.
4. (canceled)
5. A Pt(II) complex consisting of a Pt(II)-NHC ligand and 1,
3-bis(2-pyridylimino) isoindoline (BPI) ligand, wherein the
Pt(II)-NHC ligand is perpendicular to the BPI ligand, and having
the following formula: ##STR00021## wherein R.sup.1 is --CH.sub.3,
--C.sub.4H.sub.9, -nC.sub.6H.sub.13, --CH.sub.3, or --CH.sub.2Ph,
and wherein R.sup.2 is --CH.sub.3, --C.sub.4H.sub.9,
-nC.sub.6H.sub.13, --C.sub.8H.sub.17, --C.sub.16H.sub.33, or
--CH.sub.2Ph.
6. The Pt(II) complex of claim 5 wherein R.sup.1 is C.sub.4H.sub.9
and R.sup.2 is C.sub.4H.sub.9.
7. A composition comprising the Pt(II) complex of claim 5.
8. The composition of claim 7 wherein the Pt(II)-NHC ligand is
about 90.degree. to the BPI ligand.
9. The composition of claim 7 wherein the Pt(II) complex comprises
anti-tumor and/or anti-angiogenic properties.
10-18. (canceled)
Description
1. INTRODUCTION
[0001] Described herein are platinum (II) complexes containing
N-heterocyclic carbene ligand, a method of synthesis of the
platinum (II) complexes containing N-heterocyclic carbene ligand,
methods of treating and preventing cancer or tumor using the
platinum (II) complexes containing N-heterocyclic carbene ligand.
The platinum (II) complexes has a dual action including cytotoxic
to tumor growth and anti-angiogenesis. Also provided is a method of
detecting the platinum(II) complexes containing N-heterocyclic
carbene ligand by fluorescence microscopy. Also described are
therapeutic and prophylactic compositions containing a purified
platinum(II) complexes containing N-heterocyclic carbene ligand. In
certain embodiments, the methods of treating and preventing cancer
or tumor are in combination with other cancer or tumor treatment.
In certain embodiments, the cancer or tumor treatment is
chemotherapy, radiation therapy, gene therapy, surgery or a
combination thereof.
2. BACKGROUND
[0002] As stimulated by the clinical success of
cis-diamminedichloroplatinum (cisplatin), a platinum(II) complex,
for the treatment of cancers, scientists have paid great attention
to the development of metal-based anticancer drugs which target DNA
including the cisplatin analogues and some ruthenium(II)-arene
complexes [Sadler, P. J. et al. Curr. Opin. Chem. Biol. 2008, 12,
197]. However, severe side effects and the induced drug resistance
are commonly encountered and thus subsequently have hampered the
wider applications of these DNA binding agents.
[0003] Cisplatin and its derivatives are widely used as
chemotherapeutic agents for treating cancer. Yet, most of them fail
in combating with metastatic cancer, which is a big problem found
in cancer treatment. In view of this, it is important to develop
new cytotoxic agents that can at the same time regulate tumor
microenvironment which is important for governing tumor
progression, growth, angiogenesis and metastasis.
3. SUMMARY
[0004] Described herein are Pt(II)-NHC--BPI complexes, compositions
comprising Pt(II)-NHC--BPI complexes, methods of using the
Pt(II)-NHC--BPI complexes in cancer/tumor treatment, a method of
synthesis of Pt(II)-NHC--BPI complexes, and a method of detecting
the Pt(II)-NHC--BPI complexes. In one embodiment, the method of
treatment and prevention is in combination with one or more
cancer/tumor therapies.
[0005] Described herein is a Pt(II) complex comprising a Pt(II)-NHC
ligand and 1, 3-bis(2-pyridylimino) isoindoline (BPI) ligand,
wherein the Pt(II)-NHC ligand is perpendicular to the BPI
ligand.
[0006] In one embodiment, the NHC ligand and the BPI ligand have a
bond angle of about 90.degree.. In one embodiment, the Pt(II)
complex has anti-tumor or anti-angiogenic properties.
[0007] Described herein is a method of making a Pt(II) complex,
comprising reacting [Pt(BPI)Cl] with corresponding imidazolium salt
in the presence of a base to form the Pt(II) complex.
[0008] In one embodiment, provided herein is a method for cancer or
tumor treatment and prevention resulting in induction of cell
death, inhibition of cellular proliferation, inhibition of
angiogenesis, or inhibition of in vivo tumor growth. In one
embodiment, provided herein is a method comprising administering to
a subject in need thereof a composition comprising an effective
amount of a Pt(II)-NHC--BPI complex. In one embodiment, the
Pt(II)-NHC--BPI complexes is a platinum(II) complex described
herein represented by the structural formulae of I, derivatives
thereof; or a pharmaceutically acceptable salt, solvate, or hydrate
thereof,
##STR00001##
[0009] wherein R.sup.1 is --CH.sub.3, --C.sub.4H.sub.9,
-nC.sub.6H.sub.13, --CH.sub.3, or --CH.sub.2Ph, and wherein R.sup.2
is --CH.sub.3, --C.sub.4H.sub.9, -nC.sub.6H.sub.13,
--C.sub.8H.sub.17, --C.sub.16H.sub.33, or --CH.sub.2Ph. In another
embodiment, provided herein is a method for detecting an effective
amount of the Pt(II)-NHC--BPI complexes, depending on the
fluorescence changes at proper wavelength. The Pt(II)-NHC--BPI
complex is a platinum(II) complex described herein can be
represented by the structural formula of I, or an acceptable salt
thereof,
##STR00002##
wherein R.sup.1 is --CH.sub.3, --C.sub.4H.sub.9, -nC.sub.6H.sub.13,
--CH.sub.3, or --CH.sub.2Ph, and wherein R.sup.2 is --CH.sub.3,
--C.sub.4H.sub.9, -nC.sub.6H.sub.13, --C.sub.8H.sub.17,
--C.sub.16H.sub.33, or --CH.sub.2Ph.
[0010] Described herein is a Pt(II) complex which comprises a
Pt(II)-NHC ligand and 1, 3-bis(2-pyridylimino) isoindoline (BPI)
ligand, wherein the Pt(II)-NHC ligand is perpendicular to the BPI
ligand, and having the following formula:
##STR00003##
wherein R.sup.1 is --CH.sub.3, --C.sub.4H.sub.9, -nC.sub.6H.sub.13,
--CH.sub.3, or --CH.sub.2Ph, and wherein R.sup.2 is --CH.sub.3,
--C.sub.4H.sub.9, -nC.sub.6H.sub.13, --C.sub.8H.sub.17,
--C.sub.16H.sub.33, or --CH.sub.2Ph. In one embodiment, R.sup.1 is
C.sub.4H.sub.9 and R.sup.2 is C.sub.4H.sub.9.
[0011] Described herein is a composition comprising a
Pt(II)-NHC--BPI complex, which comprises a Pt(II)-NHC ligand and 1,
3-bis(2-pyridylimino) isoindoline (BPI) ligand, wherein the
Pt(II)-NHC ligand is perpendicular to the BPI ligand.
[0012] In one embodiment, the NHC ligand and the BPI ligand have a
bond angle of about 90.degree.. In one embodiment, the Pt(II)
complex comprises anti-tumor and/or anti-angiogenic properties.
[0013] Described herein is a method for treatment of tumor or
cancer in a subject comprising administering to a subject in need
thereof an effective amount of a composition comprising a Pt(II)
complex that regulates uPA/uPAR-mediated angiogenic pathway or
VEGF-induced angiogenic pathway. In one embodiment, provided herein
is a method wherein the tumor is one or more of hepatocellular
carcinoma, cervical epithelioid carcinoma, lung carcinoma, breast
cancer, colon cancer, melanoma or nasopharyngeal carcinoma. In one
embodiment, the effective amount is about 0.1 mg/kg to 50 mg/kg. In
one embodiment, the effective amount is about 2.5-5 mg/kg.
[0014] The Pt(II)-NHC--BPI complexes are stable in air and aqueous
solutions like phosphate-buffered saline (PBS) conditions. The
anti-cancer active Pt(II)-NHC--BPI complexes is also accompanied
with the release of highly fluorescent ligand. The Pt(II)-NHC--BPI
complexes display similar anti-cancer or anti-tumor activity. They
can be detected via the fluorescent ligand which makes them to be
excellent bio-probes and for prevalent biological applications.
[0015] Described herein is a method to detect the Pt(II) complex in
a subject, said method comprises administering an effective amount
of Pt(II) complex to the subject and detect the Pt(II) complex
using fluorescent detection. In one embodiment, the effective
amount of Pt(II) complex is 1 .mu.M-500 .mu.M.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1(A)-(F) show Chemical structures of the Pt(II)
complexes 1a-1j.
[0017] FIG. 2 shows perspective views of X-ray crystal structure of
1a, showing bond angle (C.sub.19--Pt.sub.1--N.sub.5) of
90.6.degree..
[0018] FIGS. 3(A)-(B) show (A) Absorption and (B) Normalized
emission spectra of 1b, 1i and 1j in degassed CH.sub.2Cl.sub.2.
[0019] FIGS. 4(A)-(C) show (A) Confocal fluorescence microscopy
images of HeLa cells incubated with 1b (5 .mu.M) for 15 min, and
subsequently co-stained with ER-Tracker.TM.. (B) Western blot
analysis of expression levels of ER stress-related proteins in
MDA-MB-231 cells treated with 1b (5 .mu.M) for indicated time. (C)
Western blot analysis of apoptosis-related proteins after treating
MDA-MB-231 cells with indicated concentration of 1b for 24 h.
[0020] FIGS. 5(A)-(I) show confocal fluorescence microscopy images
of HeLa cells incubated with 1b, ER-Tracker.TM., or both, taken
using FITC or Rhodamine filters.
[0021] FIGS. 6(A)-(I) show co-localization analysis of 1b with
ER-Tracker.TM. (upper panel), Mitotracker.RTM. (middle) and
Lysotracker.RTM. (lower panel).
[0022] FIGS. 7(A)-(C) show (A), (B) Effects of 1b on cell apoptosis
and cell cycle of MDA-MB-231 cells, as determined by flow
cytometry. Cells were treated with indicated concentrations of 1b
for 24 h. *, p<0.05 versus the control. (C) Western blot
analysis of expression levels of G0/G1 phase-related proteins in
MDA-MB-231 cells after treatment with indicated concentrations of
1b for 24 h.
[0023] FIGS. 8(A)-(D) show JC1 staining of HeLa cells. (A)-(C) HeLa
cells were treated with DMSO vehicle, 1b (5 .mu.M) and CCCP
(carbonyl cyanide m-chlorophenyl hydrazine, a mitochondrial
membrane potential disrupter; 50 .mu.M) for 2 h, and examined using
fluorescence microscope with excitation at 470 nm. (D) JC1
fluorescence intensity ratio of I.sub.580 nm/I.sub.530 nm after
treatment of HeLa cells with 1b at different concentrations.
[0024] FIGS. 9(A)-(B) show wound closure assay to determine the
effect of 1b on migration of MDA-MB-231 cells. FIGS. 9(C)-(D)
Transwell invasive assay to determine the effect of 1b on invasion
of MDA-MB-231 cells after 24 h treatment. The cells were imaged by
a phase-contrast microscope (200.times., Nikon TS 100). The
migrated and invaded cells were quantified by manual counting and
inhibition ratio was expressed as % of control (n=3; *, p<0.05;
**, p<0.01 versus the control).
[0025] FIGS. 10(A)-(L) show inhibition of MDA-MB-231 cells
migration by different concentrations of 1b at different time
points.
[0026] FIG. 11. shows the viability of MDA-MB-231 cells treated
with 1b for the indicated time intervals.
[0027] FIGS. 12(A)-(B) show (A) Tube formation assay of MS1 cells
treated with different concentrations of 1b for 3 h. (B) MTT assay
on MS1 cells after treatment with 1b for 3 h, revealing no
significant cell death at 1, 3 and 5 .mu.M of 1b.
[0028] FIGS. 13(A)-(G) show (A) Western blotting analysis of
expression levels of uPA, MMP-9 and TIMP1 in MDA-MB-231 cells
treated with different concentrations of 1b for 24 h. (B) Western
blotting analysis of expression levels of uPAR and MMP-9 in
MDA-MB-231 cells treated with 1b (2 .mu.M) for different time
intervals. (C-D) Effects of 1b on expression levels of
phosphorylated and total FAK, ERK and Akt. Cells were exposed to
(C) different concentrations of 1b for 24 h or (D) 1b (2 .mu.M)
with different incubation times. (E)-(G)) Effects of 1b, LY294002
and U0126 on inhibition of MDA-MB-231 cells (C) growth, (F)
migration and (G) invasion. For co-treatment experiments, cells
were pretreated with LY294002 or U0126 (10 or 20 .mu.M) for 1 h and
co-treated with 1b for another 24 h. All data are expressed as
means.+-.SD of triplicates.
[0029] FIGS. 14(A)-(F) show (A) Effects of 1b on secretion of
intracellular VEGF in MDA-MB-231 cells. Cells were exposed to
different concentrations of 1b for 24 h. (B)-(D) 1b inhibited
VEGF-induced HUVECs growth, migration and invasion. HUVECs were
cultured in MDA-MB-231 conditioned medium (CM, VEGF=13.4 ng/ml) and
exposed to different concentrations of 1b for 24 h. The treatment
group with VEGF (50 ng/ml) was regarded as positive control. (E) 1b
inhibited VEGF-induced tube formation of HUVECs. Cells were
pre-coated with matrigel and treated with different concentrations
of 1b for 24 h. (F) Effect of 1b on ex vivo angiogenesis as
determined by CAM assay. All data are expressed as means.+-.SD of
triplicates. Bars with different characters (A)-(D) are
statistically different at p<0.05 level.
[0030] FIGS. 15(A)-(H) show (A) The tumor volume of
MDA-MB-231-bearing mice after treatment by saline, Pt (5 mg/kg), Pt
(2.5 mg/kg), respectively. n=10, *P<0.05, **P<0.01. (B) The
weights of MDA-MB-231-bearing mice after treatment by saline, Pt (5
mg/kg), Pt (2.5 mg/kg), respectively (n=10). (C) Ki67, CD34 and
Cleavage-caspase-3 expression and TUNEL-DAPI co-staining assay of
tumor tissues after treatment with Pt (2.5 mg/kg and 5.0 mg/kg).
(D)-(H) Blood biochemistry data including liver-function markers:
AST, heart-function markers: CK, blood fat: CHOL, kidney-function
markers: BUN and blood glucose: GLU. n=3, *P<0.05,
**P<0.01.
[0031] FIG. 16 shows proposed anti-apoptotic and anti-angiogenic
pathways by 1b.
5. DETAILED DESCRIPTION
[0032] Provided herein is a new series of dual cytotoxic and
anti-angiogenic platinum(II) complexes with N-heterocylic carbene
(NHC) and 1,3-bis(2-pyridylimino)isoindoline (BPI) ligands. The NHC
ligand is found to be perpendicular to the plane of BPI ligand, as
revealed by X-ray crystallography, thus allowing these platinum(II)
complexes to target other biomolecules rather than DNA only. The
introduction of NHC ligand, which is a strong .sigma.-donor, also
renders the complexes strong luminescence in aqueous solution and
live cells, and hence their subcellular localization in endoplasmic
reticulum (ER) can be identified by fluorescence microscopy. With
their accumulation in ER, they are found to induce ER stress and
subsequent apoptotic cell death, accounting for their potent
cytotoxicity toward cancer cells.
5.1 Pt(II) Complexes
[0033] Platinum(II) compounds as exemplified by cisplatin and its
derivatives have been widely used in the treatment of
cancer..sup.[1,2] Their mechanism of action is mainly through
covalent crosslinking onto DNA, leading to cancer cell apoptosis or
cell cycle arrest..sup.[3,4] Since DNA is the primary molecular
target, cancer cells with changes in repair of DNA lesion, such as
enhanced nucleotide excision repair or deficiency in mismatch
repair, are found to show resistance to these platinum
drugs..sup.[5] Moreover, these platinum drugs generally give rise
to severe toxic side effects, probably due to the fact that DNA is
not a specific biomolecule in cancer cells..sup.[5] As a result,
there are continuing efforts on searching metal complexes with new
working mechanisms.
[0034] It should be advantageous for developing anticancer drugs to
target tumor microenvironment. Tumor microenvironment is complex
and dynamic, and is regulated by a number of mediators and
signaling transduction pathways that govern tumor progression
including tumor initiation, growth, angiogenesis and
metastasis..sup.[6,7] For example, tumor cells developing their
microenvironment by secretion of vascular endothelial growth factor
(VEGF) or cytokines to promote abnormal tumor neovasculature
formation, which provides nutrients for further tumor growth and
metastasis..sup.[8,9] In addition, binding of urokinase plasminogen
activator (uPA) to uPA receptor (uPAR) in tumor microenvironment
can trigger activation of metalloproteinases (MMPs) to degrade the
components of surrounding extracellular matrix (ECM),.sup.[10] and
hence contributes to tumor cell metastasis. Together with the fact
that over 90% of cancer deaths today are due to metastasis
formation,.sup.[11] regulations of tumor microenvironment including
inhibition of tumor growth, metastasis and VEGF-induced
angiogenesis, have been considered as effective means in combating
tumor progression..sup.[12,13]
[0035] A number of metal complexes have been reported to target
tumor microenvironment by acting as angiogenesis
inhibitors..sup.[14-16] Notably, a ruthenium(III) complex, NAMI-A,
was found to be non-cytotoxic toward solid tumor but show promising
antitumor activities by inhibition of tumor metastasis and
angiogenesis..sup.[14] In addition, platinum complexes showing dual
cytotoxic and anti-angiogenic properties have also been
explored,.sup.[17-19] and they should show improved anticancer
efficacy through decreasing acquired-drug resistance and systemic
toxicities, as compared to that of a cytotoxic or an
anti-angiogenic agent alone..sup.[20,21] However, none of them
exhibited promising in vivo antitumor and anti-angiogenic
activities.
[0036] Disclosed herein are platinum(II) complexes that exhibit
dual cytotoxic and anti-angiogenic properties, and are luminescent
in vitro so that real-time monitoring of therapeutic progress would
be feasible. Provided is an out-of-plane ancillary ligand to the
platinum(II) center for targeting biomolecules other than DNA in
order to achieve dual cytotoxic and anti-angiogenic properties. NHC
is a strong G-donor and can increase the energy level of
non-emissive ligand-field (LF) state, rendering platinum(II)
complexes strongly luminescent,.sup.[24] and this strong
luminescence feature can help to elucidate mechanism of anticancer
actions of the complexes by fluorescence microscopy. Provided is a
new series of platinum(II) complexes containing NHC ligands and
1,3-bis(2-pyridylimino)isoindoline (BPI) which has two accessible
nitrogen atoms ([Pt(BPI)(NHC)](OTf); FIG. 1). Platinum(II)
complexes with BPI and chloride or triphenylphosphine ligand was
also prepared. The complexes were found to exhibit dual cytotoxic
and anti-angiogenic activities, as revealed by proteomic data and
biochemical assays, as well as in vivo and ex vivo experiments.
[0037] Provided herein is a Pt(II)-NHC--BPI complex. In one
embodiment, the Pt(II)-NHC--BPI complexes is a platinum(II) complex
described herein represented by the structural formulae of I,
derivatives thereof; or a pharmaceutically acceptable salt,
solvate, or hydrate thereof,
##STR00004##
wherein R.sup.1 is --CH.sub.3, --C.sub.4H.sub.9, -nC.sub.6H.sub.13,
--CH.sub.3, or --CH.sub.2Ph, and wherein R.sup.2 is --CH.sub.3,
--C.sub.4H.sub.9, -nC.sub.6H.sub.13, --C.sub.8H.sub.17,
--C.sub.16H.sub.33, or --CH.sub.2Ph. Described herein is a Pt(II)
complex which comprises a Pt(II)-NHC ligand and 1,
3-bis(2-pyridylimino) isoindoline (BPI) ligand, wherein the
Pt(II)-NHC ligand is perpendicular to the BPI ligand, and having
the following formula:
##STR00005##
wherein R.sup.1 is --CH.sub.3, --C.sub.4H.sub.9, -nC.sub.6H.sub.13,
--CH.sub.3, or --CH.sub.2Ph, and wherein R.sup.2 is --CH.sub.3,
--C.sub.4H.sub.9, -nC.sub.6H.sub.13, --C.sub.8H.sub.17,
--C.sub.16H.sub.33, or --CH.sub.2Ph. In one embodiment, R.sup.1 is
C.sub.4H.sub.9 and R.sup.2 is C.sub.4H.sub.9.
[0038] Also disclosed are the synthesis of platinum(II) [Pt(II)]
complexes containing N-heterocyclic carbene ligand (NHC) and BPI
ligand, composition comprising platinum(II) [Pt(II)] complexes
containing N-heterocyclic carbene ligand (NHC) and BPI ligand,
methods of treating and preventing cancer or tumor in a subject,
and a method of detecting the Pt(II) complex. Disclosed herein is a
method of treating or preventing cancer/tumor comprising
administering a pharmaceutical composition comprising at least one
of the Pt(II)-NHC--BPI complexes in an effective amount for
anti-cancer or anti-tumor activity. In certain embodiments,
anti-cancer or anti-tumor activities includes, but are not limited
to, the induction of cell death, inhibition of cellular
proliferation, inhibition of angiogenesis, and inhibition of in
vivo tumor growth. Provided herein is a method of detecting the
Pt(II)-NHC--BPI complexes. In an embodiment, a signal is detected
depending on fluorescence changes at proper wavelength. As provided
herein, in one embodiment, Pt(II)-NHC--BPI complexes refer to a
molecule of a platinum(II) ion connected to a N-heterocyclic
carbene ligand and a BPI ligand. In one embodiment, platinum(II)
[Pt(II)] complexes containing N-heterocyclic carbene ligand (NHC)
is represented by structural formula I, derivatives thereof; or a
pharmaceutically acceptable salt, solvate, or hydrate thereof.
[0039] As used herein, the phrase "acceptable salt," as used
herein, includes salts formed from the charged Pt(II)-NHC--BPI
complex and counter-anion(s).
[0040] As used herein, the phrase "counter-anion" refers to an ion
associated with a positively charged Pt(II)-NHC--BPI complex.
Non-limiting examples of counter-ions include halogens such as
fluoride (F.sup.-), chloride (Cl.sup.-), bromide (Br.sup.-), iodide
(I.sup.-); sulfate (SO.sub.4.sup.2-); phosphate (PO.sub.4.sup.3-);
trifluoromethanesulfonate (triflate, -OTf or
CF.sub.3SO.sub.3.sup.-); acetate (.sup.-OAc); nitrate
(NO.sub.3.sup.-); perchlorate (ClO.sub.4.sup.-);
hexafluorophosphate (PF.sub.6.sup.-) and hexafluoroacetylacetonate
([CF.sub.3C(O)CHC(O)CF.sub.3].sup.-).
[0041] In one embodiment, the invention relates to the synthesis of
novel platinum(II) [Pt(II)] bearing N-heterocyclic carbene ligand
and BPI ligand.
[0042] In another embodiment, the invention relates to a
pharmaceutical composition for cancer treatment by inhibition of
the proliferation of cancer cells in vitro comprising an effective
amount of one or more of the Pt(II)-NHC--BPI complexes.
[0043] In another embodiment, the invention relates to a
pharmaceutical composition for cancer treatment by the inhibition
of tumor growth in vivo comprising an effective amount of one or
more of the Pt(II)-NHC--BPI complexes.
[0044] In another embodiment, the invention relates to fluorescent
detecting compounds, and the application in cellular imaging,
comprising an effective amount of a Pt(II)-NHC--BPI complex.
[0045] The Pt(II)-NHC--BPI complexes of this invention can be
represented by one or more of structural formula I, derivatives
thereof; or a pharmaceutically acceptable salt, solvate, or hydrate
thereof.
[0046] In one embodiment, the invention relates to a pharmaceutical
composition for treating or preventing cancer/tumor. In certain
embodiments, the treatment and prevention comprises induction of
cell death, inhibition of cellular proliferation, inhibition of
angiogenesis, and the inhibition of tumor growth in vivo. In one
embodiment, the method comprises administering an effective amount
of the Pt(II)-NHC--BPI complexes to a subject. In one embodiment,
the method comprises detecting the Pt(II) complex is a subject
comprising administering an effective amount of the Pt(II)-NHC--BPI
complexes. In one embodiment, the Pt(II) complex is detected by
fluorescence changes at proper wavelength. The Pt(II)-NHC--BPI
complex has a formula I, derivatives thereof; or a pharmaceutically
acceptable salt, solvate, or hydrate thereof,
##STR00006##
[0047] wherein R.sup.1 is --CH.sub.3, --C.sub.4H.sub.9,
-nC.sub.6H.sub.13, --CH.sub.3, or --CH.sub.2Ph, and
[0048] wherein R.sup.2 is --CH.sub.3, --C.sub.4H.sub.9,
-nC.sub.6H.sub.13, --C.sub.8H.sub.17, --C.sub.16H.sub.33, or
--CH.sub.2Ph.
5.2 Human Treatment
5.2.1 Formulations
[0049] The platinum(II) complexes provided herein can be
administered to a patient in the conventional form of preparations,
such as injections and suspensions. Suitable formulations can be
prepared by methods commonly employed using conventional, organic
or inorganic additives, such as an excipient selected from fillers
or diluents, binders, disintegrants, lubricants, flavoring agents,
preservatives, stabilizers, suspending agents, dispersing agents,
surfactants, antioxidants or solubilizers.
[0050] Excipients that may be selected are known to those skilled
in the art and include, but are not limited to fillers or diluents
(e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose,
cellulose, talc, calcium phosphate or calcium carbonate and the
like), a binder (e.g., cellulose, carboxymethylcellulose,
methylcellulose, hydroxymethylcellulose,
hydroxypropylmethylcellulose, polypropylpyrrolidone,
polyvinylpyrrolidone, gelatin, gum arabic, polyethyleneglycol or
starch and the like), a disintegrants (e.g., sodium starch
glycolate, croscarmellose sodium and the like), a lubricant (e.g.,
magnesium stearate, light anhydrous silicic acid, talc or sodium
lauryl sulfate and the like), a flavoring agent (e.g., citric acid,
or menthol and the like), a preservative (e.g., sodium benzoate,
sodium bisulfite, methylparaben or propylparaben and the like), a
stabilizer (e.g., citric acid, sodium citrate or acetic acid and
the like), a suspending agent (e.g., methylcellulose, polyvinyl
pyrrolidone or aluminum stearate and the like), a dispersing agent
(e.g., hydroxypropylmethylcellulose and the like), surfactants
(e.g., sodium lauryl sulfate, polaxamer, polysorbates and the
like), antioxidants (e.g., ethylene diamine tetraacetic acid
(EDTA), butylated hydroxyl toluene (BHT) and the like) and
solubilizers (e.g., polyethylene glycols, SOLUTOL.RTM.,
GELUCIRE.RTM. and the like). The effective amount of the
platinum(II) complexes provided herein in the pharmaceutical
composition may be at a level that will exercise the desired
effect.
[0051] In another embodiment, provided herein are compositions
comprising an effective amount of platinum(II) complexes provided
herein and a pharmaceutically acceptable carrier or vehicle,
wherein a pharmaceutically acceptable carrier or vehicle can
comprise an excipient, diluent, or a mixture thereof. In one
embodiment, the composition is a pharmaceutical composition.
[0052] Compositions can be formulated to contain a daily dose, or a
convenient fraction of a daily dose, in a dosage unit. In general,
the composition is prepared according to known methods in
pharmaceutical chemistry. Capsules can be prepared by mixing the
platinum(II) complexes provided herein with a suitable carrier or
diluent and filling the proper amount of the mixture in
capsules.
5.3 Method of Use
[0053] Solid tumor cancers that can be treated by the methods
provided herein include, but are not limited to, sarcomas,
carcinomas, and lymphomas. In specific embodiments, cancers that
can be treated in accordance with the methods described include,
but are not limited to, cancer of the breast, liver, neuroblastoma,
head, neck, eye, mouth, throat, esophagus, esophagus, chest, bone,
lung, kidney, colon, rectum or other gastrointestinal tract organs,
stomach, spleen, skeletal muscle, subcutaneous tissue, prostate,
breast, ovaries, testicles or other reproductive organs, skin,
thyroid, blood, lymph nodes, kidney, liver, pancreas, and brain or
central nervous system.
[0054] In particular embodiments, the methods for treating cancer
provided herein inhibit, reduce, diminish, arrest, or stabilize a
tumor associated with the cancer. In other embodiments, the methods
for treating cancer provided herein inhibit, reduce, diminish,
arrest, or stabilize the blood flow, metabolism, or edema in a
tumor associated with the cancer or one or more symptoms thereof.
In specific embodiments, the methods for treating cancer provided
herein cause the regression of a tumor, tumor blood flow, tumor
metabolism, or peritumor edema, and/or one or more symptoms
associated with the cancer. In other embodiments, the methods for
treating cancer provided herein maintain the size of the tumor so
that it does not increase, or so that it increases by less than the
increase of a tumor after administration of a standard therapy as
measured by conventional methods available to one of skill in the
art, such as digital rectal exam, ultrasound (e.g., transrectal
ultrasound), CT Scan, MRI, dynamic contrast-enhanced MRI, or PET
Scan. In specific embodiments, the methods for treating cancer
provided herein decrease tumor size. In certain embodiments, the
methods for treating cancer provided herein reduce the formation of
a tumor. In certain embodiments, the methods for treating cancer
provided herein eradicate, remove, or control primary, regional
and/or metastatic tumors associated with the cancer. In some
embodiments, the methods for treating cancer provided herein
decrease the number or size of metastases associated with the
cancer.
[0055] In certain embodiments, the methods for treating cancer
provided herein reduce the tumor size (e.g., volume or diameter) in
a subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, 99%, or 100%, relative
to tumor size (e.g., volume or diameter) prior to administration of
platinum(II) complexes as assessed by methods well known in the
art, e.g., CT Scan, MRI, DCE-MRI, or PET Scan. In particular
embodiments, the methods for treating cancer provided herein reduce
the tumor volume or tumor size (e.g., diameter) in a subject by an
amount in the range of about 5% to 20%, 10% to 20%, 10% to 30%, 15%
to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%,
30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 30% to
100%, or any range in between, relative to tumor size (e.g.,
diameter) in a subject prior to administration of platinum(II)
complexes as assessed by methods well known in the art, e.g., CT
Scan, MRI, DCE-MRI, or PET Scan.
[0056] In certain embodiments, the methods for treating cancer
provided herein reduce the tumor perfusion in a subject by at least
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 80%, 85%, 90%, 95%, 99%, or 100%, relative to tumor perfusion
prior to administration of platinum(II) complexes as assessed by
methods well known in the art, e.g., MRI, DCE-MRI, or PET Scan. In
particular embodiments, the methods for treating cancer provided
herein reduce the tumor perfusion in a subject by an amount in the
range of about 5% to 20%, 10% to 20%, 10% to 30%, 15% to 40%, 15%
to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%,
30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 30% to 100%, or any
range in between, relative to tumor perfusion prior to
administration of platinum(II) complexes, as assessed by methods
well known in the art, e.g., MRI, DCE-MRI, or PET Scan.
[0057] In particular aspects, the methods for treating cancer
provided herein inhibit or decrease tumor metabolism in a subject
as assessed by methods well known in the art, e.g., PET scanning.
In specific embodiments, the methods for treating cancer provided
herein inhibit or decrease tumor metabolism in a subject by at
least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 80%, 85%, 90%, 95%, or 100%, relative to tumor metabolism
prior to administration of platinum(II) complexes, as assessed by
methods well known in the art, e.g., PET scanning. In particular
embodiments, the methods for treating cancer provided herein
inhibit or decrease tumor metabolism in a subject in the range of
about 5% to 20%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%,
20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to
80%, 30% to 90%, 30% to 95%, 30% to 99%, 30% to 100%, or any range
in between, relative to tumor metabolism prior to administration of
platinum(II) complexes, as assessed by methods well known in the
art, e.g., PET scan.
5.4 Patient Population
[0058] In some embodiments, a subject treated for cancer in
accordance with the methods provided herein is a human who has or
is diagnosed with cancer. In other embodiments, a subject treated
for cancer in accordance with the methods provided herein is a
human predisposed or susceptible to cancer. In some embodiments, a
subject treated for cancer in accordance with the methods provided
herein is a human at risk of developing cancer.
[0059] In one embodiment, a subject treated for cancer in
accordance with the methods provided herein is a human infant. In
another embodiment, a subject treated for cancer in accordance with
the methods provided herein is a human toddler. In another
embodiment, a subject treated for cancer in accordance with the
methods provided herein is a human child. In another embodiment, a
subject treated for cancer in accordance with the methods provided
herein is a human adult. In another embodiment, a subject treated
for cancer in accordance with the methods provided herein is a
middle-aged human. In another embodiment, a subject treated for
cancer in accordance with the methods provided herein is an elderly
human.
[0060] In certain embodiments, a subject treated for cancer in
accordance with the methods provided herein has a cancer that
metastasized to other areas of the body, such as the bones, lung
and liver. In certain embodiments, a subject treated for cancer in
accordance with the methods provided herein is in remission from
the cancer. In some embodiments, a subject treated for cancer in
accordance with the methods provided herein that has a recurrence
of the cancer. In certain embodiments, a subject treated in
accordance with the methods provided herein is experiencing
recurrence of one or more tumors associated with cancer.
[0061] In certain embodiments, a subject treated for cancer in
accordance with the methods provided herein is a human that is
about 1 to about 5 years old, about 5 to 10 years old, about 10 to
about 18 years old, about 18 to about 30 years old, about 25 to
about 35 years old, about 35 to about 45 years old, about 40 to
about 55 years old, about 50 to about 65 years old, about 60 to
about 75 years old, about 70 to about 85 years old, about 80 to
about 90 years old, about 90 to about 95 years old or about 95 to
about 100 years old, or any age in between. In a specific
embodiment, a subject treated for cancer in accordance with the
methods provided herein is a human that is 18 years old or older.
In a particular embodiment, a subject treated for cancer in
accordance with the methods provided herein is a human child that
is between the age of 1 year old to 18 years old. In a certain
embodiment, a subject treated for cancer in accordance with the
methods provided herein is a human that is between the age of 12
years old and 18 years old. In a certain embodiment, the subject is
a male human. In another embodiment, the subject is a female human.
In one embodiment, the subject is a female human that is not
pregnant or is not breastfeeding. In one embodiment, the subject is
a female that is pregnant or will/might become pregnant, or is
breast feeding.
[0062] In some embodiments, a subject treated for cancer in
accordance with the methods provided herein is administered
platinum(II) complexes or a pharmaceutical composition thereof, or
a combination therapy before any adverse effects or intolerance to
therapies other than the platinum(II) complexes develops. In some
embodiments, a subject treated for cancer in accordance with the
methods provided herein is a refractory patient. In a certain
embodiment, a refractory patient is a patient refractory to a
standard therapy (e.g., surgery, radiation, anti-androgen therapy
and/or drug therapy such as chemotherapy). In certain embodiments,
a patient with cancer is refractory to a therapy when the cancer
has not significantly been eradicated and/or the one or more
symptoms have not been significantly alleviated. The determination
of whether a patient is refractory can be made either in vivo or in
vitro by any method known in the art for assaying the effectiveness
of a treatment of cancer, using art-accepted meanings of
"refractory" in such a context. In various embodiments, a patient
with cancer is refractory when one or more tumors associated with
cancer, have not decreased or have increased. In various
embodiments, a patient with cancer is refractory when one or more
tumors metastasize and/or spread to another organ.
[0063] In some embodiments, a subject treated for cancer accordance
with the methods provided herein is a human that has proven
refractory to therapies other than treatment with platinum(II)
complexes, but is no longer on these therapies. In certain
embodiments, a subject treated for cancer in accordance with the
methods provided herein is a human already receiving one or more
conventional anti-cancer therapies, such as surgery, drug therapy
such as chemotherapy, anti-androgen therapy or radiation. Among
these patients are refractory patients, patients who are too young
for conventional therapies, and patients with recurring tumors
despite treatment with existing therapies.
5.5 Dosage
[0064] In one aspect, a method for treating cancer presented herein
involves the administration of a unit dosage of platinum(II)
complexes or a pharmaceutical composition thereof. The dosage may
be administered as often as determined effective (e.g., once, twice
or three times per day, every other day, once or twice per week,
biweekly or monthly). In certain embodiments, a method for treating
cancer presented herein involves the administration to a subject in
need thereof of a unit dose of platinum(II) complexes that can be
determined by one skilled in the art.
[0065] In some embodiments, a unit dose of platinum(II) complexes
or a pharmaceutical composition thereof is administered to a
subject once per day, twice per day, three times per day; once,
twice or three times every other day (i.e., on alternate days);
once, twice or three times every two days; once, twice or three
times every three days; once, twice or three times every four days;
once, twice or three times every five days; once, twice, or three
times once a week, biweekly or monthly, and the dosage may be
administered orally.
5.6 Combination Therapy
[0066] Presented herein are combination therapies for the treatment
of cancer which involve the administration of platinum(II)
complexes in combination with one or more additional therapies to a
subject in need thereof. In a specific embodiment, presented herein
are combination therapies for the treatment of cancer which involve
the administration of an effective amount of platinum(II) complexes
in combination with an effective amount of another therapy to a
subject in need thereof.
[0067] As used herein, the term "in combination," refers, in the
context of the administration of platinum(II) complexes, to the
administration of platinum(II) complexes prior to, concurrently
with, or subsequent to the administration of one or more additional
therapies (e.g., agents, surgery, or radiation) for use in treating
cancer. The use of the term "in combination" does not restrict the
order in which platinum(II) complexes and one or more additional
therapies are administered to a subject. In specific embodiments,
the interval of time between the administration of platinum(II)
complexes and the administration of one or more additional
therapies may be about 1-5 minutes, 1-30 minutes, 30 minutes to 60
minutes, 1 hour, 1-2 hours, 2-6 hours, 2-12 hours, 12-24 hours, 1-2
days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2
weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9
weeks, 10 weeks, 15 weeks, 20 weeks, 26 weeks, 52 weeks, 11-15
weeks, 15-20 weeks, 20-30 weeks, 30-40 weeks, 40-50 weeks, 1 month,
2 months, 3 months, 4 months 5 months, 6 months, 7 months, 8
months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years,
or any period of time in between. In certain embodiments,
platinum(II) complexes and one or more additional therapies are
administered less than 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks,
one month, 2 months, 3 months, 6 months, 1 year, 2 years, or 5
years apart.
[0068] In some embodiments, the combination therapies provided
herein involve administering platinum(II) complexes daily, and
administering one or more additional therapies once a week, once
every 2 weeks, once every 3 weeks, once every 4 weeks, once every
month, once every 2 months (e.g., approximately 8 weeks), once
every 3 months (e.g., approximately 12 weeks), or once every 4
months (e.g., approximately 16 weeks). In certain embodiments,
platinum(II) complexes and one or more additional therapies are
cyclically administered to a subject. Cycling therapy involves the
administration of platinum(II) complexes for a period of time,
followed by the administration of one or more additional therapies
for a period of time, and repeating this sequential administration.
In certain embodiments, cycling therapy may also include a period
of rest where platinum(II) complexes or the additional therapy is
not administered for a period of time (e.g., 2 days, 3 days, 4
days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, 10 weeks, 20 weeks, 1 month, 2 months, 3 months, 4 months, 5
months, 6 months, 7 months, 8 months, 9 months, 10 months, 11
months, 12 months, 2 years, or 3 years). In an embodiment, the
number of cycles administered is from 1 to 12 cycles, from 2 to 10
cycles, or from 2 to 8 cycles.
[0069] In some embodiments, the methods for treating cancer
provided herein comprise administering platinum(II) complexes as a
single agent for a period of time prior to administering the
platinum(II) complexes in combination with an additional therapy.
In certain embodiments, the methods for treating cancer provided
herein comprise administering an additional therapy alone for a
period of time prior to administering platinum(II) complexes in
combination with the additional therapy.
[0070] In some embodiments, the administration of platinum(II)
complexes and one or more additional therapies in accordance with
the methods presented herein have an additive effect relative the
administration of platinum(II) complexes or said one or more
additional therapies alone. In some embodiments, the administration
of platinum(II) complexes and one or more additional therapies in
accordance with the methods presented herein have a synergistic
effect relative to the administration of the Compound or said one
or more additional therapies alone.
[0071] As used herein, the term "synergistic," refers to the effect
of the administration of platinum(II) complexes in combination with
one or more additional therapies (e.g., agents), which combination
is more effective than the additive effects of any two or more
single therapies (e.g., agents). In a specific embodiment, a
synergistic effect of a combination therapy permits the use of
lower dosages (e.g., sub-optimal doses) of platinum(II) complexes
or an additional therapy and/or less frequent administration of
platinum(II) complexes or an additional therapy to a subject. In
certain embodiments, the ability to utilize lower dosages of
platinum(II) complexes or of an additional therapy and/or to
administer platinum(II) complexes or said additional therapy less
frequently reduces the toxicity associated with the administration
of platinum(II) complexes or of said additional therapy,
respectively, to a subject without reducing the efficacy of
platinum(II) complexes or of said additional therapy, respectively,
in the treatment of cancer. In some embodiments, a synergistic
effect results in improved efficacy of platinum(II) complexes and
each of said additional therapies in treating cancer. In some
embodiments, a synergistic effect of a combination of platinum(II)
complexes and one or more additional therapies avoids or reduces
adverse or unwanted side effects associated with the use of any
single therapy.
[0072] The combination of platinum(II) complexes and one or more
additional therapies can be administered to a subject in the same
pharmaceutical composition. Alternatively, platinum(II) complexes
and one or more additional therapies can be administered
concurrently to a subject in separate pharmaceutical compositions.
Platinum(II) complexes and one or more additional therapies can be
administered sequentially to a subject in separate pharmaceutical
compositions. Platinum(II) complexes and one or more additional
therapies may also be administered to a subject by the same or
different routes of administration.
[0073] The combination therapies provided herein involve
administering to a subject to in need thereof platinum(II)
complexes in combination with conventional, or known, therapies for
treating cancer. Other therapies for cancer or a condition
associated therewith are aimed at controlling or relieving one or
more symptoms. Accordingly, in some embodiments, the combination
therapies provided herein involve administering to a subject to in
need thereof a pain reliever, or other therapies aimed at
alleviating or controlling one or more symptoms associated with or
a condition associated therewith.
[0074] Specific examples of anti-cancer agents that may be used in
combination with platinum(II) complexes include: a hormonal agent
(e.g., aromatase inhibitor, selective estrogen receptor modulator
(SERM), and estrogen receptor antagonist), chemotherapeutic agent
(e.g., microtubule dissembly blocker, antimetabolite, topisomerase
inhibitor, and DNA crosslinker or damaging agent), anti-angiogenic
agent (e.g., VEGF antagonist, receptor antagonist, integrin
antagonist, vascular targeting agent (VTA)/vascular disrupting
agent (VDA)), radiation therapy, and conventional surgery.
[0075] Non-limiting examples of hormonal agents that may be used in
combination with platinum(II) complexes include aromatase
inhibitors, SERMs, and estrogen receptor antagonists. Hormonal
agents that are aromatase inhibitors may be steroidal or
nonsteroidal. Non-limiting examples of nonsteroidal hormonal agents
include letrozole, anastrozole, aminoglutethimide, fadrozole, and
vorozole. Non-limiting examples of steroidal hormonal agents
include aromasin (exemestane), formestane, and testolactone.
Non-limiting examples of hormonal agents that are SERMs include
tamoxifen (branded/marketed as Nolvadex.RTM.), afimoxifene,
arzoxifene, bazedoxifene, clomifene, femarelle, lasofoxifene,
ormeloxifene, raloxifene, and toremifene. Non-limiting examples of
hormonal agents that are estrogen receptor antagonists include
fulvestrant. Other hormonal agents include but are not limited to
abiraterone and lonaprisan.
[0076] Non-limiting examples of chemotherapeutic agents that may be
used in combination with platinum(II) complexes include microtubule
disasssembly blocker, antimetabolite, topisomerase inhibitor, and
DNA crosslinker or damaging agent. Chemotherapeutic agents that are
microtubule dissemby blockers include, but are not limited to,
taxenes (e.g., paclitaxel (branded/marketed as TAXOL.RTM.),
docetaxel, abraxane, larotaxel, ortataxel, and tesetaxel);
epothilones (e.g., ixabepilone); and vinca alkaloids (e.g.,
vinorelbine, vinblastine, vindesine, and vincristine
(branded/marketed as ONCOVIN.RTM.)).
[0077] Chemotherapeutic agents that are antimetabolites include,
but are not limited to, folate anitmetabolites (e.g., methotrexate,
aminopterin, pemetrexed, raltitrexed); purine antimetabolites
(e.g., cladribine, clofarabine, fludarabine, mercaptopurine,
pentostatin, thioguanine); pyrimidine antimetabolites (e.g.,
5-fluorouracil, capcitabine, gemcitabine (GEMZAR.RTM.), cytarabine,
decitabine, floxuridine, tegafur); and deoxyribonucleotide
antimetabolites (e.g., hydroxyurea).
[0078] Chemotherapeutic agents that are topoisomerase inhibitors
include, but are not limited to, class I (camptotheca)
topoisomerase inhibitors (e.g., topotecan (branded/marketed as
HYCAMTIN.RTM.) irinotecan, rubitecan, and belotecan); class II
(podophyllum) topoisomerase inhibitors (e.g., etoposide or VP-16,
and teniposide); anthracyclines (e.g., doxorubicin, epirubicin,
Doxil, aclarubicin, amrubicin, daunorubicin, idarubicin,
pirarubicin, valrubicin, and zorubicin); and anthracenediones
(e.g., mitoxantrone, and pixantrone).
[0079] Chemotherapeutic agents that are DNA crosslinkers (or DNA
damaging agents) include, but are not limited to, alkylating agents
(e.g., cyclophosphamide, mechlorethamine, ifosfamide
(branded/marketed as IFEX.RTM.), trofosfamide, chlorambucil,
melphalan, prednimustine, bendamustine, uramustine, estramustine,
carmustine (branded/marketed as BiCNU.RTM.), lomustine, semustine,
fotemustine, nimustine, ranimustine, streptozocin, busulfan,
mannosulfan, treosulfan, carboquone,
N,N'N'-triethylenethiophosphoramide, triaziquone,
triethylenemelamine); alkylating-like agents (e.g., carboplatin
(branded/marketed as PARAPLATIN.RTM.), cisplatin, oxaliplatin,
nedaplatin, triplatin tetranitrate, satraplatin, picoplatin);
nonclassical DNA crosslinkers (e.g., procarbazine, dacarbazine,
temozolomide (branded/marketed as TEMODAR.RTM.), altretamine,
mitobronitol); and intercalating agents (e.g., actinomycin,
bleomycin, mitomycin, and plicamycin).
[0080] Non-limiting examples of other therapies that may be
administered to a subject in combination with platinum(II)
complexes include:
[0081] (1) a statin such as lovostatin (e.g., branded/marketed as
MEVACOR.RTM.);
[0082] (2) an mTOR inhibitor such as sirolimus which is also known
as Rapamycin (e.g., branded/marketed as RAPAMUNE.RTM.),
temsirolimus (e.g., branded/marketed as TORISEL.RTM.), evorolimus
(e.g., branded/marketed as AFINITOR.RTM.), and deforolimus;
[0083] (3) a farnesyltransferase inhibitor agent such as
tipifarnib;
[0084] (4) an antifibrotic agent such as pirfenidone;
[0085] (5) a pegylated interferon such as PEG-interferon
alfa-2b;
[0086] (6) a CNS stimulant such as methylphenidate
(branded/marketed as RITALIN.RTM.);
[0087] (7) a HER-2 antagonist such as anti-HER-2 antibody (e.g.,
trastuzumab) and kinase inhibitor (e.g., lapatinib);
[0088] (8) an IGF-1 antagonist such as an anti-IGF-1 antibody
(e.g., AVE1642 and IMC-A 1) or an IGF-1 kinase inhibitor;
[0089] (9) EGFR/HER-1 antagonist such as an anti-EGFR antibody
(e.g., cetuximab, panitumamab) or EGFR kinase inhibitor (e.g.,
erlotinib; gefitinib);
[0090] (10) SRC antagonist such as bosutinib;
[0091] (11) cyclin dependent kinase (CDK) inhibitor such as
seliciclib;
[0092] (12) Janus kinase 2 inhibitor such as lestaurtinib;
[0093] (13) proteasome inhibitor such as bortezomib;
[0094] (14) phosphodiesterase inhibitor such as anagrelide;
[0095] (15) inosine monophosphate dehydrogenase inhibitor such as
tiazofurine;
[0096] (16) lipoxygenase inhibitor such as masoprocol;
[0097] (17) endothelin antagonist;
[0098] (18) retinoid receptor antagonist such as tretinoin or
alitretinoin;
[0099] (19) immune modulator such as lenalidomide, pomalidomide, or
thalidomide;
[0100] (20) kinase (e.g., tyrosine kinase) inhibitor such as
imatinib, dasatinib, erlotinib, nilotinib, gefitinib, sorafenib,
sunitinib, lapatinib, or TG100801;
[0101] (21) non-steroidal anti-inflammatory agent such as celecoxib
(branded/marketed as CELEBREX.RTM.);
[0102] (22) human granulocyte colony-stimulating factor (G-CSF)
such as filgrastim (branded/marketed as NEUPOGEN.RTM.);
[0103] (23) folinic acid or leucovorin calcium;
[0104] (24) integrin antagonist such as an integrin
.alpha.5.beta.1-antagonist (e.g., JSM6427);
[0105] (25) nuclear factor kappa beta (NF-.kappa..beta.) antagonist
such as OT-551, which is also an anti-oxidant;
[0106] (26) hedgehog inhibitor such as CUR61414, cyclopamine,
GDC-0449, and anti-hedgehog antibody;
[0107] (27) histone deacetylase (HDAC) inhibitor such as SAHA (also
known as vorinostat (branded/marketed as ZOLINZA)), PCI-24781,
SB939, CHR-3996, CRA-024781, ITF2357, JNJ-26481585, or
PCI-24781;
[0108] (28) retinoid such as isotretinoin (e.g., branded/marketed
as ACCUTANE.RTM.);
[0109] (29) hepatocyte growth factor/scatter factor (HGF/SF)
antagonist such as HGF/SF monoclonal antibody (e.g., AMG 102);
[0110] (30) synthetic chemical such as antineoplaston;
[0111] (31) anti-diabetic such as rosaiglitazone (e.g.,
branded/marketed as AVANDIA.RTM.);
[0112] (32) antimalarial and amebicidal drug such as chloroquine
(e.g., branded/marketed as ARALEN.RTM.);
[0113] (33) synthetic bradykinin such as RMP-7;
[0114] (34) platelet-derived growth factor receptor inhibitor such
as SU-101;
[0115] (35) receptor tyrosine kinase inhibitorsof Flk-1/KDR/VEGFR2,
FGFR1 and PDGFR beta such as SU5416 and SU6668;
[0116] (36) anti-inflammatory agent such as sulfasalazine (e.g.,
branded/marketed as AZULFIDINE.RTM.); and
[0117] (37) TGF-beta antisense therapy.
6 EXAMPLES
Example 6.1: Preparation and Characterization of the NHC
Complexes
[0118] The following examples illustrate the synthesis and
characterization of the platinum(II) complexes.
[0119] [Pt(BPI)(NHC)](OTf) with different alkyl chains and aromatic
groups on the NHC ligands were prepared by refluxing
[Pt(BPI)Cl].sup.[25] with corresponding imidazolium salt in the
presence of base (FIG. 1; See Supporting Information for
experimental details and characterization data). The structure of
1a was further examined by X-ray crystallography (FIG. 2 and Table
1) and the NHC ligand was found to be perpendicular to the plane of
BPI ligand with bond angle (C19-Pt1-N5) of 90.6.degree..
TABLE-US-00001 TABLE 1 Crystal data and structure refinement data
for 1a. Identification code 1a Empirical formula
C.sub.23H.sub.20N.sub.7Pt CF.sub.3O.sub.3S* Formula weight 738.62
Temperature/K 100 Crystal system monoclinic Space group C2/c
a/.ANG. 24.5594 (9) b/.ANG. 14.9575 (6) c/.ANG. 15.7468 (6)
.alpha./.degree. 90.00 .beta./.degree. 119.678 (1).degree.
.gamma./.degree. 90.00 Volume/.ANG..sup.3 5025.7 (3) Z 8
.rho..sub.calc g/cm.sup.3 1.515 .mu./mm.sup.-1 11.80 F(000) 2864.0
Crystal size/mm.sup.3 0.06 .times. 0.02 .times. 0.02 Radiation
CuK.alpha. (.lamda. = 1.54178) 2.THETA. range for data
collection/.degree. 3.60 to 66.7 Index ranges -29 .ltoreq. h
.ltoreq. 28, -14 .ltoreq. k .ltoreq. 17, -18 .ltoreq. 1 .ltoreq. 18
Reflections collected 4152 Independent reflections 4152 [R.sub.int
= 0.083] Data/restraints/parameters 4390/11/380 Goodness-of-fit of
F.sup.2 1.07 Final R indexes [I >= 2.sigma. (I)] R.sub.1 =
0.054, wR.sub.2 = 0.148 Largest diff. peak/hole/e.ANG..sup.-3
1.37/-1.25 *Satisfactory disorder models for the solvent and
another triflic
[0120] The UV-visible absorption data and spectra of 1a-1j were
depicted in Table 2 and FIG. 3(A). The absorption spectra of
CH.sub.2Cl.sub.2 solutions of 1b, 1i and 1j showed intense
absorptions at 400-550 nm, arising primarily from .pi..fwdarw..pi.*
(L) intraligand (IL) and 5 d (Pt).fwdarw..pi.* (L) metal-to-ligand
charge transfer (MLCT) transitions..sup.[25] Upon photoexcitation,
1b, 1i and 1j in degassed CH.sub.2Cl.sub.2 displayed vibronic
structured emission spectra with emission maxima at 588 nm
(.PHI.=0.027, .tau.=5.3 .mu.s), 627 nm (.PHI.=0.005, .tau.=1.1
.mu.s) and 566 nm (.PHI.=0.12, .tau.=10.9 .mu.s), respectively
(FIG. 3(B)). The alkyl chain length of the NHC ligands was found to
not affect photophysical properties of the complexes significantly
(Tables 2 and 3).
TABLE-US-00002 TABLE 2 UV-visible absorption data of 1a-1j (2
.times. 10.sup.-5 mol dm.sup.-1 in CH.sub.2Cl.sub.2) Complex
.lamda..sub.abs/nm ( /dm.sup.3mol.sup.-1cm.sup.-1) 1a 247 (49186),
275 (24960), 343 (25141), 370 (11450), 408 (8265), 434 (17922), 461
(22286) 1b 247 (36940), 276 (18928), 344 (19119), 372 (9195), 406
(6510), 434 (13736), 462 (16941) 1c 247 (41946), 277 (21053), 344
(21986), 370 (10352), 408 (7396), 434 (15708), 462 (19576) 1d 247
(51933), 276 (26519), 344 (27180), 370 (12741), 408 (9239), 434
(19590), 462 (24388) 1e 247 (47280), 276 (24119), 344 (24650), 370
(11414), 408 (8207), 434 (17658), 462 (22041) 1f 247 (47522), 273
(23426), 343 (24338), 370 (11373), 408 (8365), 434 (17422), 462
(21681) 1g 247 (45308), 275 (21894), 344 (23816), 370 (11515), 408
(8674), 434 (17518), 462 (21396) 1h 248 (67601), 331 (20472), 346
(22531), 435 (14407), 459(15491) 1i 248 (42466), 277 (27255), 345
(16922), 386 (9580), 481 (10084) 1j 286 (53202), 297 (55807), 346
(21484), 405 (32808), 421 (36269), 450 (31028)
TABLE-US-00003 TABLE 3 Summary of emission data. Photoluminescence
.lamda..sub.max (nm) quantum yield (.PHI.) Lifetime (.tau.; .mu.s)
1a 588 0.029 5.5 1b 588 0.027 5.3 1c 588 0.028 5.0 d 588 0.03 5.9
1e 588 0.029 5.9 1f 588 0.023 5.7 1g 588 0.024 5.1 1h 630 0.001 0.4
1i 627 0.005 1.1 1j 566 0.12 10.9
[0121] As compared with the chloro-precursor complex [Pt(BPI)Cl]
(1i), both the absorption and emission spectra of platinum(II)
complexes with NHC ligands (1b and 1j) displayed distinct
blue-shifts (FIG. 3). With reference to previous spectroscopic work
on related platinum(II) complexes,.sup.[26] the observed
blue-shifts in [Pt(BPI)(NHC)].sup.+ were probably attributed to an
enhanced contribution from the .sup.3IL state and a reduced
.sup.3MLCT character. On the other hand, 1j, which has an extended
.pi.-conjugation through benzannulation of the pincer ligand,
showed blue-shift in both the absorption and emission spectra, as
compared to that of 1f (Table 2 and 3). This can be rationalized by
destabilization of the LUMO with successive expansion of the
.pi.-system of the pyrrolate moieties, as supported by similar
finding reported previously..sup.[27]
[0122] Interestingly, in one embodiment, proteomics data and in
vitro biochemical assays at sub-cytotoxic concentrations reveal
that a representative complex, 1b, can regulate uPA/uPAR-mediated
and VEGF-induced angiogenic pathways. Ex vivo anti-angiogenic
properties of 1b is further demonstrated by chorioallantoic
membrane (CAM) assay. More importantly, treatment of nude mice
bearing highly metastatic MDA-MB231 xenograft by 1b show
significant reduction in tumor volume. Immunohistochemical analysis
of tumor tissues from treated mice supports the promising in vivo
antitumor as well as anti-angiogenic activities of 1b, while blood
biochemistry reveal minimal systemic toxicity found in the treated
mice. All of these results indicate that these dual cytotoxic and
anti-angiogenic platinum(II) complexes are useful for treating
cancer, including non-curable highly metastatic cancer.
[0123] The luminescence properties of [Pt(BPI)(NHC)].sup.+ in live
cells were also examined. After treating human cervical epithelial
carcinoma (HeLa) cells with 1b (5 .mu.M) for 15 min, a strong green
luminescence was observed from cytoplasm of the cells (FIG. 4(A)),
demonstrating the readiness of monitoring cellular uptake and
localization of 1b by its strong luminescence in vitro. The exact
subcellular location of 1b was investigated by co-staining with
organelle-specific probes. It was noted that 1b specifically
localized in ER, as supported by high Pearson's correlation
coefficient for co-localization between 1b and ER-Tracker.TM.
(0.83; FIG. 4(A)). Control experiment showed that there was no
background signal in the rhodamine and FITC channel when the cells
were treated by 1b and ER-Tracker.TM. respectively (FIG. 5). In
addition, no significant co-localization between 1b and
mitochondria-specific Mitotracker.RTM. or lysosome-specific
Lysotracker.RTM. was observed (Pearson's correlation
coefficient=0.45 and 0.55 respectively; FIG. 6), indicating that 1b
was preferentially accumulated in ER of HeLa cells
[0124] In view of the enormous success of platinum(II) compounds
for anti-cancer treatment,.sup.[28] in vitro cytotoxicity of
[Pt(BPI)(NHC)].sup.+ towards various cancer cell lines including
HeLa, colon carcinoma (HCT116), lung cancer (NCI-H460) and highly
invasive triple-negative breast cancer (MDA-MB-231), as well as
non-tumorigenic immortalized human hepatocyte (MIHA) were examined.
1a-1h were cytotoxic against the cancer cells with IC.sub.50 (dose
required to inhibit 50% of cellular growth) ranging from
0.14.+-.0.01 to 18.21.+-.1.52 .mu.M after 72 h treatment. They were
found to be more potent in killing most of the cancer cells
investigated than cisplatin (11.75.+-.1.36 to 77.19.+-.7.82 .mu.M).
Among these platinum(II) complexes, 1b displayed relatively higher
cytotoxicity towards NCI-H460 and HCT116 cells than that towards
non-tumorigenic MIHA cells (16- and 19-fold difference in IC.sub.50
values respectively; Table 4), suggesting its selectivity on
killing cancer cells over non-tumorigenic cells. As a result, 1b
was selected as a target compound and its anti-cancer properties
were further investigated.
TABLE-US-00004 TABLE 4 In vitro cytotoxicity of 1a-1h against human
cell lines of HeLa, NCI- H460, HCT116, MDA-MB-231 and MiHa. The
IC.sub.50 (.mu.M) was determined by MTT assay upon incubation of
the live cells with the complexes for 72 h. MDA-MB- HeLa NCI-H460
HCT116 231 MiHa 1a 5.45 .+-. 0.52 2.72 .+-. 0.56 1.19 .+-. 0.06
6.39 .+-. 0.53 7.32 .+-. 3.65 1b 1.63 .+-. 0.85 0.28 .+-. 0.18 0.23
.+-. 0.02 2.34 .+-. 0.19 4.46 .+-. 0.97 1c 1.56 .+-. 0.28 0.16 .+-.
0.14 0.14 .+-. 0.01 1.62 .+-. 0.16 0.27 .+-. 0.11 1d 3.25 .+-. 0.43
1.16 .+-. 0.11 0.39 .+-. 0.06 4.44 .+-. 0.15 1.04 .+-. 0.59 1e 2.05
.+-. 0.49 2.25 .+-. 0.06 2.87 .+-. 0.41 7.45 .+-. 0.50 3.77 .+-.
1.16 1f 2.23 .+-. 0.29 1.68 .+-. 0.33 0.49 .+-. 0.10 3.78 .+-. 0.48
1.97 .+-. 1.06 1g 3.43 .+-. 0.22 0.90 .+-. 0.06 1.55 .+-. 0.38 4.15
.+-. 1.03 2.13 .+-. 0.96 1h 14.63 .+-. 1.32 13.2 .+-. 1.23 7.46
.+-. 0.97 18.21 .+-. 1.52 27.46 .+-. 12.80 Cis- 12.90 .+-. 3.84
24.9 .+-. 3.19 11.75 .+-. 1.36 77.19 .+-. 7.82 >100 platin
[0125] Since 1b was found to accumulate in ER domain as revealed by
confocal fluorescence microscopy images (FIG. 4(A)), this prompted
us to investigate any ER stress induced by 1b that accounted for
its high cytotoxicity towards cancer cells. Western blotting
analysis showed a significant up-regulation of phosphorylated
RNA-dependent protein kinase-like endoplasmic reticulum kinase
(PERK) upon treatment of MDA-MB-231 cells with 1b (5 uM) for 6, 12,
24 and 48 h (FIG. 4(B)). Also, phosphorylated eukaryotic initiation
factor 2.alpha. (eIF2.alpha.) and C/EBP homologous protein (CHOP)
were also found to be stimulated under same conditions, suggesting
that 1b could induce ER stress..sup.[29,30]
[0126] In addition to ER stress, apoptosis-related protein such as
poly(ADP-ribose) polymerase (PARP) and caspases 3 and 9 were
cleaved in MDA-MB-231 cells treated with 1b for 48 h (FIG. 4(C)),
indicative of cell apoptosis. Cell cycle analysis of MDA-MB-231
cells treated with 1b for 24 h revealed a marked accumulation in
the G0/G1 phase from 36.4% to 64% (FIG. 7(B)). The G0/G1 cell-cycle
arrest was associated with stimulation of p15 expression and
down-regulation of cyclin D1/D3 and CDK 4/6, as indicated by
western blot analysis (FIG. 7(C)). In addition, there was
dose-dependent increase (up to 8-fold) of cell population in sub-G1
phase (FIG. 7(A)), and this was a hallmark of apoptosis owing to
DNA fragmentation. On the other hand, JC1 staining.sup.[31] of
cells treated with 1b showed a decrease in ratios of orange to
green fluorescence (1580/1530) with increasing dosage of 1b (FIGS.
8(A)-(D)). This indicated that 1b could induce mitochondria
dysfunction. Collectively, in vitro assays confirm that 1b at its
cytotoxic concentrations could induce ER stress, nuclear
fragmentation and mitochondria dysfunction, leading to subsequent
apoptotic events.
[0127] To obtain a holistic insight into the mechanism of action of
1b, proteomic analysis on 1b-treated MDA-MB-231 cells was performed
using HPLC-LTQ-Orbitrap MS. A bioinformatics analysis of the
proteomic data showed that CHIP (c-terminal Hsp70-interacting
protein), c-Kit and Von Hippel-Lindau (VHL) protein pathway were
one of the most predominantly modulated pathways in MDA-MB-231
cells treated with 1b (5 .mu.M) for 5 h with high statistical
significance (Table 5). Interestingly, these three pathways were
related to angiogenic responses of cancer cells. The expression
level of CHIP is negatively correlated with VEGFR2 which is an
important receptor for initiation of angiogenesis;.sup.[32] Von
Hippel-Lindau (VHL) protein is capable of suppressing tumor growth
through down-regulation of a number of angiogenic factors;.sup.[33]
c-kit receptor regulate angiogenesis by PI3K/Akt downstream
signaling pathway..sup.[34]
[0128] In view of the regulation of angiogenesis-related pathways
by 1b as identified by proteomic data, anti-angiogenic and
anti-metastatic properties of 1b were evaluated. Wound closure
assays showed that 1b effectively inhibited migration of MDA-MB-231
cells at sub-cytotoxic concentrations (0.25-1 .mu.M) after a 24 h
treatment in a concentration- and time-dependent manner (FIGS. 9(A)
and 9(B), and 10); this effect was not due to the cytotoxicity of
1b as the cells were found to have insignificant growth inhibition
under these concentrations of 1b (FIG. 11). On the other hand,
transwell invasive assays revealed significant inhibition of
invasion of MDA-MB-231 cells by 1b at its sub-cytotoxic
concentrations after 24 h treatment (FIGS. 9(C) and (D)). In tube
formation assay, 1b displayed significant inhibition on the
angiogenesis of MS1 cells, as indicated by the loss of ability of
the endothelial cells to form three-dimensional tube-like
structures after treatment with 1b for 3 h at sub-cytotoxic
concentrations (>90% cells remained viable; FIGS. 12(A)-(B)).
All these data suggest that 1b not only can induce apoptosis and
cell cycle arrest at its cytotoxic concentrations, but also can
inhibit metastasis of highly invasive MDA-MB-231 at its
sub-cytotoxic concentrations.
TABLE-US-00005 TABLE 5 The seven signaling pathways showing highest
-log(p-value) in proteomic analysis of MDA-MB-231 cells treated
with 1b (5 .mu.M). Pathways Score p-value HIF-1alpha pathway 9.17
7.12E-05 TGF beta pathway 9.00 1.06E-04 CHIP---/Pael-R 8.71
2.08E-04 c-Kit pathway 8.22 6.42E-04 RSK1 --> MITF{pSer}{ub}
7.99 1.08E-03 VHL --> HIF-1alphadegradation 7.92 1.26E-03
Plk1cellcycleregulation 7.73 1.97E-03
[0129] To gain better insight into the anti-angiogenic properties
of 1b, effects of 1b on uPA/uPAR system were first investigated, as
this system has been found to play crucial roles in growth,
metastasis and angiogenesis of many solid malignancies, e.g. by
activation of MMPs for ECM degradation and triggering downstream
intracellular signaling for metastasis..sup.[10,35] Western
blotting experiments showed that the expression level of uPA and
MMP-9 (examples of MMPs) decreased significantly when MDA-MB-231
cells were treated with increasing concentration of 1b (FIG. 13(A))
or increasing incubation time of 1b (FIG. 13(B)). In contrast, the
expression of TIMP-1, a tissue inhibitor of MMPs,.sup.[36]
moderately increased after treatment with 1b (FIG. 13(A)). These
results suggests that 1b could slow down uPA/uPAR-mediated ECM
proteolysis process, thus inhibiting tumor progression.
[0130] In addition to the regulation of proteolysis, 1b was found
to significantly suppress phosphorylation of focal adhesion kinase
(FAK) at the site of Tyr397, and moderately inhibited levels of
phosphorylation of FAK at Tyr925 and Tyr576/577, while exhibited
little effect on total protein level of FAK (FIGS. 13(C) and (D)).
Moreover, 1b significantly inhibited phosphorylation of ERK and Akt
in a dose- and time-dependent manner (FIGS. 13(C) and (D)).
PI3K/Akt.sup.[37] and Ras/MEK/ERK.sup.[38] protein kinase pathways
have been reported to be downstream signaling pathways after
stimulation of FAK upon formation of integrin and uPAR complex, and
these pathways can facilitate cell invasion and proliferation.
Notably, PI3K/Akt pathway is also the downstream signaling pathway
of c-kit receptor which was identified as one of the predominantly
modulated pathways in proteomic studies of MDA-MB-231 cells treated
with 1b.
[0131] The effects of 1b, LY294002 (PI3K inhibitor).sup.[39] and
U0126 (ERK inhibitor).sup.[40] on PI3K and ERK signaling pathways
were further investigated. LY294002, U0126 or 1b alone displayed
insignificant cell growth inhibitory effects (1b is at
sub-cytotoxic concentration; FIG. 13(E)), but notable inhibition on
the migration and invasion of MDA-MB-231 cells (FIGS. 13(F) and
(G)). Co-treatment of MDA-MB-231 cells with 1b, and LY294002 or
U0126, was found to inhibit cell growth, migration and invasion
significantly (FIGS. 13(E), (F) and (G)). All these data indicate
the anti-angiogenic properties of 1b by inhibiting
uPA/uPAR-mediated ECM proteolysis and downstream intracellular
signaling for metastasis.
[0132] Vascular endothelial growth factor (VEGF) is another
critical mediator of angiogenesis and regulates most of the steps
in angiogenic cascade, including proliferation, migration and tube
formation of endothelial cells..sup.[41,42] Previous studies
demonstrated that MMP-9 and uPA were able to facilitate degradation
of ECM, leading to release or activation of VEGF, thus promoting
tumor growth and angiogenesis..sup.[43-45] Therefore, effect of 1b
on secretion level of VEGF in the culture media of MDA-MB-231 cells
was investigated by Quantikine.RTM. ELISA kit. It was found that
VEGF secretion level was reduced by 50% upon treating MDA-MB-231
cells with 1b for 24 h, as compared to the untreated cells (FIG.
14(A)).
[0133] To further validate anti-angiogenic properties of 1b,
effects of 1b on another aggressive cell line, human umbilical vein
endothelial cell (HUVEC) line, were studied. HUVEC is a
well-established cell line for studying angiogenesis..sup.[46] It
was found that exposure of HUVECs to VEGF (50 ng/mL) lead to
promoted cell growth, migration, invasion and tube formation (FIGS.
14(B)-(D)). In order to mimic the tumor microenvironment in vitro,
the conditioned media (CM) of MDA-MB-231 cells containing VEGF
(13.4 ng/mL) was collected for incubation of HUVECs. As shown in
FIGS. 14(B)-14(E), 1b significantly suppressed CM-mediated
migration, invasion and tube formation of HUVECs at sub-cytotoxic
concentrations. More interestingly, chorioallantoic membrane (CAM)
assay.sup.[43] showed effective ex vivo anti-angiogenic properties
of 1b (FIG. 14(F)). Taken together, these results demonstrate that
1b not only can inhibit VEGF secretion from tumor cells, but also
suppress VEGF-mediated angiogenesis.
[0134] With the promising dual cytotoxic and anti-angiogenic
properties of 1b, in vivo antitumor activity of 1b was
investigated. Treatment of nude mice bearing MDA-MB-231 xenografts
with 1b through intravenous injection once per two days resulted in
a significant growth inhibition by 81 and 64% at concentration of 5
mg/kg and 2.5 mg/kg respectively, as compared to mice treated with
solvent control (both with p<0.01; FIG. 15(A)). Importantly, 1b
did not cause any death or body weight loss of mice during the
treatment (FIG. 15(B)). Immunohistochemical analysis of tumor
tissues from treated mice revealed decrease in expression of Ki67
as compared to those from control mice, suggesting the promising
anti-proliferation effect of 1b (FIG. 15(C)). In addition, higher
levels of caspase-3 and DNA fragmentation (as shown by TUNEL assay)
were found in tumor tissues of treated mice, demonstrating the
effective killing of cancer cells by 1b in vivo (FIG. 15(C)). More
interestingly, CD34 staining, which can serve as marker for in vivo
angiogenesis,.sup.[44] showed significant inhibition of blood
vessel formation in tumors of treated mice (FIG. 15(C)). Taken
together, 1b should be the first platinum(II) compound, according
to the best of our knowledge, showing anti-proliferation,
anticancer and anti-angiogenic effects in vivo.
[0135] As a promising candidate for treating cancer in vivo, we
further investigate systemic toxicity from 1b. Blood biochemistry
of nude mice after treatment with 1b (5 mg/kg, 2.5 mg/kg) showed
low systemic toxicity of 1b (FIGS. 15(D)-(H)); plasma levels of
several organ damage indicators including aspartate
aminotransferase (AST), creatine kinase (CK) and blood fat (CHOL)
of the treated mice were lower than those of the untreated mice
bearing MDA-MB-231 xenografts (p<0.05; FIGS. 15(D), (E), (F)),
fell within the statistically relevant range of those of mice
without the xenografts. On the other hand, treated mice showed
higher level of blood urea nitrogen (BUN) and blood glucose (GLU)
level than untreated mice bearing MDA-MB-231 xenografts (FIGS.
15(G), (H)), suggesting that the treatment helped to recover the
BUN and GLU level to almost healthy level.
[0136] Discussion
[0137] Disclosed herein are dual cytotoxic and anti-angiogenic
compounds should be new candidates for treating aggressive and
highly metastatic cancers which are almost non-curable in this
moment. Although platinum(II) compounds are known for their good
potency in killing cancer cells, it is quite surprising that
anti-angiogenic platinum(II) compounds are less well known and none
of the reported platinum(II) compounds demonstrate dual cytotoxic
and anti-angiogenic activities in vivo. This can be due to the
strong binding of square-planar platinum(II) compounds onto DNA,
thus they show less tendency to interact with other biomolecules in
tumor microenvironment and hence they are not anti-angiogenic. In
order to target other biomolecules, NHC ligands were introduced
onto the complexes in order to weaken the interactions of the
complexes with DNA by the out-of-plane NHC ligands. Proteomics data
and in vitro biochemical assays indicate significant effect of 1b
on uPA/uPAR- and VEGF-mediated signaling pathway, further
suggesting that 1b can likely interact with biomolecules in tumor
microenvironment.
[0138] In addition, since NHC is a strong G-donor and can increase
the energy level of non-emissive ligand-field (LF) state, the
platinum(II) NHC complexes are strongly luminescent and displaying
vibronic structured emission spectra upon photo
excitation..sup.[24] Due to their luminescence properties, cellular
distribution of the complexes can be examined by confocal
fluorescence spectroscopy. 1b is found to preferentially accumulate
in ER domain of HeLa cells and this information helped us to
unravel the mechanism of action of these anticancer complexes:
induction of ER stress and subsequent apoptotic cell death. Also,
it is conceivable that such strong luminescence can render the
metal complexes being both diagnostic and therapeutic agents, i.e.
theranostics, and hence real-time monitoring of treatment by the
complexes can be feasible.
[0139] The promising in vitro and in vivo anticancer activities of
this class of platinum(II) complexes are attributable to their dual
cytotoxic and anti-angiogenic properties. Their high cytotoxicity
against a panel of cancer cells, including highly metastatic
MDA-MB-231, can be explained by induction of ER stress, as
supported by up regulation of phosphorylated RNA-dependent protein
kinase-like endoplasmic reticulum kinase (PERK), phosphorylated
eukaryotic initiation factor 2.alpha. (eIF2.alpha.) and C/EBP
homologous protein (CHOP) in western blotting analysis. In
addition, DNA fragmentation and mitochondria dysfunction are found
in the treated cells, as shown by G0/G1 cell-cycle arrest and
increase of cell population in sub-G1 phase, and JC1 staining assay
respectively. On the other hand, the anti-angiogenic properties of
the complexes are first revealed by proteomic analysis on
1b-treated MDA-MB-231 cells, showing that CHIP, c-Kit and VHL
protein pathway are the most predominantly modulated pathways and
they are all closely related to angiogenic responses of cancer
cells. The involvement of 1b in regulation of angiogenesis is
further supported by western blotting and in vitro experiments
including wound closure assay, transwell invasive and tube
formation assay. More importantly, 1b demonstrates excellent ex
vivo and in vivo anti-angiogenic properties in chorioallantoic
membrane (CAM) assay and immunohistochemical analysis of tumor
tissues from treated mice bearing MDA-MB-231 xenograft,
respectively. Taken together, treatment of mice bearing highly
metastatic MDA-MB-231 xenograft by 1b results in remarkable
inhibition of tumor growth, and anti-proliferation and anticancer
effects as found in immunohistochemical analysis. Interestingly,
there is no significant loss in body weight or death of mice
throughout the treatment and blood biochemistry indicates low
systemic toxicity of 1b, further demonstrating the potential of
this class of [Pt(BPI)(NHC)].sup.+ complexes for treating highly
metastatic cancers. The promising dual cytotoxic and
anti-angiogenic effects in vivo should be firstly found in
platinum(II) compounds, according to the best of our knowledge, and
the unique chemical structure of the complexes with out-of-plane
NHC ligands for prohibiting strong interactions with DNA can
probably accounted for the dual properties.
[0140] Dual cytotoxic and anti-angiogenic [Pt(BPI)(NHC)](OTf) were
synthesized and characterized. They were cytotoxic against various
cancer cells, and this was ascribed to apoptotic cell death induced
by ER-stress, mitochondria dysfunction and cell cycle arrest.
Proteomic data indicated regulation of angiogenesis by the
platinum(II) complexes. Such unique feature allows these complexes
to slow down extracellular matrix proteolysis process by inhibiting
uPA and MMP expressions. Also, this could inhibit downstream
signaling pathways of uPA/uPAR and protect native VEGF from
cleavage, thereby accounting for the promising in vitro and ex vivo
anti-angiogenic properties of the complexes (FIG. 16). Significant
inhibition of in vivo tumor growth in nude mice bearing MDA-MB-231
xenografts by 1b was also demonstrated, with minimal systemic
toxicity found as indicated by blood biochemistry.
Immunohistochemical analysis of tumor tissues from treated mice
revealed promising anti-proliferation, anticancer and
anti-angiogenic effects of 1b in vivo.
6.2 Materials and Methods
[0141] All of the starting materials for synthesis came from
commercially available resources such as Sigma Aldrich, Alfa Aesar
and Apollo Scientific companies. The solvents used were at least in
analytical grade. Elemental analysis was done by Dr. Zong of the
Institute of Chemistry at the Chinese Academy of Science located in
Beijing. 1H NMR spectra were recorded on Bruker FT-400M Hz or 300M
Hz NMR spectrometers with tetramethylsilane as the reference. Fast
atom bombardment (FAB) mass spectra were obtained on a Finnigan Mat
95 mass spectrometer. Perkin-Elmer Lambda 19 UV-vis
spectrophotometer was used for UV-vis spectral analysis.
[0142] Fluorescence images were taken using Carl Zeiss LSM 510
Meta/Axiocam confocal microscopy. For MTT and protein assays, the
absorbance was quantified by using a Perkin Elmer Fusion Reader
(Packard BioScience Company).
[0143] The BPI (1,3-Bis(2-pyridylimino)isoindoline), benz(f)BPI
(3-Bis(2-pyridylimino)benz(f)isoindoline) and Pt(BPI)Cl were
synthesized according to reported procedures. [47]
6.3 Compound Characterization
Synthesis of 1,3-Bis(2-pyridylimino)isoindoline (BPI)
##STR00007##
[0145] A mixture of phthalonitrile (1 g, 7.81 mmol),
2-aminopyridine (1.47 g, 15.62 mmol), and CaCl.sub.2 (78.6 mg, 0.7
mmol) in 1-butanol (50 mL) was refluxed for 1 day. After cooling to
room temperature, the resulting pale yellow precipitate was
filtered off. Then, the crude product was purified by
chromatography on a silica gel column using
dichloromethane/methanol (200:1, v/v) as the eluent. Yield:
54%.
Synthesis of 3-Bis(2-Pyridylimino)Benz(f)Isoindoline
(Benz(f)BPI)
##STR00008##
[0147] A mixture of naphthalene-2,3-dicarbonitrile (1.39 g, 7.81
mmol), 2-aminopyridine (1.47 g, 15.62 mmol), and CaCl.sub.2 (78.6
mg, 0.7 mmol) in 1-butanol (50 mL) was refluxed for 5 days. After
cooling to room temperature, the resulting pale yellow precipitate
was extracted to dichloromethane layer. Then, the crude product was
purified by chromatography on a silica gel column using hexane:
ethyl acetate (5:1, v/v) as the eluent. Yield: 43%. .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta.=8.65-8.64 (m, 2H), 8.60 (s, 2H),
8.09-8.06 (m, 2H), 7.81-7.77 (m, 2H), 7.64-7.61 (m, 2H), 7.51-7.49
(m, 2H), 7.16-7.13 (m, 2H).
Synthesis of Pt(BPI)Cl (1i)
##STR00009##
[0149] Silver triflate (0.138 g, 0.54 mmol) was added dropwise into
Pt(COD)Cl.sub.2 (0.1 g, 0.26 mmol) in methanol (15 mL). After
stirring for 15 min, the mixture was filtered off and added into
extend-BPI (101 mg, 0.29 mmol) in methanol (15 mL). Then,
triethylamine (27 mg, 0.26 mmol) was added into reaction mixture
and it was heated to 50.degree. C. for 24 h. After cooling to room
temperature, the crude product was extracted into dichloromethane
layer. Finally, the yellow solid was washed with ether. Yield: 64%.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=10.37-10.35 (m, 2H),
8.13-8.10 (m, 2H), 7.94-7.90 (m, 2H), 7.63-7.67 (m, 4H), 7.01-7.04
(m, 2H).
Synthesis of Pt (N{circumflex over ( )}N{circumflex over ( )}N)
Cl
##STR00010##
[0151] Silver triflate (0.138 g, 0.54 mmol) was added dropwise into
Pt(COD)Cl.sub.2 (0.1 g, 0.26 mmol) in methanol (15 mL). After
stirring for 15 min, the mixture was filtered off and added into
BPI (88 mg, 0.29 mmol) in methanol (15 mL). Then, triethylamine (27
mg, 0.26 mmol) was added into reaction mixture and it was heated to
50.degree. C. for 24 h. After cooling to room temperature, the
crude product was extracted into dichloromethane layer. Finally,
the red solid was washed with ether. Yield: 42%. .sup.1H NMR (400
MHz, CDCl.sub.3): .delta.=10.32-10.29 (m, 2H), 8.59 (s, 2H),
8.09-8.03 (m, 2H), 7.98-7.92 (m, 2H), 7.73-7.65 (m, 4H), 7.07-7.03
(m, 2H).
Synthesis of 1a
##STR00011##
[0153] A mixture of [Pt(BPI)Cl] (50 mg, 0.095 mmol), potassium
tert-butoxide (10.6 mg, 0.095 mmol) and
1,3-dimethyl-1H-imidazol-3-ium iodide (23.4 mg, 0.104 mmol) in
acetonitrile (15 mL) was heated to reflux for 12 hours. After
cooling to room temperature, silver trifluoromethanesulfonate (84
mg, 0.33 mmol) was added into reaction mixture and stirred for 30
min. After extracting the crude product into dichloromethane layer,
it was purified by column chromatography on silica gel with
CH.sub.3CN/CH.sub.2Cl.sub.2 (3:1, v/v) as eluent, and yellow powder
was obtained.
[0154] Yield 52%; .sup.1H NMR (400 MHz, CD.sub.3CN): 8=8.17-8.15
(m, 2H), 8.02 (t, 2H, J=8.0 Hz), 7.80-7.82 (d, 2H, J=4.0 Hz),
7.70-7.72 (m, 2H), 7.60 (s, 2H), 7.42-7.40 (d, 2H, J=4.0 Hz), 7.02
(t, 2H, J=8.0 Hz), 3.98 (s, 6H); MS (FAB, +ve): m/z 589
[M-OTf].sup.+; Elemental analysis calcd (%) for
C.sub.24H.sub.20F.sub.3N.sub.7O.sub.3PtS: C, 39.03, H, 2.73, N,
13.27; found: C, 38.75, H, 2.80, N, 13.22.
Synthesis of 1b
##STR00012##
[0156] The procedure is similar to that for 1a.
[0157] Yield 61%; .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.=8.17-8.16 (m, 2H), 8.06-8.02 (m, 2H), 7.84-7.82 (m, 2H),
7.74-7.72 (m, 2H), 7.62-7.61 (m, 2H), 7.44-7.42 (m, 2H), 7.04-7.00
(m, 2H), 4.38 (t, 4H, J=8.0 Hz), 1.76-1.68 (m, 4H), 1.31-1.22 (m,
4H), 0.77-0.72 (m, 6H); MS (FAB, +ve): m/z 673 [M-OTf].sup.+;
Elemental analysis calcd (%) for
C.sub.30H.sub.32F.sub.3N.sub.7O.sub.3PtS: C, 43.79, H, 3.92, N,
11.92; found: C, 43.64, H, 4.05, N, 11.82.
Synthesis of 1c
##STR00013##
[0159] The procedure is similar to that for 1a.
[0160] Yield 49%; .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.=8.20-8.14 (m, 2H), 8.08-8.02 (m, 2H), 7.88-7.82 (m, 2H),
7.78-7.72 (m, 2H), 7.62-7.60 (m, 2H), 7.42-7.38 (m, 2H), 7.02-6.96
(m, 2H), 4.35 (t, 4H, J=8.0 Hz), 1.78-1.66 (m, 4H), 1.28-1.16 (m,
4H), 1.14-1.04 (m, 8H), 0.78-0.70 (m, 6H); MS (FAB, +ve): m/z 729
[M-OTf].sup.+; Elemental analysis calcd (%) for
C.sub.35H.sub.43F.sub.3N.sub.7O.sub.3PtS.0.5H.sub.2O: C, 46.56, H,
4.91, N, 10.86; found: C, 46.38, H, 4.71, N, 11.00.
Synthesis of 1d
##STR00014##
[0162] The procedure is similar to that for 1a.
[0163] Yield 51%; .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta.=8.17-8.15 (m, 2H), 8.04-8.00 (m, 2H), 7.86-7.80 (m, 2H),
7.74-7.72 (m, 3H), 7.46-7.43 (m, 3H), 7.04-7.01 (m, 2H), 4.28 (t,
2H, J=4.0 Hz), 4.06 (s, 3H), 1.16-1.07 (m, 12H), 0.77 (t, 3H, J=8.0
Hz); MS (FAB, +ve): m/z 687 [M-OTf].sup.+; Elemental analysis calcd
(%) for C.sub.30H.sub.36F.sub.3N.sub.7O.sub.3PtS: C, 43.58, H,
4.39, N, 11.86; found: C, 43.79, H, 4.15, N, 11.53.
Synthesis of 1e
##STR00015##
[0165] The procedure is similar to that for 1a.
[0166] Yield 44%; .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta.=8.20-8.14 (m, 2H), 8.06-7.96 (m, 2H), 7.86-7.78 (m, 2H),
7.76-7.70 (m, 3H), 7.48-7.42 (m, 3H), 7.06-7.00 (m, 2H), 4.28 (t,
2H, J=6.0 Hz), 4.06 (s, 3H), 1.35-1.04 (m, 28H), 0.88 (t, 3H, J=6.0
Hz). MS (FAB, +ve): m/z 799 [M-OTf].sup.+; Elemental analysis calcd
(%) for C.sub.38H.sub.52F.sub.3N.sub.7O.sub.3PtS: C, 48.61, H,
5.58, N, 10.44; found: C, 49.01, H, 5.50, N, 10.23.
Synthesis of 1f
##STR00016##
[0168] The procedure is similar to that for 1a.
[0169] Yield 45%; .sup.1H NMR (400 MHz, CD.sub.3CN): 8=8.16-8.14
(m, 2H), 7.93-7.91 (m, 2H), 7.81-7.79 (m, 2H), 7.68-7.66 (m, 4H),
7.26-7.24 (m, 4H), 7.06-6.98 (m, 8H), 6.60-6.54 (m, 2H), 5.42 (s,
4H); MS (FAB, +ve): m/z 741[M-OTf].sup.+; Elemental analysis calcd
(%) for C.sub.36H.sub.28F.sub.3N.sub.7O.sub.3PtS.CHCl.sub.3: C,
43.99, H, 2.89, N, 9.71; found: C, 44.07, H, 2.97, N, 9.89.
Synthesis of 1 g
##STR00017##
[0171] The procedure is similar to that for 1a.
[0172] Yield 49%; .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.=8.17-8.12 (m, 2H), 8.08-8.02 (m, 2H), 7.80-7.78 (m, 2H),
7.74-7.73 (m, 2H), 7.65-7.62 (m, 1H), 7.54-7.53 (m, 1H), 7.27-7.25
(m, 2H), 7.17-7.15 (m, 2H), 7.04-6.99 (m, 3H), 6.77-6.74 (m, 2H),
5.44-5.42 (m, 2H), 4.33-4.29 (m, 2H), 1.74-1.62 (m, 2H), 1.24-1.15
(m, 2H), 0.72-0.68 (m, 3H); MS (FAB, +ve): m/z 707 [M-OTf].sup.+;
Elemental analysis calcd (%) for
C.sub.33H.sub.30F.sub.3N.sub.7O.sub.3PtS: C, 46.26, H, 3.53, N,
11.44; found: C, 46.17, H, 3.55, N, 11.14.
Synthesis of 1h
##STR00018##
[0174] Silver trifluoromethanesulfonate (24.4 mg, 0.095 mmol) was
added into a mixture of [Pt(BPI)Cl] (50 mg, 0.095 mmol) and
triphenylphosphine (30 mg, 0.114 mmol) in dichloromethane:
acetonitrile (20 mL; 1:1, v/v). The reaction mixture was stirred at
room temperature for 5 hours.
[0175] After extracting the crude product into dichloromethane
layer, it was purified by recrystallization by diffusing diethyl
ether into acetonitrile. Reddish yellow crystal was obtained.
[0176] Yield 39%; .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.=8.71-8.69 (m, 2H), 8.16-8.15 (m, 2H), 7.86-7.78 (m, 9H),
7.58-7.52 (m, 6H), 7.48-7.46 (m, 6H), 6.62-6.58 (m, 2H). .sup.31P
NMR (400 MHz, CDCl.sub.3): .delta.=12.27. Elemental analysis calcd
(%) for C.sub.37H.sub.27F.sub.3N.sub.5O.sub.3PPtS: C, 49.12, H,
3.01, N, 7.74; found: C, 48.98, H, 3.02, N, 7.99.
Synthesis of 1j
##STR00019##
[0178] The procedure is similar to that for 1a.
[0179] Yield 47%; .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=8.68
(s, 2H), 8.16-8.14 (m, 2H), 7.88-7.86 (m, 2H), 7.74-7.72 (m, 6H),
7.24-7.22 (m, 4H), 7.12-7.04 (m, 8H), 6.67-6.64 (m, 2H), 5.53 (s,
4H); MS (FAB, +ve): m/z 791 [M-OTf].sup.+; Elemental analysis calcd
(%) for C.sub.40H.sub.31F.sub.3N.sub.7O.sub.3PtS: C, 51.01, H,
3.32, N, 10.41; found: C, 50.85, H, 3.02, N, 10.15.
6.4 Experimental Procedure
6.4.1 Cell Culture
[0180] The cell lines were maintained in cell culture media
(Minimum essential medium (MEM) for HeLa; and Dulbecco's modified
eagle medium (DMEM) for MDA-MB-231 and MiHa, Roswell Park Memorial
Institute (RPMI) medium for NCI-H460 and HCT116 supplemented with
fetal bovine serum (10 vol %), streptomycin (100 .mu.g/ml) and
penicillin (100 U/ml) in an incubator (5% CO.sub.2) at 37.degree.
C.
[0181] Human umbilical vein endothelial cells (HUVEC) were cultured
in endothelial cell growth medium (ECGM): M199 medium (Life
Technologies, Invitrogen) supplemented with 15 vol % fetal bovine
serum at 37.degree. C. in a humidified (5% CO.sub.2, 95% air)
atmosphere.
6.4.2 MTT Assay
[0182] The inhibition of cell growth by different metal complexes
were determined by MTT assay. Firstly, 4.times.10.sup.3 to
8.times.10.sup.3 cells were seeded on 96-well culture plates for 24
h. Then, different concentrations of complex was added into
different wells by serial dilution and the cells were incubated
with complex for 48-72 h. After that, 10 .mu.l of MTT solution (5
mg/ml) was added per well and the plate was incubated for 4 h at
37.degree. C. in a humidified atmosphere of 5% CO.sub.2. Viable
cells with active metabolism converted MTT into a purple colored
formazan product. In order to solubilize the formazan for
absorbance readings, 100 .mu.l of SDS (0.1 g/ml, 0.01 M HCl) was
added per well and the plate was kept in a dark and humidified
chamber overnight. Finally, the absorbance at 580 nm of each well
was monitored by microtiter plate reader.
[0183] The growth inhibition by a specific complex was represented
by IC.sub.50 (concentration of a complex causing 50% inhibition of
cell growth). Each experiment was repeated three times and the
results were expressed as means.+-.standard deviation (SD).
6.4.3 Scratch Assay (Wound-Healing Assay)
[0184] MDA-MB-231 cells were cultured in 6-well plate and allowed
to form a confluent monolayer for 24 h. After serum starved for 4
h, cells were scratched by pipette tips, washed with PBS and
photographed by using a fluorescence microscope (20.times.
objective). The fresh medium supplemented with 10 vol % FBS was
added into each well with different concentrations of Pt complex.
After incubated for 24 h, cells were photographed again at three
random areas. Then the migrated cells were quantified by manual
counting and inhibition ratio was expressed as % of control.
6.4.4 Transwell Invasion Assay
[0185] Effects of Pt complex on the invasion of MDA-MB-231 or
HUVECs cells were performed on Transwell Boyden chamber (8 .mu.m
pore, Corning, Lowel, Mass.) pre-coated with matrigel for 4 h at
37.degree. C. The cell suspension (2.5.times.10.sup.5 cells/ml, 100
.mu.L) in serum free medium (SFM) was placed to the upper
compartment of chamber. The bottom chambers were supplemented with
500 .mu.l complete medium (10 vol % FBS) or conditioned medium
(with VEGF=13.4 ng/ml from MDA-MB-231 cells) containing indicated
concentrations of Pt complex. After incubated for 24 h, the
non-migrant cells from the upper face were scraped using a cotton
swab. The invaded cells on the lower face were fixed with methanol,
stained with Giemsa, photographed by a phase-contrast microscope
(200.times., Nikon TS 100). The invaded cells were quantified by
manual counting and inhibition ratio was expressed as % of
control.
6.4.5 Tube Formation Assay
[0186] The In Vitro Angiogenesis Kit (CaymanChemical) was used in
the tube formation assay. Firstly, the ECMatrix solution and
10.times. Diluent Buffer were mixed in 9:1 (v/v) ratio on ice. Then
50 .mu.L of mixture was transferred into each well of 96-well plate
and incubated at 37.degree. C. for 1 h for polymerization. Then,
around 4.times.10.sup.4 of MS-1 cells in 100 .mu.L DMEM medium was
pre-mixed with different concentrations of complex and that
cell-complex containing medium was added on the top of the
polymerized matrix. After 2 h, the tube formation was observed
under an inverted microscopy at a 50.times. magnification.
[0187] At the same time, the cell viability under the same
condition was determined by MTT assay. Again, around
4.times.10.sup.4 MS-1 cells in 100 .mu.L DMEM medium was pre-mixed
with different concentrations of complex and they were seeded into
96-well plate. After 2 h, the medium was removed and fresh medium
with 10% MTT was added per well and the plate was incubated for 4 h
at 37.degree. C. in a humidified atmosphere of 5% CO2. In order to
solubilize the formazan for absorbance readings, 100 .mu.l of SDS
(0.1 g/ml, 0.01 M HCl) was added per well and the plate was kept in
a dark and humidified chamber overnight. Finally, the absorbance at
580 nm of each well was monitored by microtiter plate reader. Each
experiment was repeated three times and the results were expressed
as means.+-.standard deviation (SD).
6.4.6 Confocal Fluorescence Microscopy
[0188] HeLa cells (2.times.10.sup.5 cells) were seeded in a one
chamber slide (Nalgene; Nunc) with culture medium (2 mL per well)
and incubated at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2/95% air for 24 h. After treating with 1b (5 .mu.M) for 1
h, ER-tracker.TM. red (1 .mu.M), Mitotracker.RTM. deep red (50 nM)
or Lysotracker.RTM. deep red (50 nM) were incubated with cells for
10 min, and then the cells were washed with PBS twice. Confocal
fluorescence images were captured using a Carl Zeiss LSM510 Meta
confocal microscope with the use of 488 and 543 nm/633 nm lasers
for the excitation of complex 1b and red/deep red tracker
respectively, under a Plan-Apochromat 63.times.1.40NA oil-immersion
objective.
6.4.7 Determination of Extracellular VEGF
[0189] The concentrations of extracellular VEGF was determined by
Quantikine.RTM. ELISA kit (R&D System). Briefly, cells were
cultured in 6-well plate for 24 h, washed with PBS twice and
replaced with fresh serum-free medium containing different
concentrations of Pt complex. After 24 h treatment, the culture
media were collected and centrifuged to eliminate cellular debris.
Then, the collected medium was added into the detected microplate
and incubates for 2 h at room temperature. After three washes, the
VEGF conjugate was added and incubated for another 2 h. After
another three washes and the addition of substrate solution and
stop solution, the detection of VEGF concentration in the culture
medium was performed by monitoring absorption at 450 nm using a
microplate spectrophotometer (VERSA max, Molecular Devices).
6.4.8 Chorioallantoic Membrane Assay
[0190] The effect of Pt complex on the ex vivo angiogenesis was
determined by chorioallantoic membrane (CAM) assay. Briefly,
fertilized chicken eggs were incubated at 37.degree. C. in a
humidified incubator with forced air circulation. After 5-6 days,
eggs were cracked open and methylcellulose discs containing
different concentrations of 1d (40 al/egg) and VEGF (50 ng/mL) were
gently implanted on top of chicken CAM. After one day incubation,
the CAM was observed under a microscope (Olympus BX 40) and
photographed. VEGF treatment group was used as a positive control.
Three eggs per group were used in each experiment and three
independent experiments were performed.
6.4.9 Flow Cytometric Analysis
[0191] The effects of 1d on the cell cycle progression and the
induction of apoptotic cell death were quantified by flow
cytometric analysis. Briefly, treated or untreated cells were
trypsinized, washed with PBS and fixed with 70% ethanol overnight
at -20.degree. C. The fixed cells were washed with PBS and
incubated with a PI working solution for 4 h in darkness. The
stained cells were analyzed by flow cytometer (Beckman Coulter,
Fullerton, Calif.). Cell cycle distribution was analyzed using
MultiCycle software (Phoenix Flow Systems, San Diego, Calif.). The
proportion of cells in G0/G1, S, and G2/M phases was represented as
DNA histograms. Apoptotic cells with hypodiploid DNA content were
measured by quantifying the sub-G1 peak in the cell cycle pattern.
For each experiment, over 10,000 events per sample were
recorded.
6.4.10 Western Blotting
[0192] MDA-MB-231 cells (5.times.10.sup.5 cells) were incubated
with Pt compound and washed with phosphate-buffered saline (PBS),
lysed with radioimmunoprecipitation assay buffer (1% Triton X-100,
10% glycerol, 150 mM NaCl, 5 mM sodium fluoride, 1 mM sodium
vanadate and protein inhibitor cocktail) for 15 min at 4.degree. C.
The cell lysates were centrifuged at 13,000 rpm for 15 min at
4.degree. C. The protein concentrations of the extracts were
determined using a BCA protein assay kit (Beyotime, Haimen, China).
Specific amount of protein sample (30 .mu.g) was then boiled for 5
min in a 5.times. sample buffer (50 mM Tris (pH 7.4), 4% sodium
dodecyl sulfate (SDS), 10% glycerol and 50 .mu.g/mL bromophenol
blue) at a volume ratio of 4:1. Protein samples were subjected to
SDS-polyacrylamide gel electrophoresis (PAGE), transferred to
polyvinylidene difluoride membranes and immunoblotted with primary
anti-bodies. After further incubation with horseradish peroxidase
(HRP)-conjugated secondary antibody, the blot was stained with a
chemiluminescent detection reagent and subsequently analyzed by
enhanced chemiluminescence. Protein expression was visualized on
Kodak Biomax X-ray film.
6.4.11 Statistic Analysis
[0193] Experiments were conducted at least three times and data was
expressed as mean.+-.standard deviation (SD). Statistical analysis
was performed on SPSS statistical program version 13 (SPSS Inc.,
Chicago, Ill.). Difference between two groups was analyzed by
two-tailed Student's t test and that between three or more groups
was analyzed by one-way ANOVA multiple comparisons. Difference with
P<0.05 (*) or P<0.01 (**) was considered to be statistically
significant.
6.4.12 Proteomic Studies
[0194] Sample preparation. MDA-MB-231 cells (8.times.10.sup.5
cells) were incubated with 5 .mu.M of 1d or DMSO for 5 h under 5%
CO.sub.2 environment at 37.degree. C. The cells were then washed
with PBS to remove excess compound and lysed with urea lysis buffer
(20 mM Tris-HCl, 8 M urea, protein phosphatase inhibitor cocktail,
pH 8.0). The cell lysates were centrifuged at 13,000 rpm for 15 min
at 4.degree. C. Specific amount of protein sample (50 .mu.g) were
then precipitated by adding 4.times. volume of ice-cold acetone and
stored at -20.degree. C. for 4 h. The precipitated proteins were
centrifuged at 13,000 rpm for 20 min at 4.degree. C. and the
acetone solvent were discarded. After that, the protein pellets
were dried by SpeedVac (Thermo Fisher Scientific) and re-suspended
in 50 .mu.L of urea buffer (100 mM Tris, 8 M urea, pH 8.5). Then,
freshly prepared DTT (final concentration: 5 mM) was added into the
sample to reduce to disulfide bond for 30 mins. Then, iodoacetamide
(final concentration: 25 mM) was added to alkylate the reduced
di-sulfide bond and the samples were kept in the dark for 30 min at
25.degree. C. In order to dilute the urea concentration down to 2
M, around 140 .mu.L of 100 mM Tris (pH 8.5) buffer was added into
the sample. Then, 1 .mu.g of trypsin was added into the sample
mixture and it was kept at 37.degree. C. overnight. About 10 .mu.L
of formic acid was added into sample mixtures to stop the
digestion. After centrifugation at 14,000 rpm for 15 min, the
supernatants were transferred to new eppendorf (can be frozen at
-80.degree. C. for long term storage). The resulting peptides were
desalted and enriched by StageTips. For each sample, three
biological replicates were prepared. The samples were re-dissolved
with H.sub.2O (containing 0.1% formic acid, v/v) for subsequent MS
analysis.
[0195] HPLC-MS/MS analysis. MS analysis was performed with a LTQ
Orbitrap Velos Orbitrap mass spectrometer (Thermo Scientific)
connected online with a HPLC. The analytical column was a
self-packed PicoTip.RTM. column (360 .mu.m outer diameter, 75 .mu.m
inner diameter, 15 .mu.m tip, New Objective) packed with 10 cm
length of C18 material (ODS-A C18 5-.mu.m beads, YMC) with a
high-pressure injection pump (Next Advance). The mobile phases of
HPLC are A (0.1% formic acid in HPLC grade H.sub.2O, volume
percentage) and B (0.1% formic acid in HPLC grade acetonitrile,
volume percentage). 3 .mu.g of sample was loaded onto the
analytical column by the auto-sampler and rinsed with 2% B for 6
min and subsequently eluted with a linear gradient B from 2% to 40%
for 120 min. For the MS analysis, LTQ-Orbitrap Velos MS was
operated in a data-dependent mode cycling through a high-resolution
(6000 at 400 m/z) full scan MS1 (300-2000 m/z) in Orbitrap followed
by CID MS2 scans in LTQ on the 20 most abundant ions from the
immediate preceding full scan. The selected ions were isolated with
a 2-Da mass window and put into an exclusion list for 60 seconds
after they were first selected for CID.
[0196] Proteins identification and quantification. The raw data
were directly used for protein identification and quantification
using MaxQuant (Version 1.5.3.30). The data were searched against
uniprot human database (27 May 2016, 70625), in which trypsin
specificity was used with up to two missed cleavages 17 allowed.
Methionine oxidation was set as a variable modification, and
iodoacetamide derivative of cysteine was set as a fixed
modification. Default settings were used for mass tolerance for MS1
and MS2. The false discovery rate (FDR) was determined by searching
against a reverse database and kept FDR at 1%. Peptides were
quantified in a label-free manner using the area under the
extracted ion chromatograph of peptides, and the protein abundances
were the sum of the peptide abundances.
[0197] Signaling pathway analysis. Lists of quantified proteins
(shown as their Protein IDs) were uploaded to the ExPlain.TM. tool
(version 3.1, BIOBASE) for further signaling pathway analysis.
Details of procedure for pathway analysis have been described
previously..sup.[48,49]
6.4.13 In Vivo Tumor Growth Inhibition Experiments
[0198] All experiments were followed to the guidelines of the
Laboratory Animal Unit of the University of Hong Kong. Ten mice
were randomly divided into two groups (5 mice for each group) for
two different treatment conditions.
[0199] Around 4.times.10.sup.6 of cancer cells in 100 .mu.L of PBS
were injected into right back flanks of the mice through
subcutaneous injection. After tumor formation (around 4 days), the
treatment group was injected with drug at the dosage of 10 mg/kg
and the control group was injected with solvent only. The size of
the tumors were measured by a ruler every 2-3 days until the mice
were sacrificed. The longest diameter (a) and shortest diameter (b)
of the tumor would be picked up and the volume of the tumor could
be calculated through the following formula:
V=0.52.times.ab.sup.2
[0200] It should be noted that the body weight of the mice were
also be recorded in order to examine the side effect of the
drugs.
[0201] To calculate the inhibition effect of the drug, the ratio of
enlargement of tumor volume between the drug treatment group and
control group would be applied in the following formula:
Inhibition = ( 1 - V t - V 0 V t ' - V 0 ' ) .times. 100 %
##EQU00001##
[0202] where V.sub.t and V.sub.t' are the tumor volumes of drug
treatment and control group respectively; V.sub.0 and V.sub.0' are
the tumor volume at the 0 day of drug treatment and control group
respectively.
[0203] The invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the
invention in addition to those described will become apparent to
those skilled in the art from the foregoing description and
accompanying figures. Such modifications are intended to fall
within the scope of the appended claims.
[0204] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
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