U.S. patent application number 15/242883 was filed with the patent office on 2018-02-22 for cobalt-polypyridyl complex for treatment of cancer, a pharmaceutical composition and a kit comprising it.
The applicant listed for this patent is Macau University of Science and Technology. Invention is credited to Kam Wai Wong, Man Chung Wong.
Application Number | 20180050044 15/242883 |
Document ID | / |
Family ID | 59399762 |
Filed Date | 2018-02-22 |
United States Patent
Application |
20180050044 |
Kind Code |
A1 |
Wong; Kam Wai ; et
al. |
February 22, 2018 |
COBALT-POLYPYRIDYL COMPLEX FOR TREATMENT OF CANCER, A
PHARMACEUTICAL COMPOSITION AND A KIT COMPRISING IT
Abstract
A method for treating a subject suffering from a cancer, in
particular a multidrug-resistant cancer includes administrating a
cobalt-polypyridyl complex to the subject. A method for suppressing
the growth of cancer cells, in particular inducing autophagy of the
cancer cells, inducing cell cycle arrest of the cancer cells and/or
inhibiting cell invasion of the cancer cells and for specifically
targeting cancer cells with multidrug-resistance includes
contacting said cancer cells with the cobalt-polypyridyl complex. A
pharmaceutical composition and a kit are provided and include the
cobalt-polypyridyl complex. Unexpectedly, the cobalt-polypyridyl
complex is especially suitable to treat cancer, in particular
multidrug-resistant cancer with an exceptionally increased
cytotoxic activity towards multidrug-resistant cancer cells.
Inventors: |
Wong; Kam Wai; (Taipa,
MO) ; Wong; Man Chung; (Taipa, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Macau University of Science and Technology |
Taipa |
|
MO |
|
|
Family ID: |
59399762 |
Appl. No.: |
15/242883 |
Filed: |
August 22, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/555 20130101;
A61K 45/06 20130101; A61K 2300/00 20130101; A61P 35/00 20180101;
A61K 31/555 20130101; A61K 31/28 20130101 |
International
Class: |
A61K 31/555 20060101
A61K031/555; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method for treating a subject suffering from cancer comprising
the step of administering an effective amount of a
cobalt-polypyridyl complex to a subject, wherein the
cobalt-polypyridyl complex comprises a of Formula (II):
##STR00017## wherein R.sub.1 and R.sub.2 are identical and
independently selected from hydrogen, a C.sub.1-C.sub.10 alkyl or a
C.sub.1-C.sub.10 alkoxy, and n is 2 or 3; and wherein the cancer is
selected from the group consisting of ovarian cancer, cervical
cancer, liver cancer, lung cancer, breast cancer, gastric cancer,
and colon cancer.
2. (canceled)
3. (canceled)
4. The method of claim 1, wherein autophagy of the cancer cells is
induced, cell cycle arrest of the cancer cells is induced and/or
cell invasion of the cancer cells is inhibited.
5. The method of claim 1, wherein the subject is a human and the
cancer is a multidrug-resistant cancer which is at least one of (i)
a multidrug-resistant ABC-protein-dependent cancer and/or (ii) a
multidrug-resistant apoptosis-deficient cancer.
6. The method of claim 5, wherein the multidrug-resistant cancer is
resistant against at least taxol and selected from a
multidrug-resistant lung cancer, multidrug-resistant breast cancer,
or multidrug-resistant colon cancer.
7. The method of claim 1, wherein R.sup.1 and R.sup.2 are identical
and selected from --H, --CH.sub.3, --C.sub.9H.sub.19 or
--OCH.sub.3.
8. The method of claim 1, wherein the cobalt-polypyridyl complex
comprises a structure selected from Formula (III), Formula (IV) or
Formula (VII): ##STR00018##
9. The method of claim 1, wherein the cobalt-polypyridyl complex is
administered in combination with: (ii) an effective amount of at
least one chemotherapeutic compound, which chemotherapeutic
compound is a compound selected from the group consisting of a
topoisomerase-II inhibitor, an anthracycline, a coordination
complex of platinum, a taxane, a protein kinase inhibitor, a vinca
alkaloid or derivative thereof, a topoisomerase-I inhibitor and a
nucleotide analog or precursor analog; (ii) radiotherapy, and/or
(iii) immunotherapy.
10. A method for suppressing the growth of cancer cells comprising
contacting the cancer cells with an effective amount of a
cobalt-polypyridyl complex, wherein the cobalt-polypyridyl complex
comprises a structure of Formula (II): ##STR00019## wherein R.sub.1
and R.sub.2 are identical and independently selected from hydrogen,
a C.sub.1-C.sub.10 alkyl or a C.sub.1-C.sub.10 alkoxy, and n is 2
or 3; and wherein the cancer cells are from one of an ovarian
cancer, a cervical cancer, a liver cancer, a lung cancer, a breast
cancer, a gastric cancer and, a colon cancer.
11. The method of claim 10, wherein autophagy of the cancer cells
is induced, cell cycle arrest of the cancer cells is induced and/or
cell invasion of the cancer cells is inhibited
12. The method of claim 10, wherein the cancer cells are
multidrug-resistant cancer cells.
13. The method of claim 12, wherein the multidrug-resistant cancer
cells are at least one of (i) multidrug-resistant
P-glycoprotein-dependent cancer cells and/or (ii)
multidrug-resistant p53-deficient cancer cells.
14. The method of claim 12, wherein the multidrug-resistant cancer
cells are at least resistant against taxol.
15. The method of claim 12, wherein the Resistant Factor of the
cobalt-polypyridyl complex towards the multidrug-resistant cancer
cells is less than 0.6.
16. The method of claim 10, wherein the IC.sub.50 of the
cobalt-polypyridyl complex against the cancer cells is at most 10
.mu.M with an IC.sub.50 against non-cancerous being at least 2
times higher.
17. (canceled)
18. The method of claim 10, wherein the cobalt-polypyridyl complex
comprises a structure of Formula (IV): ##STR00020## and wherein the
cancer cells are contacted with of from about 1 .mu.M to about 30
.mu.M of said cobalt-polypyridyl complex.
19. A pharmaceutical composition comprising an effective dose of:
(i) a cobalt-polypyridyl complex comprising a structure of Formula
(II): ##STR00021## wherein R.sub.1 and R.sub.2 are identical and
independently selected from hydrogen, a C.sub.1-C.sub.10 alkyl or a
C.sub.1-C.sub.10 alkoxy, and n is 2 or 3; and (ii) at least one
pharmaceutically acceptable excipient selected from a diluent, a
filler, a binder, a disintegrant, a lubricant, a coloring agent, a
surfactant or a preservative.
20. The pharmaceutical composition of claim 19, wherein the
cobalt-polypyridyl complex comprises a structure of Formula (IV):
##STR00022##
Description
TECHNICAL FIELD
[0001] The present invention relates in a first aspect to a method
for treating a subject suffering from a cancer, in particular a
multidrug-resistant cancer, i.e. a cancer with multidrug-resistant
phenotype and, thus, a specific subgroup of subjects with cancer.
The method comprises administrating a cobalt-polypyridyl complex to
said subject. In another aspect of the present invention, a method
for suppressing the growth of cancer cells, in particular inducing
autophagy of the cancer cells, inducing cell cycle arrest of the
cancer cells and/or inhibiting cell invasion of the cancer cells
and for specifically targeting cancer cells with
multidrug-resistance is provided comprising contacting said cancer
cells with the cobalt-polypyridyl complex. In a further aspect, the
present invention provides a pharmaceutical composition and a kit
comprising the cobalt-polypyridyl complex.
BACKGROUND OF THE INVENTION
[0002] Cancer is still a life-threatening disease affecting an
increasing number of people in the world. Platinating compounds
including cisplatin, carboplatin, and oxaliplatin are common
chemotherapeutic compounds used for treating cancer. However, a
significant number of patients have acquired or develop resistance
to these chemotherapeutic compounds after initial therapeutic
treatments. Such drug-resistance in cancer is the major impediment
to a successful treatment. Such cells display a reduced sensitivity
to chemotherapeutic compounds based on several mechanisms in
particular including an increase in drug efflux such as by an
increased expression or activity of ABC transporter proteins such
as P-glycoprotein (P-gp, MDR1, or ABCB1) or affected apoptosis
pathways such as by mutated or dysfunctionally regulated genes and
respective proteins. For example, cancer cells lacking cell death
mediators Bax and Bak have been reported to develop drug-resistance
and the high frequency of p53 mutations is expected to lead to a
drug resistance of cancer cells, too.
[0003] Therefore, different approaches have been applied to
identify novel therapeutic agents, molecular mechanisms and targets
for treating cancer which are also suitable to overcome drug
resistance.
[0004] Transition metal ions are essential for the proper functions
of organisms; examples including copper, iron, and manganese ions
work with proteins and enzymes for multiple biological processes
such as electron transfer and catalysis. As metals are involved in
redox activity, coordination, and reactivity towards organic
substrates in organisms, and are tightly regulated under normal
conditions, aberrant metal ion concentrations are associated with
pathogenesis of diseases, in particular of cancers. For instance,
enriched copper ions found in cancer tissues are suggested to
promote the angiogenesis processes in tumors.
[0005] In fact, metal-containing compounds have been used to treat
a wide range of diseases. For example, cisplatin
(cis-[PtII(NH.sub.3).sub.2Cl.sub.2]) can bind to the purine bases
of the DNA, thereby led to DNA damage resulting in apoptosis in
cancer cells. However, due to severe side effects such as
dose-dependent toxicity, allergy, effects on the kidneys and
immunity, gastrointestinal disorders, hemorrhage and loss of
hearing, the clinical use of cisplatin is limited. Acquired
resistance to cisplatin is caused by an increased efflux or
detoxification of the drug, increased rate of DNA repair, as well
as a reduced susceptibility of cancer cells in response to
drug-induced cell death. Other platinum-containing anti-cancer
analogs such as carboplatin and oxaliplatin are therefore used as
alternative to cisplatin and further transition metal complexes
including zinc(II), copper(II), gold(III), copper chelating agents,
and non-platinum metal complexes such as ruthenium-containing
compounds were studied for their potential as anti-cancer agents,
too.
[0006] The exploration and exploitation of other non-platinum
anti-cancer drugs have received considerable attention. In view of
the fact that soluble cobalt salts can adversely interfere with
cell division and bind to nucleic acids inside the cell nucleus,
one may postulate that cobalt complexes could work as anti-cancer
agents like platinum-containing analogs. However, they were also
reported for being weakly mutagenic and inducing metastasis in
animal models (Jensen A. A. and Tuchsen F., Crit Rev Toxicol, 1990,
20, 427-437). There have been some examples of cobalt(III)
complexes with equatorial tetradentate Schiff base ligands as
potent inhibitors of a wide range of zinc-dependent proteins
(Heffern, M. C. et al., Curr Opin Chem Biol, 2013, 17, 189-196,
Takeuchi, T. et al., J. Am. Chem. Soc., 1998, 120, 8555-8556,
Harney, A. S. et al., Proc Natl Acad Sci USA, 2009, 106,
13667-13672, Peterson, M. D, J Am Chem Soc, 2013, 135,
13162-13167), however, the related use of cobalt pyridine complexes
in biological applications or specifically for the development of
anti-cancer drugs remains substantially unexplored (Luis, D. V. et
al., J Biol Inorg Chem, 2014, 19, 787-803, Vignesh, G. et al., Rsc
Advances, 2014, 4, 57483-57492). On the other hand,
cobalt(II)/(III) complexes with pyridine ligands have recently been
developed as redox mediators in dye-sensitized solar cells (DSCs)
(Feldt, S. M., et al., J Am Chem Soc, 2010, 132, 16714-16724,
Klahr, B. M. and Hamann, T. W., J Phys Chem C, 2009, 113,
14040-14045, Xie, Y. and Hamann, T. W., J Phys Chem Lett, 2013, 4,
328-332, Kim, H. S. et al., Chem Commun (Camb), 2011, 47,
12637-12639, Sun, Z. et al., Acc Chem Res, 2015, 48, 1541-1550),
but not in the field of cancer treatment or the specific treatment
of multidrug-resistant cancer.
[0007] Although there has been increased research in this regard,
there remains a strong need for methods and means allowing for an
effective therapeutic treatment of cancer, especially of
multidrug-resistant cancer and cancer cells with a
multidrug-resistant phenotype, respectively. In particular,
efficacious treatment options are urgently required for
specifically treating subjects with cancer such as with
overexpression of ABC transporter proteins and/or decreased
expression of pro-apoptotic proteins, i.e. for treating said
specific subgroups of subjects with multidrug-resistant cancer
among subjects with cancer.
SUMMARY OF THE INVENTION
[0008] The present invention relates in a first aspect to a method
for treating a subject suffering from cancer, in particular
multidrug-resistant cancer, i.e. a cancer with multidrug-resistant
phenotype such as ABC-protein-dependent, in particular at least
P-glycoprotein-dependent cancer, and/or cancer being
apoptosis-deficient, in particular being at least p53-deficient.
Said method of treating the subject with cancer, in particular with
multidrug-resistant cancer, comprises the step of administering an
effective amount of a cobalt-polypyridyl complex to said
subject.
[0009] The cobalt-polypyridyl complex administered according to the
present invention comprises a cobalt ion and at least one
polypyridyl ligand of Formula (I):
##STR00001##
wherein R.sub.1 and R.sub.2 are independently selected from
hydrogen, a C.sub.1-C.sub.10 alkyl or a C.sub.1-C.sub.10 alkoxy. In
particular, R.sub.1 and R.sub.2 are identical. In particular
embodiments of the present invention, R.sub.1 and R.sub.2 are
identical and selected from --H, --CH.sub.3, --C.sub.9H.sub.19 or
--OCH.sub.3.
[0010] In particular, the cobalt-polypyridyl complex of the present
invention comprises a structure of Formula (II):
##STR00002##
wherein R.sub.1 and R.sub.2 are identical and selected from --H,
--CH.sub.3, --C.sub.9H.sub.19 or --OCH.sub.3 and wherein n is 2 or
3, i.e. the cobalt-polypyridyl complex comprises a structure of one
of Formula (III), (IV), (V), (VI), (VII) or (VIII):
##STR00003## ##STR00004##
[0011] The cobalt-polypyridyl complex of the present invention may
further comprise a counterion in particular selected from, but not
limited to, a monovalent anion such as hexafluorophosphate
(PF.sub.6.sup.-) or chloride (Cl.sup.-).
[0012] The cobalt-polypyridyl complex of the present invention may
be administered in combination with at least one further
chemotherapeutic compound, in particular selected from the group
consisting of a topoisomerase-II inhibitor, an anthracycline, a
coordination complex of platinum, a taxane, a protein kinase
inhibitor, a vinca alkaloid or derivative thereof, a
topoisomerase-I inhibitor and a nucleotide analog or precursor
analog. Alternatively or additionally, the cobalt-polypyridyl
complex of the present invention may be administered in combination
with radiotherapy and/or immunotherapy.
[0013] According to the invention is also the cobalt-polypyridyl
complex described above for use as a medicament for the treatment
of cancer, in particular multidrug-resistant cancer such as
P-glycoprotein-dependent multidrug-resistant cancer or
p53-deficient multidrug-resistant cancer. Another aspect of the
invention refers to the use of the cobalt-polypyridyl complex
described above for preparing a medicament for treatment of cancer,
in particular multidrug-resistant cancer such as
P-glycoprotein-dependent or p53-deficient multidrug-resistant
cancer. The present invention also relates to the use of the
cobalt-polypyridyl complex as described above for suppressing
cancer growth, in particular for inducing autophagy, inducing cell
cycle arrest and/or inhibiting cell invasion.
[0014] The present invention further provides a method for
suppressing the growth of cancer cells comprising contacting the
cancer cells with an effective amount of a cobalt-polypyridyl
complex, wherein the cobalt-polypyridyl complex comprises a cobalt
ion and at least one polypyridyl ligand of Formula (I):
##STR00005##
wherein R.sub.1 and R.sub.2 are independently selected from
hydrogen, a C.sub.1-C.sub.10 alkyl or a C.sub.1-C.sub.10 alkoxy.
Preferably, the method is for inducing autophagy of the cancer
cells, inducing cell cycle arrest of the cancer cells and/or
inhibiting cell invasion of the cancer cells. The cancer cells are
preferably multidrug-resistant cancer cells and more preferably at
least one of (i) multidrug-resistant ABC-protein-dependent, in
particular P-glycoprotein-dependent, multidrug-resistant cancer
cells and/or (ii) apoptosis-deficient multidrug-resistant cancer
cells, in particular p53-deficient multidrug-resistant cancer
cells. The multidrug-resistant cancer cells are preferably at least
resistant against taxol.
[0015] In another aspect of the present invention, a method for
specifically targeting cancer cells with multidrug-resistance is
provided, in particular ABC-protein-dependent such as
P-glycoprotein-dependent or apoptosis-deficient such as
p53-deficient multidrug-resistant cancer cells. Said method
comprises the step of contacting a population of cancer cells with
multidrug-resistance with a cobalt-polypyridyl complex as described
above. In particular, the growth of the multidrug-resistant cancer
cells is suppressed, preferably autophagy of the
multidrug-resistant cancer cells is induced, cell cycle arrest of
the multidrug-resistant cancer cells is induced and/or cell
invasion of the multidrug-resistant cancer cells is inhibited.
[0016] Further in accordance with the present invention is a
composition comprising the cobalt-polypyridyl complex and an
excipient such as selected from a pharmaceutically acceptable
carrier, salt, buffer, water, or a combination thereof. More
preferably, the composition is a pharmaceutical composition
comprising the cobalt-polypyridyl complex described above and at
least one pharmaceutically acceptable excipient such as selected
from a diluent, a filler, a binder, a disintegrant, a lubricant, a
coloring agent, a surfactant or a preservative.
[0017] The present invention also provides a kit comprising an
effective dose of the cobalt-polypyridyl complex as described above
and an effective dose of at least a further chemotherapeutic
compound commonly used for treating cancer, namely selected from
the group consisting of a topoisomerase-II inhibitor, an
anthracycline, a coordination complex of platinum, a taxane, a
protein kinase inhibitor, a vinca alkaloid or derivative thereof, a
topoisomerase-I inhibitor and a nucleotide analog or precursor
analog. The kit may further comprise excipients, in particular
pharmaceutically acceptable excipients, such as a carrier, salt,
buffer, water, or a combination thereof. Accordingly, the present
invention provides a novel and highly advantageous treatment option
for treating cancer from various origins. It has been found that
the cobalt-polypyridyl complex as described above, in particular a
cobalt-polypyridyl complex comprising a structure of Formula (III),
(IV) or (VII) is especially suitable for treating cancer and
especially multidrug-resistant cancer as it possesses a high
cytotoxic activity against cancer cells and specifically towards
multidrug-resistant cancer cells such as of human origin. Said
cobalt-polypyridyl complex described above, thus, allows for
effectively targeting cancer and especially multidrug-resistant
cancer, either alone or in combination with conventional
chemotherapeutic compounds. It provides an advantageous treatment
approach for cancer exceptionally suitable to specifically address
multidrug-resistant cancer.
[0018] In particular, the cobalt-polypyridyl complex comprising
structures of Formulas (III), (IV) or (VII) and in particular
essentially consisting of structures of Formulas (III), (IV) or
(VII) and optionally a counterion proved to specifically and
advantageously target P-glycoprotein-dependent multidrug-resistant
and/or p53-deficient multidrug-resistant cancer cells through
collateral sensitivity, i.e. it is especially suitable to
selectively kill these multidrug-resistant cancer cells. Namely, a
significantly lower dose is required for treating said
multidrug-resistant cancer cells compared to cancer cells of the
same cell type without a multidrug-resistant phenotype, usually the
IC.sub.50 of the cobalt-polypyridyl complex towards
multidrug-resistant cancer cells is significantly reduced.
[0019] Summing up, the herewith provided class of
cobalt-polypyridyl complexes shows highly promising anti-cancer
effects and more importantly collateral sensitivity towards
multidrug-resistant cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0021] FIGS. 1A, 1B, 1C, and 1D refer to immunofluorescence
staining patterns which show the endogenous LC3-II puncta formation
in HeLa cancer cells under the cobalt-polypyridyl complex of
Formula (IV) and in a control group. FIG. 1A is an
immunofluorescence staining pattern obtained in the control group.
FIG. 1B is an immunofluorescence staining pattern obtained under 5
.mu.M of the cobalt-polypyridyl complex of Formula (IV) with
PF.sub.6.sup.- as counterion. FIG. 1C is an immunofluorescence
staining pattern obtained under 10 .mu.M of the cobalt-polypyridyl
complex of Formula (IV) with PF.sub.6.sup.- as counterion. FIG. 1D
is an immunofluorescence staining pattern obtained under 20 .mu.M
of the cobalt-polypyridyl complex of Formula (IV) with
PF.sub.6.sup.- as counterion.
[0022] FIG. 1E is a diagram showing the endogenous LC3-II puncta
formation under different doses of the cobalt-polypyridyl complex
of Formula (IV) with PF.sub.6.sup.- as counterion.
[0023] FIG. 1F is a Western Blot showing the expression of LC3-I
and LC3-II in the control group and under rapamycin and the
cobalt-polypyridyl complex of Formula (IV) with PF.sub.6.sup.- as
counterion (10 .mu.M and 20 .mu.M). .beta.-actin was used as
control.
[0024] FIG. 2 is a graph showing the cytotoxic effect of the
cobalt-polypyridyl complex of Formula (IV) in Atg7 wild-type MEFs
and Atg7 deficient MEFs under different concentrations of the
cobalt-polypyridyl complex of Formula (IV) with PF.sub.6.sup.- as
counterion.
[0025] FIGS. 3A, 3B, 3C, 3D and 3E refer to diagrams obtained with
flow cytometry analysis and shows the cell cycle progression of
HeLa cancer cells treated with the cobalt-polypyridyl complex of
Formula (IV) with PF.sub.6.sup.- as counterion. FIG. 3A shows the
cell cycle progression of HeLa cancer cells of the control group.
FIG. 3B shows the cell cycle progression of HeLa cancer cells
treated with 5 .mu.M of the cobalt-polypyridyl complex of Formula
(IV) with PF.sub.6.sup.- as counterion. FIG. 3C shows the cell
cycle progression of HeLa cancer cells treated with 10 .mu.M of the
cobalt-polypyridyl complex of Formula (IV) with PF.sub.6.sup.- as
counterion. FIG. 3D shows the cell cycle progression of HeLa cancer
cells treated with 15 .mu.M of the cobalt-polypyridyl complex of
Formula (IV) with PF.sub.6.sup.- as counterion. FIG. 3E is a
diagram showing the cell cycle progression results in HeLa cells of
the control group and under the cobalt-polypyridyl complex of
Formula (IV) with PF.sub.6.sup.- as counterion (5 .mu.M, 10 .mu.M
and 15 .mu.M).
[0026] FIG. 4 is a diagram showing the cell invasion ability of
cancer cells H1299 upon the treatment with the cobalt-polypyridyl
complex of Formula (IV) with PF.sub.6.sup.- as counterion (0.2
.mu.M, 0.5 .mu.M and 1.0 .mu.M) and in control groups.
[0027] FIG. 5A is a graph showing the mean tumor volume after
intraperitoneal (IP) injection of the cobalt-polypyridyl complex of
Formula (IV) with PF.sub.6.sup.- as counterion and its water
soluble form (complex (IV)-Cl) at 40 mg/kg/day and in a control
group demonstrating a significant tumor inhibitory effect of up to
40% (P<0.05) reduction in tumor volume.
[0028] FIG. 5B is a graph showing the reduction in body weight in
mice treated with the cobalt-polypyridyl complex of Formula (IV)
with PF.sub.6.sup.- as counterion and its water soluble form
(complex (IV)-Cl) and in a control group.
DESCRIPTION OF THE EMBODIMENTS
[0029] The following preparations and examples are given to enable
those skilled in the art to more clearly understand and to practice
the present invention. They should not be considered as limiting
the scope of the invention, but merely as being illustrative and
for representing preferred embodiments thereof. The technical terms
used in the present patent application have the meaning as commonly
understood by a respective skilled person unless specifically
defined otherwise.
[0030] As used herein, "comprising" means including the following
elements or structures but not excluding others. "Essentially
consisting of" means that the material or compound consists of the
respective element or structure along with usually and unavoidable
impurities such as side products and components usually resulting
from the respective preparation or method for obtaining the
material such as traces of further components or solvents.
"Consisting of" means that the material solely consists of, i.e. is
formed by the respective element. As used herein, the forms "a,"
"an," and "the," are intended to include the singular and plural
forms unless the context clearly indicates otherwise.
[0031] The present invention relates in a first aspect to a method
for treating a subject suffering from cancer, in particular from a
multidrug-resistant cancer, i.e. a cancer with multidrug-resistant
phenotype. Said method of treating cancer comprises the step of
administering an effective amount of a cobalt-polypyridyl complex
to said subject. The term "cobalt-polypyridyl complex" as used
herein means a complex, i.e. a compound, formed by a cobalt ion and
polypyridyl ligands including at least one of Formula (I) with
R.sup.1 and R.sup.2 as described below and optionally comprising a
counterion. Thus, the cobalt-polypyridyl complex does not comprise
non-polypyridyl ligands or non-polypyridyl polymers. A polypyridyl
ligand generally means a ligand having at least two pyridine rings
covalently linked to each other, which may together form a larger
ring system. Polypyridine ligands include, in particular,
bipyridine, terpyridine and phenanthroline. The pyridine rings may
optionally be substituted, in particular with alkyl or alkoxy
groups such as C.sub.1-C.sub.10 alkyl or C.sub.1-C.sub.10 alkoxy
groups. The cobalt-polypyridyl complex of the present invention
comprises at least one polypyridyl ligand of Formula (I):
##STR00006##
wherein R.sub.1 and R.sub.2 are independently selected from
hydrogen, a C.sub.1-C.sub.10 alkyl or a C.sub.1-C.sub.10 alkoxy,
preferably from hydrogen, a C.sub.1-C.sub.9 alkyl or a
C.sub.1-C.sub.9 alkoxy. Preferably, R.sub.1 and R.sub.2 are
identical and most preferably selected from --H, --CH.sub.3,
--C.sub.9H.sub.19 or --OCH.sub.3. Said ligand in particular acts as
a bidentate ligand, i.e. both nitrogen atoms are coordinated to the
cobalt ion.
[0032] The term "C.sub.1-C.sub.10 alkyl" as group used in the
present invention refers to a hydrocarbyl radical having from 1 to
10 carbon atoms which includes a straight chain or branched alkyl
group. Namely, it comprises, for example, methyl, ethyl, propyl,
isopropyl, nonyl and so on. "C.sub.1-C.sub.10 alkoxy" refers to a
radical having a formula -AB wherein A is an oxygen atom and B is
C.sub.1-C.sub.10 alkyl, i.e. a straight chain or branched alkyl
group with 1 to 10 carbon atoms, including for example methoxy and
ethoxy. Preferably, the cobalt-polypyridyl complex comprises three
ligands of Formula (I) coordinated to one cobalt ion, i.e. the
cobalt-polypyridyl complex is a cobalt tris(bipyridine) system. The
cobalt ion is preferably of the oxidation state +2 or +3. The
cobalt ion is preferably octahedrally coordinated. In an octahedral
coordination geometry, the ligands are coordinated to the cobalt
ion in a symmetrical distribution, leading to the formation of an
octahedron.
[0033] A counterion might affect the solubility or other chemical
or physical properties of the cobalt-polypyridyl complex, wherein
the exact nature of the counterion is not critical as long as it is
pharmaceutically acceptable and/or not significantly toxic in the
amounts used. Counterions can in particular be anions which are
unlikely to bind directly to the cobalt ion of the
cobalt-polypyridyl complex, i.e. non-coordinating anions.
Non-coordination anions include, for example, hexafluorophosphate
(PF.sub.6.sup.-), perchlorate (ClO.sub.4.sup.-) or
tetrafluoroborate (BF.sub.4.sup.-). Examples of counterions, in
particular, include hexafluorophosphate (PF.sub.6.sup.-) or
chloride (Cl.sup.-).
[0034] The term cobalt-polypyridyl complex encompasses any
diastereomers and their mixtures, enantiomers and their mixtures
such as racemates and salts of the cobalt-polypyridyl complex, in
particular pharmaceutically acceptable salts. The skilled person is
aware of such terms and how to isolate specific diastereomers or
enantiomers. Likewise, the term cobalt-polypyridyl complex
encompasses any solvates or hydrates of the cobalt-polypyridyl
complex. The term "solvate" refers to a complex of variable
stoichiometry formed by a solute, i.e. the cobalt-polypyridyl
complex, and a solvent. If the solvent is water, the solvate formed
is a hydrate.
[0035] The method of claim 1, wherein the cobalt-polypyridyl
complex comprises a structure of Formula (II):
##STR00007##
wherein R.sub.1 and R.sub.2 are identical and as defined above.
Preferably R.sub.1 and R.sub.2 are selected from --H, --CH.sub.3,
--C.sub.9H.sub.19 or --OCH.sub.3. n corresponds to the oxidation
state of the cobalt ion and is preferably 2 or 3. The
cobalt-polypyridyl complex can optionally comprise a counterion
which is preferably but not exclusively a monovalent anion selected
from hexafluorophosphate (PF.sub.6.sup.-) or chloride
(Cl.sup.-).
[0036] More preferably, the cobalt-polypyridyl complex comprises
and in particular essentially consists of a structure of one of
Formulas (Ill), (IV), (V), (VI), (VII) or (VIII) and optionally a
counterion, in particular a monovalent counterion preferably
selected from, but not limited to, hexafluorophosphate
(PF.sub.6.sup.-) or chloride (Cl.sup.-):
##STR00008## ##STR00009##
[0037] In further preferred embodiments, the cobalt-polypyridyl
complex comprises and in particular essentially consists of a
structure selected from Formula (III), Formula (IV) or Formula
(VII) and optionally a counterion:
##STR00010##
[0038] In most preferred embodiments of the present invention, the
cobalt-polypyridyl complex comprises and in particular essentially
consists of a structure of Formula (IV) and optionally a
counterion:
##STR00011##
[0039] In particular embodiments of the present invention, the
cobalt-polypyridyl complex comprises and in particular essentially
consists of a structure of Formula (IV) and a counterion, wherein
the counterion is a monovalent anion and preferably selected from,
but not limited to, hexafluorophosphate (PF.sub.6.sup.-) or
chloride (Cl.sup.-).
[0040] The cobalt-polypyridyl complex may be administered in the
method of the present invention in combination with:
[0041] (ii) an effective amount of at least one chemotherapeutic
compound, which chemotherapeutic compound is a compound selected
from the group consisting of a topoisomerase-II inhibitor, an
anthracycline, a coordination complex of platinum, a taxane, a
protein kinase inhibitor, a vinca alkaloid or derivative thereof, a
topoisomerase-I inhibitor and a nucleotide analog or precursor
analog;
[0042] (ii) radiotherapy, and/or
[0043] (iii) immunotherapy.
[0044] The expression "effective amount" and "effective dose"
generally denote an amount sufficient to produce therapeutically
desirable results, wherein the exact nature of the result varies
depending on the specific disorder which is treated. When the
disorder is cancer, the result is usually an inhibition or
suppression of the proliferation of the cancer cells, a reduction
of cancerous cells or the amelioration of symptoms related to the
cancer cells.
[0045] The effective amount of the cobalt-polypyridyl complex of
the present invention may depend on the species, body weight, age
and individual conditions of the subject and can be determined by
standard procedures such as with cell cultures or experimental
animals. A concentration of the cobalt-polypyridyl complex such as
the cobalt-polypyridyl complex comprising a structure of Formula
(IV) may, for example, be at least 1 .mu.M, preferably at least 5
.mu.M, in particular at least 10 .mu.M or at least 20 .mu.M.
[0046] The subject can be a human or animal, in particular the
subject is a mammal, preferably a human. The subject is preferably
a human having a cancer with a multidrug-resistance. Said subject,
thus, includes human subjects having a drug resistance to
conventional therapeutic agents which induce cell death in cancer
cells, i.e. which are used to treat cancer.
[0047] The terms "cancer" and "cancerous" refer to or describe a
physiological condition in subjects in which a population of cells
are characterized by unregulated cell growth. The term "tumor"
simply refers to a mass being of benign (generally harmless) or
malignant (cancerous) growth.
[0048] The cancer can be of any origin, in particular human origin.
In particular, the cancer is selected from the group consisting of:
[0049] ovarian cancer, [0050] cervical cancer, [0051] liver cancer,
[0052] lung cancer, [0053] breast cancer [0054] gastric cancer, or
[0055] colon cancer. Preferably, the cancer is selected from
multidrug-resistant: [0056] lung cancer, [0057] breast cancer, or
[0058] colon cancer which is resistant at least against paclitaxel
(taxol).
[0059] The provided method is preferably used and particularly
effective in treating subjects whose cancer has become
"multidrug-resistant". The term "multidrug-resistance" is generally
used for an acquired or natural, i.e. intrinsic, resistance of a
cancer.
[0060] Cancers with cancer cells that have developed resistance to
or are naturally resistant to chemotherapeutic compounds, usually
to two or more chemotherapeutic compounds, are said to be
"multidrug-resistant" in the present patent application such as to
chemotherapeutic compounds selected from the group consisting of
topoisomerase-II inhibitors, anthracyclines, coordination complexes
of platinum, taxanes, protein kinase inhibitors, vinca alkaloids or
derivatives thereof, topoisomerase-I inhibitors and nucleotide
analogs or precursor analogs. In preferred embodiments of the
present invention, the multidrug-resistant cancer is a cancer
having multidrug-resistant cancer cells, i.e. cancer cells which
have developed resistance to or are naturally resistant to at least
one of paclitaxel (taxol), doxorubicin, cisplatin, etoposide and
staurosporine, in particular against one of taxol or cisplatin or
both of them, more preferably cancer cells that are resistant at
least against taxol.
[0061] A cancer is multidrug-resistant if it comprises cancer cells
which are multidrug-resistant, in particular if a significant
amount of the cancer cells, such as more than 50%, in said cancer
are multidrug-resistant. Accordingly, the multidrug-resistant
cancer will be less sensitive or more tolerant to most common
chemotherapeutic agents.
[0062] A multidrug-resistance can be detected in a subject, cancer,
tissue, or cell by administering to the subject, tissue, or cell,
compounds such as chemotherapeutic compounds and determining the
activity of the chemotherapeutic compounds such as the induction of
cell death or the inhibition of the proliferation of cancer cells
compared to a reference control, namely cells or tissue of the same
cell or tissue type, a cancer or a subject that do not have
multidrug-resistance or non-cancerous cells.
[0063] The multidrug-resistant cancer according to the present
invention preferably has multidrug-resistant cancer cells which are
at least one of: [0064] "ABC-protein-dependent", i.e. the multidrug
resistance is at least mediated by ABC transporter proteins
(hereinafter "ABC-proteins"), in particular by P-glycoprotein, i.e.
is associated with an enhanced expression and/or enhanced
functional activity of at least one ABC-protein in the
multidrug-resistant cancer cells, in particular of P-glycoprotein
also referenced as "P-glycoprotein-dependent", and/or [0065]
"apoptosis-deficient", i.e. at least associated with a decreased
expression of at least one pro-apoptotic protein and/or decreased
pro-apoptotic activity of at least one pro-apoptotic protein and/or
enhanced expression of at least one anti-apoptotic protein and/or
enhanced anti-apoptotic activity of at least one anti-apoptotic
protein in the multidrug-resistant cancer cells; in particular
apoptosis-deficient refers to at least one of decreased expression
of at least one pro-apoptotic protein and/or decreased
pro-apoptotic activity of at least one pro-apoptotic protein, which
pro-apoptotic protein is in particular p53 referenced herein as
"p53-deficient".
[0066] In one embodiment of the present invention, the
multidrug-resistance and hence the multidrug-resistant cancer is at
least ABC-protein-dependent, in particular P-glycoprotein-dependent
and/or apoptosis-deficient, in particular p53-deficient.
[0067] An enhanced expression and/or enhanced functional activity
of at least one ABC-protein, i.e. ABC-protein-dependent
multidrug-resistant cancer, means an expression and/or functional
activity exceeding, in particular significantly exceeding, the one
in normal cells or tissue, i.e. non-cancerous cells or tissue, or
cancer cells without the multidrug-resistant phenotype. The term
"enhanced expression" or "enhanced functional activity" of at least
one ABC-protein such as P-glycoprotein includes embodiments in
which the multidrug-resistant cancer cells express the ABC-protein
such as P-glycoprotein, whereas in the reference control, i.e.
cancer cells without the multidrug-resistant phenotype or
non-cancerous cells of the same cell or tissue type, said
ABC-protein such as P-glycoprotein is not expressed, at all. I.e.
when said reference control does not express the ABC-protein such
as P-glycoprotein, multidrug-resistant cancer cells having a
detectable expression or functional activity of the ABC-protein
such as P-glycoprotein are ABC-protein-dependent by definition.
[0068] Whether a multidrug-resistant cancer is an
ABC-protein-dependent multidrug-resistant cancer can be determined
by methods known to the skilled person in particular comprising
immunological methods accompanied by the use of MDR-specific
antibodies, immunocytochemistry and immunohistochemistry,
respectively, by determining respective mRNA levels such as with
Northern blots or quantitative RT-PCR, with MDR-specific antibodies
in vivo or with an ABC-protein such as P-glycoprotein efflux assay
detecting the efflux of a marker. In particular, an ABC-protein
such as P-glycoprotein efflux assay can be used for determining the
functional activity of ABC-proteins, i.e. for determining whether
multidrug-resistant cancer cells are ABC-protein-dependent. Markers
which can be used in said efflux assay include drugs which are a
substrate for the respective ABC-protein, a radionuclide or a dye.
In particular, a sample of multidrug-resistant cancer cells and,
thus, a cancer having those cells, is preferably considered for
being ABC-protein-dependent, if it comprises less cells with marker
such as dye like Rho123 as revealed by the assay compared to the
reference control which is a cell sample with ABC-protein
expression as present in cancer cells that do not have a
multidrug-resistance phenotype or non-cancerous cells of the same
cell or tissue type.
[0069] Preferably, an ABC-protein-dependent such as a
P-glycoprotein-dependent multidrug-resistant cancer is a cancer
comprising multidrug-resistant cancer cells with an expression of
ABC-protein or ABC-protein functional activity exceeding the one in
the reference control by at least 10%, in particular by at least
20%. For example, the expression or functional activity of
P-glycoprotein is at least 10% or at least 20% higher than the
expression or functional activity of P-glycoprotein in the
reference control.
[0070] The term "apoptosis-deficient" used herein refers to a
cancer having at least one of (i) decreased expression and/or
decreased pro-apoptotic activity of at least one pro-apoptotic
protein or (ii) enhanced expression and/or enhanced anti-apoptotic
activity of at least one anti-apoptotic protein or both of them,
i.e. (i) and (ii). Pro- and/or anti-apoptotic proteins in
particular include p53, mitogen-activated protein kinase
(MAPK)-family members and B cell lymphoma 2 (Bcl-2) family members.
In particular, "apoptosis-deficient" as used herein means at least
a decreased expression and/or decreased pro-apoptotic activity of
at least one pro-apoptotic protein selected from p53 or Bax or Bak
of the Bcl-2-family. The term "p53 protein" used herein includes
respective p53 isoforms encoded by the TP53 gene such as
p53.alpha., p53.beta., p53.gamma., .DELTA.40p53.alpha.,
.DELTA.40p53.beta., .DELTA.40p53.gamma., .DELTA.133p53.alpha.,
.DELTA.133p53.beta., .DELTA.133p53.gamma., .DELTA.160p53.alpha.,
.DELTA.160p53.beta., .DELTA.160p53.gamma. and the like.
[0071] Whether a multidrug-resistant cancer is apoptosis-deficient
can be determined by methods known to the skilled person, namely by
measuring the expression of the pro-apoptotic protein and/or
anti-apoptotic protein and/or by determining the apoptotic activity
of the pro-apoptotic protein and/or the anti-apoptotic protein
compared with a reference control, namely cancer cells that do not
have a multidrug-resistance phenotype or non-cancerous cells of the
same cell or tissue type. Suitable methods for determining the
expression may include immunological methods accompanied by the use
of specific antibodies, immunocytochemistry and
immunohistochemistry, respectively, or by determining respective
mRNA levels such as with Northern blots or quantitative RT-PCR. The
apoptotic activity can be determined by assays which determine the
rate of apoptosis known to the skilled person such as assays
determining cytomorphological alterations, DNA fragmentation such
as by TUNEL assay along with flow cytometry, detection of apoptosis
pathway downstream targets, cleaved substrates, regulators and
inhibitors, membrane alterations, or mitochondrial assays.
[0072] Decreased expression and/or decreased pro-apoptotic activity
of pro-apoptotic proteins in particular means an expression and/or
pro-apoptotic activity of said at least one protein which is
decreased by at least 10%, more preferably by at least 20% compared
with a reference control, namely cancer cells without the
multidrug-resistant phenotype or non-cancerous cells of the same
cell or tissue type. In particular embodiments of the present
invention, said at least one pro-apoptotic protein is not
detectably expressed in the multidrug-resistant cancer cells.
Decreased expression and/or decreased pro-apoptotic activity of at
least one pro-apoptotic protein may be caused by, for example, a
decreased expression of pro-apoptotic wild-type protein and/or by
an expression of a pro-apoptotic mutant protein with decreased
pro-apoptotic activity. A mutant pro-apoptotic protein has an amino
acid sequence different from the wild-type protein expressed in
healthy cells without a mutation in the respective encoding
genes.
[0073] In an embodiment of the present invention, the
multidrug-resistant cancer is a cancer comprising at least one of:
[0074] multidrug-resistant apoptosis-deficient cancer cells with a
decreased expression and/or decreased pro-apoptotic activity of
p53, and/or [0075] multidrug-resistant P-glycoprotein-dependent
cancer cells, in particular comprising a significant amount of such
cancer cells.
[0076] The method of the present invention may comprise further
steps before administering the cobalt-polypyridyl complex, in
particular the cobalt-polypyridyl complex of Formula (III), (IV) or
(VII) of: [0077] obtaining a sample, in particular cancer cells,
from the subject; [0078] testing said sample for at least one of
[0079] the expression of at least one ABC-protein, in particular of
P-glycoprotein; [0080] at least one ABC-protein, in particular the
P-glycoprotein, functional activity; [0081] the expression of at
least one pro- or anti-apoptotic protein, in particular p53; [0082]
the apoptotic activity of at least one pro- or anti-apoptotic
protein, in particular of p53; [0083] optionally correlating the
expression and/or functional activity of the at least one
ABC-protein and/or the expression or activity of the at least one
pro- or anti-apoptotic protein with an outcome and if conditions
are met, administrating the cobalt-polypyridyl complex to said
subject; alone or in combination with a further chemotherapeutic
compound.
[0084] According to the invention is also the cobalt-polypyridyl
complex of the present invention, in particular comprising a
structure of Formula (III), (IV) or (VII), for use as a medicament
for the treatment of cancer, in particular multidrug-resistant
cancer such as P-glycoprotein dependent multidrug-resistant cancer
or p53-deficient multidrug-resistant cancer. The cobalt-polypyridyl
complex of the present invention, in particular comprising a
structure of Formula (III), (IV) or (VII), can be used in an
effective amount for treating an animal or a human, in particular
mammal, preferably a human. Another aspect of the invention refers
to the use of the cobalt-polypyridyl complex of the present
invention, in particular comprising a structure of Formula (III),
(IV) or (VII), for preparing a medicament for treatment of cancer,
in particular multidrug-resistant cancer such as
P-glycoprotein-dependent cancer or p53-deficient
multidrug-resistant cancer. The cobalt-polypyridyl complex of the
present invention, in particular comprising a structure of Formula
(III), (IV) or (VII), may be used in combination with at least a
further chemotherapeutic compound.
[0085] The present invention also relates to a method of using the
cobalt-polypyridyl complex of the present invention, preferably
comprising and in particular essentially consisting of a structure
of Formula (III), (IV) or (VII) and optionally a counterion, for
suppressing cancer growth, in particular for inducing autophagy,
inducing cell cycle arrest and/or inhibiting cell invasion.
[0086] The suppression of cancer growth, in particular induction of
autophagy, induction of cell cycle arrest and inhibition of cell
invasion can be determined with an immunofluorescence staining with
anti-LC3 antibodies or Western Blotting of LC3-I and LC3-II, the
latter being a marker for autophagy. Cell cycle analysis can be
carried out, for example, with DNA-binding dyes such as propidium
bromide. Inhibition of cell invasion can be examined with
commercially available cell invasion assay kits. The measurements
are carried out in the presence of the cobalt-polypyridyl complex
of the present invention compared to a reference control being
cancerous cells of the same cell type in the absence of the
cobalt-polypyridyl complex. A suppression of cancer growth, in
particular induction of autophagy, induction of cell cycle arrest
and inhibition of cell invasion means a statistically significant
change compared to the reference control, preferably more than 10%
and in particular more than 20% change compared to the value
measured for the reference control.
[0087] The cobalt-polypyridyl complex of the present invention
comprising a cobalt ion in the oxidation state +2 or +3, at least
one polypyridyl ligand of Formula (I) and optionally a counterion
can be prepared by a method comprising steps of:
(i) providing a mixture of a cobalt salt with a cobalt oxidation
state of +2 and the polypyridyl ligand of Formula (I):
##STR00012##
in a solvent, wherein R.sub.1 and R.sub.2 are independently
selected from hydrogen, a C.sub.1-C.sub.10 alkyl or a
C.sub.1-C.sub.10 alkoxy and optionally a further polypyridyl
ligand; (ii) optionally adding a salt comprising the counterion;
(iii) optionally oxidizing the cobalt ion to the oxidation state
+3.
[0088] The solvent in step (i) preferably comprises an aliphatic
alcohol, which means herein an aliphatic hydrocarbon, preferably a
branched or straight chain alkane, wherein at least one hydrogen
atom of the aliphatic hydrocarbon is substituted with a hydroxyl
group, preferably one hydrogen atom is substituted with a hydroxyl
group referenced as monohydric aliphatic alcohol. More preferably,
the aliphatic alcohol is a monohydric aliphatic alcohol, still more
preferably a monohydric alcohol with 1 to 3 carbon atoms, further
preferably with 1 to 2 carbon atoms. I.e. the aliphatic alcohol is
more preferably selected from methanol, ethanol, propanol or
isopropanol or mixtures thereof and further preferably from
methanol, ethanol or mixtures thereof. More preferably, the
aliphatic alcohol is methanol. The solvent in step (i) most
preferably essentially consists of methanol.
[0089] Step (i) preferably comprises steps of:
[0090] a) preparing a mixture of the cobalt salt with a cobalt
oxidation state of +2 and the polypyridyl ligand of Formula (I) in
methanol; wherein the molar ratio of the polypyridine ligand of
Formula (I) and the cobalt salt is between 2:1 and 4:1, more
preferably about 3.3:1;
[0091] b) heating the mixture to reflux, preferably under N.sub.2
atmosphere for 1 h to 3 h, preferably for about 2 h;
[0092] c) cooling the mixture down to a temperature between
20.degree. C. and 30.degree. C., preferably to about
25.+-.2.degree. C.
[0093] The cobalt salt is preferably a cobalt chloride or hydrate
thereof, more preferably CoCl.sub.2.times.6 H.sub.2O. The ligand is
preferably of Formula (I) with R.sub.1 and R.sub.2 being identical
and selected from --H, --CH.sub.3, --C.sub.9H.sub.19, --OCH.sub.3.
Preferably, no further polypyridyl ligand is added in step (i).
[0094] Step (ii) preferably comprises adding an excess of a salt
comprising the counterion, preferably monovalent anions which are
in particular, but not exclusively, selected from PF.sub.6.sup.- or
Cl.sup.-, for example, adding NH.sub.4PF.sub.6. Step (ii) in
particular comprises steps of:
[0095] a) adding the salt comprising the counterion, in particular
in a molar ratio of at least 4:1 compared to the cobalt salt added
in step (i);
[0096] b) stirring the mixture for 30 min to 90 min, in particular
for about 60 min;
[0097] c) separating the precipitate by filtration and optional
purifying the precipitate and optional drying.
[0098] Purification in step c) may be carried out by washing with
at least one washing solvent, in particular an aliphatic alcohol
and/or an ether, still more preferably a monohydric alcohol with 1
to 3 carbon atoms, further preferably with 1 to 2 carbon atoms
and/or an ether, in particular a C.sub.2 to C.sub.6 dialkyl ether.
More preferably, the at least one washing solvent is selected from
one or more of methanol, ethanol or diethyl ether. The optional
drying may be carried out in vacuum.
[0099] Step (iii) preferably comprises steps of:
[0100] a) adding an oxidizing agent and a solvent;
[0101] b) removing the solvent for obtaining a residue;
[0102] c) optionally dissolving the residue in a solvent and adding
a salt comprising the counterion;
[0103] d) separating the precipitate by filtration and optionally
purifying the precipitate and optionally drying.
[0104] The oxidizing agent is preferably NOBF.sub.4 and used in a
molar ratio to the cobalt complex obtained in step (i) of between
0.8:1 and 1.2:1, in particular of about 1:1. The solvent is
preferably an organic nitrile, in particular acetonitrile. The
mixture in step a) is preferably left at a temperature of between
20.degree. C. and 30.degree. C., preferably about 25.+-.2.degree.
C. for about 1 h. The solvent is preferably removed under reduced
pressure. The solvent in step c) is preferably an organic nitrile,
more preferably acetonitrile. Step c) preferably comprises adding
an excess of a salt comprising the counterion, preferably but not
exclusively monovalent anions in particular selected from
PF.sub.6.sup.- or Cl.sup.-, for example, adding NH.sub.4PF.sub.6,
in particular with a molar ratio of at least 4:1 compared to the
cobalt complex obtained in step (i). Purifying the precipitate is
preferably carried out by recrystallizing the precipitate in a
recrystallization solvent comprising a ketone and/or an ether,
preferably acetone and/or diethyl ether. Drying may be carried out
in vacuum. If desired, a counterion can be readily exchanged with
another counterion by any of the methods known to one skilled in
the art, including ion exchange chromatography and other ion
exchange methods or recrystallization.
[0105] The present invention further provides a method for
suppressing the growth of cancer cells comprising contacting the
cancer cells with an effective amount of a cobalt-polypyridyl
complex, wherein the cobalt-polypyridyl complex comprises a cobalt
ion and at least one polypyridyl ligand of Formula (I):
##STR00013##
wherein R.sub.1 and R.sub.2 are independently selected from
hydrogen, a C.sub.1-C.sub.10 alkyl or a C.sub.1-C.sub.10 alkoxy.
Preferably, the method is for inducing autophagy of the cancer
cells, inducing cell cycle arrest of the cancer cells and/or
inhibiting cell invasion of the cancer cells.
[0106] The cancer cells are preferably multidrug-resistant cancer
cells and more preferably at least one of (i) multidrug-resistant
ABC-protein-dependent, in particular P-glycoprotein-dependent
multidrug-resistant cancer cells and/or (ii) apoptosis-deficient
multidrug-resistant cancer cells, in particular p53-deficient
multidrug-resistant cancer cells. The multidrug-resistant cancer
cells are preferably at least resistant against taxol. The
Resistant Factor of the cobalt-polypyridyl complex towards the
multidrug-resistant cancer cells is preferably less than 0.6.
[0107] The IC.sub.50 of the cobalt-polypyridyl complex against the
cancer cells is preferably at most 10 .mu.M with an IC.sub.50
against non-cancerous cells at least 2 times higher.
[0108] The cancer cells are preferably of human origin. The cancer
cells are preferably from one of: [0109] an ovarian cancer, [0110]
a cervical cancer, [0111] a liver cancer, [0112] a lung cancer,
[0113] a breast cancer, [0114] a gastric cancer, or [0115] a colon
cancer.
[0116] The concentration of the cobalt-polypyridyl complex used for
contacting the cancer cells may range from 0.2 .mu.M to 100 .mu.M,
preferably from 1 .mu.M to 30 .mu.M, i.e. from 1 .mu.mol/l (=1
mmol/m.sup.3) and 30 .mu.mol/l (=30 mmol/m.sup.3). The cancer cells
are preferably contacted with the cobalt-polypyridyl complex of the
present invention for at least 12 h, preferably for at least 24 h,
more preferably for at least 48 h and in particular for at least 72
h.
[0117] In especially preferred embodiments of the present
invention, the cobalt-polypyridyl complex used for contacting the
cancer cells comprises and in particular essentially consists of a
structure of Formula (III), (IV) or (VII) and optionally a
counterion:
##STR00014##
[0118] The cobalt-polypyridyl complex more preferably comprises a
structure of Formula (IV), wherein the cancer cells are contacted
with of from about 1 .mu.M to about 30 .mu.M of said
cobalt-polypyridyl complex. In particular embodiments of the
present invention, the cobalt-polypyridyl complex comprises and in
particular essentially consists of a structure of Formula (IV) and
optionally a counterion. The counterion can be a monovalent anion
and preferably but not exclusively selected from
hexafluorophosphate (PF.sub.6.sup.-) or chloride (Cl.sup.-) and
wherein the cancer cells are contacted with of from about 1 .mu.M
to about 30 .mu.M of said cobalt-polypyridyl complex. The step of
contacting the cancer cells with the cobalt-polypyridyl complex of
the present invention, in particular comprising a structure of
Formula (III), (IV) or (VII), may be carried out by applying an
incubation solution comprising the cobalt-polypyridyl complex to
said cells which incubation solution may further comprise suitable
excipients such as buffers or a suitable growth medium.
[0119] The method may further comprise contacting said cells with a
further chemotherapeutic compound; including for example,
cisplatin, doxorubicin, taxol, etoposide and staurosporine; before,
at the same time with or subsequent to the application of the
cobalt-polypyridyl complex of the present invention.
[0120] In another aspect of the present invention, a method for
specifically targeting cancer cells with multidrug-resistance is
provided, in particular ABC-protein-dependent multidrug-resistant
cancer cells such as P-glycoprotein dependent multidrug-resistant
cancer cells or apoptosis-deficient such as p53-deficient
multidrug-resistant cancer cells. Said method comprises the step of
contacting a population of cancer cells with multidrug-resistance
such as in a sample from a subject or in a subject with the
cobalt-polypyridyl complex described above. Preferably, the cancer
growth is suppressed, in particular autophagy is induced, cell
cycle arrest is induced and/or cell invasion is inhibited.
[0121] The multidrug-resistant cancer cells are in particular
ABC-protein-dependent, most preferably P-glycoprotein-dependent
and/or apoptosis-deficient such as p53-deficient.
[0122] The multidrug-resistant cancer cells can be of any origin,
in particular human origin. In particular, the multidrug-resistant
cancer cells are from a multidrug-resistant: [0123] ovarian cancer,
[0124] cervical cancer, [0125] liver cancer, [0126] lung cancer,
[0127] breast cancer [0128] gastric cancer, or [0129] colon
cancer.
[0130] In preferred embodiments of the present invention, the
multidrug-resistant cancer cells are resistant against at least one
of paclitaxel (taxol), doxorubicin, cisplatin, etoposide and
staurosporine, in particular against at least taxol.
[0131] The concentration of the cobalt-polypyridyl complex used for
contacting the multidrug-resistant cancer cells may range from 0.2
.mu.M to 100 .mu.M, preferably from 1 .mu.M to 30 .mu.M, i.e. from
1 .mu.mol/l (=1 mmol/m.sup.3) and 30 .mu.mol/l (=30 mmol/m.sup.3).
The multidrug-resistant cancer cells are preferably contacted with
the cobalt-polypyridyl complex of the present invention for at
least 12 h, preferably for at least 24 h, more preferably for at
least 48 h and in particular for at least 72 h.
[0132] Preferably, the IC.sub.50 of the cobalt-polypyridyl complex
towards the multidrug-resistant cancer cells is at most 20 .mu.M,
more preferably at most 15 .mu.M and in particular at most 5 .mu.M.
The Resistant Factor of the cobalt-polypyridyl complex of the
present invention towards the multidrug-resistant cancer cells is
preferably less than 0.8, more preferably less than 0.6 and in
particular less than 0.5 and more preferably less than 0.3. The
Resistant Factor is calculated by dividing the IC.sub.50 of the
cobalt-polypyridyl complex towards multidrug-resistant cells by its
IC.sub.50 towards cancer cells of the same cell type or tissue
which do not have a multidrug-resistance phenotype. A Resistant
Factor <1 indicates that a compound is especially effective in
multidrug-resistant cancer cells compared to cancer cells of the
same cell type or tissue which do not have a multidrug-resistant
phenotype, i.e. is especially suitable to specifically target
multidrug-resistant cancer cells.
[0133] In one embodiment of the present invention, the
multidrug-resistant cancer cells used within the method are
P-glycoprotein-dependent multidrug-resistant cancer cells and the
Resistant Factor of the cobalt-polypyridyl complex of the present
invention is at most 0.5, wherein the multidrug-resistant cancer
cells are from a lung cancer, colon cancer or breast cancer and
wherein the multidrug-resistant cancer cells are resistant against
taxol.
[0134] In another embodiment of the present invention, the
multidrug-resistant cancer cells used within the method are
p53-deficient multidrug-resistant cancer cells and wherein the
Resistant Factor of the cobalt-polypyridyl complex of the present
invention is at most 0.6 and wherein the multidrug-resistant cancer
cells are from a colon cancer.
[0135] In especially preferred embodiments of the present
invention, the cobalt-polypyridyl complex used for contacting the
multidrug-resistant cancer cells comprises and in particular
essentially consists of a structure of Formula (III), (IV) or (VII)
and optionally a counterion:
##STR00015##
[0136] The cobalt-polypyridyl complex more preferably comprises a
structure of Formula (IV) and optionally a counterion, wherein the
multidrug-resistant cancer cells are contacted with of from about 1
.mu.M to about 30 .mu.M of said cobalt-polypyridyl complex. In
particular embodiments of the present invention, the
cobalt-polypyridyl complex comprises and in particular essentially
consists of a structure of Formula (IV) and optionally a
counterion. The counterion can be a monovalent anion and preferably
but not exclusively be selected from hexafluorophosphate
(PF.sub.6.sup.-) or chloride (Cl.sup.-).
[0137] The step of contacting the cancer cells with the
cobalt-polypyridyl complex of the present invention, in particular
comprising a structure of Formula (III), (IV) or (VII), may be
carried out by applying an incubation solution comprising the
cobalt-polypyridyl complex to said cells which incubation solution
may further comprise suitable excipients such as buffers or a
suitable growth medium.
[0138] The method may further comprise contacting said cells with a
further chemotherapeutic compound; including for example,
cisplatin, doxorubicin, taxol, etoposide and staurosporine; before,
at the same time with or subsequent to the application of the
cobalt-polypyridyl complex of the present invention.
[0139] Further in accordance with the present invention is a
composition comprising the cobalt-polypyridyl complex and an
excipient such as selected from a pharmaceutically acceptable
carrier, salt, buffer, water, or a combination thereof. More
preferably, the composition is a pharmaceutical composition
comprising an effective dose of the cobalt-polypyridyl complex and
at least one pharmaceutically tolerable excipient such as selected
from at least one of a diluent, a filler, a binder, a disintegrant,
a lubricant, a coloring agent, a surfactant or a preservative.
[0140] The skilled person is able to select suitable
pharmaceutically tolerable excipients depending on the form of the
pharmaceutical composition and is aware of methods for
manufacturing pharmaceutical compositions as well as able to select
a suitable method for preparing the pharmaceutical composition
depending on the kind of pharmaceutically tolerable excipients and
the form of the pharmaceutical composition. The pharmaceutical
composition according to the invention can be present in solid,
semisolid or liquid form to be administered by an oral, rectal,
topical, parenteral or transdermal or inhalative route to a
subject, preferably a human.
[0141] Further in accordance with the present invention is a kit
comprising an effective dose of:
[0142] (i) a cobalt-polypyridyl complex as described above;
[0143] (ii) at least one chemotherapeutic compound selected from
the group consisting of a topoisomerase-II inhibitor, an
anthracycline, a coordination complex of platinum, a taxane, a
protein kinase inhibitor, a vinca alkaloid or derivative thereof, a
topoisomerase-I inhibitor and a nucleotide analog or precursor
analog.
[0144] The kit may comprise excipients, in particular
pharmaceutically tolerable excipients, such as a carrier, salt,
buffer, water, or a combination thereof. The skilled person is able
to select suitable excipients. Still further, the kit may comprise
at least one container.
[0145] Preferably, the cobalt-polypyridyl complex in the
pharmaceutical composition or the kit comprises and in particular
essentially consists of a structure of Formula (III), (IV) or (VII)
and optionally a counterion:
##STR00016##
[0146] The cobalt-polypyridyl complex more preferably comprises a
structure of Formula (IV). In particular embodiments of the present
invention, the cobalt-polypyridyl complex comprises and in
particular essentially consists of a structure of Formula (IV) and
optionally a counterion, which can be a monovalent anion and
preferably but not exclusively be selected from hexafluorophosphate
(PF.sub.6.sup.-) or chloride (Cl.sup.-).
[0147] Still another aspect of the present invention concerns the
use of the pharmaceutical composition or the kit described above
for suppressing the growth of cancer cells, in particular
multidrug-resistant cancer cells, more preferably for inducing
autophagy of the cancer cells, inducing cell cycle arrest of the
cancer cells and/or inhibiting cell invasion of the cancer
cells.
EXAMPLES
Example 1
Preparation of Cobalt-Polypyridyl Complexes of the Present
Invention
[0148] Cobalt-polypyridyl complexes of Formula (V) and (VI) with a
counterion were synthesized according to the method below. Other
cobalt-polypyridyl complexes were prepared by the modification of
the reported procedure.
Preparation of [Co.sup.II(N N).sub.3](PF.sub.6).sub.2
(Cobalt-Polypyridyl Complex of Formula (V) with PF.sub.6.sup.- as
Counterion)
[0149] A mixture of CoC1.sub.2.6H.sub.2O (4 mmol) and
2,2'-bipyridine (13.2 mmol) in methanol (100 mL) was heated to
reflux under N.sub.2 for 2 h. After cooling down to room
temperature, NH.sub.4PF.sub.6 (20 mmol) was added to the reaction
mixture and the reaction mixture was stirred for another 1 h. The
precipitate was filtrated and washed with methanol and then diethyl
ether.
Preparation of [Co.sup.III(N N).sub.3](PF.sub.6).sub.3
(Cobalt-Polypyridyl Complex of Formula (VI) with PF.sub.6.sup.- as
Counterion)
[0150] Oxidation of [Co.sup.II(N N).sub.3](PF6).sub.2 (2 mmol) was
carried out by the treatment of NOBF.sub.4 (2 mmol) in acetonitrile
(40 mL) at room temperature for 1 h. After removing the solvent
under reduced pressure, the residue was dissolved in acetonitrile
(10 mL) and NH.sub.4PF.sub.6 (10 mmol) was added. The precipitate
was collected by filtration and the solid was purified by
recrystallization of acetone/diethyl ether to afford the crystal
form of the desired product.
[Co.sup.II(4,4'-Me.sub.2-bpy).sub.3](PF.sub.6).sub.2
(Cobalt-Polypyridyl Complex of Formula (III) with PF.sub.6.sup.- as
Counterion)
[0151] This complex has been obtained as yellow solid in 95% yield.
Positive-ion ESI-MS ion cluster at m/z (%): 756.20
[M-PF.sub.6.sup.-].sup.+. IR (KBr) v/cm.sup.-1: 843
(PF.sub.6.sup.-). Anal. Calcd for
CoH.sub.36H.sub.36N.sub.6F.sub.2P.sub.12.H.sub.2O (%): C, 47.02; H,
4.17; N, 9.14. Found: C, 46.88; H, 4.14; N, 9.13.
[Co.sup.III(4,4'-Me.sub.2-bpy).sub.3](PF.sub.6).sub.3
(Cobalt-Polypyridyl Complex of Formula (IV) with PF.sub.6.sup.- as
Counterion)
[0152] This complex has been obtained as yellow crystals in 88%
yield (through two steps). .sup.1H NMR (300 MHz, DMSO-d.sub.6,
298K, TMS)/ppm: .delta. 8.91 (s, 2H, H3 of bpy), 7.61 (d, 2H, J=4.8
Hz, H6 of bpy), 7.27 (d, 2H, J=4.8 Hz, H5 of bpy), 2.63 (s, 6H,
CH.sub.3). .sup.13C NMR (75 MHz, DMSO-d.sub.6, 298K, TMS)/ppm:
.delta. 156.7, 155.2, 150.4, 132.2, 127.9, 21.5. IR (KBr)
v/cm.sup.-1: 853 (PF.sub.6). Positive-ion ESI-MS ion cluster at m/z
901.2 [M-PF.sub.6.sup.-].sup.+, 305.6
[M-2.times.PF.sub.6.sup.-].sup.2+, 302.7
[M-3.times.PF.sub.6.sup.-].sup.3+. Anal. Calcd for
CoH.sub.36H.sub.36N.sub.6F.sub.3P.sub.18.MeOH (%): C, 41.20; H,
3.74; N, 7.79. Found: C, 41.09; H, 4.09; N, 7.82.
[Co.sup.III(4,4'-(OMe).sub.2-bpy).sub.3](PF.sub.6).sub.3
(Cobalt-Polypyridyl Complex of Formula (VII) with PF.sub.6.sup.- as
Counterion)
[0153] This complex has been obtained as orange crystals in 87%
yield (through two steps). .sup.1H NMR (300 MHz, DMSO-d.sub.6,
298K, TMS)/ppm: .delta. 8.68 (s, 2H, H3 of bpy), 7.32 (d, 2H, J=5.1
Hz, H6 of bpy), 7.21 (d, 2H, J=5.1 Hz, H5 of bpy), 4.09 (s, 6H,
OCH.sub.3). .sup.13C NMR (75 MHz, DMSO-d.sub.6, 298K, TMS)/ppm:
.delta. 170.6, 156.8, 151.6, 117.4, 114.1, 58.2. IR (KBr)
v/cm.sup.-1: 845 (PF.sub.6.sup.-). Positive-ion ESI-MS ion cluster
at m/z 997.1 [M-PF.sub.6.sup.-].sup.+, 426.1
[M-2.times.PF.sub.6.sup.-].sup.+, 235.7
[M-3.times.PF.sub.6.sup.-].sup.+. Anal. Calcd for
CoH.sub.36H.sub.36O.sub.6N.sub.6F.sub.3P.sub.18.2H.sub.2O (%): C,
36.39; H, 3.42; N, 7.13. Found: C, 36.66; H, 3.50; N, 7.24.
[Co.sup.III(4,4'-(C.sub.9H.sub.19).sub.2-bpy).sub.3](PF.sub.6).sub.3
(Cobalt-Polypyridyl Complex of Formula (VIII) with PF.sub.6.sup.-
as Counterion)
[0154] This complex has been obtained as yellow crystals in 85%
yield (through two steps). .sup.1H NMR (300 MHz, DMSO-d.sub.6,
298K, TMS)/ppm: .delta. 8.95 (s, 2H, H3 of bpy), 7.64 (d, 2H, J=4.8
Hz, H6 of bpy), 7.24 (d, 2H, J=4.8 Hz, H5 of bpy), 2.88 (t, 4H,
J=5.7 Hz, CH.sub.2), 1.71-1.67 (m, 4H, CH.sub.2), 1.31-1.20 (m,
24H, CH.sub.2), 0.87 (t, 6H, J=5.1 Hz, CH.sub.3). .sup.13C NMR (75
MHz, DMSO-d.sub.6, 298K, TMS)/ppm: .delta. 160.6, 155.5, 150.8,
131.3, 127.2, 35.0, 31.7, 29.7, 29.4, 29.2, 29.1, 22.6, 14.4. IR
(KBr) v/cm.sup.-1: 835 (PF.sub.6.sup.-). Positive-ion ESI-MS ion
cluster at m/z 1574.9 [M-PF.sub.6.sup.-].sup.+, 714.5
[M-2.times.PF.sub.6.sup.-].sup.2+, 428.0
[M-3.times.PF.sub.6.sup.-].sup.3+. Anal. Calcd for
CoH.sub.84H.sub.132N.sub.6F.sub.3P.sub.18.CH.sub.3CH.sub.2OCH.sub.2CH.sub-
.3 (%): C, 58.92; H, 7.98; N, 4.68. Found: C, 59.03; H, 7.93; N,
4.65.
Example 2
Cytotoxicity Assays
[0155] All prepared cobalt-polypyridyl complexes were dissolved in
DMSO at a final concentration of 50 mmol/L and stored at
-20.degree. C. before use. Cytotoxicity was assessed by using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(5.0 mg/ml) assay as previously described (Wong, V. K. et al., Cell
Death Dis, 2013, 4, e720). Briefly, 4.times.10.sup.3 cells per well
were seeded in 96-well plates before drug treatments. After
overnight culture, the cells were then exposed to different
concentrations of selected compounds (0.039-100 .mu.mol/L) for 72
h. Cells without drug treatment were used as control. Subsequently,
MTT (10 .mu.L) solution was added to each well and incubated at
37.degree. C. for 4 h followed by the addition of 100 .mu.L
solubilization buffer (10% SDS in 0.01 mol/L HCl) and overnight
incubation. A.sub.570 nm was then determined in each well on the
next day. The percentage of cell viability was calculated using the
following formula: Cell viability
(%)=A.sub.treated/A.sub.control.times.100. Data were obtained from
three independent experiments. Cells include HeLa cells (cervical
cancer), MCF-7 (breast cancer), H1299, H1975, A549 and LLC-1 (lung
cancer), HepG2 and Hep3B (liver cancer) and LO2 (normal human
hepatocytes).
[0156] The prepared cobalt-polypyridyl complexes displayed
considerable cytotoxicity against cancer cells from different
origins. Among the six cobalt-polypyridyl complexes, the
cobalt-polypyridyl complexes of Formula (III), (IV) and (VII) (with
PF.sub.6.sup.- as counterion) demonstrated the highest anti-cancer
potency with mean IC.sub.50 values ranging from 2.79-18.6 .mu.M,
the lower the IC.sub.50 values, the higher the anti-cancer potency.
By contrast, the cytotoxicity was lower in LO2 normal human
hepatocytes. Complexes of Formula (V) and (VI) showed moderate
anti-cancer potency with mean IC.sub.50 values ranging from
6.92-37.2 .mu.M, whereas complex (VIII) possessed the lowest
anti-cancer potency with mean IC.sub.50 values ranged from
10.4-82.8 .mu.M. The cobalt-polypyridyl complexes in different
oxidation states (or in different spin states) with the same
substituents on the bipyridine ligand showed similar cytotoxicity
on the cancer cells. Although the Co.sup.2+ and Co.sup.3+ complexes
are considered as inert d.sup.5 and lable d.sup.6 species,
respectively, for bioreductive activation as redox-activated
prodrugs through ligand modification, there is no direct
relationship in the nature of spin state on the cytotoxic effect.
Cobalt-polypyridyl complex of Formula (VII) having OCH.sub.3 groups
generally exhibited higher anti-cancer potency compared to
cobalt-polypyridyl complex of Formula (VI) with an un-substituted
bipyridine ligand which was less effective compared to
cobalt-polypyridyl complexe of Formula (IV) with CH.sub.3 groups.
On the other hand, the larger coordination sphere size and higher
lipophilicity may account for the high IC.sub.50 values of complex
(VIII). By scrutinizing their anti-cancer potencies, it is
noteworthy that complex (IV) was found to exhibit specific
cytotoxic effects towards cancer cells by demonstrating the lowest
mean IC.sub.50 values in various types of cancer cell lines.
Results were summarized in Table 1.
TABLE-US-00001 TABLE 1 Mean IC.sub.50 values [.mu.M] of the
prepared cobalt-polypyridyl complexes in various cancer cell lines
complex (III) (IV) (V) (VI) (VII) (VIII) HeLa 10.1 11.9 24.2 35.9
12.8 21.3 MCF-7 15.4 6.03 15.8 10.1 10.8 37.4 H1299 3.43 2.84 9.4
14.9 8.71 26.7 H1975 15.4 8.84 17.0 21.0 9.31 24.2 A549 9.45 7.86
26.3 37.2 18.6 82.8 LLC-1 2.79 3.39 17.2 29.2 8.71 10.4 HepG2 7.39
5.44 13.3 22.7 15.4 26.0 Hep 3B 4.06 3.7 6.92 13.8 5.33 54.1 LO2
13.5 13.2 33.6 43.7 20.6 40.2
Example 3
Collateral Sensitivity in Taxol-Resistant and p53-Deficient Cancer
Cells
[0157] All prepared cobalt-polypyridyl complexes were dissolved in
DMSO at a final concentration of 50 mmol/L and stored at
-20.degree. C. before use. Cytotoxicity was assessed by using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(5.0 mg/ml) assay as previously described (Wong, V. K. et al., Cell
Death Dis, 2013, 4, e720). Briefly, 4.times.10.sup.3 cells per well
were seeded in 96-well plates before drug treatments. After
overnight culture, the cells were then exposed to different
concentrations of selected compounds (0.039-100 .mu.mol/L) for 72
h. Cells without drug treatment were used as control. Subsequently,
MTT (10 .mu.L) solution was added to each well and incubated at
37.degree. C. for 4 h followed by the addition of 100 .mu.L
solubilization buffer (10% SDS in 0.01 mol/L HCl) and overnight
incubation. A.sub.570 nm was then determined in each well on the
next day. The percentage of cell viability was calculated using the
following formula: Cell viability
(%)=A.sub.treated/A.sub.control.times.100. Data were obtained from
three independent experiments.
[0158] As shown in Table 2, nearly all cobalt-polypyridyl complexes
displayed lower mean IC.sub.50 values in taxol-resistant cancers
which are P-glycoprotein-dependent among others when compared to
their taxol-sensitive cells in MCF-7, HCT8 and A549 cancer cells,
with a Resistant Factor (IC.sub.50 values of taxol-resistant cells
over the IC.sub.50 values of taxol-sensitive cells) below 0.8,
suggesting that these cobalt-polypyridyl complexes exhibit
collateral sensitivity towards the taxol-resistant cancers.
Compared with the cytotoxicity effect to the drug-sensitive cancer,
the cobalt-polypyridyl complexes of Formula (III), (IV) and (VII)
(with PF.sub.6.sup.- as counterion) demonstrated the most potent
cytotoxic effect towards these taxol-resistant cancers. In
addition, cisplatin-sensitive and -resistant pair of cancer cells
including A2780 ovarian cancer, SGC-7901 gastric cancer, OV2008 and
C13 cervical cancer were further adopted to examine the collateral
sensitivity of the cobalt-polypyridyl complexes as presented in
Table 3.
[0159] Only the cobalt-polypyridyl complexes of Formula (III), (IV)
and (VII) (with PF.sub.6.sup.- as counterion) were found to display
collateral sensitivity in p53-deficient colon cancer with a
Resistant Factor ranging from 0.52 to 0.59 as presented in Table 4,
whereas the cobalt-polypyridyl complexes of Formula (III), and (IV)
demonstrated the most potent and lowest IC.sub.50 values in
p53-deficient colon cancer.
TABLE-US-00002 TABLE 2 Collateral sensitivity of the prepared
cobalt-polypyridyl complexes in taxol-resistant cancer cells
IC.sub.50 value, .mu.M IC.sub.50 value, .mu.M Cancer
Taxol-sensitive Taxol-resistant Resistant complex Types cancer
cancer Factor (III) MCF-7 4.62 2.51 0.54 HCT8 3.13 1.16 0.37 A549
9.59 2.37 0.25 (IV) MCF-7 4 2.71 0.68 HCT8 3.39 1.22 0.36 A549 11.5
2.85 0.25 (V) MCF-7 13.4 16.8 1.25 HCT8 11.4 7.78 0.68 A549 29.9
7.85 0.26 (VI) MCF-7 8.02 5.01 0.62 HCT8 18.5 10.2 0.55 A549 51.7
11.1 0.21 (VII) MCF-7 9.45 6.28 0.66 HCT8 4.3 1.04 0.24 A549 15
2.85 0.19 (VIII) MCF-7 28.5 11.1 0.39 HCT8 56.4 41.1 0.73
TABLE-US-00003 TABLE 3 Collateral sensitivity of the prepared
cobalt-polypyridyl complexes in cisplatin-resistant cancer cells
IC.sub.50 value, .mu.M IC.sub.50 value, .mu.M Cancer cisplatin-
cisplatin-resistant Resistant Types sensitive cancer cancer Factor
(III) A2780 11.9 11.2 0.94 SGC-7901 11.7 17.6 1.50 OV2008 7.06 --
2.21 OV2008/C13 -- 15.6 (IV) A2780 12.3 10.7 0.87 SGC-7901 11.1
12.6 1.14 OV2008 7.62 -- 1.0 OV2008/C13 -- 7.59 (V) A2780 28.8 31.6
1.10 SGC-7901 29.9 29.9 1.00 OV2008 18.9 -- 0.79 OV2008/C13 -- 14.9
(VI) A2780 55 54.1 0.98 SGC-7901 41.1 41.7 1.01 OV2008 42.5 -- 0.88
OV2008/C13 -- 37.4 (VII) A2780 24 27 1.13 SGC-7901 21.6 20.7 0.96
OV2008 14 -- 1.45 OV2008/C13 -- 20.3 (VIII) A2780 30.2 29.1 0.96
SGC-7901 36.3 35.9 0.99 OV2008 49.4 -- 1.87
TABLE-US-00004 TABLE 4 Collateral sensitivity of the prepared
cobalt-polypyridyl complexes in p53 mutant colon cancer cells
(IC.sub.50 values) Resistant HCT116 p53.sup.+/+ HCT116 p53.sup.+/+
Factor (III) 17.1 8.89 0.52 (IV) 15.4 9.07 0.59 (V) 34.3 29.1 0.85
(VI) 45.1 45 1.00 (VII) 77.8 41.3 0.53
Example 4
Inhibition of Cancer Cell Growth Via Autophagy Induction, Cell
Cycle Arrest and Cell Invasion Inhibition
Endogenous Autophagy Detection
[0160] The detection of endogenous LC3 was conducted using
immunofluorescence staining method as described below. HeLa cancer
cells treated with the cobalt-polypyridyl complex of Formula (IV)
(with PF.sub.6.sup.- as counterion) on cover slips were fixed with
4% paraformaldehyde (Sigma) for 20 min at room temperature and then
rinsed with PBS. Coverslips were immersed in methanol at room
temperature for 2 min. After washing with PBS, the cells were then
incubated with anti-LC3 (1:200) in TBST (100 mM TrisHCl, pH 7.5,
150 mM NaCl, 0.05% Tween 20 and 5% BSA) overnight at 4.degree. C.
After washing with PBS, the cells were incubated with anti-mouse
secondary antibody (TRITC) (1:200) in TBST containing 5% BSA at
37.degree. C. for 1 h in the dark. The coverslips were then mounted
with FluorSave.TM. mounting media (Calbiochem, San Diego, Calif.,
USA) for fluorescence imaging and localization of LC3
autophagosomes were captured under the API Delta Vision Live-cell
Imaging System (Applied Precision Inc., GE Healthcare Company,
Washington, USA). To quantify autophagy, guidelines of autophagy
quantitation were followed (Klionsky, D. J. et al., Autophagy,
2016, 12, 1-222). The percentage of cells with autophagic induction
was calculated by the number of the cells with increased formation
of punctate LC3 fluorescence dots (.gtoreq.10 dots/cell) over the
total number of immunofluorescence-positive cells in the same
field. A minimum of 1000 cells from randomly selected fields were
scored.
Protein Extraction and Western Blotting
[0161] After drug treatment, adherent and floating cells were lysed
with RIPA lysis buffer. Protein concentrations were determined
using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc.,
Hercules, Calif., USA). The cell lysates of samples were subjected
to electrophoresis on SDS polyacrylamide gels and transferred to
Hybond enhanced chemiluminescence nitrocellulose membranes
(Amersham Biosciences, Piscataway, N.J.), which were then blocked
with 5% non-fat dry milk protein for 1 h. Membranes were then
incubated with the indicated primary antibodies overnight at
4.degree. C. The binding of the antibody was visualized by
peroxidase-coupled secondary antibody using the ECL Western
Blotting Detection Reagents (Invitrogen, Paisley, Scotland, UK).
Band intensities were quantified by using the software ImageJ (NIH,
Bethesda, Md., USA).
Cell Culture
[0162] All cells were obtained from the American Type Culture
Collection (Rockville, Md., USA) unless otherwise specified.
Immortalized wild-type and Atg7-deficient mouse embryonic
fibroblasts (MEFs) were provided by Professor Masaaki Komatsu
(Juntendo University, School of Medicine, Japan). Taxol-resistant
types of A549, MCF-7, HCT-8 cancer cells, cisplatin-resistant
SGC7901 cancer cells were purchased from KeyGEN BioTECH, China.
[0163] Cisplatin-sensitive or -resistant A2780, OV2008 and C13
cancer cells were provided by Prof. Benjamin Tsang (Department of
Obstetrics and Gynecology and Department of Cellular and Molecular
Medicine, University of Ottawa, Canada). HCT116 p53.sup.+/+ and
p53.sup.-/- isogenic human colon cancer cells were provided by
Professor Bert Vogelstein (Ludwig Center at Johns Hopkins, Howard
Hughes Medical Institute, USA). All media were supplemented with
10% fetal bovine serum and the antibiotics penicillin (50 U/ml) and
streptomycin (50 .mu.g/ml; Invitrogen, Paisley, Scotland, UK). All
cell cultures were incubated at 37.degree. C. in a 5% humidified
CO.sub.2 incubator.
Cell Cycle Analysis
[0164] For cell cycle analysis, the cells were harvested and washed
with ice-cold phosphate-buffered saline (PBS), and then suspended
and permeabilized with 70% ethanol for overnight at 4.degree. C.
For detecting DNA content and cell cycle, cells were incubated with
the freshly prepared propidium iodide (PI) staining buffer for 30
min at room temperature in dark. Fractions of the cells in G1, S,
and G2/M phase were analyzed using Modfit software 3.1.
Cell Invasion Assay
[0165] The cancer cell invasion assay was performed in a Cell
Invasion Chamber, a 24-well tissue culture plate with cell culture
inserts that contain an 8 mm pore size polycarbonate membrane over
a thin layer of dried ECMatrix.TM. (CHEMICON).
[0166] As shown in FIGS. 1A to 1D and 1E, the most potent
cobalt-polypyridyl complex of Formula (IV) (with PF.sub.6.sup.- as
counterion) was found to induce autophagy in dose-dependent manner
as indicated by increased percentages of red endogenous LC3-II
puncta formation (red TRITC signal), whereas the cells without
treatment (Ctrl) showed only slight or no red puncta formation. In
addition, the protein conversion from LC3-I to LC3-II is the marker
for autophagy induction. Here, is has been demonstrated that the
positive control drug, rapamycin (Rapa) increased the LC3-II
conversion, whereas the cobalt-polypyridyl complex of Formula (IV)
also dose-dependently increased the LC3-II protein conversion (FIG.
1F). In order to further confirm that the cobalt complex-induced
autophagy leads to its anti-cancer effect, the cytotoxic effect of
the cobalt-polypyridyl complex of Formula (IV) has been examined in
both autophagy-wild type and -deficient cells respectively.
Therefore, both autophagy related gene (Atg) 7 wild-type and
deficient mouse embryonic fibroblasts (MEFs) (Wong, V. K. et al.,
Cell Death Dis, 2013, 4, e720) were used in this study. The
cobalt-polypyridyl complex of Formula (III) showed much lower
IC.sub.50 value in Atg7 wild-type MEFs (mean IC.sub.50, 4.84 .mu.M)
(FIG. 2), whereas less toxic effect in autophagy deficient cells
(Atg7 deficient MEFs, mean IC.sub.50, 14 .mu.M) was observed. In
view of the fact that the cobalt-polypyridyl complex of Formula
(IV)-mediated cytotoxicity was markedly abrogated from the failure
of autophagy induction in Atg7-/- deficient cells, these findings
implicated that the cobalt-polypyridyl complex of Formula
(IV)-induced autophagy would ultimately led to autophagic cell
death.
[0167] To investigate whether the cobalt-polypyridyl complexes
could suppress cancer cell growth by arresting cell cycle
progression, HeLa cancer cells were treated with different
concentrations of the cobalt-polypyridyl complex of Formula (IV)
(with PF.sub.6.sup.- as counterion). Apparently, low concentration
of the cobalt-polypyridyl complex of Formula (IV) showed negligible
effect on cell cycle progression, On the other hand, high
concentration of the cobalt-polypyridyl complex of Formula (IV) was
found to arrest HeLa cancer cell in S-phase, as determined by the
increased percentage of cell population accumulated in S-phase
(FIG. 3A to 3E), suggesting that this cobalt complex may affect the
DNA replication process in cancer cells.
[0168] To determine the inhibitory effect of cobalt-polypyridyl
complexes of the present invention in cancer cell invasion, human
H1299 lung cancer cells with well-known cell invasion ability were
treated with the cobalt-polypyridyl complex of Formula (IV) in its
sub-lethal (non-toxic) doses. FIG. 4 shows the invasion assay of
cancer cell H1299 upon the treatment with the cobalt-polypyridyl
complex of Formula (IV). The H1299 cancer cells treated with DMSO
control demonstrated a significant number of invaded cells, as
determined by the increased fluorescence intensity. It is
noteworthy that the cobalt-polypyridyl complex of Formula (IV)
(with PF.sub.6.sup.- as counterion) was found to dose-dependently
decrease the cell fluorescence signal, suggesting the
cobalt-polypyridyl complex of Formula (IV) could function to
suppress cancer cell invasion, and metastasis.
Example 5
Suppression of Cancer Growth in a Mouse Lung Cancer Xenograft Model
without Observable Adverse Effects
[0169] The mice were maintained under specific pathogen-free
condition in a 12-h light-dark cycle. Experimental conditions were
followed in accordance to the "Institutional AnimalCare and User
Committee guidelines" of the Macau University of Science and
Technology. Xenograft mouse model was constructed as previously
described (Wong, V. K. et al., Oncotarget, 2016, 7, 9907-9924).
LLC-1 Lewis lung cancer cells (1.times.10.sup.6 cells) were
subcutaneously injected in the right dorsal region of the mice.
After tumors grew to about 100 mm.sup.3, the mice were randomly
divided into 3 groups: 1) the control group, 2) 40 mg/kg
cobalt-polypyridyl complex of Formula (IV) group, and 3) 40 mg/kg
cobalt-polypyridyl complex of Formula (IV) as chloride, also
referenced as (IV)-Cl (water soluble form) group. The
cobalt-polypyridyl complex of Formula (IV) was dissolved in 100
.mu.L buffer (PEG400:EtOH:ddH.sub.2O=6:1:3), whereas (IV)-Cl (water
soluble form) was dissolved in ddH.sub.2O. The complexes were then
intraperitoneal injected in mice every day. The body weight of each
mouse was recorded with the tumor volume determined by Vernier
caliper using the formula of L.times.W.sup.2.times.0.52, where L is
the longest diameter of tumor and W is the shortest diameter of
tumor. Mice with tumor implants were sacrificed after 10 days when
the tumor size of vehicle control group reached above 1000 mm.sup.3
due to animal ethical issue.
[0170] The cobalt-polypyridyl complex of Formula (IV) was further
assessed in a lung cancer xenograft model in order to investigate
its in vivo anti-tumor effect. As shown in FIG. 5A, intraperitoneal
(IP) injection of the cobalt-polypyridyl complex of Formula (IV) or
its water soluble form (IV)-Cl at 40 mg/kg/day demonstrated
significant tumor inhibitory effect of up to 40% (P<0.05) of
reduction in tumor volume. Treatment with the cobalt-polypyridyl
complex of Formula (IV) or (IV)-Cl showed no significant reduction
in body weight (FIG. 5B), suggesting the less or non-toxic nature
of the cobalt complex. Collectively, the anti-cancer effect of the
cobalt-polypyridyl complex of Formula (IV) not only has been
validated in different cancer and multidrug-resistant cancer
models, but also in animal system. The results therefore have
provided the solid evidence to support that the cobalt complexes
represent highly potential anti-cancer drugs.
* * * * *