Copper (II) complexes of bidentate ligands exhibit potent anti-cancer activity regardless of platinum sensitivity status

Article abstract of DOI:10.1007/s10637-017-0488-2

Insensitivity to platinum, either through inherent or acquired resistance, is a major clinical problem in the treatment of many solid tumors. Here, we explored the therapeutic potential of diethyldithiocarbamate (DDC), pyrithione (Pyr), plumbagin (Plum), 8-hydroxyquinoline (8-HQ), clioquinol (CQ) copper complexes in a panel of cancer cell lines that differ in their sensitivity to platins (cisplatin/carboplatin) using a high-content imaging system. Our data suggest that the copper complexes were effective against both platinum sensitive (IC₅₀?~?1?μM platinum) and insensitive (IC₅₀?>?5?μM platinum) cell lines. Furthermore, copper complexes of DDC, Pyr and 8-HQ had greater therapeutic activity compared to the copper-free ligands in all cell lines; whereas the copper-dependent activities of Plum and CQ were cell-line specific. Four of the copper complexes (Cu(DDC)₂, Cu(Pyr)₂, Cu(Plum)₂ and Cu(8-HQ)₂) showed IC₅₀ values less than that of cisplatin in all tested cell lines. The complex copper DDC (Cu(DDC)₂) was selected for in vivo evaluation due to its low nano-molar range activity in vitro and the availability of an injectable liposomal formulation. Liposomal (Cu(DDC)₂) was tested in a fast-growing platinum-resistant A2780-CP ovarian xenograft model and was found to achieve a statistically significant reduction (50%; p?

Full text of DOI:10.1007/s10637-017-0488-2

Invest New Drugs
DOI 10.1007/s10637-017-0488-2
PRECLINICAL STUDIES
Copper (II) complexes of bidentate ligands exhibit potent
anti-cancer activity regardless of platinum sensitivity status
Mohamed Wehbe1,2 & Cody Lo1 & Ada W. Y. Leung1 & Wieslawa H. Dragowska1
Gemma M. Ryan1 & Marcel B. Bally1,2,3,4
&
Received: 19 May 2017 /Accepted: 28 June 2017
#
The Author(s) 2017. This article is an open access publication
Summary Insensitivity to platinum, either through inherent
or acquired resistance, is a major clinical problem in the treat-
ment of many solid tumors. Here, we explored the therapeutic
potential of diethyldithiocarbamate (DDC), pyrithione (Pyr),
plumbagin (Plum), 8-hydroxyquinoline (8-HQ), clioquinol
(CQ) copper complexes in a panel of cancer cell lines that
differ in their sensitivity to platins (cisplatin/carboplatin) using
a high-content imaging system. Our data suggest that the cop-
per complexes were effective against both platinum sensitive
(IC50 ~ 1 μM platinum) and insensitive (IC50 > 5 μM plati-
num) cell lines. Furthermore, copper complexes of DDC, Pyr
and 8-HQ had greater therapeutic activity compared to the
copper-free ligands in all cell lines; whereas the copper-
dependent activities of Plum and CQ were cell-line specific.
Four of the copper complexes (Cu(DDC)2, Cu(Pyr)2,
Cu(Plum)2 and Cu(8-HQ)2) showed IC50 values less than that
of cisplatin in all tested cell lines. The complex copper DDC
(Cu(DDC)2) was selected for in vivo evaluation due to its low
nano-molar range activity in vitro and the availability of an
injectable liposomal formulation. Liposomal (Cu(DDC)2) was
tested in a fast-growing platinum-resistant A2780-CP ovarian
xenograft model and was found to achieve a statistically sig-
nificant reduction (50%; p < 0.05) in tumour size. This work
supports the potential use of copper-based therapeutics to treat
cancers that are insensitive to platinum drugs.
.
Keywords Cisplatin Platinum drugs, platinum resistant
.
.
.
.
cancer Diethyldithiocarbamate Plumbagin Pyrithione
.
.
.
8-hydroxyquinoline Clioquinol Carboplatin Copper
.
complexes Copper based therapeutics
Introduction
Platinum (Pt) drugs are the most successful class of inorganic
medicinal compounds used to treat cancer [1, 2]. They are a
mainstay in cancer therapy, being utilized in approximately
50% of chemotherapeutic regimens [2, 3]. Cisplatin (CDDP)
was first used to treat leukemia in the 1960s, but through
several inorganic medicinal chemistry programs, other Pt-
based drugs (Carboplatin (CBDCA), Oxaliplatin, Paraplatin
etc.) have been produced and approved by the FDA [2, 4].
Mechanistically, these drugs are known to act by forming Pt-
DNA complexes that cause DNA damage that accumulate to a
point that is beyond repair, ultimately leading to cell death [3,
5]. Pt drugs are currently used as first-line therapy in blood,
lung, ovarian, testicular, and head and neck cancers. While
some cancers are Pt sensitive and thus respond to Pt drugs
[2], many others are Pt-insensitive due to inherent or acquired
resistance [3, 5]. There is a need to define drugs capable of
treating Pt insensitive cancers [6].
* Mohamed Wehbe
mwehbe@bccrc.ca
1
Experimental Therapeutics, British Columbia Cancer Agency, 675
West 10th Avenue, Vancouver, BC V5Z 1L3, Canada
2
Faculty of Pharmaceutical Sciences, University of British Columbia,
2146 East Mall, Vancouver, BC V6T 1Z3, Canada
While Pt drugs have been successful for many patients, they
also produce serious side effects including nephrotoxicity, neu-
rotoxicity and ototoxicity [7]. To address these adverse effects,
many inorganic medicinal chemistry investigations have fo-
cused on developing alternatives to Pt-based drugs by replacing
3
Department of Pathology and Laboratory Medicine, University of
British Columbia, 2211 Wesbrook Mall, Vancouver, BC V6T 2B5,
Canada
4
Center for Drug Research and Development, Vancouver, BC V6T
1Z4, Canada

Invest New Drugs
Pt with other divalent metals, such as copper [8]. Copper-based
therapeutics are generally less toxic owing to the physiological
processes that detoxifying excess of copper [9]. In line with this
approach, many groups have demonstrated in vitro that copper
complexes of natural compounds have anticancer properties;
some examples include dithiocarbamates and analogues of 8-
hydroxyquinoline [10, 11]. To date, however, no copper com-
plex has transitioned into clinical use for any indication in
humans. While the platins that are used in the clinic are water
soluble, one major challenge of copper-based therapeutics is
their inherently poor aqueous solubility [12]. This problem
has made in vivo testing very challenging and required solvents
containing mixtures of DMSO and Cremphor which are not
clinic applicable due their high toxicity [13, 14]. Recently, we
solved the solubility issue of copper-based therapeutics by syn-
thesising copper complexes within liposomes [12]. This tech-
nology, referred herein as Metaplex™, was used to examine the
effects of copper DDC (Cu(DDC)2) in animals bearing a xeno-
graft tumours [15].
In these studies, we selected five bidentate copper ligands
(Scheme 1) and screened both the ligand and the correspond-
ing copper complex in a panel of eight cancer cells. As indi-
cated above diethyldithiocarbamate (DDC) [16, 17],
Pyrithione (Pyr) [13], Plumbagin (Plum) [18, 19], 8-
hydroquinoline (8-HQ) [17, 20, 21] and Clioquinol (CQ)
[10, 17, 22, 23] have all been shown to have anticancer activ-
ity but have never been tested directly against Pt-resistant
cancers. Here we compare the activity of these complexes to
the activity of CDDP and CBDCA (Scheme 2). The screen
showed that many copper complexes are more active in vitro
than the Pt controls. We also demonstrate that a Metaplex™
formulation of copper DDC (Cu(DDC)2) was active in
Scheme 2 Structures of commonly used platinum drugs. a Cisplatin and
(b) Carboplatin
animals bearing Pt-resistant A2780-CP xenograft tumours.
The results highlight the potential of the copper-based thera-
peutics as candidates to treat Pt resistant cancers.
Materials and methods
Materials
Plumbagin was obtained from Plumbago indica. Pyrithione
(2-Mercaptopyridine N-oxide sodium salt), 8-
Hydroxyquinoline, Sodium Diethyldithiocarbamate
trihydrate, Copper Sulfate, Clioquinol (5-Chloro-7-iodo-8-
quinolinol), and all other chemicals were purchased from
Sigma Aldrich. Carboplatin (CBDCA) and Cisplatin
(CDDP) were obtained from Hospira. 1,2-distearoyl-sn-
glycero-3-phosphocholine (DSPC) and Cholesterol (Chol)
were purchased from Avanti Polar Lipids (Alabaster, AL).
Cell lines
The A549, FaDu, Cal-27, SCC-25, and MV-4-11 cell lines
were purchased from ATCC. A2780-S and A2780-CP cell
Scheme 1 Ligands and their
respective copper complex
structures. a DDC and Cu(DDC)2
(b) Pyr and Cu(Pyr)2. (c)Plum
and Cu(Plum)2. (d) 8-HQ and
Cu(8-HQ)2 and (e) CQ and
Cu(CQ)2

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lines were obtained from Dr. Mark W. Nachtigal at the
University of Manitoba (Winnipeg, Canada) and the H1933
cells were provided by Dr. William Lockwood at the BC
Cancer Agency’s Vancouver Research Centre (Vancouver,
Canada). All cell lines were used for up to eighteen passages.
A549 and H1933 cells were maintained in RPMI (Gibco).
A2780-S, A2780-CP and SCC-25 were maintained in
DMEM/F12 (Gibco). MV-4-11, Cal-27, and FaDu cells were
maintained in IMDM (Gibco), DMEM (Gibco), and MEM
(Gibco), respectively. Media for all cell lines was supplement-
ed with 2 mM L-glutamine (Gibco) and 10% fetal bovine
serum (Gibco) and maintained at 37 °C and 5% CO2. Media
for the SCC-25 cell line was also supplemented with 400 ng/
mL hydrocortisone. All cell lines tested negative for myco-
plasma when obtained by the lab. Prior to drug treatment, the
cells were seeded into 384 well plates and incubated 24 h in
media prior to treatment.
freezer and desiccated for 2 h prior to being weighed and
dissolved in chloroform at a mol:mol ratio of 55:45
(DSPC:Chol). A non-exchangeable and non-metabolizable
lipid marker, 3H- cholesteryl hexadecyl ether, was incorporat-
ed into the chloroform mixture and the solution was dried of
chloroform using nitrogen gas and a thin film was generated
using a high vacuum for 2 h. The lipid film was rehydrated
using 300 mM CuSO4 and extruded for at least 10 passes
through a high pressure LIPEX extruder (Evonik Transferra
Nanosciences, BC, Canada) using 0.1 μm polycarbonate fil-
ters. The final liposome size was determined by quasi-electric
light scattering with a ZetaPALS analyzer (Brookhaven). The
unencapsulated copper was removed via size exclusion chro-
matography (SEC; Sephadex G50) and replaced with a buffer
containing sucrose (300 mM), HEPES (20 mM) and EDTA
(15 mM) (SHE buffer, pH 7.4). The resulting copper-free li-
posomes were dialyzed against a sucrose (300 mM) and
HEPES (20 mM) buffer (SH buffer, pH 7.4) and used for
Cu(DDC)2 synthesis within the liposomes.
Cu(DDC)2 synthesis inside the liposomes was performed
at 25 °C in a round bottom flask. Copper-containing lipo-
somes (20 mM) were mixed with DDC (10 mM) for 1 h. As
DDC permeates across the liposomal lipid bilayer it interacts
with the encapsulated copper to form Cu(DDC)2. The free, un-
reacted, DDC was removed by SEC and the Cu(DDC)2 con-
taining liposomes, collected in the excluded volume were used
for in vivo tumour efficacy studies.
Cytotoxicity assay of Pt drugs, ligands and copper
complexes
For in vitro testing, copper complexes were synthesized prior to
cell treatment by mixing CuSO4 and the ligand at a fixed 2:1
ratio in DMSO (50%). The ligands DDC, CQ, 8-HQ, Plum were
solubilised in DMSO and Pyr was dissolved in sterile water.
CBDCA and CDDP were dissolved in sterile saline (0.9%).
The adherent cell lines (A549, A2780-S, A2780-CP, FaDu,
Cal-27, SCC-25, H1933) were exposed to the indicated com-
pounds in triplicate wells for 72 h. Following treatment, cells
were stained in situ with Hoechst 33,342 and ethidium
homodimer-I to differentiate between viable (Hoechst-posi-
tive/ethidium homodimer-negative) and dead cells that had
lost membrane integrity (Hoechst-positive/ethidium-positive).
Cells were imaged with the IN Cell Analyzer 2200 (GE
Healthcare Life Sciences) and 4 images/well were collected.
Images were analyzed with the ToolBox Developer 1.9 soft-
ware (GE Healthcare Life Sciences) to obtain viable and dead
cell counts based on differential staining of cell nuclei. The
suspension cell line (MV-4-11) was treated for 72 h and via-
bility was assessed using the Presto Blue™ assay (Life
Technologies) following the manufacturer’s instructions. The
viability data were normalized to vehicle control (0.5%
DMSO in media) and expressed as fraction affected where
value of 1 corresponded to 100% loss of cell viability relative
to vehicle controls and 0 corresponds to a viability comparable
to control cells in culture.
In vivo efficacy study
A2780-CP cells that were used for subcutaneous (sc) implan-
tation were between passages 3–10 and maintained in
DMEM/F12 (Gibco) at a confluence of 80–90%. NRG mice
(7 per group) were inoculated sc with 1 × 106 cells in a volume
of 50 μL using a 28-gauge needle. Treatment was initiated on
day four and treatment groups included the vehicle control
(SH buffer), CuSO4-liposomes (1.7 mg/kg copper, 50 mg/kg
lipid), and Cu(DDC)2 liposomes (8 mg/kg Cu(DDC)2 and
50 mg/kg lipid) on a Monday, Wednesday and Friday ×2
weeks dosing schedule. The copper dose of the CuSO4 lipo-
some control was equivalent to that of Cu(DDC)2.
Animals were monitored at least three times weekly for body
weight and tumour growth which was measured with calipers
and tumour volumes were calculated based on a formula of
(tumour length x tumour width2)/2. Animals were terminated
by isoflorane followed by CO2 asphyxiation when animals
reached a humane endpoint for these studies was defined when
tumours exceeded 800 mm3 or when tumours ulcerated.
Cu(DDC)2 liposome preparation
For efficacy studies, copper complexes were synthesized in-
side liposomes, as previously described [12]. Liposomes were
prepared using the extrusion method which has been well
documented by others [24]. In brief, lipids were removed from
Statistical analysis
All data were plotted as mean SEM or mean SD, as de-
scribed in the figure legends. The IC50 values and 95%

Invest New Drugs
confidence intervals (CI) were extrapolated from nonlinear re-
gression (curve fit) of the cytotoxicity curves using Prism 6.0
(GraphPad software). To determine whether the cytotoxic ef-
fects of copper complexes are associated with platinum sensi-
tivity, the IC50 values of each copper complex was plotted
against the CDDP IC50 for each cell line. Each data point rep-
resents one cell line. The Pearson Correlation coefficient and
corresponding two-tail p-values were then determined using
Prism 6.0 (GraphPad software). Tumour volumes between dif-
ferent treatment groups were compared using one-way
ANOVA followed by Tukey adjustments to correct for multiple
comparisons using Prism 6.0. An adjusted P-value <0.05 was
considered statistically significant.
Activity of copper complexes in cancer cells with different
Pt sensitivity
The IC50 values 95% confidence intervals for the ligands and
their corresponding copper complexes are shown in Fig. 2.
Cells were treated with the ligand/drug or complex (2:1
ligand:Cu) for 72 h. All cell lines were also exposed to
CuSO4 as a control to ensure that copper alone did not impact
viability (IC50 > 10 μM for all cell lines, data not shown). The
results in Fig. 2a indicate that treatment of different cells with
the ligand DDC results in an IC50 ranging from 5 to 300 μM
DDC. In contrast, the IC50 for the Cu(DDC)2 ranged from
0.02–0.15 μM corresponding to differences of 90–11,000 fold
(Fig. 2a). The IC50 of Pyr ranged from 1 to 30 μM, and when
used as a copper complex (Cu(Pyr)2), the IC50 decreased to
0.1–7.4 μM (Fig. 2b). The IC50 of Plum ranged from 1.5–
11 μM while the IC50 of and Cu(Plum)2 Ranged from 0.8–
3 μM in the tested cell lines. In some cell lines (A2780-S,
A2780-CP, MV-4-11, Cal-27, FaDu) there was only approxi-
mately a 2-fold difference in activity between Plum and
Cu(Plum)2 (Fig. 2c). Considering the 2:1 (ligand:Cu) com-
plexation ratio, these data indicate that the copper Plum com-
plex is as active as the uncomplexed ligand, suggesting that
the cytotoxic effects observed for Plum is copper-indepen-
dent. 8-HQ was more active as a copper complex (IC50 for
Cu(8-HQ)2 ranged between 0.2–4.5 μM compared to 1.5-
30 μM for 8-HQ); however, in A2780-S and FaDu cells, the
toxicity of 8-HQ appeared to be copper independent (Fig. 2d).
The IC50 for CQ and Cu(CQ)2 ranged from 10 to 350 μM and
20-60 μM, respectively. Here the dependence on copper com-
plexation for CQ cytotoxicity appears to be cell line-specific:
copper-dependent activity was observed in A2780-S, A2780-
CP and A549 cells, whereas in MV-4-11, SCC-25 and H1933,
Results
Pt-resistance does not impact sensitivity of cancer cells
to DDC copper complexes
CDDP and CBDCA, two commonly used Pt-based therapeu-
tics were tested in a pair of isogenic ovarian cancer cells:
A2780-S cells are the parental cells that are sensitive to
CDDP while A2780-CP cells are platinum-resistant. As
shown in Fig. 1a and b, the IC50 of CDDP and CBDCA was
3.7-and 8.5-fold greater, respectively, in the Pt resistant cells
when compared to Pt sensitive cells. To determine if Pt resis-
tance impacts activity of copper complexes, A2780-S and
A2780-CP cells were treated with Cu(DDC)2. The data in
Fig. 1c show that the IC50 value of the copper complex was
not different for Pt sensitive or resistant cells. These data sug-
gest that copper complexes may be effective regardless of Pt
sensitivity status.
Fig. 1 Cytotoxicity profiles of Pt
sensitive A2780-S (●) and
resistant A2780-CP (■) ovarian
cancer cells (or) following 72 h
treatment with (a) CDDP, (b)
CBDCA and (c) Cu(DDC)2.
Fraction affected cells was
assessed based on viability data
normalized to vehicle controls.
(d) The IC50 95% CI for
A2780-S (black) and A2780-CP
(white) after treatment with
CDDP, CBDCA and Cu(DDC)2.
Data is presented as the mean of 3
independent experiments SEM

Invest New Drugs
DDC and Cu (DDC)2
11595
130
(A)
1000
92
700
120
182
3982
515
100
10
1
0.1
0.01
-CP
A549
FaDu
H1933
V-4-11
Cal-27
SCC-25
M
A2780-S
A2780
Pyr and Cu (Pyr)2
Plum and Cu(Plum)
2
(B)
(C)
4.0
100
100
5.0
4.8
26
3.3
2.2
2.3
1.9
18
3.4
87
10
2.2
10
30
1.7
5.1
7.0
1
1
0.1
0.1
P
P
25
SCC-
549
A
-C
4-11
1933
H
1933
H
A549
FaDu
FaDu
Cal-27
Cal-27
SCC-25
MV-
MV-4-11
A2780-S
A2780-S
A2780-C
A2780
8-HQ and Cu(8-HQ)
CQ and Cu(CQ)
2
2
(D)
(E)
19
100
1000
100
10
1.1
5 .2
23
0 .3
2.9
0 .2
51
1 .2
2.9
2 .5
5 .0
1 .4
10
2.1
4.1
7.2
1
0.1
1
P
0-S
933
1
549
A
A549
0-CP
FaDu
FaDu
H
H1933
Cal-27
V-4-11
M
Cal-27
780-C
2
SCC-25
SCC-25
MV-4-11
A2780-S
A278
A
A278
Fig. 2 Ligand/drug and copper complex cytotoxicity. A panel of 8 cancer
cell lines (A2780-S, MV-4-11, A549, SCC-25, Cal-27, A2780-CP, FaDu
and H1933) were treated for 72 h with the ligand/drug (■) or copper
complex (□): (a) DDC/Cu(DDC)2 , (b) Pyr/Cu(Pyr)2 ,(c)
IC50 values were obtained based on the viability data acquired with IN
Cell 2200 or PrestoBlue® (M-V-411). The fold difference between the
IC50 of the ligand and respective copper complex is shown above the
histogram bars
Plum/Cu(Plum)2, (d) 8-HQ/Cu(8-HQ) and (e) CQ/Cu(CQ) 2. The
2
the cytotoxicity of CQ was copper-independent. Moreover,
CQ appears to be more cytotoxic than Cu(CQ)2 in Cal-27
and FaDu cells (Fig. 2e).
study show no correlation with Pt CDDP sensitivity in tested
cell lines (Table 1). The heatmap indicates that (Cu(DDC)2 is
the most effective complex tested against Pt-resistant cancers.
The IC50s obtained from cytotoxicity assays for each cell
line were arranged as a heat-map in order of Pt sensitivity to
CDDP (Fig. 3). An IC50 cut-off of 10 μM was used to distin-
guish more potent agents from those that were regarded as
pharmaceutically inactive. In general, the ligands appear to
be less active in the Pt resistant cell lines whereas the copper
complexes augmented anti-cancer activity considerably. The
Pyr, Plum and 8-HQ ligands showed activity in most cell
lines, however, their activity was enhanced in the tested cell
lines when used as complexes with copper; where the greatest
increases were observed for Cu(Pyr)2. DDC and CQ were the
least active ligands. The Cu(CQ)2 complex did not show im-
provement in cytotoxicity whereas the copper complex of
DDC was the most active in all cell lines. In general, except
for Cu(Pyr)2, the activity of the copper complexes used in our
Efficacy of liposomal cu(DDC)2 in a Pt-resistant
A2780-CP tumour xenograft model
Cu(DDC)2 has represented a formulation challenge because of
its poor aqueous solubility. Previously we developed a strate-
gy where the Cu(DDC)2 was synthesized inside a liposomal
formulation [12]. Here we used this formulation (see
Methods) to assess the activity of the Cu(DDC)2 in a Pt-
resistant A2780-CP subcutaneous tumour xenograft model.
The data, shown in Fig. 4, indicate that Cu(DDC)2 engendered
a statistically (p < 0.05) significant ~50% reduction in tumour
burden when compared to the activity of the vehicle control.
These data provide proof-of-concept results suggesting that
liposomal copper complexes have the potential to be

Invest New Drugs
Fig. 3 Screen of copper based
therapeutics in cancer cells with
different Pt sensitivities. IC50
values for Pt drugs (CDDP and
CBDCA), ligands (DDC, Pyr,
Plum, 8-HQ, CQ) and respective
copper complexes are shown for
cancer cell lines of differing
origin arranged in order of
sensitivity to CDDP. IC50 values
were calculated from viability
data (n = 3 experiments/each cell
line) obtained with IN Cell
Analyzer 2200 platform or
PrestoBlue™ assay (MV-4-11
cells). The agents with IC50
values >10 uM (black) are
considered inactive
developed as a novel class of therapeutic agents for use in the
treatment of Pt-resistant cancers.
new therapeutic entities that are active in Pt resistant cancers is
a priority to improve treatment outcome for patients.
The ovarian cancer cell line A2780-CP had a clear resis-
tance to CDDP and CBDCAwhen compared to the activity of
these drugs in A2780-S cells. In order to test the utility of
copper based therapies in platinum-resistant cancer cells, pre-
liminary studies investigated Cu(DDC)2 in Pt sensitive and
insensitive cell lines. Cu(DDC)2 was active irrespective of Pt
sensitivity at equivalent doses. This Pt sensitivity-independent
activity could be explained by the Cu(DDC)2 mechanism of
toxicity which overcomes resistance mechanisms commonly
seen in Pt-resistant cancers. Cu(DDC)2 is able to accumulate
in cancer cells irrespective of CTR1 expression [32] and
Discussion
Pt drugs are standard first-line therapies for the treatment of
lung, ovarian, and head and neck cancers [25, 26]. While
some cancers are inherently resistant, the widespread use of
Pt-based therapies has resulted in the development of resistant
cancers, often in the relapse setting where prognosis is poor [6,
25]. In vitro, cell lines can be developed to replicate this effect
(e.g. A2780-CP), wherein cells are treated with low dose Pt
drugs such as CDDP over extended periods of time until re-
sistance is observed through changes in IC50 values [27, 28].
The two major mechanisms responsible for Pt resistance are
DNA repair and Pt drug transport into cancer cells [2, 29].
Through extensive characterization, it was determined that at
equivalent levels of drug accumulation, the A2780-CP cell
line is 2-fold more efficient at repairing Cisplatin-DNA le-
sions when compared to the parental, Pt-sensitive cell line
(A2780-S) [29]. The copper transporter CTR1 is the major
influx transporter for cisplatin both in vitro and in vivo and
has been reported to be reduced in cisplatin resistant cancers
[25, 30]. This mechanism was attributed to cisplatin influx
resulting in the rapid degradation of CTR1 [31]. Identifying
Table 1 Correlation between CDDP and copper complex sensitivity
based on the IC50 values
Fig. 4 In vivo activity of liposomal Cu(DDC)2 in animals bearing Pt
resistant A2780-CP tumours. 1 × 106 A2780-CP cells were inoculated
sc into immune-compromised NRG mice which were subsequently treat-
ed intravenously with vehicle (SH buffer), CuSO4-liposomes (1.7 mg/kg
copper, 50 mg/kg lipid), and Cu(DDC)2 liposomes (8 mg/kg Cu(DDC)2
and 50 mg/kg lipid) using a 3 x per week for 2 weeks dosing schedule.
Mean tumour volume was determined on day 18 (the humane endpoint
for vehicle treated animals). Data represents the mean SEM (n = 7). *
indicates statistical difference (p < 0.05)
Drug
Cu(DDC)2 Cu(Pyr)2 Cu(Plum)2 Cu(8-HQ)2 Cu(CQ)2
Pearson R2 0.0680
0.6966
0.0100
Yes
0.1030
0.4382
No
0.0880
0.4755
No
0.4365
0.0745
No
P-value 0.0.5325
Significant No

Invest New Drugs
cytotoxicity is mediated through proteosome inhibition [16].
These results suggest that CBTs use mechanisms of cytotox-
icity outside of those used by platinums and warrant further
investigations in resistant cancers.
DMSO cannot be included at concentrations above 0.5% which
would be needed to administer the complexes at relevant doses
[39]. While the ligands used in this work are approved agents,
the copper complexes themselves have never been adminis-
tered as therapeutic agents. Thus, in vivo testing of selected
copper complexes should be done in conjunction with cytotox-
icity studies in healthy human cells. This was previously done
for Cu(DDC)2 and both ligand as well as the copper complex
exhibited IC50 values greater than 10 μM when added to pri-
mary (normal) human bronchial epithelial cells [12].
We have previously disclosed the Metaplex™ technology as
a platform approach to formulate copper complexes inside li-
posomes in order to overcome solubility issues and to allow for
parenteral administration of copper complexed ligands [12].
Thus, as a proof-of-concept we tested a liposomal formulation
of Cu(DDC)2 in a xenograft tumour model of A2780-CP Pt-
resistant ovarian cancer. In this study, we showed that liposo-
mal Cu(DDC)2 (8 mg/kg) but not Cu-liposomes produced a
statistically significant ~50% reduction in tumour burden when
compared to the vehicle control. This difference is not consid-
ered clinically relevant (based on RECIST criteria [40]) but the
results does suggest that copper complexes are a class of ther-
apeutic that should be further investigated. Cu(DDC)2 pharma-
cokinetics for the liposomal product show rapid release from
the liposome and degradation in the plasma, and as such would
require further development to improve circulation lifetime and
stability [12]. Also the use of this formulated Cu(DDC)2 prep-
aration in combination with other agents typically combined
with platinums (eg. the taxanes [41]) is warranted. The other
complexes discussed are proposed as future developments to
identify those which have activity as single agents in Pt resis-
tant cancers and as those which can augment therapy in com-
bination with traditional chemotherapeutics.
Five copper binding ligands (DDC, Pyr, Plum, 8-HQ and
CQ) were tested in a panel of eight cancer cell lines of differ-
ent origins (blood, lung, ovarian, head and neck) and differing
sensitivity to Pt-based drugs exemplified by CDDP/CBDCA
activity. Our in vitro data suggest that the cytotoxicity of li-
gands DDC, Pyr and 8-HQ appeared to be copper-dependent
whereas the cytotoxicity of Plum and CQ were copper inde-
pendent. This finding is important as both DSF (metabolized
to DDC in vivo) + Cu-Gluconate and CQ were involved in
clinical trials with no listed benefit [33–35]. By knowing the
relationship between Cu and the activity of the complex, it can
be reasoned that ligands such as DDC, Pyr and 8-HQ should
be administered as copper complexes to ensure that the active
(copper complexed) species reaches the cancer cells. This is
supported by work performed by Katano et al. which showed
that Pt resistant cancers have lower basal levels of copper,
suggesting that the uncomplexed ligands would be less active
[30]. In the current study, H1933 was the least sensitive to Pt
as reflected by a > 10 μM IC50 cytotoxicity to all the ligands
tested. In contrast, Plum and CQ could be used as single
agents and pharmaceutical challenges associated with their
solubility may be overcome using the Metaplex™ approach
that we have described previously to prepare a nanomedicine
formulation of Cu(DDC)2 with no loss in activity [12, 15].
Cu(II) dominates the coordination chemistry of copper and
was the focus of this work but Cu(I) complexes with thera-
peutic activity do exist [9]. For example, Han et al. tested the
Cu(I)-DDC complex in a subcutaneous tumour model of ad-
enocarcinoma with ~60% reduction in tumour burden by day
25 [36]. This research focused on Cu(II) species for two rea-
sons. First, Cu(I) is not stable in aqueous solutions and un-
dergoes conversion to Cu(II) [37]. The creation of the Cu(I)
complexes in aqueous solutions would require characteriza-
tion to ensure that the Cu(I) and not the Cu(II) complexes was
being synthesized. Second, Cu(I) and Cu(II) preferentially
coordinate with different atoms. Cu(II) is able to complex
Bhard^ donors such as nitrogen and oxygen, whereas Cu(I)
is known to coordinate with thiols and thioesters [38].
Conclusion
Pt drugs have been widely successful in the treatment of can-
cer. The treatment of Pt-resistant cancers has been a pharma-
ceutical focus that requires new therapeutics with mechanisms
of action differing from those invoked by Pt-based therapies.
Copper-based therapeutics represents a new class of drugs that
can address many of the challenges associated with Pt-
resistant cancers. Our data show that four out of five screened
ligands that form copper complexes reached IC50 values be-
low 10 μM in the eight cancer cell lines regardless of their Pt
sensitivity. Additionally, we tested one of these complexes
(Cu(DDC)2) in vivo in a Pt-resistant ovarian cancer xenograft
model and attained a 50% reduction in tumor volume when
compared to vehicle-treated control mice. These data not only
provide in vivo validation that copper-based therapeutics
could be used to treat Pt-resistant cancer but also provides
proof-of-concept that the Metaplex™ technology could be
Our data support the utility of copper-based therapeutics in
the treatment of Pt-insensitive cancer. To date, not a single
copper-based therapeutic has received FDA approval. This is
in part due to the solubility challenges associated with these
complexes. All copper complexes studied as well as the ligands
Plum, 8-HQ and CQ are water insoluble (<0.1 mg/mL); in
contrast, DDC and Pyr are water soluble as ligands but their
therapeutic activity is significantly enhanced in the presence of
Cu. All copper complexes had to be dissolved in DMSO (final
concentration 0.5%) to allow for in vitro testing. The ICH
guidelines for residual solvents in a product indicate that

Invest New Drugs
cancer cells. Breast Cancer Research : BCR 7(6):R897–R908. doi:
10.1186/bcr1322
used to develop formulations of inherently insoluble copper
complexes for pre-clinical and clinical evaluations.
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Acknowledgements The authors would like to acknowledge N. Dos
Santos and the staff of the Investigational Drug Program at the BC Cancer
Agency for their contributions to the toxicity, pharmacokinetic and effi-
cacy studies.
Compliance with ethical standards
13. Liu NN, Liu CJ, Li XF, Liao SY, Song WB, Yang CS, Zhao C,
Huang HB, Guan LX, Zhang PQ, Liu ST, Hua XL, Chen X, Zhou P,
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Conflict of interest All authors declare no conflict of interests.
Funding The research described in this original paper was supported
by grant funding from the Canadian Cancer Society Research Institute
(Award # 702491 and 705,290). Additional funding was obtained from
the BC Cancer Foundation and the Centre for Drug Research and
Development.
Ethical approval Studies involving the use of animals were completed
under an Animal Care Protocol approved by the University of British
Columbia’s Animal Care Committee. Health assessment was completed
using a standard operating procedure (SOP), approved by the Institutional
Animal Care Committee.
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L, Bally MB (2017) Development and optimization of an injectable
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Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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