A Lipophilic Pt(IV) Oxaliplatin Derivative Enhances Antitumor Activity

Article abstract of DOI:10.1021/acs.jmedchem.6b00955

Side effects and acquired resistance by cancer cells limit the use of platinum anticancer drugs. Modification of oxaliplatin (OXA) into a lipophilic Pt(IV) complex [Pt(DACH)(OAc)(OPal)(ox)] (1), containing both lipophilic and hydrophilic axial ligands, was applied to improve performance and facilitate incorporation into polymeric nanoparticles. Complex 1 exhibited unique potency against a panel of cancer cells, including cisplatin-resistant tumor cells. [Pt(DACH)(OAc)(OPal)(ox)] incorporated nanoparticles (2) presented a mean diameter of 146 nm with encapsulation yields above 95% as determined by HPLC. Complexes 1 and 2 showed enhanced in vitro cellular Pt accumulation, DNA platination, and antiproliferative effect compared to OXA. Results of an orthotopic intraperitoneal model of metastatic ovarian cancer (SKOV-3) and a xenograft subcutaneous model of colon (HCT-116) tumor in SCID-bg mice showed that the activity of 1 and 2 significantly decreased tumor growth rates compared to control and OXA treatment groups. Consequently, these findings warrant further development toward clinical translation.

Full text of DOI:10.1021/acs.jmedchem.6b00955

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Article
A Lipophilic Pt(IV) Oxaliplatin Derivative Enhances Antitumor Activity
Aiman Abu Ammar, Raji Raveendran, Dan Gibson, Taher Nassar, and Simon Benita
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00955 • Publication Date (Web): 07 Sep 2016
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A Lipophilic Pt(IV) Oxaliplatin Derivative Enhances Antitumor activity
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Aiman Abu Ammar, Raji Raveendran, Dan Gibson, Taher Nassar† and Simon Benita†*
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The Hebrew University of Jerusalem, Institute for Drug Research of the School of
Pharmacy, Faculty of Medicine, POB 12065, Jerusalem, 9112100, Israel
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ABSTRACT
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Side effects and acquired resistance by cancer cells limit the use of Platinum (Pt)
anticancer drugs. Modification of oxaliplatin (OXA) into a lipophilic Pt(IV) complex
[Pt(DACH)(OAc)(OPal)(ox)] (1), containing both lipophilic and hydrophilic axial
ligands, was applied to improve performance and facilitate incorporation into
polymeric nanoparticles. Complex 1 exhibited unique potency against a panel of
cancer cells, including cisplatinꢀresistant tumor cells. [Pt(DACH)(OAc)(OPal)(ox)]
incorporated nanoparticles (2) presented a mean diameter of 146 nm with
encapsulation yields above 95% as determined by HPLC. 1 and 2 showed enhanced in
vitro cellular Pt accumulation, DNA platination and antiꢀproliferative effect compared
to OXA. Results of an orthotopic intraperitoneal model of metastatic ovarian cancer
(SKOVꢀ3) and a xenograft subcutaneous model of colon (HCTꢀ116) tumor in SCIDꢀ
bg mice showed that the activity of 1 and 2 significantly decreased tumor growth rates
compared to control and OXA treatment groups. Consequently, these findings warrant
further development towards clinical translation.
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Keywords: Nanoparticles; Oxaliplatin; Lipophilicity; Prodrug; Pt(IV) derivative
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1. INTRODUCTION
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Cancer is considered to be one of the main causes of death in the world. While
chemotherapy continues to be the mainstay treatment for cancer, it is closely linked
with considerable mortality and morbidity rates especially due to nonꢀspecific damage
to normal cells1. The clinical success of cisplatin has played an instrumental role in
the development of Pt anticancer compounds. With the purpose of searching for safer
and more effective Ptꢀbased compounds, numerous efforts have been made in the past
decades. However, only a few of these complexes have entered clinical trials. Two of
these, carboplatin and oxaliplatin (OXA) have been approved by the FDA and are
currently being used worldwide for malignant tumor chemotherapy2.
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OXA is a diaminocyclohexane (DACH) derivative of cisplatin however, the spectrum
of activity of OXA differs from cisplatin and includes several lines of colon, ovarian,
and lung cancer, as well as cell lines that are resistant to Pt therapy. OXA was the first
Ptꢀbased drug approved for use against metastatic colorectal cancer in combination
with fluorouracil and leucovorin. OXA interferes with DNA replication and
transcription systems through nuclear DNA adduct formation. Despite its better
tolerability compared to other Pt compounds, OXA is also associated with side effects
such as neurotoxicity, hematologic and gastrointestinal tract toxicities, neutropenia,
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nausea, and vomiting that limit the range of usable doses . Hence, novel Pt
complexes displaying increased antiꢀtumor activity and reduced crossꢀresistance are
still being sought
.
Pt(IV) complexes are a new generation of Ptꢀbased drugs, possessing markedly
different properties from those of Pt(II) compounds. The kinetic inertness prevents the
conversion to offꢀtarget biological substances before the Pt complex can reach the
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nuclear DNA. Furthermore, the two additional ligands can be utilized and tuned to
adjust the chemical properties and facilitate the incorporation of Pt compounds into
nanoparticles. These features can potentially improve both the efficacy and
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tolerability profile of Pt complexes . The strategy for the preparation of Pt(IV)
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complexes involves oxidative addition of the square planar Pt(II) complexes, usually
with hydrogen peroxide, to yield axial hydroxide ligands which can be carboxylated
or carbamated with the aim of introducing a versatile axial ligands 5ꢀ7. The reduction
to Pt(II) is a paramount demand for binding to the targeted biological sites. Thus, the
reduction potential of the Pt(IV) complexes as well as the redox status of the tumor
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environment have a strong impact on the activity of Pt(IV) anticancer drugs . Pt(IV)
drugs are generally regarded as prodrugs due to the disparity in reactivity prior to and
following reduction4. Solubility and lipophilicity have a crucial role in designing new
Pt(IV) compounds. Numerous studies have shown a good correlation between
lipophilicity and cytotoxicity 9ꢀ11. Even though it has a critical role in enhancing the
cytotoxic activity of the prodrug, lipophilicity is not the main factor, which
determines the overall activity. Moreover, high lipophilicity usually leads to low
water solubility, making the drug formulation in an aqueous solution difficult. The
low aqueous solubility has been always considered a stumbling block to the
development and clinical use of anticancer compounds, due to the intravenous (i.v.)
infusion route of their administration. Poorly waterꢀsoluble anticancer drugs need to
be formulated in coꢀsolvents and/or surfactant solutions to enhance their solubility,
but many of these solutiuons, which have been used for solubilization such drugs, are
responsible for the severe side effects. The classic examples for these formulations are
the marketed taxane products, paclitaxel and docetaxel which have had a profound
effect in a wide variety of malignancies. In the case of paclitaxel, a polyoxylꢀ35 castor
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oil surfactant vehicle is used in the commercial formulation. Its presence triggers
severe hypersensitive reactions in patients and it has been reported that 30–40% of the
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patients receiving paclitaxel suffer from abnormal lipoprotein patterns, aggregation of
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erythrocytes, severe hypersensitive reactions and peripheral neuropathy
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Similarly, ethanol and polysorbate 80, used for docetaxel solubilization also lead to
adverse effects 15ꢀ17. Thus, there is a clear need to compromise between lipophilicity
and solubility. Wellꢀdesigned drug delivery systems are essential if we wish to
continue to clinical practice18.
Nanoparticles (NPs) are particulate dispersions or solid particles possessing
dimensions of 1ꢀ1000 nm, with the capability to transport and deliver medicinal
substances to selected targets within biological systems19. A large number of
nanotherapeutics have been designed and evaluated over the years, relying on
liposomes, polymers, micelles, NPs and antibodies as carrier materials 20. Due to their
miniscule and colloidal nature, polymerꢀbased NPs are considered an ideal transport
vehicle for drugs and other therapeutic agents as the agent can be encapsulated within
or conjugated onto the NP's surface21. Several methods for polymeric NP preparation
have been developed. Amongst them, the nanoprecipitation technique, also known as
the solvent displacement method, exhibits manifest advantages, essentially because it
is a oneꢀstep process and easy to perform at room temperature 22. Owing to their size,
NPs preferentially target solid tumors rather than healthy tissues by exploiting
vasculature inadequacy 23. One of the clinically successful candidates was the
albuminꢀbased formulation of paclitaxel NPs. It was developed with the aim of
overcoming the very low solubility of paclitaxel, to form nabꢀpaclitaxel NPs with a
mean particle size of 130 nm. The resulting marketed paclitaxel nanoparticulate
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formulation is waterꢀdispersible and was first approved by the FDA in 2005 for the
treatment of metastatic breast cancer.
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This study presents the development and characterization of a drug delivery system
loaded with a potent Pt(IV) prodrug of OXA, [Pt(DACH)(OAc)(OPal)(ox)] (1)
(Scheme 1), as well as handling properties of NPs before and after freeze drying and
storage. The cytotoxicity, Pt accumulation, and DNA platination of 2 was evaluated in
vitro in comparison to 1 and OXA. Furthermore, two in vivo studies were conducted
in SCIDꢀbg mice including: an orthotopic intraperitoneal (i.p.) model of metastatic
ovarian cancer, and a subcutaneous xenograft model of colon cancer.
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Scheme 1. Structure of OXA and lipophilic Pt(IV) compound 1.
2. RESULTS
2.1. In vitro cytotoxic activity
The antiꢀproliferative activities of 1 and OXA were tested in PCꢀ3, PCꢀ3ꢀluc (human
prostate cancer), BxPCꢀ3 (human pancreatic cancer), OVCARꢀ8, SKOVꢀ3 (human
ovarian cancer), CTꢀ26ꢀluc (murine colorectal cancer) cell lines by the MTT assay.
The cells were treated continuously over 120h. The 50% growth inhibitory
concentration (IC50) values were calculated and summarized in Table 1. Complex 1
showed a unique potency against different cancer cell lines, with higher cytotoxicity
than OXA.
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The activity of OXA and 1 against a pair of cisplatinꢀsensitive and resistant ovarian
cancer cell lines, A2780 and A2780ꢀcisR, was determined as a function of the
concentration. The resistance of A2780cisR to cisplatin is achieved by reducing the
uptake, improving DNA repair and increasing glutathione levels.
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Table 1. IC50 values of OXA and 1 in various cancer cell lines.
IC50 (µM)[a]
Cell line
OXA
1
9.2±0.4
27.7±9.1
3.4±0.9
9.4±4.4
58.3±6.3
5.6±0.6
0.35±0.01
0.46±0.11
0.35±0.01
0.46±0.03
0.54±0.03
0.62±0.24
PC-3
PC-3-luc
BxPC-3
OVCAR-8
SKOV-3
CT-26-luc
[a] Values are meansꢁ±ꢁSD obtained from three independent experiments.
The resistance factor (RF) is defined as the ratio of IC50 value in the resistant cell line
to that in the parent cell line. The RF values of cisplatin, OXA, and 1 were 15.53,
5.21, and 0.93, respectively (Table S1). The results show that 1 has the lowest RF
values, which indicates that 1 can overcome acquired resistance to cisplatin and
exhibit efficient activity against the A2780ꢀcisR line and probably against other
cancer cell lines that are typically resistant to Pt therapy.
2.2. Preparation and characterization of 2
The preparation of 2 was carried out using the wellꢀestablished solvent interfacial
deposition method resulting in the formation of spherical NPs (Figure 1). The mean
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diameter of 2 was about 146nm with polydispersity index (PDI) of 0.1, and the zeta
potential value of 2 was approximately ꢀ49mV. Furthermore, optimal encapsulation
yields were achieved (>95% as determined by HPLC) and incorporation into the NPs
ranged from 21.5% to 22.7% w/w. However, because NPs are unstable within
aqueous mediums, their clinical use in delivery systems is significantly limited.
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Freezeꢀdrying is a reliable method to maintain longꢀterm stability for a wide range of
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biological products such as proteins, peptides or drugꢀloaded NPs or liposomes
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Therefore, after drug encapsulation, formulation 2 was freezeꢀdried using five cryoꢀ
and lyoꢀprotectants; sucrose, (+) trehalose, D (ꢀ) mannitol, hydroxypropylꢀβꢀ
cyclodextrin (HPβCD), and xylitol at different concentrations. Among these,
lyophilization of 2 using sucrose (5% w/v) resulted in physically stable NPs. The
lyophilized formulation of 2 was stable following incubation at 4°C over a period of 4
weeks, and following incubation at ꢀ20°C over a period of 16 weeks (Figure S3).
Considering possible clinical issues, the procedures established to freezeꢀdry 2 appear
to be appropriate for the successful development of drugꢀloaded nanoparticulate
formulations intended for parenteral administration. Each batch of 2 was triplicated
and the results were similar indicating a good reproducibility of the manufacturing
process of the nanoparticles and control of the experimental parameters.
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F
D
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Figure 1. Morphological evaluation of 2: (A) SEM secondary electron image. (B) SEM backscattered
electron image. (C,D) TEM micrographs of uranyl acetate negatively stained NPs at different areas of
the grid, scale bar = 200 nm. (E,F) 2D and 3D AFM images of NPs.
Once 1 was successfully encapsulated into NPs, the cytotoxic activity was evaluated
in SKOVꢀ3 ovarian cancer cells. The results showed that the cytotoxic activity of 1
and 2 were more pronounced than the toxic activity of OXA, as depicted in Figure S1
and Table S2.
2.3. Cellular uptake, DNA platination and intracellular localization
To examine the cellular accumulation of platinum of OXA, 1 and 2 in cancer cells,
the total Pt content was measured by ICPꢀMS in SKOVꢀ3 cells following their 24h
exposure to the compounds at a concentration of 35µM. The data, normalized to the
total cellular protein, are presented in Figure 2A. Moreover, the platination of DNA
was assessed. Analysis of the extent of DNA platination measured after 24h exposure
of SKOVꢀ3 cells to 25µg/mL of OXA (62.5µM), 1 and 2 (35µM), indicated higher
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levels of DNAꢀplatination for 1 and 2 in comparison to OXA (Figure 2B). Further
cellular uptake studies were carried out to measure the distribution of Pt species in
different subcellular fractions. SKOVꢀ3 cells were treated with 35ꢂM of OXA, 1 and
2 for 24h and the cells were fractionated into four compartments: membrane, cytosol,
nucleus, and cytoskeleton. The Pt levels in each compartment were measured by ICPꢀ
MS. The results are presented in Figure 2C.
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Figure 2. (A) Platinum accumulation in whole cells following incubation with 35µM of OXA, 1 and 2
following a 24h exposure to SKOVꢀ3 cells as measured by ICPꢀMS. Values are the means ± SD
obtained from three independent experiments. Statistical analysis revealed that the observed difference
in platinum accumulation values was significant (p<0.01, one-way ANOVA). (B) The extent of DNA
platination following 24h exposure to SKOVꢀ3 cells as measured by ICPꢀMS. Values are mean ± SD
obtained from seven independent experiments. Statistical analysis revealed that the observed difference
in DNA platination values was significant (p<0.01, one-way ANOVA). (C) Cellular distribution of Pt
compounds in different fractions. The amount of Pt per 50x103 cells in cell membrane, cytosol, nucleus
and cytoskeleton fractions. Cells were exposed to 35ꢂM of Pt compounds for 24h. Values are
meansꢁ±ꢁSD. Nuclear fraction described by the manufacturer as follows: Total nucleus soluble proteins,
including the nuclear membrane proteins. Cytosol Fraction: Total cellular soluble proteins from
cytoplasm. Membrane/Particulate: Total cellular membrane proteins including cellular organelles and
organelle membrane proteins (excluding the nuclear membrane proteins). Cytoskeletal Fraction: Total
cellular insoluble proteins, genomic DNA.
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2.4. Cellular uptake of NPs in SKOV-3 cells by CLSM
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Localization and cellular uptake of the NPs were evaluated 15 min following
incubation (Figure 3A) and was most evident after 6h (Figure 3B). The nucleus was
stained by DAPI (blue) whereas the NPs, fluorescently labeled using PE Lissamine
Rhodamine B (red), were easily visualized within the cytoplasm. Indeed, increased
cytoplasmic accumulation of NPs was observed as a function of dilution (1:100 versus
1:200) and time, with a more pronounced perinuclear and nuclear localization
pattern(Figure 3B).
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(A)
(B)
Figure 3. Intracellular uptake of NPs visualized by CLSM. (A) Intracellular uptake of PE Lissamine
Rhodamine Bꢀloaded NPs (red) by SKOVꢀ3 cells was evaluated following 0ꢀ60min. incubation with
two formulation dilutions 1:100 and 1:200, nuclei were stained with DAPI (blue). (B) Zꢀstack confocal
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imaging of SKOVꢀ3 cells incubated with PE Lissamine Rhodamine Bꢀloaded NPs (red) for 6h (dilution
1:100).
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2.5. In vivo evaluation of 1 and 2 for the treatment of solid tumors
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For in vivo evaluation of 1, 2 and OXA, the mice were divided into three treatment
groups including: solution of 1 in polyoxylꢀ35 castor oil /ethanol diluted by 10 in
water for injection, free OXA and control (polyoxylꢀ35 castor oil /ethanol diluted by
10 in water for injection), and 2. In fact, when diluted in 90% water for injection,
solution of 1 in polyoxylꢀ35 castor oil / ethanol produces micelles of approximately
13 nm diameter, indicating that the administered solution could behave like a
transparent tiny suspension of micelleꢀtype nanocarrier, with potential accumulation
ability at RES organs and tumor tissue (Figure S2). A pharmacological evaluation was
performed to verify the therapeutic superiority of 1 and 2 compared to OXA using a
CCCD camera to detect the luminescence emitted from the tumor following luciferin
i.p. administration. The tumor bioluminescence evaluation (at longitudinal view) of
the tumor within the same groups of animals exhibit both sensitive and quantitative
measurement of tumor response in vivo.
Metastatic ovarian tumors were induced by luciferase transfected SKOV3ꢀlucꢀD3
cells injected into the peritoneal cavity of SCIDꢀbg mice. Tumor development and
progression was validated by bioluminescent imaging. Tumor signals from the images
were quantified using LivingImage software version 4.3.1, and luminescence group
averages were plotted over time. Thirty days following cells' injection, treatments
were injected into the tail vein once a week over four weeks. It can be clearly seen
from Figure 4A and D, that 1 and 2 were significantly most efficient in ovarian tumor
growth inhibition in comparison to OXA and the control. Although the tumor was not
completely eradicated, 1 and 2 reached the tumor more efficiently than the OXA, and
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the subsequent therapeutic effect was significantly greater. Ventral images of a
representative mouse from each group taken on weeks 4, 7, 9 and 10 following tumor
inoculation are presented in Figure 4D. Importantly, no signs of systemic toxicities
[weight loss (Figure 4B), weakness, pain or discomfort] and no local reactions at the
injection site were observed following 1 and 2 administrations.
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We were interested in evaluating the effect of larger tolerated doses of 1 than with
OXA based on molar calculations. The molar equivalence dose of OXA (5mg/kg) for
1, is 9mg/kg while the dose injected was 15mg/kg. However, the results of a small
pilot study where 15mg/kg of 1 was compared to 9mg/kg equivalent to the dose
injected of OXA of 5mg/kg, showed no significant difference in tumor growth
inhibition and therefore it was decided to proceed with only 9mg/kg of 1 either in free
solution or in NPs for the other cancer models.
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Figure 4. Efficacy study. (A) Longitudinal detection and quantification of SKOV3ꢀlucꢀD3 ovarian
tumor growth in live mice by the bioluminescent luciferase imaging assay using a CCCD camera.
Tumor size is designated by the total luminescence intensity of the dorsal and ventral images,
expressed in radiance units (photons/s/cm2/sr). Arrows indicate the treatment schedules on the 30th,
37th, 45th and 52nd day. Results are presented as mean ± SE. Statistical analysis revealed that the
observed differences at days 62 and 72 were significant (p<0.01, oneꢀway ANOVA). Post hoc analysis
showed that there are significant differences between the control group and 1 and 2 treatment groups
[p<0.01, Dunnett test (2ꢀsided)]. (B) Body weight followꢀup beginning from tumor inoculation (day 0)
through the study period. Changes were recorded as a percentage of the initial body weight observed on
the day of tumor cells injection (100% at day 0). Results are presented as mean ± SD. (C) Kaplanꢀ
Meier survival curve from day of tumor cells injection until death. Death was recorded once animals
fulfilled predetermined euthanasia criteria. ∗∗∗∗ p < 0.0001. The study was terminated on the 93rd day.
(D) Representative ventral images of mice taken on weeks 4, 7, 9 and 10 following tumor inoculation
are given.
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Animals treated with 1 and 2 exhibited the longest survival rate compared to OXA
and control groups (Figure 4C). On the 73rd day, 100% of mice in the 2 group were
still alive, whereas all mice in the control and OXA groups had died and 75% survival
proportions were recorded for the 1 group. The mean survival time in the 1 and 2
groups, as analyzed by the Kaplan–Meier statistics, was 88 and 84 days, respectively,
whereas for OXA and the control the mean survivals were 70 and 71 days,
respectively. Survival differences were statistically significant.
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We next monitored the effect of 1, 2 and OXA on tumor growth over time in a mouse
xenograft model of colon cancer by noninvasive in vivo luminescence imaging. Mice
were subcutaneously injected with HCT 116ꢀluc2 cells, which stably expressed firefly
luciferase, and treatment was administered once every fourth day by i.v. injection
(tail) as described previously. There was an increase in tumor growth in the groups of
the control and OXA. However, in the groups of 1 and 2 in an equivalent
concentration to OXA, tumor growth was significantly impaired with a decrease in
tumor volume as compared to the control (Figure 5A). In contrast to the other animal
studies, the animals in the treated groups initially lost weight but recovered and even
increased in weight with time (Figure 5B).
Animals treated with 2 exhibited the longest survival rate compared to other treatment
groups (Figure 5C). On the 63rd day, 50% of mice in the 2 treatment group were still
alive, whereas all mice in the 1, control and OXA groups had died. The mean survival
time in the 2 treatment group, as analyzed by the Kaplan–Meier statistics, was 69
days, whereas for 1, OXA and control the mean survivals were 55, 55, and 47 days,
respectively. Survival differences were statistically significant, even when 2 treated
mice were only compared to 1 treated mice, reinforcing the added value provided by
the nanoꢀdelivery system (Figure 5C).
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Figure 5. Efficacy study. (A) Longitudinal detection and quantification of HCT 116ꢀluc2 colon tumor
growth in live mice by the bioluminescent luciferase imaging assay using a CCCD camera. Tumor size
is designated by the luminescence intensity of the dorsal images, expressed in radiance units
(photons/s/cm2/sr). Results are presented as mean ± SE. Statistical analysis revealed that the observed
differences in radiance values on the 33rd day were significant (p<0.01, oneꢀway ANOVA). Post hoc
analysis showed significant differences between 1, 2 and control on the 33rd day [p<0.01, Dunnett test
(2ꢀsided)], whereas there was no significant difference between OXA and control. (B) Body weight
followꢀup beginning from tumor inoculation (day 0) through the study period. Changes were recorded
as a percentage of the initial body weight observed on the day of tumor cells injection (100% at day 0).
Results are presented as mean ± SD. (C) KaplanꢀMeier survival curve from tumor cells injection day
until death. Death was recorded once animals fulfilled predetermined euthanasia criteria. ∗∗∗∗ p <
0.0001.
3. DISCUSSION
Ptꢀbased complexes are widely used in the treatment of various solid tumors, although
exhibiting serious sideꢀeffects. Furthermore, it is well known from the literature that
the therapeutic outcome of Ptꢀbased chemotherapy can be impaired by acquired
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resistance which is one of the major limitations of Pt anticancer chemotherapy. The
present study aimed to develop and characterize a drug delivery system loaded with a
novel potent Pt(IV) prodrug of OXA for improved treatment of solid tumors.
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Because of the drawbacks of Pt(II) drugs, there is an incentive to synthesize lipophilic
Pt(IV) complexes in an attempt to improve efficacy and overcome resistance as
already reported by other authors, while trying to enhance the lipophilicity of cisplatin
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and OXA 29ꢀ31. Due to their octahedral coordination geometry, platinum(IV)
complexes are preferred over their platinum(II) counterparts. The presence of two
additional ligands facilitates their ability to detect and attach to specified targets. In
contrast to previous approaches, in which symmetric Pt(IV) compounds were
designed with the aim of tuning lipophilicity 32ꢀ34, in the present study efforts
concentrated on enhancing the lipophilicity of OXA using a different approach based
on combining adversative ligands, by means of aqueous solubility and lipophilicity on
the same Pt(IV) scaffold resulting in an asymmetric compound, namely 1.
The antiproliferative activities of the OXA and 1 were assessed in several cancer cell
lines, as depicted in Table 1. The cytotoxicity of 1 was substantially greater (more
than two orders of magnitude) than that of OXA in both Pt sensitive and resistant cell
lines, perhaps as a result of its enhanced accumulation because of its lipophilic
character, as shown previously with other Pt compounds 29, 35ꢀ37
.
Formulation 2 formed using the interfacial deposition method in the presence of lipoid
E80 and OCA. PLGA was selected for this study because of its biocompatibility,
biodegradability and FDA approval for biomedical applications 38. The formed
particles typically displayed a remarkable loading rate in contrast to other
nanoparticulate formulations where low loading rates (less than 6%w/w) of the active
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ingredients were obtained, confirming the adequate optimization of the actual
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formulation. Fatemeh et al. reported preparation of cisplatin and carboplatinꢀloaded
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polybutylcyanoacrylate NPs with loading percentages of 4 and 6 %, respectively
.
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Indeed, the low drug loading is common for Pt NPs, and can be attributed to their
physicoꢀchemical characteristics 40, 41
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.
2, 42, 43
It should be emphasized that in contrast to other studies
where the
incorporation of Ptꢀbased agents within NPs enhanced their cytotoxic activity owing
to improved internalization, in the present case 1 displayed similar activity to 2. This
is apparently due to its lipophilic character and intrinsic properties of the free active
molecule allowing for significant penetration within the cytosol as shown in Figure 2
where the amounts of 1 in the various cellular fractions were much higher than the
amount of 2, with the exception of the cytoskeletal fraction where the amount was not
significantly different. A plausible explanation for why these differences in
concentrations in the various cellular fractions are not reflected in the cytotoxic
activity should be attributed to the gradual release of 1 from NPs over a prolonged
period of time into the target nucleus and cytoskeletal fraction compensating for the
smaller concentration which remains active. Moreover, an alternative explanation
cannot be excluded, i.e. the formation of free 1 molecules assembling into large
micelles following the preparation of 1 solution and dilution by cell culture media.
Nevertheless, it is still worth continuing the development of 2 because the presence of
two additional ligands in 1 modified the chemical properties compared with the parent
drug (OXA), making it more lipophilic and difficult to formulate in an aqueous
solution.
Delivery of a lipophilic drug to its target has always been a challenge and a subject of
active research. The reduced solubility of drug molecules may hinder their ability to
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cross the physiological aqueous environments encompassing a cell and to penetrate
the cell membrane. Furthermore, complications such as embolisms and local toxicity
are likely to occur if the lipophilic or hydrophobic molecules tend to precipitate once
i.v. injected at the site of administration44. Thus, such compounds need to be
formulated in surfactant solutions to enhance the solubility and prevent potential
precipitation. However, it is known from literature that surfactant agents such as
polyoxylꢀ35 castor oil and polysorbate 80, widely used in the marketed cytotoxic
products, can cause many adverse effects including pain, inflammation, neutropenia,
hypersensitivity and even anaphylactic shock 12, 15, 17. Therefore, in the present case
and in order to avoid the adverse effects normally elicited by these excipients as well
as future complications, it was decided to formulate the novel compound in PLGA
NPs. This is a promising approach to overcome the poor water solubility of a
lipophilic drug, by encapsulation in a NPꢀbased carrier. Additionally, in the present
study it was anticipated that the incorporation of 1 in NPs would not enhance the
cytotoxic activity by accumulation of drugꢀloaded NPs in the cancer cells via
endocytosis, as already reported in other studies 45ꢀ47, because it was assumed that like
other lipophilic prodrugs under dilution of the parent drug, it would form micellar
nanocarriers per se 48. Nevertheless, cellular internalization of NPs into cancer cells
was observed within minutes and gradually increased (Figure 3).
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Pt(II) complexes are kinetically labile, and one advantage of Pt(IV) prodrugs is their
4
relatively greater kinetic inertness . This reduced reactivity is expected to translate
into prolonged circulation time in vivo, because of the protection of Pt(II)
pharmacophore from deactivation by biological nucleophiles. Once the complex
enters the reducing environment of the cancer cell, it should reduce, forming the
active Pt(II) agent 49. We propose that incorporation of 1 into NPs further protects it
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from reduction by physically preventing interaction between reducing agents and the
encapsulated 1. It was recently reported that encapsulation of the Pt(IV) anticancer
compound, mitaplatin, prolonged retention of Pt in the bloodstream more than
unencapsulated mitaplatin. Moreover, while both mitaplatin alone and mitaplatinꢀ
loaded NPs displayed the same longꢀterm tumor growth inhibition in mice carrying
MDAꢀMBꢀ468 tripleꢀnegative breast cancer xenografts, the nanodelivery of the Pt
agent significantly shifted the biodistribution of Pt in treated animals away from
accumulation in the kidneys, a traditional site of Ptꢀinduced toxicity 50.
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In the present study, an in vivo experiment was conducted, in which SKOVꢀ3ꢀluc cells
were inoculated in SCIDꢀbg mice. Due to the lack of an anatomical barrier between
the ovaries and the peritoneal cavity, metastatic resistance of patients with ovarian
cancer is severely reduced, and cancer cells are able to escape from the ovaries and
form tumor nodules on the peritoneal surface51. To closely mimic the disease, we
established an i.p. disease model by injecting SKOVꢀ3 cells intraperitoneally and
evaluating the efficacy of 1, 2 and OXA accordingly. The antitumor activity of 1 and
2 indicated significantly decreased tumor growth compared to the control and OXA
treatment groups (Figure 4).
The similar antitumor activity exhibited by 1 and 2 irrespective of the animal cancer
model might be due to micelles formation resulting from dilution of 1 in polyoxylꢀ35
castor oil / ethanol solution in 90% water for injection, suggesting that the
administered solution is in fact a micellar solution displaying an EPR effect (Figure
S2). Additionally, in vivo findings reinforce in vitro results obtained for the tested cell
lines (Table 1 and Figure 2), in which IC50 values of 1 and 2 were lower than those of
OXA, and respective Pt accumulation values were significantly greater than OXA
elicited values.
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Moreover, animals treated with 1 and 2 exhibited the longest survival rate compared
to OXA and control groups in animal models. In ovarian cancer model, on the 73rd
day, 100% of mice in 2 treatment group were still alive, whereas all mice in the
control and OXA groups had died and 75% survival proportions were recorded for 1
treatment group (Figure 4C). Moreover, on the 63rd day, 50% of mice in the 2
treatment group were still alive, whereas all mice in the 1, control and OXA groups
had died in the subcutaneous model of colon cancer (HCTꢀ116) in SCIDꢀbg mice
(Figure 5C). These findings reinforce the advantage of 1 molecule per se and 2 over
OXA in the treatment of ovarian and colon cancer. Tumor development and
progression were also validated by bioluminescent imaging. The results revealed that
1 and 2 were equally and significantly more efficient in tumor growth inhibition
compared to OXA and the control (Figure 5). It should be emphasized that 2
exhibited a remarkable survival advantage over other groups in the colon cancer
animal model since the final surviving mouse lived for 90 days, whereas all the mice
in the other groups survived up to 63 days, indicating the added value of 2 (Figure
5C). The findings of this study support the improved treatment hypothesis and
substantially enhance tumor growth inhibition elicited by 1 and 2 over OXA, although
no complete tumor eradication was achieved. This could be attributed to several
factors, one being that the cancer cells applied in the in vivo experiments are of human
origin, yielding very aggressive tumor models with a rapid growth rate, which is
unlikely to be seen in the clinics. Indeed, while the efficacy of the novel complex as a
singleꢀagent therapy was demonstrated, combination therapy is the mainstay treatment
of cancer in clinical practice, particularly for advanced stages of the disease.
Moreover, the results of this study are encouraging and reveal considerable potential
for further development of this formulation.
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So far, only a few reports of nano delivery systems of OXA have been reported
.
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Among them, to the best of our knowledge, there are no reports describing the
asymmetric Pt(IV) prodrug of OXA incorporated into PLGA nanospheres.
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4. CONCLUSIONS
Pt drugs are still among the most essential drugs in chemotherapy, but their lack of
cancerꢀcell selectivity leads to toxicity issues. Here we report a novel Pt(IV) prodrug
of OXA, namely 1, containing both lipophilic and hydrophilic axial ligands, with
increased efficiency in killing cancer cells. The overall results indicate that 1 and 2
lead to significant tumor growth inhibition in comparison to OXA, and were effective
in both orthotopic and xenograft tumor models. Moreover, complex 1 was
incorporated in PLGA NPs delivery vehicles with the aim of mitigating the high
systematic toxicity and severe side effects associated with Pt drugs. Such a NP
treatment could become an attractive candidate for further investigation as a
chemotherapeutic treatment for solid tumors.
5. EXPERIMENTAL SECTION
5.1. Materials
Oxaliplatin was acquired from AK Scientific, Inc. (Union City, CA, US); Cisplatin,
Palmitic acid, polyoxylꢀ35 castor oil (Cremophor®EL) and Thiazolyl Blue
Tetrazolium Bromide were purchased from SigmaꢀAldrich (Jerusalem, Israel). Lipoid
E80 was acquired from Lipoid GmbH (Ludwigshafen, Germany). PLGA (MW 50 kD,
Resomer RG 504 H) was purchased from Boehringer Ingelheim GmbH (Ingelheim,
48
Germany). Oleyl cysteineamide (OCA) was synthesized as published previously
.
Solutol® HS 15 was donated by BASF (Ludwigshafen, Germany). Firefly Dꢀluciferin,
potassium salt was obtained from Regis Technologies, Morton Grove, IL, USA. All
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organic solvents were HPLC grade and purchased from J.T Baker (Deventer,
Holland). The PCꢀ3, BxPCꢀ3, OVCARꢀ8 and SKOVꢀ3 cell lines were purchased from
American Type Culture Collection (ATCC), Rockville, MD, USA. PCꢀ3ꢀluc,
SKOV3ꢀlucꢀD3, and HCT 116ꢀluc2 were purchased from Caliper Life Sciences
(Hopkinton, MA, USA). CTꢀ26ꢀluc cell line was kindly supplied by Dr. Gilad
Bachrach from Hebrew University. A2780 and A2780ꢀcisR cells line were kindly
supplied by Prof. B. Keppler from University of Vienna. Most of the cells were
purchased during the last three years and normally when received, they were deꢀ
freezed, subcultured according to the manufacturer protocol to the appropriate
passage and stored as aliquots in liquid nitrogen. Every two months, the cells were
tested for mycoplasma contamination according to the manufacturer protocol. All
tissue culture products were from Biological Industries Ltd (Beit Ha Emek, Israel).
Luciferase transfected cell lines were obtained from Caliper Life Sciences
(Hopkinton, MA). Pt concentrations were determined by an Agilent 7500ce
inductively coupled plasma mass spectroscopy (ICPꢀMS). Tumor signals from the
images of in vivo studies were quantified using LivingImage software version 4.3.1.
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5.2. Instrumentation
All NMR data were collected on a Bruker AVANCE IIITM HD 500 MHz
spectrometer using a 500 Mhz SmartProbe. 195Pt NMR data were collected at 107.47
MHz using sw= 217391.3 Hz aq=0.002sec and rd=0.001sec. The data were processed
1
using MestreNova with a 300 Hz line broadening and backward linear prediction. H
spectra were recorded with standard parameters. Analytical HPLC analysis was
performed on a standard Dionex UPLC system equipped with a Kinetex 5µ XBꢀC18
100A New Column 250x4.6mm with detection at 220nm. Preparative purification
was carried out on a Dionex semiꢀ preparative HPLC using Phenomenex Luna 10µ
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C18 100A 250x21.20mm column at 220nm. Platinum concentrations were determined
by an Agilent 7500ce inductively coupled plasma mass spectroscopy (ICPꢀMS). The
mean diameter and the zeta potential of the NPs were characterized using Malvern's
Zetasizer (Nano series, NanosꢀZS, UK) at 25°C and using water as diluent.
Morphological evaluation was performed on carbonꢀcoated Cu grids (300ꢀmesh),
using an electron microscope (CM12 TEM, Philips) at 100 kV, after negative staining
using 2% uranyl acetate. Cellular uptake evaluation of NPs was done by Zeiss LSM
710 Spectral Confocal Laser Scanning Microscope (CLSM). MS analysis was
performed with a TSQ Quantum Access Max mass spectrometer with a heated
electrospray ionization (HꢀESI) interface (Thermo Scientific, San Jose, CA, USA).
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5.3. Synthesis and characterization of 1
5.3.1. Synthesis of [Pt(DACH)(OAc)(OH)(ox)]: OXA (1.5g, 3.8mmol) was taken in
100mL acetic acid and 50µL of 30% H2O2 was added. The reaction mixture was
stirred at room temperature for 4ꢀ5h and the reaction was monitored by 195Pt NMR.
Excess acetic acid was removed by vacuum evaporation and the resulting white solid
was washed with diethyl ether and acetone and dried under vacuum. The product was
further purified by preparative HPLC (5%ACN:95% H2O as mobile phase), RT=
1
7.6min. Yield: 1.456g, 80.94%. 195Pt nmr (in acetic acid),
(D2O)
2.75(b, 2H)
δ
(ppm)= +1389. H NMR
δ
= 1.02ꢀ1.17(b,2H), 1.32ꢀ1.54(b,4H), 1.89(s,3H), 2.06ꢀ2.18 (B,2H), 2.66ꢀ
5.3.2. Synthesis of Palmitic anhydride: Palmitic acid (500mg, 1.95mmol) was
dissolved in 20mL dichloromethane and N,N'ꢀDicyclohexylcarbodiimide (0.5eq.,
201.2mg, 0.975mmol) was added. The reaction mixture was stirred at room
temperature for 12h. Progress of the reaction was monitored by TLC with ethyl
acetate as the mobile phase. N,N'ꢀDicyclohexylurea that formed was filtered off from
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the reaction mixture and excess dichloromethane was removed by evaporation. The
white solid obtained after the removal of dichloromethane was reꢀdissolved in a small
amount of dichloromethane and the N,N'ꢀDicyclohexylurea that precipitated was
filtered off again. This step was repeated four times to remove N,N'ꢀDicyclohexylurea
completely. Palmitic anhydride was characterized by1H NMR. Yield: 858 mg 89%.
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1H NMR (CDCl3):
4H).
δ= 0.8ꢀ0.9(t,6H), 1.16ꢀ1.36(m,48H), 1.56ꢀ1.69(q,4H), 2.30ꢀ2.46(t,
5.3.3. Synthesis of 1: [Pt(DACH)(OAc)(OH)(ox)] (1g, 2.1mmol) was dissolved in
5mL DMF and palmitic anhydride (1.57g. 3.17mmol) was added. The reaction
mixture was stirred at 40°C overnight and monitored by 195Pt NMR. Unreacted
anhydride (precipitated upon cooling) was filtered off from the reaction mixture and
DMF was removed by vacuum evaporation yielding a lightꢀyellow solid. In order to
purify the compound, 1mL methanol was added to the solid obtained after the
evaporation of DMF and the mixture was shaken well. The insoluble white precipitate
was collected by centrifugation and washed twice with small portions of methanol and
1
dried in vacuum. The compound obtained was characterized by 195Pt NMR and H
1
NMR. Yield: 1.156g, 76.97%, 195Pt NMR (DMF)
δ
= 1589. H NMR (DMSOꢀD6)
δ
=: 0.79ꢀ0.87(t,3H), 1.13ꢀ1.27(b,24H), 1.30ꢀ1.54(b,5H), 1.93(S,3H), 2.02ꢀ2.15(m,2H),
2.17 ꢀ2.28(m,2H), 2.76ꢀ2.94(b,2H), 8.20ꢀ8.6(b,4H). m/z (calculated) = 711.75, m/z
(found)=712.19. The calculated elemental analysis for 1 is C, 43.87; H, 6.80; N, 3.94.
The actual analysis of the prepared material was C, 43.69; H, 6.61; N, 3.89. HPLC
chromatogram of 1 (100%Acetonitrile), RT= 3.6 min. According to these findings,
the purity of 1 is higher than 98%. The purity of 1 was further confirmed by the
HPLC method previously described in the instrumentation section following injection
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of high concentrations (200 µg/ml). The purity was higher than 95% based on the
various peaks areas detected in the chromatogram.
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5.4. Cell lines and cell incubation conditions
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All cell culture mediums and all the cell lines were tested and authenticated from any
mycoplasma contamination. A Mycoplasma contamination test was done prior to the
use of cells by a polymerase chain reaction (PCR) method (EZꢀPCR Mycoplasma kit,
Biological Industries, Israel). The in vitro experiments were performed in human cell
lines of prostate cancer (PCꢀ3 and PCꢀ3ꢀluc), pancreatic cancer (BxPCꢀ3), ovarian
cancer (SKOVꢀ3, A2780, A2780ꢀcisR and OVCARꢀ8), and murine colorectal cancer
CTꢀ26ꢀluc cells. BxPCꢀ3 and OVCARꢀ8 cells were grown in RPMI medium
supplemented with 10% fetal bovine serum (Gibco), 1% Lꢀglutamine, 100U/mL
penicillin, 100ug/mL streptomycin and 0.13% w/w gentamycin. PCꢀ3 and PCꢀ3ꢀluc
cells were cultured in DMEM plus 10% fetal bovine serum, 1% Lꢀglutamine,
100U/mL penicillin, 100µg/mL streptomycin and 1% pyrovate. SKOVꢀ3 cells were
cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum,
100U/mL penicillin, and 100ug/mL streptomycin. A2780 and A2780ꢀcisR cells were
grown in RPMI medium supplemented with 10% fetal bovine serum, 1% Lꢀglutamine
and 0.1% w/w gentamycin, and the acquired resistance in A2780ꢀcisR cell line was
maintained by supplementing the medium with 1uM of cisplatin every second
passage. CTꢀ26ꢀluc cells were grown in RPMI supplemented with 10% fetal bovine
serum, 1% Lꢀglutamine, 100U/mL penicillin, 100ug/mL streptomycin, and 100ug/mL
neomycin. SKOV3ꢀlucꢀD3 and HCT 116ꢀluc2 cells were cultured in McCoy’s 5A
medium supplemented with 10% heat inactivated fetal bovine serum, 100U/mL
penicillin, and 100ug/mL streptomycin.
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The cell lines were maintained at 37°C under a moist atmosphere containing 5% CO2
and 95% air. All cell culture products were obtained from Biological Industries (Beit
Ha Emek, Israel).
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5.5. Preparation and characterization of 2
5.5.1. Preparation of 2
Formulation 2 was prepared using the solvent deposition method 22. Briefly, 7mg of 1,
14mg PLGA 50kDa, 7mg lipoid E80 and 1.5mg Oleyl cysteineamide (OCA) were
dissolved in 4mL (1:20) ethanol/acetone mixture. The organic phase was added to
2.5mL aqueous solution containing 0.1% w/v Solutol® HS 15. The suspension was
stirred at 900rpm over 15min, and then concentrated by solvents’ evaporation to a
final volume of 2.5mL, followed by centrifugation for 10min at 4400rpm.
The resulting drugꢀloaded NPs consisted of spherical nanomatrices of PLGA, lipoid
Eꢀ80 and OCA embedding 1, and dispersed in 0.1% of Solutol HS15, solution were
entitled 2.
5.5.2. Content determination of 1 in NPs
To determine the amount of drug loaded in the NPs, formulation 2 was dissolved in
acetonitrile and the drug concentration was determined using analytical Dionex HPLC
system equipped with a Kinetex 5u XBꢀC18 100A Column 250x4.6mm. 1 was
detected at a wavelength of 220nm. Acetonitrile 100% was used as a mobile phase
and the flow rate was 1mL/min.
5.5.3. Physicochemical and morphological characterization
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The particle size distribution and zeta potential of 2 were characterized by dynamic
light scattering (DLS, Malvern Zetasizer Nano ZS, UK). Morphological and size
evaluation of 2 was carried out using XHR SEM (model: Magellan 400L, FEI,
Germany). The samples were fixed on a SEMꢀstub using doubleꢀsided adhesive tape
and then made electrically conductive following standard coating by gold sputtering
(Pilaron E5100) under vacuum. Morphological evaluation of the NPs was performed
on carbonꢀcoated Cu grids (300ꢀmesh), using an electron microscope (CM12 TEM,
Philips) at 100kV, after negative staining using 2% uranyl acetate. A diluted
suspension of 2 (1:1x106) in water (HPLC grade, Baker) was placed onto the surface
of cover glass (Marienfeld cover glass, 100, 22 x 22 mm). Samples were left to dry.
The morphology and surface states were investigated using Scanning Probe
Microscope Dimension 3100, Nanoscope V (Veeco, CA, USA).
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5.5.4. Lyophilization of 2
Finally, to ensure the prolonged physical stability of the NPs, five types of sugars
were evaluated as cryoꢀ and lyoprotectants for the freezeꢀdrying process of 2: sucrose,
(+) trehalose, D (ꢀ) mannitol, xylitol, and hydroxypropylꢀβꢀcyclodextrin (HPβCD).
The NP suspension was freezeꢀdried using an Epsilon 2ꢀ6d Martin Christ lyophilizer
(Gef., Germany) to obtain a dry powder.
5.6. In vitro cytotoxicity assay
Cytotoxicity was determined by the colorimetric MTT (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ
2,5 diphenyltetrazolium bromide) assay as described previously48. In brief, cells were
seeded in a sterile, 24ꢀwell plate and incubated in the presence of the drug for 120h at
37°C, 5% CO2. In the first 24h the cells were allowed to settle and resume exponential
growth. Then, cells were incubated for 120h with treatments dispersed in full growth
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medium at different drug concentrations (0ꢀ50ꢂg/mL). Cells were extensively washed
with HBSS and incubated with MTT solution (0.5mg/ml) in HBSS, 2, 37°C).
Following another washing step, the dye was solubilized with DMSO and the
absorbance was determined at 540nm.
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5.7. Determination of platinum accumulation in whole cell
SKOVꢀ3 cells were seeded in triplicate 24ꢀwell plates (50x103 cell/well), allowed to
reach 80–90% confluence, and treated with OXA, 1 and 2 at concentrations of 35µM
for 24h at 37°C. Following incubation, the drugꢀcontaining medium was removed,
and cells were rinsed twice with cold PBS. The cells were harvested and centrifuged
into a pellet. 1mL of concentrated nitric acid was added to cell pellets, and incubated
at 70°C on a hot plate until the samples dried. The samples were dissolved in 0.1%
SDS and 1% nitric acid, and analyzed for Pt content by ICPꢀMS. Protein
concentration analysis was conducted by BCA assay. Briefly, the cells were incubated
in the same mentioned conditions and concentrations. Following incubation, the drugꢀ
containing medium was removed, and each well was rinsed twice with cold PBS,
trypsinized, and the cells were centrifuged into a pellet (4400rpm, 5min). The pellet
was rinsed one more time with PBS and NaOH (0.1N) was added. After diluting the
samples using water, they were incubated at 70ºC for 1h and the absorbance was
measured at 562nm. The protein concentration of the sample was calculated according
to the BSA standard curve.
5.8. Determination of platinum content binding to DNA (DNA platination)
SKOVꢀ3 cells were seeded in triplicate 24ꢀwell plates (50x103 cell/well), allowed to
reach 80–90% confluence, and treated with OXA, 1 and 2 at concentrations of
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