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Liposome formulation of co-encapsulated vincristine and quercetin enhanced antitumor activity in a trastuzumab-insensitive breast tumor xenograft model
POTENTIAL CLINICAL RELEVANCE Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 834 – 840
Research Article
nanomedjournal.com
Liposome formulation of co-encapsulated vincristine and quercetin enhanced antitumor activity in a trastuzumab-insensitive breast tumor xenograft model Man-Yi Wong, BSc (Hons), Gigi N.C. Chiu, PhD⁎ Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore Received 13 August 2010; accepted 3 February 2011
Abstract Hormone- and trastuzumab-insensitive breast cancer has limited and ineffective clinical treatment options. This study sought to develop a liposome formulation containing a synergistic combination of vincristine and quercetin, with prolonged drug circulation times and coordinated drug release in vivo, to develop effective treatments against this subtype of breast cancer. The 2:1 molar ratio of vincristine/ quercetin showed strong synergism in the hormone- and trastuzumab-insensitive JIMT-1 cells. Liposome co-encapsulation prolonged plasma circulation of the two drugs and maintained the synergistic drug ratio in vivo. Furthermore, the co-encapsulated liposome formulation demonstrated the most effective tumor growth inhibition in the JIMT-1 human breast tumor xenograft in comparison with vehicle control, free quercetin, free vincristine and free vincristine/quercetin combinations. Specifically, only the co-encapsulated liposome formulation exhibited significant antitumor activity at two-thirds of the maximum tolerated dose of vincristine, without significant body weight loss in the animals. From the Clinical Editor: In this study, a novel liposome formulation containing a synergistic combination of vincristine and quercetin was utilized in the treatment of breast cancer. Prolonged drug circulation times and coordinated drug release characterize this effective treatment, which may find its way to clinical applications in the near future. © 2011 Elsevier Inc. All rights reserved. Key words: Liposomes; Quercetin; Vincristine; Trastuzumab; Breast cancer
Globally, breast cancer is the most prevalent cancer,1 and is the leading cause of cancer-related deaths in women.2 Breast cancer can be characterized by the presence or the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth-factor receptor-2 (HER-2). Antihormonal treatments and HER-2 targeted therapies such as trastuzumab have increased the therapeutic options and have improved the survival rates of patients who are responsive to these treatments.2 However, patients who are unresponsive to antihormonal and trastuzumab treatments are limited to the use of high doses of single drug regimens3 or combination regimens of various chemotherapeutic agents.4 These approaches are either too toxic and have resulted in more treatment-related deaths3 or lacking therapeutic efficacy with
This work was supported by the Singapore Ministry of Education via the National University of Singapore Academic Research Fund (grant # R-148050-077-101 & R-148-050-077-133). Man-Yi Wong is a recipient of a research scholarship from Singapore Ministry of Education. There are no conflicts of interest in connection with this article. ⁎Corresponding author. E-mail address: [email protected] (G.N.C. Chiu).
no increase in disease-free survival.5 This highlights the need to develop safe and effective treatment options against ER-, PR- and trastuzumab-insensitive breast cancers. The problems of toxicity and lack of efficacy could be resolved by the use of liposomes, which are small spherical vesicles formed by a lipid bilayer enclosing an aqueous compartment.6 The toxicity of the liposome-encapsulated chemotherapeutic drug could be reduced, as the drug could not exert its activity when sequestered in liposomes during bloodstream circulation.7,8 In addition, liposomes have been shown to enhance therapeutic efficacy of anticancer drugs by increasing drug exposure in the tumor resulting from the prolonged circulation times of the drugs,9 and by preferential accumulation of the anticancer drugs as a result of the enhanced permeability and retention effect in the tumor.10 Efficacy of chemotherapeutic agents can be increased through the administration of synergistic drug combinations in fixed molar drug ratios.11-13 Owing to the differences in pharmacokinetic profiles of the drugs in a combination regimen, the synergistic drug ratios may not be attained in the tumor site. The use of liposomes has been shown to maintain the desirable, synergistic drug ratios as
1549-9634/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.02.001 Please cite this article as: M.-Y. Wong, G.N.C. Chiu, Liposome formulation of co-encapsulated vincristine and quercetin enhanced antitumor activity in a trastuzumab-insensitive breast .... Nanomedicine: NBM 2011;7:834-840, doi:10.1016/j.nano.2011.02.001
M.-Y. Wong, G.N.C. Chiu / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 834–840
well as to coordinate the release of the encapsulated drug combinations to achieve increased antitumor activity.11-13 In view of these advantages, our approach is to deliver a combination regimen with synergistic drug ratios through liposomes to develop potentially effective treatment options for breast-cancer subtypes that are unresponsive to hormonal and trastuzumab treatments. The drug combination to be co-encapsulated in liposomes is vincristine and quercetin. The cytotoxicity of vincristine can be enhanced by quercetin, which reduced vincristine efflux from the cancer cells.14 This is further supported by our previous findings that the vincristine/quercetin combination, given at molar ratios of 4:1, 2:1 and 1:1, showed moderate to strong synergism, with the molar ratio of 2:1 being the most synergistic in the hormone receptor-negative and trastuzumab-insensitive cell line MDA-MB-231.15 Based on these results, a liposome formulation has been developed that consists of vincristine encapsulated in egg sphingomyelin (ESM)/Cholesterol/Npalmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000] (PEG2000-ceramide)/quercetin (72.5:17.5:5:5 mole ratio) with the vincristine/quercetin molar ratio of 2:1. This liposome formulation coordinated the in vitro release of vincristine and quercetin in the synergistic ratio of 2:1 over 72 hours and displayed a combination index (CI) of 0.113 in MDA-MB-231 cells.15 In this study, we sought to 1) determine if the synergism could be demonstrated in another hormone- and trastuzumabinsensitive cell line, JIMT-1; 2) assess whether liposome encapsulation would prolong the in vivo circulation times of vincristine and quercetin; and 3) assess whether a coordinated release of the drug combination could be achieved in vivo and thereby improve the antitumor efficacy in the human JIMT-1 breast-cancer xenograft model. Methods Materials All lipids were obtained from Avanti Polar Lipids (Alabaster, Alabama). JIMT-1 cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). The materials 3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO) and chloroform were purchased from MP Biomedicals Asia Pacific Pte Ltd (Singapore). Balb/c mice were supplied via the Centre for Animal Resources, National University of Singapore (Singapore), and SCID mice were supplied via the Biological Resource Centre, Biopolis, Singapore. All other materials were purchased from SigmaAldrich (St. Louis, Missouri). In vitro cytoxicity assay In vitro cytotoxicity was assessed by MTT assay.16 JIMT-1 human breast-cancer cells were grown in DMEM/F-12 media supplemented with 10% fetal bovine serum and seeded at 4000 cells/well in 96-well plates. Cells were incubated at 37°C with 5% CO2 for 24 hours to allow adherence to the cell culture plates before treatment with serial dilutions of either single drugs (quercetin, vincristine) or drug combinations (vincristine/
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quercetin at molar ratios of 4:1, 2:1, 1:1 and 1:2) for 72 hours. Subsequently, 50 μL of MTT reagent (1 mg/mL) was added to each well, and was incubated with the cells for 4 hours. At the end of the incubation period, the cell culture medium was aspirated, and 150 μL of DMSO was added to each well. The 96well plates were shaken for 20 minutes to solubilize the cells and subsequently were read on the Tecan Sunrise microplate reader (Männedorf, Switzerland) with an absorbance wavelength of 570 nm. Cell survival at the end of treatment was calculated from the absorbance readings as a percentage of the control. All assays were performed in triplicate. Median-effect analysis for drug combinations Drug combination analyses were performed using CalcuSyn (Biosoft, Cambridge, United Kingdom), which is a software program developed using the median-effect principle described by Chou and Talalay.17 For studies on the combined effects of vincristine and quercetin, fixed ratios of vincristine and quercetin (4:1, 2:1, 1:1 and 1:2) were used. The CalcuSyn software generates the CI value for a particular combination of vincristine/ quercetin based on the cell-survival data from the MTT assay, whereby additivity, synergy and antagonism were reflected by CI values of 0.9–1.1, b 0.9 and N 1.1, respectively. Liposome preparation Liposomes were prepared by the thin-film hydration method.18 Briefly, the lipids dissolved in chloroform were mixed with quercetin dissolved in ethanol. The preparation was subsequently dried under a stream of nitrogen gas, and the resulting lipid film was placed under vacuum to remove organic solvents. The dried lipid films were hydrated with 300 mM manganese sulfate (pH 3.4) for 1 hour at 60°C. The resulting preparation was extruded 15 times at 60°C through stacked 0.1-μm pore size polycarbonate filters (Northern Lipids Inc., Vancouver, British Columbia, Canada) with an extruder apparatus (Northern Lipids Inc.). The mean diameter of the extruded liposomes was determined using the Zetasizer 3000HS operating at 633 nm, and was found to have diameters of ∼130 nm. Vincristine (drug-to-lipid mole ratio of 0.1:1) was subsequently loaded into the liposomes using an ionophore-mediated proton gradient.19 The encapsulated drug was separated from the free drug using 1-mL Sephadex G-50 spin columns. Vincristine and quercetin were quantified spectroscopically based on their absorbance at 297 nm20 and 376 nm,21 respectively, after solubilization of the liposomes in n-octyl-D-glucopyranoside and ethanol, respectively. Animal studies The studies were approved by the National University of Singapore Institutional Animal Care and Use Committee (NUS IACUC), and the procedures followed were in accordance with NUS IACUC guidelines. The animals were under humane care throughout the studies and were housed in micro-isolator cages with free access to food and water. All mice used were between 20–22 g and were quarantined for 7 days before the study was initiated.
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Pharmacokinetic study Drugs in free form or in liposome formulation were injected intravenously into the lateral tail vein of female Balb/c mice (4 mice per time point). Vincristine was given at 1.33 mg/kg and quercetin was given at 0.24 mg/kg. These doses corresponded to a 2:1 molar drug ratio of vincristine and quercetin. At 10 minutes, 0.5 hour, 1 hour, 2 hours, 4 hours and 24 hours after drug administration, the animals were euthanized by CO2 asphyxiation. Blood samples were collected by cardiac puncture and centrifuged for 10 minutes at 1000 × g to isolate plasma, which was stored at −20°C until analysis of drug concentrations by UPLC. In vivo efficacy study Tumors were established in SCID mice by a single subcutaneous injection of 5 × 106 JIMT-1 cells in the upper back area. Tumor progression was monitored by caliper measurements of the tumors along the length and width thrice a week. Tumor volumes were calculated by the following formula: Tumor volume = (length × width2)/2. When the tumor size reached 200–300 mm3, randomization of animals was performed to give 5 groups of 5 animals each. The study groups included 1) vehicle control, 2) free quercetin, 3) free vincristine, 4) free vincristine/quercetin combination, and 5) co-encapsulated liposomal vincristine/quercetin groups. The dose of vincristine was 1.33 mg/kg (two-thirds of the maximum tolerated dose in SCID mice20) and that of quercetin was 0.24 mg/kg. At these values, the molar ratio of vincristine/quercetin was 2:1. Tumor size and body weight of the mice was monitored thrice weekly until 60 days post tumor inoculation. Animal euthanization was performed when tumor size reached 1000 mm3, when tumor ulcerations developed, or with body weight loss of N 5%. UPLC method development and drug extraction A Waters UPLC AcQuity UPLC system (Milford, Massachusettes) was used to quantify vincristine and quercetin in plasma samples, using a Waters AcQuity UPLC, BEH C18, 2.1×50 mm, 1.7-μM column protected by a guard column. The assay was performed at 25°C with 0.1% formic acid as weak solvent and acetonitrile/0.1% formic acid as strong solvent. The initial phase comprised 75% of 0.1% formic acid and 25% of acetonitrile/0.1% formic acid. The final phase comprised 5% of 0.1% formic acid and 95% of acetonitrile/0.1% formic acid at 3.8 minutes, with a flow rate of 0.5 mL/minute. The detection wavelengths of quercetin and vincristine were 376 nm and 297 nm, respectively. Extraction of quercetin and vincristine from plasma was modified from previously reported methods.22-23 Apigenin was used as the internal standard. Briefly, 100 μL of plasma was mixed with 50 μL of β-glucuronidase, sulfatase and 25 μL of ascorbic acid. The mixture was incubated at 37°C for 1 hour anaerobically. The serum was acidified with 10 μL of 0.1 N HCl, and extracted 4 times with 100 μL of ethyl acetate. The ethyl acetate layer was collected and evaporated to dryness under nitrogen gas and reconstituted with acetonitrile/0.1% formic acid for UPLC analysis. The supernatant was injected into the UPLC
Figure 1. CI values at ED50 for vincristine/quercetin exposed to JIMT-1 breast cancer cells at molar ratios of vincristine/quercetin of 4:1, 2:1, 1:1, and 1:2. Each value represents the mean ± SEM from 3 independent experiments. Additivity, synergy and antagonism are reflected by CI values of 0.9–1.1, b 0.9 and N 1.1, respectively.
system at 1 μL. The concentrations of quercetin and vincristine in the samples were determined by comparing the peak area ratios of the samples versus a calibration curve obtained from spiking known amounts of quercetin and vincristine in pooled mouse plasma. The linearity of the standard curve was more than 0.998, and the limits of detection for vincristine and quercetin were 0.008 nmoles/mL and 0.05 nmoles/mL, respectively. Pharmacokinetic analyses were performed using non-compartmental analysis with WinNonlin software standard version 1.0 (Sunnyvale, California). Statistics All statistical analyses were performed using the NCSS 2004 software (Kaysville, Utah). All experimental data were expressed as mean ± SEM. One way ANOVA and the post hoc Tukey test were used. Kaplan-Meier survival analysis with log-rank significance test was conducted to determine whether survival differed among the treatment groups. P values of b 0.05 were considered to be statistically significant. Results In vitro combination effects of free vincristine and quercetin Previously, we showed that the most synergistic molar ratio of vincristine and quercetin was 2:1 in MDA-MB-231 cells.15 Given that it is important to demonstrate consistent display of synergy across different cell lines for a specific ratio of chemotherapeutic agents,24 it was our aim to assess if a similar trend could be observed in JIMT-1 cells. The in vitro combination effects of vincristine and quercetin were compared among the molar drug ratios of 4:1, 2:1, 1:1 and 1:2. Figure 1 shows the CI values at different molar ratios of vincristine/ quercetin at the median effective dose (ED50). The CI values were 0.699, 0.0000223, 0.00102 and 0.354 for the molar drug ratios of 4:1, 2:1, 1:1 and 1:2, respectively. Therefore, the ratio of 2:1 for vincristine/quercetin was the most synergistic. Similar trends were observed at other ED values, but ED50 values were
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Figure 2. Concentrations of vincristine and quercetin needed to achieve 50% cell kill when incorporated into liposomes. Data were analyzed using the CalcuSyn software based on the median effect principle developed by Chou and Talalay. Each value represents the mean ± SEM from 3 independent experiments. Quercetin, solid bars; vincristine, open bars.
reported because the aim of cancer treatment is to eradicate cancer cells and the doses needed to attain high cell kills are more clinically relevant in comparison with doses needed to attain lower cell kills.25 Liposomal co-encapsulation of vincristine and quercetin Vincristine (drug-to-lipid molar ratio of 0.1) was loaded into ESM/cholesterol/PEG2000-ceramide/quercetin (72.5:17.5:5:5 molar ratio) liposomes. The encapsulation efficiencies for vincristine and quercetin were 78.3% and 78.5%, respectively. Optimization of this formulation have been described in our previous work.15 The final vincristine/quercetin ratio in the liposomes was 2:1. In vitro combination effects of liposomal vincristine and quercetin In vitro cytotoxicity studies were first conducted with empty liposomes on the JIMT-1 cells to determine whether the liposomes would contribute to the cytotoxicity. The concentration of lipid tested was matched to the amount of lipid used in the drug-loaded formulation. At the concentrations of lipids used, cell viability ranged from 94.0% to 105.9% of untreated control, indicating that the liposomes alone did not contribute to significant cell kill. When vincristine and quercetin were co-encapsulated in liposomes at a molar ratio of 2:1, the concentrations of vincristine and quercetin required to attain 50% cell kill were reduced approximately by 1.5fold for vincristine and 10-fold for quercetin, in comparison with the liposomal monotherapy (Figure 2). The CI value of the coencapsulated liposomal formulation was 0.0900 (very synergistic) at ED50. Similar trends for CI were observed at other ED values.
Figure 3. (A) Concentrations of vincristine and quercetin in plasma after intravenous administration of the free vincristine/quercetin combination or the liposomal co-encapsulated vincristine/quercetin combination into Balb/c mice. Free quercetin, ♦; free vincristine, ▲; liposomal quercetin, ⋄; liposomal vincristine, △. (B) Comparison of the ratio of vincristine/quercetin over time for free vincristine/quercetin combination (△) and liposomal co-encapsulated vincristine/quercetin (♦) in plasma. *P b 0.05, one way ANOVA and post-hoc Tukey test. The x-axis is in log scale to facilitate visualization of the data points. Each value represents the mean ± SEM from 4 samples.
Balb/c mice. Figure 3, A shows that vincristine and quercetin levels in plasma were undetectable after 60 minutes and 120 minutes, respectively, for the free drug combination group. In contrast, the plasma levels of vincristine and quercetin in the co-encapsulated liposome formulation remained detectable throughout the entire study. In addition, the co-encapsulated liposome formulation displayed higher Cmax, AUClast, t1/2 and MRTlast with lower Cl, in comparison with the free drug combination (Table 1). Finally, Figure 3, B shows that the initial vincristine/quercetin ratio of 2:1was maintained over the entire study period for the co-encapsulated liposome formulation but not for the free drug combination group (P b 0.05).
Plasma elimination profiles of free and liposomal combination of vincristine and quercetin
In vivo antitumor effects against the JIMT-1 xenograft
The plasma elimination profiles of vincristine and quercetin either as free or liposomal drug combinations were determined in
The antitumor efficacy against the JIMT-1 xenograft was evaluated by comparing 1) vehicle control, 2) free quercetin, 3)
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Table 1 Summary of pharmacokinetic parameters for vincristine and quercetin in mice⁎ Free †
Cmax (nmol/mL) AUClast (μmol.h/mL) t1/2 (h) Cl (mL/h) MRTlast (h) V (mL/g) r
Liposomal
Quercetin
Vincristine
Quercetin
Vincristine
2.07 6.65 1.49 0.47 1.20 1.00 −0.82
0.19 0.33 0.48 16.90 0.40 12.19 −0.92
19.20 604.16 14.20 0.01 9.22 0.08 −0.84
34.10 1240.54 16.00 0.01 8.26 0.09 −0.96
⁎ Mice (n = 4) received either 1.33 mg/kg vincristine together with 0.24 mg/ kg quercetin (2:1 vincristine: quercetin molar ratio) as free drug combination or 1.33 mg/kg vincristine and 0.24 mg/kg quercetin co-encapsulated in liposomes. Injection volume was 200 μL. † Pharmacokinetic parameters were calculated based on pooled data from 4 mice. Cmax, maximum plasma concentration of drug; AUClast, area under the curve computed to the last time point; t1/2, half life; Cl, total body clearance; MRTlast, mean residence time up to the last time point; V, volume of distribution; r, Pearson correlation coefficient.
free vincristine, 4) free vincristine/quercetin combination, and 5) co-encapsulated liposome vincristine/quercetin formulation. Although all treatment groups (groups 2–5) showed initial tumor regression, only the liposome co-encapsulated formulation showed tumor regression over the 60-day study period (Figure 4, A). Table 2 shows the time taken for tumors to reach 500 mm3 with various treatments. Statistically significant difference was observed between the co-encapsulated liposome formulation and the other study groups (P b 0.05). In addition, free quercetin monotherapy and free vincristine monotherapy were not statistically different from the vehicle control in terms of the time for tumors to reach 500 mm3 (P N 0.05). Table 2 also lists the percentages of tumor-growth inhibition, which shows a trend similar to that of the time for tumors to reach 500 mm3. Percentage weight change at nadir was used as an indicator of the toxicity of the treatment,26 and statistical significance (P b 0.05) was observed between the free vincristine/ quercetin combination as compared to the co-encapsulated liposome formulation (Table 2). Finally, Kaplan-Meier survival analysis with log-rank significance test was conducted, and the co-encapsulated liposome formulation was statistically different from the vehicle control, free quercetin monotherapy and free vincristine monotherapy (P b 0.05, Figure 4, B). No significant difference was observed between the coencapsulated liposome formulation and the free drug combination (P N 0.05).
respectively, indicating antagonism when the two drugs were administered freely without a carrier.
In vitro evaluation of CI values in the ratios of free vincristine and quercetin
Discussion
The ratios of vincristine and quercetin in plasma following the administration of the free drug combination were 1:11, 1:12 and 1:6. The ratios of vincristine/quercetin 1:12 and 1:6 were tested in vitro, and the drug combination effects were analyzed to determine the CI values at ED50. The CI values were 2.02 and 1.33 for the vincristine/quercetin ratios of 1:12 and 1:6,
We will discuss our work from the formulation and clinical perspectives. From the formulation perspective, we have demonstrated that the administration of free drug combination in the absence of a drug carrier led to antagonistic CI values in vivo. This highlights the need for a delivery vehicle to attain optimal antitumor efficacy for the vincristine/quercetin
Figure 4. (A) In vivo antitumor effects of various treatment groups against JIMT-1 tumor xenograft in SCID mice (n = 5). The mice were treated via tail vein injections with vehicle control (blue), free quercetin (pink), free vincristine (orange), free vincristine/quercetin combination (green) and coencapsulated liposomal vincristine/quercetin (purple) groups. The doses of vincristine and quercetin given were 1.33 mg/kg and 0.24 mg/kg, respectively (2:1 vincristine/quercetin mole ratio). A total of 3 doses were administered on days 17, 20 and 23. (B) Kaplan-Meier survival curves of the different treatment groups (n = 5). Vehicle control, blue; free quercetin, pink; free vincristine, orange; free vincristine/quercetin combination, green; coencapsulated liposomal vincristine/quercetin, purple. Log-rank significance test was conducted.
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Table 2 Summary of in vivo antitumor efficacy studies in the JIMT-1 breast-cancer xenograft in SCID mice Treatment group
Time for tumor to reach 500 mm3 (days ± SD)
Tumor growth inhibition at 500 mm3 (%)⁎
Percentage weight change at nadir†
Control Free quercetin Free vincristine Free vincristine/quercetin combination Co-encapsulated liposomal vincristine/quercetin
29.0 ± 3.4 30.0 ± 1.2 36.0 ± 2.2 50.0 ± 9.5‡ N60 days‡
NA 87 68 49 36
5.43 ± 0.28 5.68 ± 0.55 −3.43 ± 0.91 −4.72 ± 0.32§ 2.75 ± 0.37§
⁎ Tumor growth inhibition = median tumor volume in the treated group / median tumor volume in the control group ×100%. † Percentage weight change at nadir = (initial weight before treatment – weight at nadir) / initial weight before treatment ×100%. ‡ P b .05, statistically significant difference as compared to the control group. § P b .05, statistically significant difference between free and liposomal drug combination.
combination. Liposomal co-encapsulation of vincristine and quercetin maintained the optimal synergistic drug ratio in plasma to be delivered to the tumor site and prolonged the circulation time of the two drugs in comparison with free drugs. In contrast, previous work on poly (D,L-lactideco-glycolide) nanoparticle (NP) co-encapsulating vincristine and quercetin failed to demonstrate coordinated release of the two agents, with ∼50% of vincristine and quercetin released by 5 hours post administration27 and did not test the in vivo efficacy of the formulation. Rapid and uncoordinated release of vincristine and quercetin from the NPs could prevent the drug combination from exhibiting its full antitumor potential. This is illustrated in Figure 1, where a slight shift of vincristine/quercetin molar ratio from 2:1 to 4:1 led to a 3 log-fold difference in CI values, indicating a reduction in the extent of synergism between vincristine and quercetin. In addition, the rapid release of vincristine and quercetin from the carrier could prevent the accumulation of the drugs in the tumor through the enhanced permeability and retention effect, as the drugs released from the carrier could leak out from the tumor interstitium back to the bloodstream due to their small molecular sizes.10 From a clinical perspective, this is the first attempt at exploring the use of vincristine and quercetin for HER-2 overexpressing breast cancer. The co-encapsulated liposomal formulation showed good antitumor efficacy and low toxicity in the JIMT-1 breast cancer xenograft, which is a hormone receptor-negative and trastuzumab-insensitive breast-cancer subtype that currently lacks effective treatment options. In addition, we have shown that the optimal ratio of vincristine/ quercetin was 2:1 in both MDA-MB-23115 and JIMT-1 breastcancer cells. The CI value in MDA-MB-231 cells was 0.113,15 and that of JIMT-1 cells was 0.0900. The lower CI value observed in JIMT-1 cells indicates that the vincristine/quercetin combination was more synergistic in these cells in comparison with MDA-MB-231 cells. Therefore, the in vivo antitumor efficacy was assessed in the JIMT-1 human breast-cancer xenograft in this study. Nevertheless, the low CI values in both cell lines indicate that the vincristine/quercetin combination is effective against two distinct subtypes of breast cancers. This suggests the clinical potential of the co-encapsulated liposome formulation, which is not specific to a particular subtype of breast cancer. Among the different treatment groups, the best antitumor activity was observed in the co-encapsulated liposome formu-
lation, as reflected by the time taken for tumors to reach 500 mm3 and the tumor growth inhibition values. The drug-evaluation branch of the division of cancer treatment of the National Cancer Institute considers treatments with tumor growth inhibition values of ≤ 42% as having significant antitumor activity.28 With this criterion, only the co-encapsulated liposome formulation exhibits significant antitumor activity. Considering the pharmacokinetic data from this study, the increased antitumor efficacy from the co-encapsulated liposome formulation could be explained by the longer t1/2 of quercetin and vincristine that increased drug exposure of the tumor 29,30 and by the maintenance of the optimal synergistic ratio of vincristine/ quercetin of 2:1 in the circulation. This is further supported by the antagonistic combination indices obtained from the administration of free vincristine/quercetin combination, whereby antagonistic drug ratios in the bloodstream were likely to reach the tumor site, resulting in a compromise of the therapeutic efficacy of the drug combination. Significantly, the higher in vivo antitumor activity of the co-encapsulated liposome formulation was attained at two-thirds of the maximum tolerated dose of vincristine in SCID mice that highlights the potential of this formulation to achieve antitumor efficacy while reducing concentration-dependent side effects. The log-rank significance test showed no statistical difference in animal survival between the free vincristine/quercetin combination and the liposome coencapsulated formulation. However, given that the tumor size of the free drug combination group was double that of the coencapsulated formulation, a statistical difference in survival might be attained if the study period were prolonged. Finally, the percentage weight loss at nadir was used to evaluate the toxicity of the treatments.31 The percentage weight change between the free drug combination was statistically different (P b 0.05) in comparison with that of the liposome co-encapsulated formulation. Although higher plasma levels of vincristine and quercetin were obtained from the liposomal formulation in comparison with the free drug combination, this did not lead to an increase in toxicity. A possible explanation for this could be due to the sequestration of the two drugs in the liposomes,32 whereby the two drugs would not be able to exert their biological effects and thus minimized weight loss at nadir.8 This study demonstrated that the co-encapsulation of vincristine and quercetin in liposomes at synergistic ratio showed good antitumor efficacy and low toxicity in ER-, PR-
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and trastuzumab-insensitive xenograft models, with a dose of vincristine given at two-thirds of its maximum tolerated dose. Therefore, the co-encapsulated liposome formulation has the potential to be developed further into a possible clinical treatment option for HER-2 overexpressing breast cancer. This enhancement in antitumor efficacy could be due to the delivery of the drugs from the blood to the tumor in synergistic ratios and/ or the prolonged circulation lifetime of the agents. The promising results warrant future studies which involve the determination of the tumor levels of vincristine and quercetin as well as the comparison of the efficacy of individually encapsulated quercetin, and individually encapsulated vincristine with that of the co-encapsulated liposome formulation to determine which of the two factors play a more important role in the enhancement of antitumor efficacy.
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References 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global Cancer Statistics, 2002. CA Cancer J Clin 2005;55:74-108. 2. Hennessy BT, Pusztai L. Adjuvant therapy for breast cancer. Minerva Ginecol 2005;57:305-26. 3. Dizdar O, Altundag K. Current and emerging treatment options in triplenegative breast cancer. Oncol Rev 2010;4:5-13. 4. Chen JQ, Russo J. ER-negative and triple negative breast cancer: molecular features and potential therapeutic approaches. Biochim Biophys Acta - Rev Cancer 2009;1796:162-75. 5. Heinemann V. Definition of an optimal first-line chemotherapy in metastatic breast cancer. Breast Cancer Res Treat 2003;81:43-8. 6. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 1965;13:238-52. 7. Zamboni WC. Liposomal, Nanoparticle, and conjugated formulations of anticancer agents. Clin Cancer Res 2005;11:8230-4. 8. Lee RJ. Liposomal delivery as a mechanism to enhance synergism between anticancer drugs. Mol Cancer Ther 2006;5:1639-40. 9. Allen T, Stuart D. Liposomal pharmacokinetics. Classical, stericallystabilized, cationic liposomes and immunoliposomes. In: Janoff AS, editor. Liposomes: Rational design. New York: Marcel Dekker; 1999. p. 63-7. 10. Maeda H. Enhanced permeability and retention (EPR) effect: basis for drug targeting to tumor. In: Muzykantov V, Torchilin V, editors. Biomedical aspects of drug targeting. 1st ed. Norwell, MA: Kluwer Academic Publishers; 2002. p. 211-28. 11. Tardi P, Johnstone S, Harasym N, Xie S, Harasym T, Zisman N, et al. In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances therapeutic efficacy. Leuk Res 2009;33:129-39. 12. Tardi PG, Gallagher RC, Johnstone S, Harasym N, Webb M, Bally MB, et al. Coencapsulation of irinotecan and floxuridine into low cholesterolcontaining liposomes that coordinate drug release in vivo. Biochim Biophys Acta (BBA) - Biomembranes 2007;1768:678-87. 13. Mayer LD, Harasym TO, Tardi PG, Harasym NL, Shew CR, Johnstone SA, et al. Ratiometric dosing of anticancer drug combinations: controlling drug ratios after systemic administration regulates therapeutic activity in tumor-bearing mice. Mol Cancer Ther 2006;5:1854-63. 14. Leslie EM, Mao Q, Oleschuk CJ, Deeley RG, Cole SPC. Modulation of multidrug resistance protein 1 (MRP1/ABCC1) transport and ATPase
21.
22.
23.
24.
25.
26.
27.
28.
29.
30. 31.
32.
activities by interaction with dietary flavonoids. Mol Pharmacol 2001;59:1171-80. Wong MY, Chiu GNC. Simultaneous liposomal delivery of quercetin and vincristine for enhanced estrogen-receptor-negative breast cancer treatment. Anti-Cancer Drugs 2010;21:401-10. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63. Chou TC, Talaly P. A simple generalized equation for the analysis of multiple inhibitions of Michaelis-Menten kinetic systems. J Biol Chem 1977;252:6438-42. Mayer LD, Tai LC, Bally MB, Mitilenes GN, Ginsberg RS, Cullis PR. Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients. Biochim Biophys Acta 1990;1025:143-51. Fenske DB, Wong KF, Maurer E, Maurer N, Leenhouts JM, Boman N, et al. Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients. Biochim Biophys Acta (BBA) - Biomembranes 1998;1414:188-204. Waterhouse DN, Madden TD, Cullis PR, Bally MB, Mayer LD, Webb MS. Preparation, characterization, and biological analysis of liposomal formulations of vincristine. In: Düzgünes N, editor. Liposomes, Part E. 1st ed. San Diego, CA: Elsevier Academic Press; 2005. p. 40-57. Goniotaki M, Hatziantoniou S, Dimas K, Wagner M, Demetzos C. Encapsulation of naturally occurring flavonoids into liposomes: physicochemical properties and biological activity against human cancer cell lines. J Pharm Pharmacol 2004;56:1217-24. Yang CY, Hsiu SL, Wen KC, Lin SP, Tsai SY, Hou YC, et al. Bioavailability and metabolic pharmacokinetics of rutin and quercetin in rats. J Food Drug Anal 2005;13:244-50. Park K, de Oca G, Bonello P, Lee KJ, Dabrowski K. Determination of quercetin concentrations in fish tissues after feeding quercetin-containing diets. Aquacult Int 2009;17:537-44. Harasym TO, Liboiron BD, Mayer LD. Drug ratio-dependent antagonism: a new category of multidrug resistance and strategies for its circumvention. Methods Mol Biol 2010;596:291-323. Harasym TO, Tardi PG, Johnstone SA, Mayer LD. Fixed drug ratio liposome formulations of combination cancer therapeutics. In: Gregoriadis G, editor. Liposome Technology. New York: Informa Healthcare; 2007. p. 25-48. Laster Jr WR, Schabel Jr FM, Skipper HE, Wilcox WS, Thomson JR. Experimental evaluation of potential anticancer agents. IV. Host weight loss as it relates to false positives in drug evaluation. Cancer Res 1961;21:895-906. Song X, Zhao Y, Hou S, Xu F, Zhao R, He J, et al. Dual agents loaded PLGA nanoparticles: systematic study of particle size and drug entrapment efficiency. Eur J Pharm Biopharm 2008;69:445-53. Bissery MC, Guenard D, Gueritte-Voegelein F, Lavelle F. Experimental antitumor activity of taxotere (RP 56976, NSC 628503), a taxol analogue. Cancer Res 1991;51:4845-52. Horton JK, Houghton PJ, Houghton JA. Relationships between tumor responsiveness, vincristine pharmacokinetics and arrest of mitosis in human tumor xenografts. Biochem Pharmacol 1988;37:3995-4000. Jackson Jr DV, Bender RA. Cytotoxic thresholds of vincristine in a murine and a human leukemia cell line in vitro. Cancer Res 1979;39:4346-9. Corbett TH, Polin L, Roberts BJ. Transplantable syngeneic rodent tumors: Solid tumors in mice. In: Teicher BA, editor. Tumor Models in Cancer Research. 1st ed. Totowa, NJ: Humanapress.com; 2002. p. 41-73. Krishna R, Webb M, St.Onge G, Mayer LD. Liposomal and nonliposomal drug pharmacokinetics after administration of liposomeencapsulated vincristine and their contribution to drug tissue distribution properties. J Pharmacol Exp Ther 2001;298:1206-12.
Research Article
nanomedjournal.com
Liposome formulation of co-encapsulated vincristine and quercetin enhanced antitumor activity in a trastuzumab-insensitive breast tumor xenograft model Man-Yi Wong, BSc (Hons), Gigi N.C. Chiu, PhD⁎ Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore Received 13 August 2010; accepted 3 February 2011
Abstract Hormone- and trastuzumab-insensitive breast cancer has limited and ineffective clinical treatment options. This study sought to develop a liposome formulation containing a synergistic combination of vincristine and quercetin, with prolonged drug circulation times and coordinated drug release in vivo, to develop effective treatments against this subtype of breast cancer. The 2:1 molar ratio of vincristine/ quercetin showed strong synergism in the hormone- and trastuzumab-insensitive JIMT-1 cells. Liposome co-encapsulation prolonged plasma circulation of the two drugs and maintained the synergistic drug ratio in vivo. Furthermore, the co-encapsulated liposome formulation demonstrated the most effective tumor growth inhibition in the JIMT-1 human breast tumor xenograft in comparison with vehicle control, free quercetin, free vincristine and free vincristine/quercetin combinations. Specifically, only the co-encapsulated liposome formulation exhibited significant antitumor activity at two-thirds of the maximum tolerated dose of vincristine, without significant body weight loss in the animals. From the Clinical Editor: In this study, a novel liposome formulation containing a synergistic combination of vincristine and quercetin was utilized in the treatment of breast cancer. Prolonged drug circulation times and coordinated drug release characterize this effective treatment, which may find its way to clinical applications in the near future. © 2011 Elsevier Inc. All rights reserved. Key words: Liposomes; Quercetin; Vincristine; Trastuzumab; Breast cancer
Globally, breast cancer is the most prevalent cancer,1 and is the leading cause of cancer-related deaths in women.2 Breast cancer can be characterized by the presence or the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth-factor receptor-2 (HER-2). Antihormonal treatments and HER-2 targeted therapies such as trastuzumab have increased the therapeutic options and have improved the survival rates of patients who are responsive to these treatments.2 However, patients who are unresponsive to antihormonal and trastuzumab treatments are limited to the use of high doses of single drug regimens3 or combination regimens of various chemotherapeutic agents.4 These approaches are either too toxic and have resulted in more treatment-related deaths3 or lacking therapeutic efficacy with
This work was supported by the Singapore Ministry of Education via the National University of Singapore Academic Research Fund (grant # R-148050-077-101 & R-148-050-077-133). Man-Yi Wong is a recipient of a research scholarship from Singapore Ministry of Education. There are no conflicts of interest in connection with this article. ⁎Corresponding author. E-mail address: [email protected] (G.N.C. Chiu).
no increase in disease-free survival.5 This highlights the need to develop safe and effective treatment options against ER-, PR- and trastuzumab-insensitive breast cancers. The problems of toxicity and lack of efficacy could be resolved by the use of liposomes, which are small spherical vesicles formed by a lipid bilayer enclosing an aqueous compartment.6 The toxicity of the liposome-encapsulated chemotherapeutic drug could be reduced, as the drug could not exert its activity when sequestered in liposomes during bloodstream circulation.7,8 In addition, liposomes have been shown to enhance therapeutic efficacy of anticancer drugs by increasing drug exposure in the tumor resulting from the prolonged circulation times of the drugs,9 and by preferential accumulation of the anticancer drugs as a result of the enhanced permeability and retention effect in the tumor.10 Efficacy of chemotherapeutic agents can be increased through the administration of synergistic drug combinations in fixed molar drug ratios.11-13 Owing to the differences in pharmacokinetic profiles of the drugs in a combination regimen, the synergistic drug ratios may not be attained in the tumor site. The use of liposomes has been shown to maintain the desirable, synergistic drug ratios as
1549-9634/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.02.001 Please cite this article as: M.-Y. Wong, G.N.C. Chiu, Liposome formulation of co-encapsulated vincristine and quercetin enhanced antitumor activity in a trastuzumab-insensitive breast .... Nanomedicine: NBM 2011;7:834-840, doi:10.1016/j.nano.2011.02.001
M.-Y. Wong, G.N.C. Chiu / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 834–840
well as to coordinate the release of the encapsulated drug combinations to achieve increased antitumor activity.11-13 In view of these advantages, our approach is to deliver a combination regimen with synergistic drug ratios through liposomes to develop potentially effective treatment options for breast-cancer subtypes that are unresponsive to hormonal and trastuzumab treatments. The drug combination to be co-encapsulated in liposomes is vincristine and quercetin. The cytotoxicity of vincristine can be enhanced by quercetin, which reduced vincristine efflux from the cancer cells.14 This is further supported by our previous findings that the vincristine/quercetin combination, given at molar ratios of 4:1, 2:1 and 1:1, showed moderate to strong synergism, with the molar ratio of 2:1 being the most synergistic in the hormone receptor-negative and trastuzumab-insensitive cell line MDA-MB-231.15 Based on these results, a liposome formulation has been developed that consists of vincristine encapsulated in egg sphingomyelin (ESM)/Cholesterol/Npalmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000] (PEG2000-ceramide)/quercetin (72.5:17.5:5:5 mole ratio) with the vincristine/quercetin molar ratio of 2:1. This liposome formulation coordinated the in vitro release of vincristine and quercetin in the synergistic ratio of 2:1 over 72 hours and displayed a combination index (CI) of 0.113 in MDA-MB-231 cells.15 In this study, we sought to 1) determine if the synergism could be demonstrated in another hormone- and trastuzumabinsensitive cell line, JIMT-1; 2) assess whether liposome encapsulation would prolong the in vivo circulation times of vincristine and quercetin; and 3) assess whether a coordinated release of the drug combination could be achieved in vivo and thereby improve the antitumor efficacy in the human JIMT-1 breast-cancer xenograft model. Methods Materials All lipids were obtained from Avanti Polar Lipids (Alabaster, Alabama). JIMT-1 cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). The materials 3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO) and chloroform were purchased from MP Biomedicals Asia Pacific Pte Ltd (Singapore). Balb/c mice were supplied via the Centre for Animal Resources, National University of Singapore (Singapore), and SCID mice were supplied via the Biological Resource Centre, Biopolis, Singapore. All other materials were purchased from SigmaAldrich (St. Louis, Missouri). In vitro cytoxicity assay In vitro cytotoxicity was assessed by MTT assay.16 JIMT-1 human breast-cancer cells were grown in DMEM/F-12 media supplemented with 10% fetal bovine serum and seeded at 4000 cells/well in 96-well plates. Cells were incubated at 37°C with 5% CO2 for 24 hours to allow adherence to the cell culture plates before treatment with serial dilutions of either single drugs (quercetin, vincristine) or drug combinations (vincristine/
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quercetin at molar ratios of 4:1, 2:1, 1:1 and 1:2) for 72 hours. Subsequently, 50 μL of MTT reagent (1 mg/mL) was added to each well, and was incubated with the cells for 4 hours. At the end of the incubation period, the cell culture medium was aspirated, and 150 μL of DMSO was added to each well. The 96well plates were shaken for 20 minutes to solubilize the cells and subsequently were read on the Tecan Sunrise microplate reader (Männedorf, Switzerland) with an absorbance wavelength of 570 nm. Cell survival at the end of treatment was calculated from the absorbance readings as a percentage of the control. All assays were performed in triplicate. Median-effect analysis for drug combinations Drug combination analyses were performed using CalcuSyn (Biosoft, Cambridge, United Kingdom), which is a software program developed using the median-effect principle described by Chou and Talalay.17 For studies on the combined effects of vincristine and quercetin, fixed ratios of vincristine and quercetin (4:1, 2:1, 1:1 and 1:2) were used. The CalcuSyn software generates the CI value for a particular combination of vincristine/ quercetin based on the cell-survival data from the MTT assay, whereby additivity, synergy and antagonism were reflected by CI values of 0.9–1.1, b 0.9 and N 1.1, respectively. Liposome preparation Liposomes were prepared by the thin-film hydration method.18 Briefly, the lipids dissolved in chloroform were mixed with quercetin dissolved in ethanol. The preparation was subsequently dried under a stream of nitrogen gas, and the resulting lipid film was placed under vacuum to remove organic solvents. The dried lipid films were hydrated with 300 mM manganese sulfate (pH 3.4) for 1 hour at 60°C. The resulting preparation was extruded 15 times at 60°C through stacked 0.1-μm pore size polycarbonate filters (Northern Lipids Inc., Vancouver, British Columbia, Canada) with an extruder apparatus (Northern Lipids Inc.). The mean diameter of the extruded liposomes was determined using the Zetasizer 3000HS operating at 633 nm, and was found to have diameters of ∼130 nm. Vincristine (drug-to-lipid mole ratio of 0.1:1) was subsequently loaded into the liposomes using an ionophore-mediated proton gradient.19 The encapsulated drug was separated from the free drug using 1-mL Sephadex G-50 spin columns. Vincristine and quercetin were quantified spectroscopically based on their absorbance at 297 nm20 and 376 nm,21 respectively, after solubilization of the liposomes in n-octyl-D-glucopyranoside and ethanol, respectively. Animal studies The studies were approved by the National University of Singapore Institutional Animal Care and Use Committee (NUS IACUC), and the procedures followed were in accordance with NUS IACUC guidelines. The animals were under humane care throughout the studies and were housed in micro-isolator cages with free access to food and water. All mice used were between 20–22 g and were quarantined for 7 days before the study was initiated.
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Pharmacokinetic study Drugs in free form or in liposome formulation were injected intravenously into the lateral tail vein of female Balb/c mice (4 mice per time point). Vincristine was given at 1.33 mg/kg and quercetin was given at 0.24 mg/kg. These doses corresponded to a 2:1 molar drug ratio of vincristine and quercetin. At 10 minutes, 0.5 hour, 1 hour, 2 hours, 4 hours and 24 hours after drug administration, the animals were euthanized by CO2 asphyxiation. Blood samples were collected by cardiac puncture and centrifuged for 10 minutes at 1000 × g to isolate plasma, which was stored at −20°C until analysis of drug concentrations by UPLC. In vivo efficacy study Tumors were established in SCID mice by a single subcutaneous injection of 5 × 106 JIMT-1 cells in the upper back area. Tumor progression was monitored by caliper measurements of the tumors along the length and width thrice a week. Tumor volumes were calculated by the following formula: Tumor volume = (length × width2)/2. When the tumor size reached 200–300 mm3, randomization of animals was performed to give 5 groups of 5 animals each. The study groups included 1) vehicle control, 2) free quercetin, 3) free vincristine, 4) free vincristine/quercetin combination, and 5) co-encapsulated liposomal vincristine/quercetin groups. The dose of vincristine was 1.33 mg/kg (two-thirds of the maximum tolerated dose in SCID mice20) and that of quercetin was 0.24 mg/kg. At these values, the molar ratio of vincristine/quercetin was 2:1. Tumor size and body weight of the mice was monitored thrice weekly until 60 days post tumor inoculation. Animal euthanization was performed when tumor size reached 1000 mm3, when tumor ulcerations developed, or with body weight loss of N 5%. UPLC method development and drug extraction A Waters UPLC AcQuity UPLC system (Milford, Massachusettes) was used to quantify vincristine and quercetin in plasma samples, using a Waters AcQuity UPLC, BEH C18, 2.1×50 mm, 1.7-μM column protected by a guard column. The assay was performed at 25°C with 0.1% formic acid as weak solvent and acetonitrile/0.1% formic acid as strong solvent. The initial phase comprised 75% of 0.1% formic acid and 25% of acetonitrile/0.1% formic acid. The final phase comprised 5% of 0.1% formic acid and 95% of acetonitrile/0.1% formic acid at 3.8 minutes, with a flow rate of 0.5 mL/minute. The detection wavelengths of quercetin and vincristine were 376 nm and 297 nm, respectively. Extraction of quercetin and vincristine from plasma was modified from previously reported methods.22-23 Apigenin was used as the internal standard. Briefly, 100 μL of plasma was mixed with 50 μL of β-glucuronidase, sulfatase and 25 μL of ascorbic acid. The mixture was incubated at 37°C for 1 hour anaerobically. The serum was acidified with 10 μL of 0.1 N HCl, and extracted 4 times with 100 μL of ethyl acetate. The ethyl acetate layer was collected and evaporated to dryness under nitrogen gas and reconstituted with acetonitrile/0.1% formic acid for UPLC analysis. The supernatant was injected into the UPLC
Figure 1. CI values at ED50 for vincristine/quercetin exposed to JIMT-1 breast cancer cells at molar ratios of vincristine/quercetin of 4:1, 2:1, 1:1, and 1:2. Each value represents the mean ± SEM from 3 independent experiments. Additivity, synergy and antagonism are reflected by CI values of 0.9–1.1, b 0.9 and N 1.1, respectively.
system at 1 μL. The concentrations of quercetin and vincristine in the samples were determined by comparing the peak area ratios of the samples versus a calibration curve obtained from spiking known amounts of quercetin and vincristine in pooled mouse plasma. The linearity of the standard curve was more than 0.998, and the limits of detection for vincristine and quercetin were 0.008 nmoles/mL and 0.05 nmoles/mL, respectively. Pharmacokinetic analyses were performed using non-compartmental analysis with WinNonlin software standard version 1.0 (Sunnyvale, California). Statistics All statistical analyses were performed using the NCSS 2004 software (Kaysville, Utah). All experimental data were expressed as mean ± SEM. One way ANOVA and the post hoc Tukey test were used. Kaplan-Meier survival analysis with log-rank significance test was conducted to determine whether survival differed among the treatment groups. P values of b 0.05 were considered to be statistically significant. Results In vitro combination effects of free vincristine and quercetin Previously, we showed that the most synergistic molar ratio of vincristine and quercetin was 2:1 in MDA-MB-231 cells.15 Given that it is important to demonstrate consistent display of synergy across different cell lines for a specific ratio of chemotherapeutic agents,24 it was our aim to assess if a similar trend could be observed in JIMT-1 cells. The in vitro combination effects of vincristine and quercetin were compared among the molar drug ratios of 4:1, 2:1, 1:1 and 1:2. Figure 1 shows the CI values at different molar ratios of vincristine/ quercetin at the median effective dose (ED50). The CI values were 0.699, 0.0000223, 0.00102 and 0.354 for the molar drug ratios of 4:1, 2:1, 1:1 and 1:2, respectively. Therefore, the ratio of 2:1 for vincristine/quercetin was the most synergistic. Similar trends were observed at other ED values, but ED50 values were
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Figure 2. Concentrations of vincristine and quercetin needed to achieve 50% cell kill when incorporated into liposomes. Data were analyzed using the CalcuSyn software based on the median effect principle developed by Chou and Talalay. Each value represents the mean ± SEM from 3 independent experiments. Quercetin, solid bars; vincristine, open bars.
reported because the aim of cancer treatment is to eradicate cancer cells and the doses needed to attain high cell kills are more clinically relevant in comparison with doses needed to attain lower cell kills.25 Liposomal co-encapsulation of vincristine and quercetin Vincristine (drug-to-lipid molar ratio of 0.1) was loaded into ESM/cholesterol/PEG2000-ceramide/quercetin (72.5:17.5:5:5 molar ratio) liposomes. The encapsulation efficiencies for vincristine and quercetin were 78.3% and 78.5%, respectively. Optimization of this formulation have been described in our previous work.15 The final vincristine/quercetin ratio in the liposomes was 2:1. In vitro combination effects of liposomal vincristine and quercetin In vitro cytotoxicity studies were first conducted with empty liposomes on the JIMT-1 cells to determine whether the liposomes would contribute to the cytotoxicity. The concentration of lipid tested was matched to the amount of lipid used in the drug-loaded formulation. At the concentrations of lipids used, cell viability ranged from 94.0% to 105.9% of untreated control, indicating that the liposomes alone did not contribute to significant cell kill. When vincristine and quercetin were co-encapsulated in liposomes at a molar ratio of 2:1, the concentrations of vincristine and quercetin required to attain 50% cell kill were reduced approximately by 1.5fold for vincristine and 10-fold for quercetin, in comparison with the liposomal monotherapy (Figure 2). The CI value of the coencapsulated liposomal formulation was 0.0900 (very synergistic) at ED50. Similar trends for CI were observed at other ED values.
Figure 3. (A) Concentrations of vincristine and quercetin in plasma after intravenous administration of the free vincristine/quercetin combination or the liposomal co-encapsulated vincristine/quercetin combination into Balb/c mice. Free quercetin, ♦; free vincristine, ▲; liposomal quercetin, ⋄; liposomal vincristine, △. (B) Comparison of the ratio of vincristine/quercetin over time for free vincristine/quercetin combination (△) and liposomal co-encapsulated vincristine/quercetin (♦) in plasma. *P b 0.05, one way ANOVA and post-hoc Tukey test. The x-axis is in log scale to facilitate visualization of the data points. Each value represents the mean ± SEM from 4 samples.
Balb/c mice. Figure 3, A shows that vincristine and quercetin levels in plasma were undetectable after 60 minutes and 120 minutes, respectively, for the free drug combination group. In contrast, the plasma levels of vincristine and quercetin in the co-encapsulated liposome formulation remained detectable throughout the entire study. In addition, the co-encapsulated liposome formulation displayed higher Cmax, AUClast, t1/2 and MRTlast with lower Cl, in comparison with the free drug combination (Table 1). Finally, Figure 3, B shows that the initial vincristine/quercetin ratio of 2:1was maintained over the entire study period for the co-encapsulated liposome formulation but not for the free drug combination group (P b 0.05).
Plasma elimination profiles of free and liposomal combination of vincristine and quercetin
In vivo antitumor effects against the JIMT-1 xenograft
The plasma elimination profiles of vincristine and quercetin either as free or liposomal drug combinations were determined in
The antitumor efficacy against the JIMT-1 xenograft was evaluated by comparing 1) vehicle control, 2) free quercetin, 3)
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Table 1 Summary of pharmacokinetic parameters for vincristine and quercetin in mice⁎ Free †
Cmax (nmol/mL) AUClast (μmol.h/mL) t1/2 (h) Cl (mL/h) MRTlast (h) V (mL/g) r
Liposomal
Quercetin
Vincristine
Quercetin
Vincristine
2.07 6.65 1.49 0.47 1.20 1.00 −0.82
0.19 0.33 0.48 16.90 0.40 12.19 −0.92
19.20 604.16 14.20 0.01 9.22 0.08 −0.84
34.10 1240.54 16.00 0.01 8.26 0.09 −0.96
⁎ Mice (n = 4) received either 1.33 mg/kg vincristine together with 0.24 mg/ kg quercetin (2:1 vincristine: quercetin molar ratio) as free drug combination or 1.33 mg/kg vincristine and 0.24 mg/kg quercetin co-encapsulated in liposomes. Injection volume was 200 μL. † Pharmacokinetic parameters were calculated based on pooled data from 4 mice. Cmax, maximum plasma concentration of drug; AUClast, area under the curve computed to the last time point; t1/2, half life; Cl, total body clearance; MRTlast, mean residence time up to the last time point; V, volume of distribution; r, Pearson correlation coefficient.
free vincristine, 4) free vincristine/quercetin combination, and 5) co-encapsulated liposome vincristine/quercetin formulation. Although all treatment groups (groups 2–5) showed initial tumor regression, only the liposome co-encapsulated formulation showed tumor regression over the 60-day study period (Figure 4, A). Table 2 shows the time taken for tumors to reach 500 mm3 with various treatments. Statistically significant difference was observed between the co-encapsulated liposome formulation and the other study groups (P b 0.05). In addition, free quercetin monotherapy and free vincristine monotherapy were not statistically different from the vehicle control in terms of the time for tumors to reach 500 mm3 (P N 0.05). Table 2 also lists the percentages of tumor-growth inhibition, which shows a trend similar to that of the time for tumors to reach 500 mm3. Percentage weight change at nadir was used as an indicator of the toxicity of the treatment,26 and statistical significance (P b 0.05) was observed between the free vincristine/ quercetin combination as compared to the co-encapsulated liposome formulation (Table 2). Finally, Kaplan-Meier survival analysis with log-rank significance test was conducted, and the co-encapsulated liposome formulation was statistically different from the vehicle control, free quercetin monotherapy and free vincristine monotherapy (P b 0.05, Figure 4, B). No significant difference was observed between the coencapsulated liposome formulation and the free drug combination (P N 0.05).
respectively, indicating antagonism when the two drugs were administered freely without a carrier.
In vitro evaluation of CI values in the ratios of free vincristine and quercetin
Discussion
The ratios of vincristine and quercetin in plasma following the administration of the free drug combination were 1:11, 1:12 and 1:6. The ratios of vincristine/quercetin 1:12 and 1:6 were tested in vitro, and the drug combination effects were analyzed to determine the CI values at ED50. The CI values were 2.02 and 1.33 for the vincristine/quercetin ratios of 1:12 and 1:6,
We will discuss our work from the formulation and clinical perspectives. From the formulation perspective, we have demonstrated that the administration of free drug combination in the absence of a drug carrier led to antagonistic CI values in vivo. This highlights the need for a delivery vehicle to attain optimal antitumor efficacy for the vincristine/quercetin
Figure 4. (A) In vivo antitumor effects of various treatment groups against JIMT-1 tumor xenograft in SCID mice (n = 5). The mice were treated via tail vein injections with vehicle control (blue), free quercetin (pink), free vincristine (orange), free vincristine/quercetin combination (green) and coencapsulated liposomal vincristine/quercetin (purple) groups. The doses of vincristine and quercetin given were 1.33 mg/kg and 0.24 mg/kg, respectively (2:1 vincristine/quercetin mole ratio). A total of 3 doses were administered on days 17, 20 and 23. (B) Kaplan-Meier survival curves of the different treatment groups (n = 5). Vehicle control, blue; free quercetin, pink; free vincristine, orange; free vincristine/quercetin combination, green; coencapsulated liposomal vincristine/quercetin, purple. Log-rank significance test was conducted.
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Table 2 Summary of in vivo antitumor efficacy studies in the JIMT-1 breast-cancer xenograft in SCID mice Treatment group
Time for tumor to reach 500 mm3 (days ± SD)
Tumor growth inhibition at 500 mm3 (%)⁎
Percentage weight change at nadir†
Control Free quercetin Free vincristine Free vincristine/quercetin combination Co-encapsulated liposomal vincristine/quercetin
29.0 ± 3.4 30.0 ± 1.2 36.0 ± 2.2 50.0 ± 9.5‡ N60 days‡
NA 87 68 49 36
5.43 ± 0.28 5.68 ± 0.55 −3.43 ± 0.91 −4.72 ± 0.32§ 2.75 ± 0.37§
⁎ Tumor growth inhibition = median tumor volume in the treated group / median tumor volume in the control group ×100%. † Percentage weight change at nadir = (initial weight before treatment – weight at nadir) / initial weight before treatment ×100%. ‡ P b .05, statistically significant difference as compared to the control group. § P b .05, statistically significant difference between free and liposomal drug combination.
combination. Liposomal co-encapsulation of vincristine and quercetin maintained the optimal synergistic drug ratio in plasma to be delivered to the tumor site and prolonged the circulation time of the two drugs in comparison with free drugs. In contrast, previous work on poly (D,L-lactideco-glycolide) nanoparticle (NP) co-encapsulating vincristine and quercetin failed to demonstrate coordinated release of the two agents, with ∼50% of vincristine and quercetin released by 5 hours post administration27 and did not test the in vivo efficacy of the formulation. Rapid and uncoordinated release of vincristine and quercetin from the NPs could prevent the drug combination from exhibiting its full antitumor potential. This is illustrated in Figure 1, where a slight shift of vincristine/quercetin molar ratio from 2:1 to 4:1 led to a 3 log-fold difference in CI values, indicating a reduction in the extent of synergism between vincristine and quercetin. In addition, the rapid release of vincristine and quercetin from the carrier could prevent the accumulation of the drugs in the tumor through the enhanced permeability and retention effect, as the drugs released from the carrier could leak out from the tumor interstitium back to the bloodstream due to their small molecular sizes.10 From a clinical perspective, this is the first attempt at exploring the use of vincristine and quercetin for HER-2 overexpressing breast cancer. The co-encapsulated liposomal formulation showed good antitumor efficacy and low toxicity in the JIMT-1 breast cancer xenograft, which is a hormone receptor-negative and trastuzumab-insensitive breast-cancer subtype that currently lacks effective treatment options. In addition, we have shown that the optimal ratio of vincristine/ quercetin was 2:1 in both MDA-MB-23115 and JIMT-1 breastcancer cells. The CI value in MDA-MB-231 cells was 0.113,15 and that of JIMT-1 cells was 0.0900. The lower CI value observed in JIMT-1 cells indicates that the vincristine/quercetin combination was more synergistic in these cells in comparison with MDA-MB-231 cells. Therefore, the in vivo antitumor efficacy was assessed in the JIMT-1 human breast-cancer xenograft in this study. Nevertheless, the low CI values in both cell lines indicate that the vincristine/quercetin combination is effective against two distinct subtypes of breast cancers. This suggests the clinical potential of the co-encapsulated liposome formulation, which is not specific to a particular subtype of breast cancer. Among the different treatment groups, the best antitumor activity was observed in the co-encapsulated liposome formu-
lation, as reflected by the time taken for tumors to reach 500 mm3 and the tumor growth inhibition values. The drug-evaluation branch of the division of cancer treatment of the National Cancer Institute considers treatments with tumor growth inhibition values of ≤ 42% as having significant antitumor activity.28 With this criterion, only the co-encapsulated liposome formulation exhibits significant antitumor activity. Considering the pharmacokinetic data from this study, the increased antitumor efficacy from the co-encapsulated liposome formulation could be explained by the longer t1/2 of quercetin and vincristine that increased drug exposure of the tumor 29,30 and by the maintenance of the optimal synergistic ratio of vincristine/ quercetin of 2:1 in the circulation. This is further supported by the antagonistic combination indices obtained from the administration of free vincristine/quercetin combination, whereby antagonistic drug ratios in the bloodstream were likely to reach the tumor site, resulting in a compromise of the therapeutic efficacy of the drug combination. Significantly, the higher in vivo antitumor activity of the co-encapsulated liposome formulation was attained at two-thirds of the maximum tolerated dose of vincristine in SCID mice that highlights the potential of this formulation to achieve antitumor efficacy while reducing concentration-dependent side effects. The log-rank significance test showed no statistical difference in animal survival between the free vincristine/quercetin combination and the liposome coencapsulated formulation. However, given that the tumor size of the free drug combination group was double that of the coencapsulated formulation, a statistical difference in survival might be attained if the study period were prolonged. Finally, the percentage weight loss at nadir was used to evaluate the toxicity of the treatments.31 The percentage weight change between the free drug combination was statistically different (P b 0.05) in comparison with that of the liposome co-encapsulated formulation. Although higher plasma levels of vincristine and quercetin were obtained from the liposomal formulation in comparison with the free drug combination, this did not lead to an increase in toxicity. A possible explanation for this could be due to the sequestration of the two drugs in the liposomes,32 whereby the two drugs would not be able to exert their biological effects and thus minimized weight loss at nadir.8 This study demonstrated that the co-encapsulation of vincristine and quercetin in liposomes at synergistic ratio showed good antitumor efficacy and low toxicity in ER-, PR-
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and trastuzumab-insensitive xenograft models, with a dose of vincristine given at two-thirds of its maximum tolerated dose. Therefore, the co-encapsulated liposome formulation has the potential to be developed further into a possible clinical treatment option for HER-2 overexpressing breast cancer. This enhancement in antitumor efficacy could be due to the delivery of the drugs from the blood to the tumor in synergistic ratios and/ or the prolonged circulation lifetime of the agents. The promising results warrant future studies which involve the determination of the tumor levels of vincristine and quercetin as well as the comparison of the efficacy of individually encapsulated quercetin, and individually encapsulated vincristine with that of the co-encapsulated liposome formulation to determine which of the two factors play a more important role in the enhancement of antitumor efficacy.
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References 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global Cancer Statistics, 2002. CA Cancer J Clin 2005;55:74-108. 2. Hennessy BT, Pusztai L. Adjuvant therapy for breast cancer. Minerva Ginecol 2005;57:305-26. 3. Dizdar O, Altundag K. Current and emerging treatment options in triplenegative breast cancer. Oncol Rev 2010;4:5-13. 4. Chen JQ, Russo J. ER-negative and triple negative breast cancer: molecular features and potential therapeutic approaches. Biochim Biophys Acta - Rev Cancer 2009;1796:162-75. 5. Heinemann V. Definition of an optimal first-line chemotherapy in metastatic breast cancer. Breast Cancer Res Treat 2003;81:43-8. 6. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 1965;13:238-52. 7. Zamboni WC. Liposomal, Nanoparticle, and conjugated formulations of anticancer agents. Clin Cancer Res 2005;11:8230-4. 8. Lee RJ. Liposomal delivery as a mechanism to enhance synergism between anticancer drugs. Mol Cancer Ther 2006;5:1639-40. 9. Allen T, Stuart D. Liposomal pharmacokinetics. Classical, stericallystabilized, cationic liposomes and immunoliposomes. In: Janoff AS, editor. Liposomes: Rational design. New York: Marcel Dekker; 1999. p. 63-7. 10. Maeda H. Enhanced permeability and retention (EPR) effect: basis for drug targeting to tumor. In: Muzykantov V, Torchilin V, editors. Biomedical aspects of drug targeting. 1st ed. Norwell, MA: Kluwer Academic Publishers; 2002. p. 211-28. 11. Tardi P, Johnstone S, Harasym N, Xie S, Harasym T, Zisman N, et al. In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances therapeutic efficacy. Leuk Res 2009;33:129-39. 12. Tardi PG, Gallagher RC, Johnstone S, Harasym N, Webb M, Bally MB, et al. Coencapsulation of irinotecan and floxuridine into low cholesterolcontaining liposomes that coordinate drug release in vivo. Biochim Biophys Acta (BBA) - Biomembranes 2007;1768:678-87. 13. Mayer LD, Harasym TO, Tardi PG, Harasym NL, Shew CR, Johnstone SA, et al. Ratiometric dosing of anticancer drug combinations: controlling drug ratios after systemic administration regulates therapeutic activity in tumor-bearing mice. Mol Cancer Ther 2006;5:1854-63. 14. Leslie EM, Mao Q, Oleschuk CJ, Deeley RG, Cole SPC. Modulation of multidrug resistance protein 1 (MRP1/ABCC1) transport and ATPase
21.
22.
23.
24.
25.
26.
27.
28.
29.
30. 31.
32.
activities by interaction with dietary flavonoids. Mol Pharmacol 2001;59:1171-80. Wong MY, Chiu GNC. Simultaneous liposomal delivery of quercetin and vincristine for enhanced estrogen-receptor-negative breast cancer treatment. Anti-Cancer Drugs 2010;21:401-10. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63. Chou TC, Talaly P. A simple generalized equation for the analysis of multiple inhibitions of Michaelis-Menten kinetic systems. J Biol Chem 1977;252:6438-42. Mayer LD, Tai LC, Bally MB, Mitilenes GN, Ginsberg RS, Cullis PR. Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients. Biochim Biophys Acta 1990;1025:143-51. Fenske DB, Wong KF, Maurer E, Maurer N, Leenhouts JM, Boman N, et al. Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients. Biochim Biophys Acta (BBA) - Biomembranes 1998;1414:188-204. Waterhouse DN, Madden TD, Cullis PR, Bally MB, Mayer LD, Webb MS. Preparation, characterization, and biological analysis of liposomal formulations of vincristine. In: Düzgünes N, editor. Liposomes, Part E. 1st ed. San Diego, CA: Elsevier Academic Press; 2005. p. 40-57. Goniotaki M, Hatziantoniou S, Dimas K, Wagner M, Demetzos C. Encapsulation of naturally occurring flavonoids into liposomes: physicochemical properties and biological activity against human cancer cell lines. J Pharm Pharmacol 2004;56:1217-24. Yang CY, Hsiu SL, Wen KC, Lin SP, Tsai SY, Hou YC, et al. Bioavailability and metabolic pharmacokinetics of rutin and quercetin in rats. J Food Drug Anal 2005;13:244-50. Park K, de Oca G, Bonello P, Lee KJ, Dabrowski K. Determination of quercetin concentrations in fish tissues after feeding quercetin-containing diets. Aquacult Int 2009;17:537-44. Harasym TO, Liboiron BD, Mayer LD. Drug ratio-dependent antagonism: a new category of multidrug resistance and strategies for its circumvention. Methods Mol Biol 2010;596:291-323. Harasym TO, Tardi PG, Johnstone SA, Mayer LD. Fixed drug ratio liposome formulations of combination cancer therapeutics. In: Gregoriadis G, editor. Liposome Technology. New York: Informa Healthcare; 2007. p. 25-48. Laster Jr WR, Schabel Jr FM, Skipper HE, Wilcox WS, Thomson JR. Experimental evaluation of potential anticancer agents. IV. Host weight loss as it relates to false positives in drug evaluation. Cancer Res 1961;21:895-906. Song X, Zhao Y, Hou S, Xu F, Zhao R, He J, et al. Dual agents loaded PLGA nanoparticles: systematic study of particle size and drug entrapment efficiency. Eur J Pharm Biopharm 2008;69:445-53. Bissery MC, Guenard D, Gueritte-Voegelein F, Lavelle F. Experimental antitumor activity of taxotere (RP 56976, NSC 628503), a taxol analogue. Cancer Res 1991;51:4845-52. Horton JK, Houghton PJ, Houghton JA. Relationships between tumor responsiveness, vincristine pharmacokinetics and arrest of mitosis in human tumor xenografts. Biochem Pharmacol 1988;37:3995-4000. Jackson Jr DV, Bender RA. Cytotoxic thresholds of vincristine in a murine and a human leukemia cell line in vitro. Cancer Res 1979;39:4346-9. Corbett TH, Polin L, Roberts BJ. Transplantable syngeneic rodent tumors: Solid tumors in mice. In: Teicher BA, editor. Tumor Models in Cancer Research. 1st ed. Totowa, NJ: Humanapress.com; 2002. p. 41-73. Krishna R, Webb M, St.Onge G, Mayer LD. Liposomal and nonliposomal drug pharmacokinetics after administration of liposomeencapsulated vincristine and their contribution to drug tissue distribution properties. J Pharmacol Exp Ther 2001;298:1206-12.