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Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative
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IJP 14664 1–8 International Journal of Pharmaceutics xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Pharmaceutical nanotechnology
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Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative
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Giovanna Lollo a,b,1, Pablo Hervella b , Pilar Calvo c, Pablo Avilés c , Maria Jose Guillén c , Marcos Garcia-Fuentes a,b , Maria José Alonso a,b , Dolores Torres b, * a Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Campus Vida, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain b Department of Pharmaceutics and Pharmaceutical Technology, School of Pharmacy and Heath Research Institute of Santiago de Compostela (IDIS), Campus Vida, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain c PharmaMar S.A, Colmenar Viejo, Madrid, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2014 Received in revised form 8 February 2015 Accepted 10 February 2015 Available online xxx
The focus of this study is to disclose a new delivery carrier intended to improve the pharmacokinetic characteristics of the anticancer drug plitidepsin and to favor its accumulation within the tumor. These nanocarriers named as nanocapsules, consist of an oily core surrounded by a highly PEGylated polyglutamic acid (PGA-PEG) shell loaded with plitidepsin. They showed a size of around 190 nm, a zeta potential of 24 mV and were able to encapsulate a high percentage (85%) of plitidepsin. In vivo studies, following intravenous injection in healthy mice, indicated that the encapsulation of the drug within PGA-PEG nanocapsules led to an important increase in its area under the curve (AUC) which is related to the important decrease of the clearance, as compared to the values observed for the drug dissolved in a Cremophor1 EL solution. This improvement of the pharmacokinetic profile of the encapsulated plitidepsin was accompanied by a high increase (2.5-fold) of the maximum tolerated dose (MTD) in comparison to that of plitidepsin Cremophor1 EL solution. The efficacy study performed in a xenograft tumor mice model evidenced the capacity of PGA-PEG nanocapsules to significantly reduce tumor growth. These promising results highlight the potential of PGA-PEG nanocapsules as an effective drug delivery system for cancer therapy. ã 2015 Published by Elsevier B.V.
Keywords: Nanomedicine Antitumor drugs Stealth properties Polyglutamic acid Plitidepsin Cancer therapy
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1. Introduction
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Over the last decades, nanopharmaceuticals have a particular impact in improving cancer therapeutics (Blanco et al., 2011; Wang and Thanou, 2010). Most anticancer drugs used in conventional chemotherapy are rapidly cleared from the blood circulation and, because they do not differentiate between cancerous and normal tissues, their use generally leads to major systemic side effects (Shi et al., 2011). Nanocarriers with long-circulating times offer the potential advantage of being accumulated and entrapped within tumors due to the high permeability of tumoral vasculature and frequently poor lymphatic drainage (Fang et al., 2011; Bertrand et al., 2014). A large number of nanocarriers containing cytotoxic drugs have been examined in clinical trials and been approved for
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* Corresponding author. Tel.: +34 8814880; fax: +34 8814880. E-mail address: [email protected] (D. Torres). 1 Present address: LUNAM Université – INSERM U1066, IBS-CHU, F-49933 Angers, France.
use in humans. In particular, the FDA approval of Doxil1, PEGylated liposomes containing doxorubicin, has opened the door for the clinical development of other long circulating nanocarriers including micelles, nanoparticles or conjugates (Shi et al., 2011). Despite these advances, there is a recognized need to further improve the design and development of advanced nanocarriers that should be able to improve the therapeutic benefits of Q3 oncologicals (González-Aramundiz et al., 2012). Among the biomaterials used in the development of nanooncologicals, polyaminoacids and, in particular poly(L-glutamic acid) (PGA), have raised great expectancy because of their biodegradability and acceptable regulatory profile (Li and Wallace, 2008). In fact, PGA conjugated with paclitaxel (Opaxio1, before Xyotax1) has already reached phase III clinical trials (Singer, 2005), while other combinations, i.e., PGA conjugated with camptothecin, are in earlier clinical development (clinical phase I and II) (Singer, 2005; Ng et al., 2012). An additional interesting property of polyaminoacids relies on the possibility to conjugate them with poly(ethylene glycol) (PEG), thus, rendering their surface more hydrophilic and flexible to prevent the uptake by the mononuclear
http://dx.doi.org/10.1016/j.ijpharm.2015.02.028 0378-5173/ ã 2015 Published by Elsevier B.V.
Please cite this article in press as: Lollo, G., et al., Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.02.028
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Instruments, UK). The morphology of the nanocapsules was analyzed by transmission electron microscopy using a Philips CM-12 microscope (FEI Company, Eindhoven). Samples were stained with phosphotungstic acid solution (2% w/v) and placed on a copper grid with Formvar1 films for analysis. Plitidepsin encapsulation efficiency in PGA-PEG nanocapsules was calculated indirectly by the difference between the total amount of plitidepsin in the system and the non-encapsulated drug measured after the isolation of loaded-nanocapsules:
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- The total amount of drug was estimated from a fresh aliquot of
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the nanocapsules formulation by dissolving the colloidal suspension in acetonitrile. These samples were centrifuged (4000 g, 20 min, 20 C) and the supernatant was analyzed by HPLC. - The free drug was quantified by the HPLC following separation of the nanocapsules by ultracentrifugation (27,400 g, 1 h, 15 C).
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Therefore, the encapsulation efficiency (E.E.) was calculated as follows (Eq. (1)): AB (1) 100 E:E:% ¼ A
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phagocytic system (MPS). The PEG-modified PGA nanosystems will have the opportunity to perform as long circulating carriers, and then ideally to decrease the access to the MPS organs and maximize their presence in the tumor (Huynh et al., 2010). Recently, we reported the design of a novel nanocapsule-type carrier made of PGA and PGA-PEG, with a lower PEG content, which is particularly attractive for accommodating hydrophobic anticancer drugs such as plitidepsin (Gonzalo et al., 2013). Plitidepsin is a hydrophobic antitumor drug originally isolated from the Mediterranean tunicate Aplidium albicans and currently produced by chemical synthesis (PharmaMar S.A., Spain). It is currently in phase II clinical trials for solid and haematological malignant neoplasias like T cell lymphoma and in phase III clinical trials for multiple myeloma (Geoerger et al., 2012; Ribrag et al., 2013). Because of its extremely low aqueous solubility, instability in the biological environment, and non-selective distribution, we hypothesized that this drug could benefit from its formulation in the form of nanocapsules. The results showed that encapsulation within the nanocapsules provided the drug with a prolonged blood circulation and a significantly reduced toxicity. Based on this previous experience, this work was aimed at developing a new version of these PGA-PEG nanocapsules, in which the shell is made of a di-block copolymer with a high PEG content (57% w/w). The purpose was to obtain further improvements in the biodistribution profile of plitidepsin, thus, achieving a better toxicity-efficacy profile in comparison with plitidepsin dissolved in a Cremophor1 EL solution as reference formulation.
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2. Materials and Methods
where A is the experimental total drug amount and B is the unloaded drug amount. The HPLC system consisted of an Agilent 1100 series instrument equipped with UV detector set at 225 nm. The analytic method employed was previously validated by PharmaMar S.A. (Spain) (Mohammad Shahin, 2014).
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2.1. Chemicals
2.4. In vitro release study of plitidepsin from PGA-PEG nanocapsules
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The release study of plitidepsin was performed by incubating an aliquot of the nanocapsules formulation in PBS (pH 7.4) at an appropriate concentration (0.5 mg/mL) to assure sink conditions (drug concentration below 4.9 mg/mL). The vials were placed in an incubator at 37 C with horizontal shaking. At different intervals (1 h, 3 h, 6 h and 24 h), 3 mL of the suspension diluted in PBS were collected and ultracentrifuged in Herolab1 tubes (27,400 g, 1 h, 15 C). The plitidepsin released at each time was calculated indirectly by the difference between the total amount of drug present in the system and the free plitidepsin in the infranatant after the ultracentrifugation, both determined by HPLC, as described in the previous section.
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Plitidepsin was provided by PharmaMar S.A. (Spain). Miglyol1 812, a neutral oil formed by esters of capric and caprilyc fatty acid and glycerol, was a gift sample from Sasol Germany GmbH (Germany). Epikuron1 170, a phosphatidylcholine enriched fraction of soybean lecithin, was kindly provided by Cargill (Spain). Benzalkonium chloride, Poloxamer 188 (Pluronic1 F68) and D-(+)-trehalose dehydrate were purchased from Sigma–Aldrich (Spain). Poly-L-glutamic acid-polyethylene glycol (PGA-PEG Mw 35 kDa) was synthetized and supplied by Alamanda Polymers (USA). PGA-PEG was a diblock copolymer with a PEG content of 57% w/w; PEG chains length was 20 kDa and the PGA chains length was about 15 kDa.
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2.2. Preparation of PGA-PEG nanocapsules
2.5. Stability study of plitidepsin-loaded PGA-PEG nanocapsules upon storage
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The stability of plitidepsin-loaded nanocapsules was evaluated upon storage in sealed tubes at 4 C. Size, polydispersity index, zeta potential and leakage of the drug from the nanocapsules were measured for a period of 1 month.
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2.6. Freeze-drying study of PGA-PEG nanocapsules
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The selected variables for the lyophilisation study of blank PGA-PEG nanocapsules were the nanocapsules concentration (1, 0.75 and 0.5% w/v) and the trehalose concentration (5 and 10% w/v). For this purpose, 1 mL of the diluted blank PGA-PEG nanocapsules containing trehalose was placed in glass vials and frozen in liquid nitrogen. The freeze-drying program consisted of an initial drying step at 35 C, and a secondary drying where the temperature was finally equilibrated at 20 C over a period of 60 h (Labconco Corp., USA). PGA-PEG nanocapsules were resuspended by adding 1 mL of ultrapure water to the freeze-dried powders followed by manual
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The preparation of unloaded PGA-PEG nanocapsules was based on a modification of the solvent displacement technique which involved the ionic interaction between the PGA-PEG and a cationic surfactant after the solvent diffusion (Gonzalo et al., 2013). Briefly, an organic phase made of 30 mg of Epikuron1 170, 0.125 mL of Miglyol1, 9.5 mL of acetone, 0.5 mL of ethanol and 7 mg of benzalkonium chloride was poured over an aqueous phase containing 10 mg of the polymer PGA-PEG and 50 mg of Poloxamer 188. Solvents were evaporated from the suspension under vacuum to a final volume of 10 mL. Plitidepsin-loaded PGA-PEG nanocapsules were obtained as previously described but dissolving 20 mg of the hydrophobic drug in the 0.5 mL of ethanol and then following the mentioned procedure. 2.3. Characterization of PGA-PEG nanocapsules Particle size and z potential of colloidal systems were determined, respectively, by photon correlation spectroscopy and laser Doppler anemometry, using a Zetasizer Nano ZS (Malvern
Please cite this article in press as: Lollo, G., et al., Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.02.028
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stirring. Finally, their size distribution was measured after resuspension. An additional freeze-drying study was done with drug-loaded nanocapsules at a concentration of 1% w/v with a 10% w/v of trehalose.
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2.7. In vivo studies
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2.7.1. Animals Female athymic nu/nu mice and CD-1 male mice between 4 and 6 weeks of age and ranging in weight from 21 to 30 g were purchased from Harlan Laboratories Models, S.L. (Barcelona, Spain). Animals were housed in individually ventilated cages (Sealsafe1 Plus, Techniplast S.P.A.), 10 mice per cage, on a 12 h light-dark cycle at 21–23 C and 40–60% relative humidity. Mice were allowed free access to irradiated standard rodent diet (Tecklad 2914C) and sterilized water. Animals were acclimated for five days prior to being individually tattoo-identified. Animal protocols were reviewed and approved according to regional Institutional Animal Care and Use Committees (Spain).
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2.7.2. Pharmacokinetic evaluation The pharmacokinetic study of plitidepsin was performed in CD-1 healthy male mice (n = 4 animals each time point) after administration of a single i.v. dose of plitidepsin encapsulated in PGA-PEG nanocapsules and compared with i.v. administration of plitidepsin dissolved in a Cremophor1 EL reference solution (Cremophor1 EL/ethanol/water 15/15/70% by weight); the plitidepsin dose was 0.1 mg/kg of body weight and the volume administered was 150 mL. On the day of dosing, blood samples were drawn via cardiac puncture at 9 pre-established time points: 5, 15, 30 min and 1, 2, 4, 8, 24, 48 h post-injection (two animals were used per time point and formulation (n = 36)). Blood samples were transferred into tubes containing EDTA as anticoagulant. The blood was kept in darkness on ice until it was centrifuged at 3000 rpm for 15 min at 5 C. The plasma obtained was frozen at 20 C and maintained in darkness until analysis. Plitidepsin concentrations were determined in mouse plasma samples using a modified HPLC/MS/MS method previously described (Brandon et al., 2005). Briefly, plitidepsin and the internal standard (IS), PM91105, were extracted from plasma by liquid solid extraction. 200 mL of urea:glycine buffer 1:1 v/v were added to plasma samples (100 mL) containing IS (25 mL; 20 ng/mL) and this solution was extracted with 1500 mL of tert-butylmethylether (TBME)/hexane 1:1 v/v. The analysis was carried out in a gradient reversed phase chromatography followed by positive ion electrospray tandem mass spectrometry (ESI/MS/MS) detection using multiple reaction monitoring (MRM). The HPLC system consisted of a Shimadzu LC-10ADvp solvent delivery unit, on-line degasser, gradient mixer and system controller (Shimadzu Scientific, Columbia, MD, USA). A CTC-PAL Lcap autosampleer (LEAP Technologies, Carrboro, NC, USA) was used to inject samples. A PE Sciex API 4000 triple quadrupole mass spectrometer (Toronto, ON, Canada), equipped with a Turbo Ion Spay interface, was used. The analytical column was a Waters Atlantis T3, 3 mm, 20 2.1 mm. The mobile phase was acetonitrile (0.1% formic acid)/water (0.1% formic acid) with a flow rate of 600 mL/min. The column oven temperature was set at 50 C and the sample injection volume was 20 mL. The assay was linear over the concentration range 0.05–50 ng/ mL of plitidepsin. The calibration curve was defined by a slope of 0.23 and an intercept of 0.006 (R = 0.9958). The coefficient of variations for low (0.1 ng/mL), mid (5 ng/mL) and high (30 ng/mL) quality control samples of plitidepsin were 12, 18.5 and 9.5%, respectively. The pharmacokinetic parameters of plitidepsin were
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obtained using a non-compartmental pharmacokinetic method with WinNonlin 5.2 software (Brandon et al., 2005).
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2.7.3. Toxicity studies Acute toxicity of the formulations was determined by assessing the MTD (maximum tolerated dose after single administration) and the MTMD (maximum tolerated dose after multiple administration) of plitidepsin-loaded PGA-PEG nanocapsules in healthy CD-1 male mice following i.v. injection (150 mL; n = 10/group). Toxicity of plitidepsin dissolved in the Cremophor1 EL reference solution (Cremophor1 EL/ethanol/water 15/15/70% by weight) was also investigated. MTD and MTMD were defined as the highest dose not causing significant lethality (as death) or any prominent observable changes during the experiment (14 days) according to the standard ethical issues. For the MTD evaluation, the formulations were administered as a single i.v. bolus in the lateral vein of the tail, whereas for the MTMD, they were intravenously administered following 2 cycles of 5 consecutive treatment days spaced by 4 free days (Oliveira et al., 2014). Plitidepsin-loaded PGA-PEG and the reference plitidepsin solution were tested from 1.5 to 0.1 mg/kg. The MTD of blank systems could not be determined as the toxic dose was not reached with the tested concentrations.
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2.8. Efficacy studies
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2.8.1. Xenograft model MRI-H-121 is a human renal carcinoma originally obtained from the DCT Tumor Bank, developed by Dr. A.E. Bogden, Mason Research Institute, MA, and maintained as a serial transplanted tumor line in athymic nude mice. The original tissue came from a patient at University of Massachusetts Medical Center (USA).
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2.8.2. In vivo antitumor activity For the study, 4–6 week-old athymic nu/nu mice were subcutaneously implanted in their right flank with MRI-H121 tissue from serially transplanted donor mice using a 13G trocar. The tissue was debrided of membrane, haemorrhagic and necrotic areas and 3 mm3 fragments were implanted. When tumors reached 150–200 mm3, tumor-bearing animals (n = 10/group) were randomly allocated into the following treatment groups: (i) plitidepsin dissolved in the Cremophor1 EL reference solution, (ii) plitidepsin-loaded PGA-PEG nanocapsules, and (iii) serum saline as a control. The doses of plitidepsin and schedules were selected based on MTMD determination (i.e., 0.15 and 0.3 mg/kg for plitidepsinloaded PGA-PEG nanocapsules and Cremophor1 EL reference solution, respectively), having a final dose of 3 mg/kg for both formulations. The formulations were injected intravenously in the tail veins of the mice:
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(i) Plitidepsin dissolved in Cremophor1 EL was injected at a dose
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of 0.3 mg/kg during 2 cycles of 5 consecutive treatment days and then 4 days free. (ii) Plitidepsin-loaded PGA-PEG nanocapsules were injected daily at a dose of 0.15 mg/kg during 20 days.
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The lower dose used for the nanocapsules formulation was fixed according to the MTMD value, which clearly is related with their long circulating properties and their accumulation in blood circulation for extended periods of time. Tumor volume and mice body weight were measured 2–3 times per week starting from the first day of treatment (day 0). Treatments that produced 20% lethality and/or 20% of net body weight loss were considered toxic.
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Tumor volume was calculated using the equation (Eq. (2)): 2
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2.8.3. Experimental design and statistical analysis Design, randomization and monitoring of body weight and tumor measurements were performed using NewLab Oncology Software (version 2.25.06.00). Tumor volume data are presented as medians and interquartile range (IQR). Tumor volumes of the treated groups on day 0 and day X (T0 TX) and those of the control group (C0 CX) were used to determine the activity rating as follows (Eq. (3)): DT T0 TX %¼ (3) 100 C0 CX DC
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Activity rating:
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DT/DC > 50% inactive (). DT/DC > 25–50% tumor inhibition (+). DT/DC < 25% and TX/T0 > 75–125% tumor stasis (++) or TX/T0 > 10–75% partial regression (+++).
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TX/T0: tumor volume of the compound-treated group on day X and on day 0. Tumor volume data from groups following the 1st, 2nd and 3rd study weeks were compared using a two-tailed Mann–Whitney U test. In all cases, p < 0.05 was accepted as denoting a statistical difference.
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3. Results and discussion
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We have recently developed a new type of nanocapsules made of PGA and PGA-PEG (PEG-grafted copolymer; 24% w/w of PEG) that were able to provide the anticancer drug plitidepsin with halflives appreciably prolonged, and increased AUC values in comparison with the drug-loaded uncoated cores. In addition, we could also appreciate the favorable effect of the polymer PEGylation on plitidepsin’s pK profile. Based on those results, the main goal of the present work has been to develop a more advanced prototype of PGA-PEG nanocapsules by increasing their PEG content, with the final aim of maximizing its passive accumulation at the tumor site. This advanced prototype consists of an oily core and a polymeric shell made of a highly PEGylated PGA-PEG diblock copolymer (57% w/w of PEG). By selecting this diblock copolymer we could easily double the PEG content in comparison with the previous grafted copolymer. Taking into account the extreme low solubility of plitidepsin and the necessity of using solvents to make its i.v. administration feasible, this drug should clearly benefit from this new technology. The pharmacokinetic parameters and therapeutic efficacy of the loaded systems were evaluated and compared for first time with those of
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3.1. Preparation and characterization of plitidepsin-loaded PGA-PEG nanocapsules
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PGA-PEG nanocapsules were prepared by the solvent displacement technique (Gonzalo et al., 2013). By using this method, the deposition of a polymer coating onto the oily core is produced once the organic solvent diffuses immediately into the polymer aqueous solution. In our case, the attachment of the PGA-PEG to the oily core was driven by the inclusion of the positively charged surfactant, benzalkonium chloride, in the oily phase. Benzalkonium chloride was selected on the basis of its acceptable toxicological profile and it was used in the minimum amount that allowed the formation of stable systems. The polymer chosen was a block copolymer that contains a high percentage, around 57% w/w, of high Mw PEG (20 kDa). It is expected that its disposition on the external layer create a complete PEG-hydrated corona around the particles. This disposition is expected to endow the system with improved stealth properties (Mosqueira et al., 2001) which could also to contribute to a better access to the tumor area. The physicochemical properties of blank and plitidepsin-loaded PGA-PEG nanocapsules are summarized in Table 1. It can be noted that the use of adequate concentrations of polymer and cationic surfactant results in the formation of homogenous populations of nanocapsules around 180–190 nm. The results showed that the incorporation of the drug into PGA-PEG nanocapsules did not affect the size and the z potential of the systems. Both empty and loaded systems showed negative zeta potential values, which indicates the inversion from the positive values of the uncoated nanoemulsion (+38 mV), confirming the success in the formation of the PGA-PEG coating. Furthermore, an encapsulation efficiency value around of 85% indicated that the oily core could easily allocate the drug plitidepsin. The morphological appearance of the nanocapsules was observed by transmission electron microscopy (Fig. 1). The micrographs indicated that PGA-PEG nanocapsules have a round shape and a size below than 200 nm, similar to that obtained by photon correlation spectroscopy. In a second step, we evaluated the release pattern of plitidepsin-loaded PGA-PEG nanocapsules under “sink conditions”. The in vitro release profile of loaded nanocapsules (Fig. 2) indicated that the system follows a biphasic profile characterized by an initial burst of about 80% of the drug payload, followed by a second phase in which no further drug release was observed. This profile, typical of other nanocapsules and nanoemulsions, indicates that the release process is highly dependent on the partition of the drug between the oily cores and the great volume of the external aqueous phase (Oliveira et al., 2014). Even though these results cannot be extrapolated to the in vivo situation, the fact that a fraction of the drug remained encapsulated despite the “sink conditions” is an indication of the affinity of plitidepsin for the oily core and/or the shell of the nanocapsules.
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(2)
where a and b were the longest and shortest diameters, respectively. Animals were euthanized when their tumors reached a volume of 2000 mm3 and/or severe necrosis was seen. The antitumor effect was calculated by using DT/DC (%), defined as the percent change in tumor volume for each treated (T) and placebo (C) group. DT/DC was calculated on days 7, 14 and 21. The data are presented as medians and interquartile range (IQR). Treatment tolerability was assessed by monitoring body weight evolution and clinical signs as well as evidence of local damage at the injection site. All placebo-treated animals died or were sacrificed for ethical reasons from day 0 to 21.
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plitidepsin dissolved in a Cremophor1 EL solution, used as reference.
Table 1 Physicochemical characteristics of blank and plitidepsin-loaded PGA-PEG nanocapsules (mean S.D.; n = 3). NCs: nanocapsules; P.I.: polydispersity index; E.E.: encapsulation efficiency. Formulation
Size (nm)
P.I.
z potential (mV) E.E. (%)
Blank PGA-PEG NCs NCs Plitidepsin-loaded PGA-PEG NCs
180 4 190 15
0.1 0.1
20 4 24 5
– 85 4
Please cite this article in press as: Lollo, G., et al., Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.02.028
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Fig. 1. TEM images of plitidepsin-loaded PGA-PEG nanocapsules.
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3.2. Stability and freeze-drying studies of PGA-PEG nanocapsules
3.3. In vivo studies
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The stability of the plitidepsin-loaded nanocapsules under storage at 4 C in terms of size, zeta potential and leakage of the drug, was assessed during a period of 1 month. There was no modification on the particle size neither on the zeta potential of the nanocapsules, which maintained their original values throughout the study. Additionally, no leakage of plitidepsin could be observed, which demonstrates the effectiveness of PGA-PEG nanocapsules as carriers for the hydrophobic anticancer drug plitidepsin. In a second step, we explored the optimal freeze-drying conditions for the conversion of the aqueous suspension of nanocapsules into a powder. The blank nanocapsules were freezedried at two different trehalose concentrations (5 and 10% w/v) and the concentration of blank nanocapsules was tested at three levels (0.5–0.75–1% w/v). The results indicated that the recovery of the initial properties of PGA-PEG nanocapsules upon freezedrying and reconstitution was dependent on the cryoprotectant and nanosystem concentration. PGA-PEG nanocapsules could be freeze-dried using a trehalose concentration of 10% at any of the tested concentrations tested, remaining the size close to the initial values (Fig. 3). The lower concentration of trehalose led to sizes slightly higher after reconstitution. When drug-loaded nanocapsules at a 1% w/v concentration were freeze-dried with a 10% w/v of trehalose, no changes in size or drug integrity were found.
3.3.1. Pharmacokinetic evaluation The plasma disposition characteristics of plitidepsin formulated in PGA-PEG nanocapsules and in the Cremophor1 EL reference solution were evaluated in health CD-1 mice. The formulations were administered i.v. through a single bolus injection (0.1 mg/kg plitidepsin). As shown in Fig. 4, encapsulating the drug within PGA-PEG nanocapsules led to a great prolongation of its presence in the blood circulation. In fact, approximately a 10% of the injected
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Fig. 2. Plitidepsin release in PBS (pH 7.4, 37 C) from PGA-PEG nanocapsules (mean SD; n = 3).
Fig. 3. Particle size of the reconstituted freeze-dried blank PGA-PEG nanocapsules (NCs). Different concentrations (w/v) of nanocapsules were lyophilized using trehalose at 5% (&) or 10% (&) w/v (mean S.D.; n = 3).
Fig. 4. Plasma concentration–time profiles of plitidepsin following i.v. injection in mice of plitidepsin-loaded PGA-PEG nanocapsules (^) and plitidepsin Cremophor1 EL solution (&). Data represent mean S.D.
Please cite this article in press as: Lollo, G., et al., Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.02.028
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Table 2 Pharmacokinetic parameters of plitidepsin-loaded PGA-PEG nanocapsules and plitidepsin Cremophor1 EL reference solution after single i.v. injection in mice. PK parameters are average values.
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Formulation
t1/2 (h)
AUC0!48h/dose (ng h/mL/mg)
CL (mL/min/kg)
V (L/kg)
MRT (h)
Cremophor1 EL solution PGA-PEG NCs
8.2 17.0
57.9 274.7
157.0 52.7
106.9 77.7
10.5 20.8
dose of plitidepsin-loaded nanocapsules remained in circulation at 48 h after injection, whereas plitidepsin formulated in the Cremophor1 EL solution was below the limit of detection after the sampling time of 8 h. Pharmacokinetic analysis of the plitidepsin levels upon administration of the nanocapsules or the reference formulation is shown in Table 2. Plitidepsin in PGA-PEG nanocapsules exhibited a 2-fold higher half-life (17.0 h) and MRT (20.8 h) compared to plitidepsin dissolved in the Cremophor1 EL solution. The plasma AUC0–24h of plitidepsin-loaded nanocapsules was about 5-fold greater than that obtained with the reference formulation. Moreover, plitidepsin in PGA-PEG nanocapsules showed a 3-fold lower plasmatic cleareance (52.7 mL/min/kg) as compared to the plitidepsin reference solution (157.0 mL/min/kg). In a recent work, plitidepsin-loaded micelles were developed and their pharmacokinetics was also compared with that of the drug dissolved in the Cremophor1 EL solution (Oliveira et al., 2014). These micellar structures, especially those made of poly (trimethylene carbonate)-block-poly(glutamic acid) were able to increase the AUC0–24h of plitidepsin, in an extent almost attaining the double of the reference value. However the half-life was hardly modified (from 8 to 8.6 h), being notably shorter than that showed in the present work. Overall, the pharmacokinetic parameters of plitidepsin-loaded PGA-PEG nanocapsules highlight the long-circulating properties of PGA-PEG in comparison with the reference formulation. It seems clear, as with other PEGylated nanocarriers containing hydrophobic antitumor drugs, that the long circulating properties and high AUC values are related to an important decrease in the clearance of the antitumor drug. This was observed, for example, for PEGylated lipid nanocapsules containing docetaxel, when compared with the commercial formulation Taxotere1 (Khalid et al., 2006), or for PEGylated polymeric micellar and liposomal nanoformulations containing paclitaxel when compared with Taxol1 (Yang et al., 2007; Xiao et al., 2012; Zhang et al., 2012). Moreover, if we examine our results and compare them with those previously reported by us for plitidepsin-loaded PGA-PEG nanocapsules with a low PEG content, it is important to note that the plitdepsin’s AUC was considerably increased and the clearance considerably decreased (a 3-fold change for each parameter) (Gonzalo et al., 2013), demonstrating the effect of the higher PEGylation on the pharmacokinetic behavior. These changes were not reflected, however, in an increase in the half-life or MRT values, as might be expected. In fact, these values remained almost unchanged (t1/2 18 h and MRT 20 h for both high and low PEGylated nanocapsules). The different values of the plitidepsin distribution volumes observed for the two nanoformulations (106.9 L for high-PEGylated and 265.8 L for low-PEGylated nanocapsules) would explain these results. Reports in the literature
suggest that long circulating times and a smaller distribution volume of taxanes are consistent with less extensive antitumor drug distribution in tissues such as liver, spleen or kidneys (Khalid et al., 2006; Yoshizawa et al., 2011). Interestingly, this increased plasmatic residence and lower tissue concentrations were also accompanied by a significant increase in the tumor accumulation measured in tumor-bearing mice (Yoshizawa et al., 2011). On the other hand, these results are in agreement with extensive data in the literature that correlates the long residence time in plasma of taxanes after being administered in a PEGylated nanostructure with an increased deposition of the drug in the tumor via passive targeting (Yang et al., 2007; Senthilkumar et al., 2008; Jing et al., 2014).
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3.3.2. Toxicological evaluation The toxicological study was aimed at establishing the MTD and the MTMD of plitidepsin-loaded PGA-PEG nanocapsules following i.v. injection. The results were compared with those of plitidepsin in Cremophor1 EL reference solution. As observed in Table 3, the MTD of PGA-PEG nanocapsules was 2.5 times higher than that of Cremophor1 EL solution, and higher
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Fig. 5. Evolution of tumor volume median following i.v. multiple administration of plitidepsin-loaded PGA-PEG nanocapsules (~), Cremophor1 EL reference solution (^) and saline serum (&) in a subcutaneously implanted MRI-H-121 human renal xenograft mouse model (total plitidepsin dose was 0.3 mg/kg).
Table 4 Tumor volumes (TV) and mortality in mice bearing MRI-H-121 xenografts treated with multiple i.v. plitidepsin-loaded PGA-PEG nanocapsules and the plitidepsin Cremophor1 EL reference solution. Formulation 1
Table 3 MTD (maximum tolerated dose after single administration) and the MTMD (maximum tolerated dose after multiple administration) of plitidepsin-loaded PGA-PEG nanocapsules and plitidepsin dissolved in the Cremophor1 EL reference solution, in healthy CD-1 male mice following i.v. injection (n = 10/group). Formulation
MTD (mg/kg)
MTMD (mg/kg)
PGA-PEG nanocapsules Cremophor1 EL reference solution
0.75 0.30
0.15 0.30
Cremophor
PGA-PEG NCs
EL solution
Day
TV mm3 median (IQR)
p
Mortality
7 14 21
149 (133.9–268.6) 80.1 (52.7–127.7) 181.3 (144.0–264.9)
0.0001 <0.0001 <0.0001
0/10 0/10 1/10
7 14 21
426.1 (376.5–561.6) 358.8 (275.8–486.0) 154.5 (80.5–236.8)
NS <0.0001 <0.0001
0/10 0/10 0/10
Data are presented as median and interquartile range (IQR). p value for Mann–Whitney U test (control group compared against the rest). NS: not statistically significance; NCs: nanocapsules.
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Table 5 Antitumor effect parameters and activity ranking of plitidepsin-loaded PGA-PEG nanocapsules and plitidepsin Cremophor1 EL formulation after i.v. multiple injection in mice bearing MRI-H121 xenograft during a period of 20 days. NCs: nanocapsules. Formulation
Cremophor1 EL solution PGA-PEG NCs
Dose (mg/kg)
0.30 0.15
DT/DC%
Activity rating
Day 7
Day 14
Day 21
Day 7
Day 14
Day 21
5.8 55.0
9.1 17.2
0.3 1.3
++ –
+++ +
++ ++
Tumor inhibition (+); tumor stasis (++); partial regression (+++).
DT/DC: difference of volumes of the treated groups on day 0 and day X (T0 TX) and those of control group (C0 CX) as reported in Section 2. DT/DC > 50% inactive (). DT/DC > 25–50% tumor inhibition (+). DT/DC < 25% and TX/T0 > 75–125% tumor stasis (++) or TX/T0 > 10–75% partial regression (+++). TX/T0: tumor volume of the compound-treated group on day X and on day 0.
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than their MTMD value. Besides, the MTMD of drug-loaded particles is lower than the value obtained for the Cremophor1 EL formulation (0.15 vs 0.30 mg/kg). The MTD results indicate that nanocapsules are better tolerated than the reference formulation and could be administered at higher unique doses. However, in the case of MTMD values, the opposite occurs, this being in accordance with the pharmacokinetic evaluation above reported. The long circulation properties of plitidepsin-encapsulated into PGA-PEG nanocapsules help us to explain this MTMD reduction. The drug exposure is higher, plitidepsin remains for longer time into the blood circulation and it is eliminated slowly. When plitidepsin is dissolved in Cremophor1 EL, the pharmacokinetic behavior is different, leading to a rapid elimination of the drug. To allow an easier comparison between the two formulations for the in vivo efficacy study, plitidepsin was used around their MTMD values (0.15 and 0.3 mg/kg). 3.3.3. Antitumor activity The in vivo antitumor efficacy of plitidepsin-loaded PGA-PEG nanocapsules was evaluated in a human renal xenograft mouse model (MRI-H-121). A comparative study was performed by dividing animals into 3 groups according to the treatment received (plitidepsin-loaded PGA-PEG nanocapsules, reference formulation and serum) and the schedule of administration established. A strong antitumor activity was seen after the treatment with both plitidepsin formulated in Cremophor1 EL and PGA-PEG nanocapsules (Fig. 5). Moreover, there is a significant difference (p < 0.001) in tumor volume at the day 14 and 21 for plitidepsin-loaded PGA-PEG nanocapsules and the reference Cremophor1 EL solution with the respect to the control, indicating that both formulations had a similar antitumor activity (Table 4). In fact, tumor stasis was recovered at the end of the experiment after the treatment with plitidepsin-loaded PGA-PEG nanocapsules and the reference solution (Table 5). However, neither mortality nor significant changes in body weight were observed throughout the study when the nanocapsules were administered, whereas one animal was found dead in the reference formulation group on day 15. The activity rating and the antitumor effect calculated as DT/DC, and reported in Table 5, highlight the differences in the mechanism of action of the two formulations. Plitidepsin dissolved in the Cremophor1 EL formulation showed a rapid onset of action (delayed tumor growth), but this effect decreased once the treatment was finished, at day 21. The trend of the plitidepsinloaded nanocapsules was slightly different, showing a tendency not only to achieve the stasis of the tumor at the end of the treatment (day 21; Table 4) but also to stop the tumor growth toward the end of the study (day 26; Fig. 5). These different behaviors could be clearly related with the different pharmacokinetic profiles of both formulations, showing the nanocapsules a
more prolonged plasma residence and hence a delayed but apparently more efficient response. Considering the antitumor activity, we suggest that the improved biodistribution profile observed for PGA-PEG nanocapsules as compared to that of the Cremophor1 EL formulation, might be responsible for the passive accumulation of the drug in the pathological site. These results are in agreement with those described for PEGylated nanocarriers which have shown enhancements of the therapeutic index of different antitumor drugs (Hureaux et al., 2010; Kim et al., 2001).
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4. Conclusions
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High PEGylated PGA-PEG nanocapsules were developed as novel carriers for the antitumor hydrophobic drug plitidepsin. Results from the present study demonstrated that the coating of nanocapsules with a high PEGylated PGA-PEG diblock copolymer improved the pharmacokinetic profile of the drug as compared to the reference formulation consisting of a Cremophor1 EL solution. Moreover, plitidepsin-loaded PGA-PEG nanocapsules were better tolerated than plitidepsin formulated in the Cremophor1 EL solution. In vivo antitumor activity in a xenograft tumor model in mice also revealed an important suppression of tumor growth upon multiple i.v. administration, an effect that was comparable to that of the reference formulation. All these data indicate the interest of PGA-PEG nanocapsules as a novel delivery platform for cancer chemotherapy.
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Acknowledgements
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The authors would like to acknowledge financial support from Q5 CENIT-NANOFAR XS53 project, PharmaMar, Spain; the Xunta de Galicia (Competitive Reference Groups-FEDER Funds Ref 2014/ 043) and the European Commission FP7 EraNet-EuroNanoMed Program-Instituto Carlos III (Lymphotarg proyect, Ref. PS09/ 02670). Giovanna Lollo was a recipient of a FPU fellowship from the Ministry of Education of Spain. Marcos Garcia-Fuentes was a recipient Isidro Parga Pondal Fellowship from Xunta de Galicia.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
1
Pharmaceutical nanotechnology
2
Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative
3
4 Q1 5 6 7 8 9 10
Giovanna Lollo a,b,1, Pablo Hervella b , Pilar Calvo c, Pablo Avilés c , Maria Jose Guillén c , Marcos Garcia-Fuentes a,b , Maria José Alonso a,b , Dolores Torres b, * a Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Campus Vida, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain b Department of Pharmaceutics and Pharmaceutical Technology, School of Pharmacy and Heath Research Institute of Santiago de Compostela (IDIS), Campus Vida, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain c PharmaMar S.A, Colmenar Viejo, Madrid, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2014 Received in revised form 8 February 2015 Accepted 10 February 2015 Available online xxx
The focus of this study is to disclose a new delivery carrier intended to improve the pharmacokinetic characteristics of the anticancer drug plitidepsin and to favor its accumulation within the tumor. These nanocarriers named as nanocapsules, consist of an oily core surrounded by a highly PEGylated polyglutamic acid (PGA-PEG) shell loaded with plitidepsin. They showed a size of around 190 nm, a zeta potential of 24 mV and were able to encapsulate a high percentage (85%) of plitidepsin. In vivo studies, following intravenous injection in healthy mice, indicated that the encapsulation of the drug within PGA-PEG nanocapsules led to an important increase in its area under the curve (AUC) which is related to the important decrease of the clearance, as compared to the values observed for the drug dissolved in a Cremophor1 EL solution. This improvement of the pharmacokinetic profile of the encapsulated plitidepsin was accompanied by a high increase (2.5-fold) of the maximum tolerated dose (MTD) in comparison to that of plitidepsin Cremophor1 EL solution. The efficacy study performed in a xenograft tumor mice model evidenced the capacity of PGA-PEG nanocapsules to significantly reduce tumor growth. These promising results highlight the potential of PGA-PEG nanocapsules as an effective drug delivery system for cancer therapy. ã 2015 Published by Elsevier B.V.
Keywords: Nanomedicine Antitumor drugs Stealth properties Polyglutamic acid Plitidepsin Cancer therapy
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1. Introduction
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Over the last decades, nanopharmaceuticals have a particular impact in improving cancer therapeutics (Blanco et al., 2011; Wang and Thanou, 2010). Most anticancer drugs used in conventional chemotherapy are rapidly cleared from the blood circulation and, because they do not differentiate between cancerous and normal tissues, their use generally leads to major systemic side effects (Shi et al., 2011). Nanocarriers with long-circulating times offer the potential advantage of being accumulated and entrapped within tumors due to the high permeability of tumoral vasculature and frequently poor lymphatic drainage (Fang et al., 2011; Bertrand et al., 2014). A large number of nanocarriers containing cytotoxic drugs have been examined in clinical trials and been approved for
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* Corresponding author. Tel.: +34 8814880; fax: +34 8814880. E-mail address: [email protected] (D. Torres). 1 Present address: LUNAM Université – INSERM U1066, IBS-CHU, F-49933 Angers, France.
use in humans. In particular, the FDA approval of Doxil1, PEGylated liposomes containing doxorubicin, has opened the door for the clinical development of other long circulating nanocarriers including micelles, nanoparticles or conjugates (Shi et al., 2011). Despite these advances, there is a recognized need to further improve the design and development of advanced nanocarriers that should be able to improve the therapeutic benefits of Q3 oncologicals (González-Aramundiz et al., 2012). Among the biomaterials used in the development of nanooncologicals, polyaminoacids and, in particular poly(L-glutamic acid) (PGA), have raised great expectancy because of their biodegradability and acceptable regulatory profile (Li and Wallace, 2008). In fact, PGA conjugated with paclitaxel (Opaxio1, before Xyotax1) has already reached phase III clinical trials (Singer, 2005), while other combinations, i.e., PGA conjugated with camptothecin, are in earlier clinical development (clinical phase I and II) (Singer, 2005; Ng et al., 2012). An additional interesting property of polyaminoacids relies on the possibility to conjugate them with poly(ethylene glycol) (PEG), thus, rendering their surface more hydrophilic and flexible to prevent the uptake by the mononuclear
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Please cite this article in press as: Lollo, G., et al., Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.02.028
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Instruments, UK). The morphology of the nanocapsules was analyzed by transmission electron microscopy using a Philips CM-12 microscope (FEI Company, Eindhoven). Samples were stained with phosphotungstic acid solution (2% w/v) and placed on a copper grid with Formvar1 films for analysis. Plitidepsin encapsulation efficiency in PGA-PEG nanocapsules was calculated indirectly by the difference between the total amount of plitidepsin in the system and the non-encapsulated drug measured after the isolation of loaded-nanocapsules:
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- The total amount of drug was estimated from a fresh aliquot of
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the nanocapsules formulation by dissolving the colloidal suspension in acetonitrile. These samples were centrifuged (4000 g, 20 min, 20 C) and the supernatant was analyzed by HPLC. - The free drug was quantified by the HPLC following separation of the nanocapsules by ultracentrifugation (27,400 g, 1 h, 15 C).
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Therefore, the encapsulation efficiency (E.E.) was calculated as follows (Eq. (1)): AB (1) 100 E:E:% ¼ A
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phagocytic system (MPS). The PEG-modified PGA nanosystems will have the opportunity to perform as long circulating carriers, and then ideally to decrease the access to the MPS organs and maximize their presence in the tumor (Huynh et al., 2010). Recently, we reported the design of a novel nanocapsule-type carrier made of PGA and PGA-PEG, with a lower PEG content, which is particularly attractive for accommodating hydrophobic anticancer drugs such as plitidepsin (Gonzalo et al., 2013). Plitidepsin is a hydrophobic antitumor drug originally isolated from the Mediterranean tunicate Aplidium albicans and currently produced by chemical synthesis (PharmaMar S.A., Spain). It is currently in phase II clinical trials for solid and haematological malignant neoplasias like T cell lymphoma and in phase III clinical trials for multiple myeloma (Geoerger et al., 2012; Ribrag et al., 2013). Because of its extremely low aqueous solubility, instability in the biological environment, and non-selective distribution, we hypothesized that this drug could benefit from its formulation in the form of nanocapsules. The results showed that encapsulation within the nanocapsules provided the drug with a prolonged blood circulation and a significantly reduced toxicity. Based on this previous experience, this work was aimed at developing a new version of these PGA-PEG nanocapsules, in which the shell is made of a di-block copolymer with a high PEG content (57% w/w). The purpose was to obtain further improvements in the biodistribution profile of plitidepsin, thus, achieving a better toxicity-efficacy profile in comparison with plitidepsin dissolved in a Cremophor1 EL solution as reference formulation.
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2. Materials and Methods
where A is the experimental total drug amount and B is the unloaded drug amount. The HPLC system consisted of an Agilent 1100 series instrument equipped with UV detector set at 225 nm. The analytic method employed was previously validated by PharmaMar S.A. (Spain) (Mohammad Shahin, 2014).
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2.1. Chemicals
2.4. In vitro release study of plitidepsin from PGA-PEG nanocapsules
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The release study of plitidepsin was performed by incubating an aliquot of the nanocapsules formulation in PBS (pH 7.4) at an appropriate concentration (0.5 mg/mL) to assure sink conditions (drug concentration below 4.9 mg/mL). The vials were placed in an incubator at 37 C with horizontal shaking. At different intervals (1 h, 3 h, 6 h and 24 h), 3 mL of the suspension diluted in PBS were collected and ultracentrifuged in Herolab1 tubes (27,400 g, 1 h, 15 C). The plitidepsin released at each time was calculated indirectly by the difference between the total amount of drug present in the system and the free plitidepsin in the infranatant after the ultracentrifugation, both determined by HPLC, as described in the previous section.
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Plitidepsin was provided by PharmaMar S.A. (Spain). Miglyol1 812, a neutral oil formed by esters of capric and caprilyc fatty acid and glycerol, was a gift sample from Sasol Germany GmbH (Germany). Epikuron1 170, a phosphatidylcholine enriched fraction of soybean lecithin, was kindly provided by Cargill (Spain). Benzalkonium chloride, Poloxamer 188 (Pluronic1 F68) and D-(+)-trehalose dehydrate were purchased from Sigma–Aldrich (Spain). Poly-L-glutamic acid-polyethylene glycol (PGA-PEG Mw 35 kDa) was synthetized and supplied by Alamanda Polymers (USA). PGA-PEG was a diblock copolymer with a PEG content of 57% w/w; PEG chains length was 20 kDa and the PGA chains length was about 15 kDa.
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2.2. Preparation of PGA-PEG nanocapsules
2.5. Stability study of plitidepsin-loaded PGA-PEG nanocapsules upon storage
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The stability of plitidepsin-loaded nanocapsules was evaluated upon storage in sealed tubes at 4 C. Size, polydispersity index, zeta potential and leakage of the drug from the nanocapsules were measured for a period of 1 month.
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2.6. Freeze-drying study of PGA-PEG nanocapsules
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The selected variables for the lyophilisation study of blank PGA-PEG nanocapsules were the nanocapsules concentration (1, 0.75 and 0.5% w/v) and the trehalose concentration (5 and 10% w/v). For this purpose, 1 mL of the diluted blank PGA-PEG nanocapsules containing trehalose was placed in glass vials and frozen in liquid nitrogen. The freeze-drying program consisted of an initial drying step at 35 C, and a secondary drying where the temperature was finally equilibrated at 20 C over a period of 60 h (Labconco Corp., USA). PGA-PEG nanocapsules were resuspended by adding 1 mL of ultrapure water to the freeze-dried powders followed by manual
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The preparation of unloaded PGA-PEG nanocapsules was based on a modification of the solvent displacement technique which involved the ionic interaction between the PGA-PEG and a cationic surfactant after the solvent diffusion (Gonzalo et al., 2013). Briefly, an organic phase made of 30 mg of Epikuron1 170, 0.125 mL of Miglyol1, 9.5 mL of acetone, 0.5 mL of ethanol and 7 mg of benzalkonium chloride was poured over an aqueous phase containing 10 mg of the polymer PGA-PEG and 50 mg of Poloxamer 188. Solvents were evaporated from the suspension under vacuum to a final volume of 10 mL. Plitidepsin-loaded PGA-PEG nanocapsules were obtained as previously described but dissolving 20 mg of the hydrophobic drug in the 0.5 mL of ethanol and then following the mentioned procedure. 2.3. Characterization of PGA-PEG nanocapsules Particle size and z potential of colloidal systems were determined, respectively, by photon correlation spectroscopy and laser Doppler anemometry, using a Zetasizer Nano ZS (Malvern
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stirring. Finally, their size distribution was measured after resuspension. An additional freeze-drying study was done with drug-loaded nanocapsules at a concentration of 1% w/v with a 10% w/v of trehalose.
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2.7. In vivo studies
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2.7.1. Animals Female athymic nu/nu mice and CD-1 male mice between 4 and 6 weeks of age and ranging in weight from 21 to 30 g were purchased from Harlan Laboratories Models, S.L. (Barcelona, Spain). Animals were housed in individually ventilated cages (Sealsafe1 Plus, Techniplast S.P.A.), 10 mice per cage, on a 12 h light-dark cycle at 21–23 C and 40–60% relative humidity. Mice were allowed free access to irradiated standard rodent diet (Tecklad 2914C) and sterilized water. Animals were acclimated for five days prior to being individually tattoo-identified. Animal protocols were reviewed and approved according to regional Institutional Animal Care and Use Committees (Spain).
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2.7.2. Pharmacokinetic evaluation The pharmacokinetic study of plitidepsin was performed in CD-1 healthy male mice (n = 4 animals each time point) after administration of a single i.v. dose of plitidepsin encapsulated in PGA-PEG nanocapsules and compared with i.v. administration of plitidepsin dissolved in a Cremophor1 EL reference solution (Cremophor1 EL/ethanol/water 15/15/70% by weight); the plitidepsin dose was 0.1 mg/kg of body weight and the volume administered was 150 mL. On the day of dosing, blood samples were drawn via cardiac puncture at 9 pre-established time points: 5, 15, 30 min and 1, 2, 4, 8, 24, 48 h post-injection (two animals were used per time point and formulation (n = 36)). Blood samples were transferred into tubes containing EDTA as anticoagulant. The blood was kept in darkness on ice until it was centrifuged at 3000 rpm for 15 min at 5 C. The plasma obtained was frozen at 20 C and maintained in darkness until analysis. Plitidepsin concentrations were determined in mouse plasma samples using a modified HPLC/MS/MS method previously described (Brandon et al., 2005). Briefly, plitidepsin and the internal standard (IS), PM91105, were extracted from plasma by liquid solid extraction. 200 mL of urea:glycine buffer 1:1 v/v were added to plasma samples (100 mL) containing IS (25 mL; 20 ng/mL) and this solution was extracted with 1500 mL of tert-butylmethylether (TBME)/hexane 1:1 v/v. The analysis was carried out in a gradient reversed phase chromatography followed by positive ion electrospray tandem mass spectrometry (ESI/MS/MS) detection using multiple reaction monitoring (MRM). The HPLC system consisted of a Shimadzu LC-10ADvp solvent delivery unit, on-line degasser, gradient mixer and system controller (Shimadzu Scientific, Columbia, MD, USA). A CTC-PAL Lcap autosampleer (LEAP Technologies, Carrboro, NC, USA) was used to inject samples. A PE Sciex API 4000 triple quadrupole mass spectrometer (Toronto, ON, Canada), equipped with a Turbo Ion Spay interface, was used. The analytical column was a Waters Atlantis T3, 3 mm, 20 2.1 mm. The mobile phase was acetonitrile (0.1% formic acid)/water (0.1% formic acid) with a flow rate of 600 mL/min. The column oven temperature was set at 50 C and the sample injection volume was 20 mL. The assay was linear over the concentration range 0.05–50 ng/ mL of plitidepsin. The calibration curve was defined by a slope of 0.23 and an intercept of 0.006 (R = 0.9958). The coefficient of variations for low (0.1 ng/mL), mid (5 ng/mL) and high (30 ng/mL) quality control samples of plitidepsin were 12, 18.5 and 9.5%, respectively. The pharmacokinetic parameters of plitidepsin were
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obtained using a non-compartmental pharmacokinetic method with WinNonlin 5.2 software (Brandon et al., 2005).
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2.7.3. Toxicity studies Acute toxicity of the formulations was determined by assessing the MTD (maximum tolerated dose after single administration) and the MTMD (maximum tolerated dose after multiple administration) of plitidepsin-loaded PGA-PEG nanocapsules in healthy CD-1 male mice following i.v. injection (150 mL; n = 10/group). Toxicity of plitidepsin dissolved in the Cremophor1 EL reference solution (Cremophor1 EL/ethanol/water 15/15/70% by weight) was also investigated. MTD and MTMD were defined as the highest dose not causing significant lethality (as death) or any prominent observable changes during the experiment (14 days) according to the standard ethical issues. For the MTD evaluation, the formulations were administered as a single i.v. bolus in the lateral vein of the tail, whereas for the MTMD, they were intravenously administered following 2 cycles of 5 consecutive treatment days spaced by 4 free days (Oliveira et al., 2014). Plitidepsin-loaded PGA-PEG and the reference plitidepsin solution were tested from 1.5 to 0.1 mg/kg. The MTD of blank systems could not be determined as the toxic dose was not reached with the tested concentrations.
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2.8. Efficacy studies
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2.8.1. Xenograft model MRI-H-121 is a human renal carcinoma originally obtained from the DCT Tumor Bank, developed by Dr. A.E. Bogden, Mason Research Institute, MA, and maintained as a serial transplanted tumor line in athymic nude mice. The original tissue came from a patient at University of Massachusetts Medical Center (USA).
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2.8.2. In vivo antitumor activity For the study, 4–6 week-old athymic nu/nu mice were subcutaneously implanted in their right flank with MRI-H121 tissue from serially transplanted donor mice using a 13G trocar. The tissue was debrided of membrane, haemorrhagic and necrotic areas and 3 mm3 fragments were implanted. When tumors reached 150–200 mm3, tumor-bearing animals (n = 10/group) were randomly allocated into the following treatment groups: (i) plitidepsin dissolved in the Cremophor1 EL reference solution, (ii) plitidepsin-loaded PGA-PEG nanocapsules, and (iii) serum saline as a control. The doses of plitidepsin and schedules were selected based on MTMD determination (i.e., 0.15 and 0.3 mg/kg for plitidepsinloaded PGA-PEG nanocapsules and Cremophor1 EL reference solution, respectively), having a final dose of 3 mg/kg for both formulations. The formulations were injected intravenously in the tail veins of the mice:
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(i) Plitidepsin dissolved in Cremophor1 EL was injected at a dose
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of 0.3 mg/kg during 2 cycles of 5 consecutive treatment days and then 4 days free. (ii) Plitidepsin-loaded PGA-PEG nanocapsules were injected daily at a dose of 0.15 mg/kg during 20 days.
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The lower dose used for the nanocapsules formulation was fixed according to the MTMD value, which clearly is related with their long circulating properties and their accumulation in blood circulation for extended periods of time. Tumor volume and mice body weight were measured 2–3 times per week starting from the first day of treatment (day 0). Treatments that produced 20% lethality and/or 20% of net body weight loss were considered toxic.
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Tumor volume was calculated using the equation (Eq. (2)): 2
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2.8.3. Experimental design and statistical analysis Design, randomization and monitoring of body weight and tumor measurements were performed using NewLab Oncology Software (version 2.25.06.00). Tumor volume data are presented as medians and interquartile range (IQR). Tumor volumes of the treated groups on day 0 and day X (T0 TX) and those of the control group (C0 CX) were used to determine the activity rating as follows (Eq. (3)): DT T0 TX %¼ (3) 100 C0 CX DC
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Activity rating:
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DT/DC > 50% inactive (). DT/DC > 25–50% tumor inhibition (+). DT/DC < 25% and TX/T0 > 75–125% tumor stasis (++) or TX/T0 > 10–75% partial regression (+++).
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TX/T0: tumor volume of the compound-treated group on day X and on day 0. Tumor volume data from groups following the 1st, 2nd and 3rd study weeks were compared using a two-tailed Mann–Whitney U test. In all cases, p < 0.05 was accepted as denoting a statistical difference.
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3. Results and discussion
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We have recently developed a new type of nanocapsules made of PGA and PGA-PEG (PEG-grafted copolymer; 24% w/w of PEG) that were able to provide the anticancer drug plitidepsin with halflives appreciably prolonged, and increased AUC values in comparison with the drug-loaded uncoated cores. In addition, we could also appreciate the favorable effect of the polymer PEGylation on plitidepsin’s pK profile. Based on those results, the main goal of the present work has been to develop a more advanced prototype of PGA-PEG nanocapsules by increasing their PEG content, with the final aim of maximizing its passive accumulation at the tumor site. This advanced prototype consists of an oily core and a polymeric shell made of a highly PEGylated PGA-PEG diblock copolymer (57% w/w of PEG). By selecting this diblock copolymer we could easily double the PEG content in comparison with the previous grafted copolymer. Taking into account the extreme low solubility of plitidepsin and the necessity of using solvents to make its i.v. administration feasible, this drug should clearly benefit from this new technology. The pharmacokinetic parameters and therapeutic efficacy of the loaded systems were evaluated and compared for first time with those of
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3.1. Preparation and characterization of plitidepsin-loaded PGA-PEG nanocapsules
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PGA-PEG nanocapsules were prepared by the solvent displacement technique (Gonzalo et al., 2013). By using this method, the deposition of a polymer coating onto the oily core is produced once the organic solvent diffuses immediately into the polymer aqueous solution. In our case, the attachment of the PGA-PEG to the oily core was driven by the inclusion of the positively charged surfactant, benzalkonium chloride, in the oily phase. Benzalkonium chloride was selected on the basis of its acceptable toxicological profile and it was used in the minimum amount that allowed the formation of stable systems. The polymer chosen was a block copolymer that contains a high percentage, around 57% w/w, of high Mw PEG (20 kDa). It is expected that its disposition on the external layer create a complete PEG-hydrated corona around the particles. This disposition is expected to endow the system with improved stealth properties (Mosqueira et al., 2001) which could also to contribute to a better access to the tumor area. The physicochemical properties of blank and plitidepsin-loaded PGA-PEG nanocapsules are summarized in Table 1. It can be noted that the use of adequate concentrations of polymer and cationic surfactant results in the formation of homogenous populations of nanocapsules around 180–190 nm. The results showed that the incorporation of the drug into PGA-PEG nanocapsules did not affect the size and the z potential of the systems. Both empty and loaded systems showed negative zeta potential values, which indicates the inversion from the positive values of the uncoated nanoemulsion (+38 mV), confirming the success in the formation of the PGA-PEG coating. Furthermore, an encapsulation efficiency value around of 85% indicated that the oily core could easily allocate the drug plitidepsin. The morphological appearance of the nanocapsules was observed by transmission electron microscopy (Fig. 1). The micrographs indicated that PGA-PEG nanocapsules have a round shape and a size below than 200 nm, similar to that obtained by photon correlation spectroscopy. In a second step, we evaluated the release pattern of plitidepsin-loaded PGA-PEG nanocapsules under “sink conditions”. The in vitro release profile of loaded nanocapsules (Fig. 2) indicated that the system follows a biphasic profile characterized by an initial burst of about 80% of the drug payload, followed by a second phase in which no further drug release was observed. This profile, typical of other nanocapsules and nanoemulsions, indicates that the release process is highly dependent on the partition of the drug between the oily cores and the great volume of the external aqueous phase (Oliveira et al., 2014). Even though these results cannot be extrapolated to the in vivo situation, the fact that a fraction of the drug remained encapsulated despite the “sink conditions” is an indication of the affinity of plitidepsin for the oily core and/or the shell of the nanocapsules.
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(2)
where a and b were the longest and shortest diameters, respectively. Animals were euthanized when their tumors reached a volume of 2000 mm3 and/or severe necrosis was seen. The antitumor effect was calculated by using DT/DC (%), defined as the percent change in tumor volume for each treated (T) and placebo (C) group. DT/DC was calculated on days 7, 14 and 21. The data are presented as medians and interquartile range (IQR). Treatment tolerability was assessed by monitoring body weight evolution and clinical signs as well as evidence of local damage at the injection site. All placebo-treated animals died or were sacrificed for ethical reasons from day 0 to 21.
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plitidepsin dissolved in a Cremophor1 EL solution, used as reference.
Table 1 Physicochemical characteristics of blank and plitidepsin-loaded PGA-PEG nanocapsules (mean S.D.; n = 3). NCs: nanocapsules; P.I.: polydispersity index; E.E.: encapsulation efficiency. Formulation
Size (nm)
P.I.
z potential (mV) E.E. (%)
Blank PGA-PEG NCs NCs Plitidepsin-loaded PGA-PEG NCs
180 4 190 15
0.1 0.1
20 4 24 5
– 85 4
Please cite this article in press as: Lollo, G., et al., Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.02.028
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Fig. 1. TEM images of plitidepsin-loaded PGA-PEG nanocapsules.
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3.2. Stability and freeze-drying studies of PGA-PEG nanocapsules
3.3. In vivo studies
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The stability of the plitidepsin-loaded nanocapsules under storage at 4 C in terms of size, zeta potential and leakage of the drug, was assessed during a period of 1 month. There was no modification on the particle size neither on the zeta potential of the nanocapsules, which maintained their original values throughout the study. Additionally, no leakage of plitidepsin could be observed, which demonstrates the effectiveness of PGA-PEG nanocapsules as carriers for the hydrophobic anticancer drug plitidepsin. In a second step, we explored the optimal freeze-drying conditions for the conversion of the aqueous suspension of nanocapsules into a powder. The blank nanocapsules were freezedried at two different trehalose concentrations (5 and 10% w/v) and the concentration of blank nanocapsules was tested at three levels (0.5–0.75–1% w/v). The results indicated that the recovery of the initial properties of PGA-PEG nanocapsules upon freezedrying and reconstitution was dependent on the cryoprotectant and nanosystem concentration. PGA-PEG nanocapsules could be freeze-dried using a trehalose concentration of 10% at any of the tested concentrations tested, remaining the size close to the initial values (Fig. 3). The lower concentration of trehalose led to sizes slightly higher after reconstitution. When drug-loaded nanocapsules at a 1% w/v concentration were freeze-dried with a 10% w/v of trehalose, no changes in size or drug integrity were found.
3.3.1. Pharmacokinetic evaluation The plasma disposition characteristics of plitidepsin formulated in PGA-PEG nanocapsules and in the Cremophor1 EL reference solution were evaluated in health CD-1 mice. The formulations were administered i.v. through a single bolus injection (0.1 mg/kg plitidepsin). As shown in Fig. 4, encapsulating the drug within PGA-PEG nanocapsules led to a great prolongation of its presence in the blood circulation. In fact, approximately a 10% of the injected
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Fig. 2. Plitidepsin release in PBS (pH 7.4, 37 C) from PGA-PEG nanocapsules (mean SD; n = 3).
Fig. 3. Particle size of the reconstituted freeze-dried blank PGA-PEG nanocapsules (NCs). Different concentrations (w/v) of nanocapsules were lyophilized using trehalose at 5% (&) or 10% (&) w/v (mean S.D.; n = 3).
Fig. 4. Plasma concentration–time profiles of plitidepsin following i.v. injection in mice of plitidepsin-loaded PGA-PEG nanocapsules (^) and plitidepsin Cremophor1 EL solution (&). Data represent mean S.D.
Please cite this article in press as: Lollo, G., et al., Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.02.028
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Table 2 Pharmacokinetic parameters of plitidepsin-loaded PGA-PEG nanocapsules and plitidepsin Cremophor1 EL reference solution after single i.v. injection in mice. PK parameters are average values.
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Formulation
t1/2 (h)
AUC0!48h/dose (ng h/mL/mg)
CL (mL/min/kg)
V (L/kg)
MRT (h)
Cremophor1 EL solution PGA-PEG NCs
8.2 17.0
57.9 274.7
157.0 52.7
106.9 77.7
10.5 20.8
dose of plitidepsin-loaded nanocapsules remained in circulation at 48 h after injection, whereas plitidepsin formulated in the Cremophor1 EL solution was below the limit of detection after the sampling time of 8 h. Pharmacokinetic analysis of the plitidepsin levels upon administration of the nanocapsules or the reference formulation is shown in Table 2. Plitidepsin in PGA-PEG nanocapsules exhibited a 2-fold higher half-life (17.0 h) and MRT (20.8 h) compared to plitidepsin dissolved in the Cremophor1 EL solution. The plasma AUC0–24h of plitidepsin-loaded nanocapsules was about 5-fold greater than that obtained with the reference formulation. Moreover, plitidepsin in PGA-PEG nanocapsules showed a 3-fold lower plasmatic cleareance (52.7 mL/min/kg) as compared to the plitidepsin reference solution (157.0 mL/min/kg). In a recent work, plitidepsin-loaded micelles were developed and their pharmacokinetics was also compared with that of the drug dissolved in the Cremophor1 EL solution (Oliveira et al., 2014). These micellar structures, especially those made of poly (trimethylene carbonate)-block-poly(glutamic acid) were able to increase the AUC0–24h of plitidepsin, in an extent almost attaining the double of the reference value. However the half-life was hardly modified (from 8 to 8.6 h), being notably shorter than that showed in the present work. Overall, the pharmacokinetic parameters of plitidepsin-loaded PGA-PEG nanocapsules highlight the long-circulating properties of PGA-PEG in comparison with the reference formulation. It seems clear, as with other PEGylated nanocarriers containing hydrophobic antitumor drugs, that the long circulating properties and high AUC values are related to an important decrease in the clearance of the antitumor drug. This was observed, for example, for PEGylated lipid nanocapsules containing docetaxel, when compared with the commercial formulation Taxotere1 (Khalid et al., 2006), or for PEGylated polymeric micellar and liposomal nanoformulations containing paclitaxel when compared with Taxol1 (Yang et al., 2007; Xiao et al., 2012; Zhang et al., 2012). Moreover, if we examine our results and compare them with those previously reported by us for plitidepsin-loaded PGA-PEG nanocapsules with a low PEG content, it is important to note that the plitdepsin’s AUC was considerably increased and the clearance considerably decreased (a 3-fold change for each parameter) (Gonzalo et al., 2013), demonstrating the effect of the higher PEGylation on the pharmacokinetic behavior. These changes were not reflected, however, in an increase in the half-life or MRT values, as might be expected. In fact, these values remained almost unchanged (t1/2 18 h and MRT 20 h for both high and low PEGylated nanocapsules). The different values of the plitidepsin distribution volumes observed for the two nanoformulations (106.9 L for high-PEGylated and 265.8 L for low-PEGylated nanocapsules) would explain these results. Reports in the literature
suggest that long circulating times and a smaller distribution volume of taxanes are consistent with less extensive antitumor drug distribution in tissues such as liver, spleen or kidneys (Khalid et al., 2006; Yoshizawa et al., 2011). Interestingly, this increased plasmatic residence and lower tissue concentrations were also accompanied by a significant increase in the tumor accumulation measured in tumor-bearing mice (Yoshizawa et al., 2011). On the other hand, these results are in agreement with extensive data in the literature that correlates the long residence time in plasma of taxanes after being administered in a PEGylated nanostructure with an increased deposition of the drug in the tumor via passive targeting (Yang et al., 2007; Senthilkumar et al., 2008; Jing et al., 2014).
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3.3.2. Toxicological evaluation The toxicological study was aimed at establishing the MTD and the MTMD of plitidepsin-loaded PGA-PEG nanocapsules following i.v. injection. The results were compared with those of plitidepsin in Cremophor1 EL reference solution. As observed in Table 3, the MTD of PGA-PEG nanocapsules was 2.5 times higher than that of Cremophor1 EL solution, and higher
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Fig. 5. Evolution of tumor volume median following i.v. multiple administration of plitidepsin-loaded PGA-PEG nanocapsules (~), Cremophor1 EL reference solution (^) and saline serum (&) in a subcutaneously implanted MRI-H-121 human renal xenograft mouse model (total plitidepsin dose was 0.3 mg/kg).
Table 4 Tumor volumes (TV) and mortality in mice bearing MRI-H-121 xenografts treated with multiple i.v. plitidepsin-loaded PGA-PEG nanocapsules and the plitidepsin Cremophor1 EL reference solution. Formulation 1
Table 3 MTD (maximum tolerated dose after single administration) and the MTMD (maximum tolerated dose after multiple administration) of plitidepsin-loaded PGA-PEG nanocapsules and plitidepsin dissolved in the Cremophor1 EL reference solution, in healthy CD-1 male mice following i.v. injection (n = 10/group). Formulation
MTD (mg/kg)
MTMD (mg/kg)
PGA-PEG nanocapsules Cremophor1 EL reference solution
0.75 0.30
0.15 0.30
Cremophor
PGA-PEG NCs
EL solution
Day
TV mm3 median (IQR)
p
Mortality
7 14 21
149 (133.9–268.6) 80.1 (52.7–127.7) 181.3 (144.0–264.9)
0.0001 <0.0001 <0.0001
0/10 0/10 1/10
7 14 21
426.1 (376.5–561.6) 358.8 (275.8–486.0) 154.5 (80.5–236.8)
NS <0.0001 <0.0001
0/10 0/10 0/10
Data are presented as median and interquartile range (IQR). p value for Mann–Whitney U test (control group compared against the rest). NS: not statistically significance; NCs: nanocapsules.
Please cite this article in press as: Lollo, G., et al., Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.02.028
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Table 5 Antitumor effect parameters and activity ranking of plitidepsin-loaded PGA-PEG nanocapsules and plitidepsin Cremophor1 EL formulation after i.v. multiple injection in mice bearing MRI-H121 xenograft during a period of 20 days. NCs: nanocapsules. Formulation
Cremophor1 EL solution PGA-PEG NCs
Dose (mg/kg)
0.30 0.15
DT/DC%
Activity rating
Day 7
Day 14
Day 21
Day 7
Day 14
Day 21
5.8 55.0
9.1 17.2
0.3 1.3
++ –
+++ +
++ ++
Tumor inhibition (+); tumor stasis (++); partial regression (+++).
DT/DC: difference of volumes of the treated groups on day 0 and day X (T0 TX) and those of control group (C0 CX) as reported in Section 2. DT/DC > 50% inactive (). DT/DC > 25–50% tumor inhibition (+). DT/DC < 25% and TX/T0 > 75–125% tumor stasis (++) or TX/T0 > 10–75% partial regression (+++). TX/T0: tumor volume of the compound-treated group on day X and on day 0.
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than their MTMD value. Besides, the MTMD of drug-loaded particles is lower than the value obtained for the Cremophor1 EL formulation (0.15 vs 0.30 mg/kg). The MTD results indicate that nanocapsules are better tolerated than the reference formulation and could be administered at higher unique doses. However, in the case of MTMD values, the opposite occurs, this being in accordance with the pharmacokinetic evaluation above reported. The long circulation properties of plitidepsin-encapsulated into PGA-PEG nanocapsules help us to explain this MTMD reduction. The drug exposure is higher, plitidepsin remains for longer time into the blood circulation and it is eliminated slowly. When plitidepsin is dissolved in Cremophor1 EL, the pharmacokinetic behavior is different, leading to a rapid elimination of the drug. To allow an easier comparison between the two formulations for the in vivo efficacy study, plitidepsin was used around their MTMD values (0.15 and 0.3 mg/kg). 3.3.3. Antitumor activity The in vivo antitumor efficacy of plitidepsin-loaded PGA-PEG nanocapsules was evaluated in a human renal xenograft mouse model (MRI-H-121). A comparative study was performed by dividing animals into 3 groups according to the treatment received (plitidepsin-loaded PGA-PEG nanocapsules, reference formulation and serum) and the schedule of administration established. A strong antitumor activity was seen after the treatment with both plitidepsin formulated in Cremophor1 EL and PGA-PEG nanocapsules (Fig. 5). Moreover, there is a significant difference (p < 0.001) in tumor volume at the day 14 and 21 for plitidepsin-loaded PGA-PEG nanocapsules and the reference Cremophor1 EL solution with the respect to the control, indicating that both formulations had a similar antitumor activity (Table 4). In fact, tumor stasis was recovered at the end of the experiment after the treatment with plitidepsin-loaded PGA-PEG nanocapsules and the reference solution (Table 5). However, neither mortality nor significant changes in body weight were observed throughout the study when the nanocapsules were administered, whereas one animal was found dead in the reference formulation group on day 15. The activity rating and the antitumor effect calculated as DT/DC, and reported in Table 5, highlight the differences in the mechanism of action of the two formulations. Plitidepsin dissolved in the Cremophor1 EL formulation showed a rapid onset of action (delayed tumor growth), but this effect decreased once the treatment was finished, at day 21. The trend of the plitidepsinloaded nanocapsules was slightly different, showing a tendency not only to achieve the stasis of the tumor at the end of the treatment (day 21; Table 4) but also to stop the tumor growth toward the end of the study (day 26; Fig. 5). These different behaviors could be clearly related with the different pharmacokinetic profiles of both formulations, showing the nanocapsules a
more prolonged plasma residence and hence a delayed but apparently more efficient response. Considering the antitumor activity, we suggest that the improved biodistribution profile observed for PGA-PEG nanocapsules as compared to that of the Cremophor1 EL formulation, might be responsible for the passive accumulation of the drug in the pathological site. These results are in agreement with those described for PEGylated nanocarriers which have shown enhancements of the therapeutic index of different antitumor drugs (Hureaux et al., 2010; Kim et al., 2001).
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4. Conclusions
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High PEGylated PGA-PEG nanocapsules were developed as novel carriers for the antitumor hydrophobic drug plitidepsin. Results from the present study demonstrated that the coating of nanocapsules with a high PEGylated PGA-PEG diblock copolymer improved the pharmacokinetic profile of the drug as compared to the reference formulation consisting of a Cremophor1 EL solution. Moreover, plitidepsin-loaded PGA-PEG nanocapsules were better tolerated than plitidepsin formulated in the Cremophor1 EL solution. In vivo antitumor activity in a xenograft tumor model in mice also revealed an important suppression of tumor growth upon multiple i.v. administration, an effect that was comparable to that of the reference formulation. All these data indicate the interest of PGA-PEG nanocapsules as a novel delivery platform for cancer chemotherapy.
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Acknowledgements
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The authors would like to acknowledge financial support from Q5 CENIT-NANOFAR XS53 project, PharmaMar, Spain; the Xunta de Galicia (Competitive Reference Groups-FEDER Funds Ref 2014/ 043) and the European Commission FP7 EraNet-EuroNanoMed Program-Instituto Carlos III (Lymphotarg proyect, Ref. PS09/ 02670). Giovanna Lollo was a recipient of a FPU fellowship from the Ministry of Education of Spain. Marcos Garcia-Fuentes was a recipient Isidro Parga Pondal Fellowship from Xunta de Galicia.
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