Research paperIn vitro investigation of multidrug nanoparticles for combined therapy with gemcitabine and a tyrosine kinase inhibitor: Together is not better
Graphical abstract
Introduction
Despite the progresses in oncology achieved in the last decade, advancements in the therapy of pancreatic ductal adenocarcinoma (i.e., PDAC, the most common form of pancreatic cancer) [1] have remained extremely limited resulting in a currently relative median survival at 5 years of only 7% [2]. In 2015, with 48960 estimated new cases and 40560 estimated deaths, PDAC still remained the fourth main cause of death in the USA [2]. Although surgery represents a curative option, only about 20% of pancreatic cancer patients is eligible to surgery while for the others, diagnosed at an unresectable, advanced and metastatic stage, only palliative treatments are available at the moment [3]. Since its FDA approval in 1996, gemcitabine (Gem), a nucleoside analogue which stops DNA replication, is the treatment of choice in the clinic, although only a limited response rate and survival benefits are observed [4], [5], [6]. The limited efficacy of gemcitabine mainly arises from the rapid blood metabolization of this drug, which dramatically decreases its plasma half-life [7], the downregulation of its membrane transporters responsible for a limited cellular internalization [8], [9], and the insurgence of various resistance phenomena [10], [11]. In addition, the formation of a dense stroma [12], the limited tumor tissue vascularization and the heterogeneity of pancreatic cancer cells, displaying several genetic alterations [13], dramatically hamper drug efficacy and drug bioavailability in the tumor [3]. According to the complex physiology of the pancreatic tumor and the surrounding microenvironment, improvement of the therapeutic outcomes might be achieved with the simultaneous co-administration of two or more drugs (i.e., combined therapy).
The co-delivery of drugs with distinct mechanism of action and the targeting of diverse cell types and different signaling pathways involved in tumor growth and metastatic dissemination may induce synergistic effects. Such approach may reduce the effective dose required for each single drug thus decreasing the associated undesired side effects. Accordingly, combined protocol therapies of platinum based agents [14], taxanes [15], topoisomerase or tyrosine-kynase inhibitors (TKIs) [16], [17] with gemcitabine have been tested in randomized clinical trials for treatment of PDAC, leading to variable outcome improvements [18], [19], [20]. Among them, TKIs appear as an attractive therapeutic strategy due their broad spectrum of action and the capacity to affect angiogenesis as well as tumor progression and metastatic dissemination. This results from the inhibition of the activation of several signaling transduction cascades following competition with ATP for binding to the intracellular pocket of various tyrosine kinase receptors whose ligands are growth factors such as the epidermal (EGF), the vascular endothelial (VEGF) and the platelet derived (PDGF) ones [21], [22], [23]. Overexpression of these receptors and their ligands are both hallmarks of aggressiveness and poor prognosis in pancreatic cancer [24], [25].
Combined therapy of gemcitabine and erlotinib (Tarceva®), a TKI specifically acting on the epidermal growth factor receptor-1, resulted in a significant increase of the overall survival and has been approved by FDA for pancreatic cancer treatment in 2005 [17], [26]. In parallel, attention has also focused on sunitinib (Sun), a multitargeted TKI which acts on the VEGF types 1 and 2, the PDGF α and β, the FLT3 (Fms-like tyrosine kinase 3) and the SCF (stem-cell factor) receptors [27]. Sunitinib antitumor efficacy has been observed in various tumor types and results from both antiangiogenic and antiproliferative activity [27], [28], [29], [30]. Sunitinib is currently approved in monotherapy for oral treatment of advanced cell renal carcinoma and imatinib-refractory gastrointestinal stromal tumors [31]. In combined therapy with gemcitabine it has been proposed for the treatment of pancreatic ductal adenocarcinoma in preclinical [32], [33] and in clinical studies [34]. However, sunitinib therapy is associated to the insurgence of a plethora of side effects including gastrointestinal toxicity (nausea, diarrhea, stomatitis), dermatological (e.g., skin discoloration, hand-foot syndrome) and hematological events (e.g., anemia), cardiac effects (hypertension), fatigue [35], [36], [37]. Despite the possible interest of combined therapies in order to reduce sunitinib dosage and the resulting adverse effects, the safety profile of any combination may represent a real challenge.
In this context the delivery of drugs using nanoscale systems (i.e., nanomedicines) constitutes a valuable option by controlling drug release, distribution, bioavailability and thus improving the therapeutic index [38], [39]. Whereas variously engineered sunitinib and sunitinib analogues-loaded nanocarriers have been already designed [40], [41], [42], nanodevices could be further used for the development of a combined therapy [43] enabling the co-existence in a single nanocarrier of drugs with different mechanisms of action and pharmacokinetic profiles and finely tuning their release rate overcoming the rapid clearance often observed with free drugs [44]. Moreover, this approach holds the promise to modify favorably the biodistribution of each drug and improve their therapeutic efficacy.
In this perspective, we have designed a novel multidrug nanocarrier for combined delivery of sunitinib and gemcitabine, which could yield to a maximal benefit thanks to the distinct mechanism of action of these two drugs. The so called squalenoylation approach, (i.e., synthesis of bioconjugates by covalently coupling the drug molecule to the squalene and their further self-assembly as nanoparticles) has been already successfully applied to the gemcitabine (squalene-gemcitabine bioconjugate, SQGem) [45] with dramatic improvement in the anticancer activity in various pre-clinical models [46], [47], [48]. This procedure has been used herein to synthetize a squalene-derivative of sunitinib (SQSun) by conjugation to the squalene via a hemiaminal spacer bound to the oxindole moiety [49]. Then, multidrug nanoparticles (SQGem/SQSun NPs) have been constructed by self-assembly of the two bioconjugates in water.
We formulated the initial hypothesis that the co-administration of gemcitabine and sunitinib in form of a multidrug squalene-based NP may give rise to increased anticancer activity. Thus the cytotoxicity of these multidrug SQGem/SQSun NPs has been tested in vitro on pancreatic cancer cells and compared to monodrug NPs to investigate whether the co-delivery of the two drugs could offer an overall improvement of the response to the treatment.
Section snippets
Chemicals and instruments
Gemcitabine (2′,2′-difluorodeoxycytidine, Gem (1)) hydrochloride and sunitinib malate (2) were bought from Sequoia Research Products Ltd. (Pangbourne, UK). 4-(N)-trisnorsqualenoyl-gemcitabine (SQGem, (3)) was synthesized as previously reported [50]. Squalene (SQ), p-toluenesulfonic acid, cesium carbonate, chloromethoxy-trisopropylsilane, tetrabutylammonium fluoride and all other reagents were obtained by Sigma Aldrich (France). All solvents (analytical grade) were purchased from VWR (France).
Results and discussion
With the aim to realize a concerted delivery of gemcitabine (1) and sunitinib (2), the squalenoylation approach was applied to both drugs (3 and 4) (Fig. 1).
The SQSun conjugate 4 was designed with an easily cleavable hemiaminal linker. As previously described [49], 4 was obtained through a three-step sequence from sunitinib base (5) involving: (i) alkylation of the nitrogen atom of the oxindole ring with chloromethoxy-triisopropylsilane in the presence of cesium carbonate, (ii) deprotection of
Conclusion
Multidrug nanoparticles for combined delivery of gemcitabine and sunitinib according to a metronomic schedule have been successfully formulated by simple self-assembly of the squalenoylated derivatives of the two drugs. Exposure to SQGem/SQSun NPs resulted in higher inhibition of cell proliferation compared to the NPs in monotherapy. However, combined therapy with multidrug NPs (SQGem/SQSun NPs) was as effective as the physical mixture of monodrug NPs (SQGem NPs + SQSun NPs), probably as a
Acknowledgements
The research leading to these results has received funding from the European Research Council under the European Community's Seventh Framework Programme FP7/2007-2013 Grant Agreement N°249835. This work was also supported by MIUR - University of Turin “Fondi Ricerca Locale (ex-60%)”. The authors acknowledge the Università Italo Francese/Université Franco Italienne for the PhD co-tutoring agreement to S.V. The authors thank the service of electron microscopy (IBPS FR 3631) for the cryo-TEM
References (67)
- et al.
Tumour-stroma interactions in pancreatic ductal adenocarcinoma: rationale and current evidence for new therapeutic strategies
Cancer Treat. Rev.
(2014) - et al.
Second-line treatment in advanced pancreatic cancer: a comprehensive analysis of published clinical trials
Ann. Oncol.
(2013) - et al.
SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo
Blood
(2003) - et al.
Antitumour activity of sunitinib in combination with gemcitabine in experimental pancreatic cancer
HPB Oxf.
(2011) - et al.
Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib
Lancet
(2007) - et al.
Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial
Lancet
(2006) - et al.
Nanotechnology-based combinational drug delivery: an emerging approach for cancer therapy
Drug Discov. Today
(2012) - et al.
Squalenoylation: a generic platform for nanoparticular drug delivery
J. Control. Release
(2012) - et al.
Squalenoyl gemcitabine nanomedicine overcomes the low efficacy of gemcitabine therapy in pancreatic cancer
Nanomedicine
(2011) - et al.
Synthesis and cytotoxic activity of self-assembling squalene conjugates of 3-[(Pyrrol-2-yl)methylidene]-2,3-dihydro-1H-indol-2-one anticancer agents
Eur. J. Org. Chem.
(2015)
Nanocapsule formation by interfacial polymer deposition following solvent displacement
Int. J. Pharm.
Polyisoprenoyl gemcitabine conjugates self assemble as nanoparticles, useful for cancer therapy
Cancer Lett.
Simultaneous determination of gemcitabine and gemcitabine-squalene by liquid chromatography-tandem mass spectrometry in human plasma
J. Chromatogr. B
Metronomic scheduling: the future of chemotherapy?
Lancet Oncol.
SU11248 (sunitinib) sensitizes pancreatic cancer to the cytotoxic effects of ionizing radiation, Int J Radiat
Oncol. Biol. Phys.
Sunitinib malate (SU-11248) alone or in combination with low-dose docetaxel inhibits the growth of DU-145 prostate cancer xenografts
Cancer Lett.
Interaction of an amphiphilic squalenoyl prodrug of gemcitabine with cellular membranes
Eur. J. Pharm. Biopharm.
Transmembrane diffusion of gemcitabine by a nanoparticulate squalenoyl prodrug: an original drug delivery pathway
J. Control. Release
Pancreatic cancer
N. Engl. J. Med.
Cancer statistics, 2015
CA Cancer J. Clin.
Pancreatic adenocarcinoma
N. Engl. J. Med.
Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial
J. Clin. Oncol.
First-line treatment for advanced pancreatic cancer. Highlights from the “2011 ASCO Gastrointestinal Cancers Symposium”. San Francisco, CA, USA. January 20-22, 2011
JOP
First-line treatment for advanced pancreatic cancer
JOP
Preclinical, pharmacologic, and phase I studies of gemcitabine
Semin. Oncol.
Equilibrative-sensitive nucleoside transporter and its role in gemcitabine sensitivity
Cancer Res.
Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines
Cancer Res.
Mechanisms underlying gemcitabine resistance in pancreatic Cancer and sensitisation by the iMiD™ lenalidomide
Anticancer Res.
Gemcitabine resistance in pancreatic cancer: picking the key players
Clin. Cancer Res.
Core signaling pathways in human pancreatic cancers revealed by global genomic analyses
Science
Gemcitabine in combination with oxaliplatin compared with gemcitabine alone in locally advanced or metastatic pancreatic Cancer: results of a GERCOR and GISCAD phase III trial
J. Clin. Oncol.
Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine
N. Engl. J. Med.
Irinotecan plus gemcitabine results in No survival advantage compared with gemcitabine monotherapy in patients with locally advanced or metastatic pancreatic Cancer despite increased tumor response rate
J. Clin. Oncol.
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