Elsevier

Journal of Controlled Release

Volume 276, 28 April 2018, Pages 140-149
Journal of Controlled Release

Readily prepared biodegradable nanoparticles to formulate poorly water soluble drugs improving their pharmacological properties: The example of trabectedin

https://doi.org/10.1016/j.jconrel.2018.03.005Get rights and content

Abstract

The improvement of the pharmacological profile of lipophilic drug formulations is one of the main successes achieved using nanoparticles (NPs) in medicine. However, the complex synthesis procedure and numerous post-processing steps hamper the cost-effective use of these formulations. In this work, an approach which requires only a syringe to produce self-assembling biodegradable and biocompatible poly(caprolactone)-based NPs is developed. The effective synthesis of monodisperse NPs has been made possible by the optimization of the block-copolymer synthesized via a combination of ring opening polymerization and reversible addition-fragmentation chain transfer polymerization. These NPs can be used to formulate lipophilic drugs that are barely soluble in water, such as trabectedin, a potent anticancer therapeutic. Its biodistribution and antitumor activity have been compared with the commercially available formulation Yondelis®. The results indicate that this trabectedin NP formulation performs with the same antitumor activity as Yondelis®, but does not have the drawback of severe local vascular toxicity in the injection site.

Introduction

Polyester-based nanoparticles (NPs) have gained attention in the last decades as biodegradable delivery systems for lipophilic therapeutics in the treatment of a large variety of diseases, e.g. cancer [1,2]. In the treatment of solid tumors, the improvement in the therapeutic index of the drug is achieved by the enhanced permeability and retention (EPR) effect [3]. The leaky vasculature of the tumor blood vessels allows for the passage of macromolecules bigger than 40,000 g/mol, while the impaired lymphatic drainage leads to their retention in the extracellular environment. However, the progress of the nanotechnology has been hampered by the heterogeneity of the solid tumors in the patients [4]. In fact, the pore of the vessel tissues differs both within the same and different types of tumors. In addition, several portions of the tumor tissue may experience hypoxia and completely absence of vasculature or compressed blood vessels, making it impossible to take advantage of the EPR effect. The accumulation of the NPs developed so far in solid tumors is still limited to a median of 0.7% of the administered dose [5]. Target agents are often added to increase the selectivity of the NPs, but the improvement is only minimal. For this reason, several nano-based therapeutics approved by the Food and Drug Administration (FDA), such as Doxil®, have resulted in only a marginal improvement in the overall survival of the patients [6]. The most important results in the NP-based therapeutics approved so far rely on the reduction of the overall toxicity of the formulation by removing toxic excipients and/or solvents that are used to improve their solubility and by reducing the drug related side effects. An example is the paclitaxel (PTX) where the toxic cremophor EL of the Taxol® has been removed in the Abraxane® and Genexol® new formulations [7,8]. However, the complexity related to the NP manufacturing process can lead to an increase in the final cost of these “re-formulations” [9] and problems in batch to batch reproducibility, significantly reducing the availability of these treatments among the patient population. For example, the manufacturing difficulties in the synthesis of Doxil® have led to a drug shortage in the market that the approval of nano-generics have only partially solved [10]. In fact, even in the production of an analogous NP formulation, the same level of quality controls, high-trained specialists and good manufacturing practices (GMPs) are necessary to assure pharmaceutical, clinical and physical equivalence of these nanotherapeutics. These technical issues often dissuade pharmaceutical companies from entering the nanotechnological field [11,12]. For this reason, a proper balance between simplicity and complexity of the drug delivery systems must be taken into account from the very first step of the NP design and development [10]. This is particularly true in the case of polyester based-NPs, which are one of the most diffused class of nanotherapeutics studied in literature due to their biodegradability and biocompatibility [13], but that have resulted in only few products being investigated in clinical trials [11] and entering into the market. They are preferentially synthesized via nanoprecipitation [14] where amphiphilic copolymer and a drug are dissolved in a water-miscible organic solvent that is, in turn, added dropwise to an aqueous solution. The rapid diffusion of the solvent in the bulk phase under turbulent conditions generates polymer NPs able to encapsulate a lipophilic therapeutic in a “one-step” procedure with a relatively high encapsulation efficiency. However, this method presents negative aspects, such as (i) the need of using high amount of organic solvent and sometimes toxic surfactants, (ii) the poor control over the NP size and size distribution, and (iii) the reproducibility issues. In particular, the turbulent conditions in the mixing step have a significant impact on the NP quality [15] and, for this reason, several devices have been developed to improve the contact efficiency between the aqueous and organic phase. These devices are generally micro-channels in which the organic and the aqueous phases are fed in a countercurrent and intimately mixed under turbulent conditions; one example is the T-mixer [16]. In order to eliminate the large amount of organic solvent and the unloaded drug in the system after the drug-loaded NPs are obtained, dialysis must be performed to prevent undesired effects from the cytotoxicity of the solvent and uncontrollable release of the drug. Moreover, after this step, an appropriate storage of the final product is needed. Even though a colloidal suspension is generally stable, some agglomeration can occur, especially when the time between drug loading and administration of the drug-loaded NPs is too long [13]. In addition, water must be removed to avoid NP degradation from hydrolysis of the ester bond, which would compromise the physicochemical stability of the colloids. Among the various methods to increase the stability of the NPs, one of the most commonly used is lyophilization [17]. After complete desiccation, NPs are obtained in the form of a dry powder that is easily handled and stored; in most cases the freeze-dried particles are readily dispersible in aqueous solutions. It is also true, however, that freezing is the most aggressive step of the freeze-drying operation; in order to improve the resistance of the NPs and avoid alteration of the suspension, the addition of a cryoprotectant is required [18]. Consequently, there is a clear need to develop a simpler and reliable method to produce NPs that avoids the use of sophisticated devices and the above mentioned post-production methods, such as dialysis and lyophilization. It is noteworthy to mention that all the above reported post-processing steps are required for the large majority of the nanotherapeutics developed so far, independently from their nature and composition [10], leading to the establishment of complex quality control systems and difficulties in the application of GMPs.

In this work, a library of amphiphilic biodegradable block-copolymers has been developed to formulate lipophilic drugs into NPs directly at the bed of the patient via a simplified nanoprecipitation method that requires only a syringe and a small amount of an organic solvent (i.e. DMSO). This has been possible via the fine-tuning of the hydrophilic/lipophilic balance (HLB) of these self-assembling “comb-like” materials and the optimization of the various components of the final formulation: the drug, the organic solvent, the aqueous medium, and the polymeric surfactant. In particular, the combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and the ring opening polymerization (ROP) have allowed for an additional degree of freedom in the common linear polyester-polyethylen glycol (PEG) block-copolymers [19], which could pave the way to greater control over their structure and molecular weight. The impact of turbulent conditions on the NP formation has been studied by comparing the quality of the NPs obtained via the syringe method and the ones produced via a set-up already reported in literature that uses a mixing device [16]. Then this method has been used to load trabectedin (ET-743) into NPs without the need of any purification steps in order to provide a formulation that can be easily produced by the end-user from the native block-copolymer.

ET-743 is marine alkaloid first isolated from the Caribbean Ecteinascidia turbinate that has been developed in the clinic because of its striking antitumor activity in several preclinical models and its unique mechanism of action [[20], [21], [22]]. It binds to the N2 position of guanines in the minor groove of DNA and affects transcription in a gene and promoter-specific fashion [23]. The drug causes cancer cell death and modifies the tumor microenvironment by reducing the number of tumor associated macrophages and inhibiting the production of inflammatory and angiogenic factors [24,25]. It is approved in Europe for the second line therapy of soft tissue sarcoma and in combination with pegylated liposomal doxorubicin in ovarian cancer patients, and in US for the second line therapy of leiomyosarcomas and liposarcomas. ET-743 cannot be injected in a peripheral vein because it causes severe local toxicity with painful erythema along the blood vessel and sclerotic phlebitis at the site of injection. It therefore requires administration through a central venous catheter [26], a procedure associated with increased health care costs, morbidity and possible serious medical complications, e.g. bloodstream infections. For this reason, ET-743 has been chosen as candidate for this technological platform. The NP-based formulation contains <1.5 vol.% of DMSO and presents the same antitumor activity as the currently approved clinical formulation (Yondelis®), but with a better toxicological profile. In particular, it shows the ability to prevent local toxicity, leaving no sign of phlebitis at the site of the injection and allowing for a classic i.v. administration of the drug compared to the ones currently based on venous catheters. This formulation is expected to reduce the ET-743 related side effects and to significantly improve patient compliance to the treatment.

Section snippets

Materials

ε-caprolactone (CL, 97%), 2-hydroxyethyl methacrylate (HEMA, 97%), stannous octoate [Sn(Oct)2, 98%], Sodium sulfate (Na2SO4, 99%), poly(ethylene glycol) methyl ether methacrylate (PEG45MA, Mn 2000 g/mol, 80 wt.% in water), 4,40-azobis(4-cyanovaleric acid) (ACVA, 98%), 4-cyano-4-(phenylcarbonothioylthio)-pentanoic acid (CPA, >97%), ethanol (EtOH, 99,8%), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF, ≥99.9%), chloroform-d were purchased from Sigma-Aldrich (St. Louis, Missouri, United States)

NP synthesis and formulation optimization

PCL-based block-copolymers were synthesized via two subsequent RAFT polymerizations of a PEG-based methacrylate and of a PCL-based methacrylate, the latter one obtained via the ring opening polymerization of CL in the presence of HEMA as initiator. These block-copolymers are made up of hydrophilic part (PEG) and a biodegradable lipophilic one, so that they can self-assemble into NPs once nanoprecipitated in an aqueous phase. The main advantage of this system compared to the linear PCL-PEG

Conclusions

In conclusion, a nanoprecipitation procedure to formulate lipophilic anticancer therapeutics into NPs directly at the bed of the patient has been developed. This method consists of the use of a syringe with a needle, a small amount of organic solvent and of an amphiphilic block-copolymer. The effective synthesis of NPs has been made possible due to the optimization of the block-copolymer via a combination of RAFT and ROP polymerization and the fine tuning of the main parameters of the final

Conflict of interest

Umberto Capasso Palmiero, Massimo Morbidelli, Maurizio D'Incalci and Davide Moscatelli filed a patent application regarding this technological platform.

Acknowledgements

We acknowledge support from AIRC (10016) Special Program Molecular Clinical Oncology “5 per mille”.

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