Polymer-drug conjugates as nano-sized medicines

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Polymer–drug conjugates as nano-sized medicines Fabiana Canal, Joaquin Sanchis and Marı´a J Vicent Polymer Therapeutics have enormously evolved in the past decades. Several polymeric drugs as well as polymer–protein conjugates have been in the market since the 90s, but although polymer–drug conjugates are already in clinical trials they still need to reach this final goal. There are four main convergent strategies to move this platform technology further. First, exploitation of new molecular targets in cancer therapy and design of polymer–drug conjugates as treatments for other diseases. Second, the development of combination therapy. Third, attempts to improve polymer chemistry, including the use of new well-defined architectures and the optimization of the advanced characterization techniques essential to transform a promising conjugate into a candidate for clinical evaluation. Finally, increased understanding of polymer conjugate features that govern clinical risk–benefit is leading to an appreciation of clinical biomarkers that will open new possibilities for personalized therapy. Address Polymer Therapeutics Laboratory, Centro de Investigacio´n Prı´ncipe Felipe, Av. Autopista del Saler 16, E-46012 Valencia, Spain Corresponding author: Vicent, Marı´a J ([email protected])

Current Opinion in Biotechnology 2011, 22:894–900 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Luis Angel Ferna´ndez and Serge Muyldermans Available online 1st July 2011 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.06.003

main strategies: 1) design of innovative polymer conjugates targeted to new molecular targets, 2) the search for better physico-chemical characterization methods paying special attention to conformational issues in solution, 3) the synthesis of new polymeric carriers with defined architecture and, 4) the use of polymer-based combination therapy. One of the main goals, as with all other nanopharmaceutics, is achieving better individualized therapies [6]. Although polymer–drug conjugates have been explored for more than 30 years, they have not yet reached the market. We believe that this landmark will soon be achieved following on from the success of PEGylated proteins. Important lessons have been learnt and the exponentially growing number of compounds entering clinical trials clearly reflects the strong scientific foundations of this discipline [3,4] (Box 2). This fact, together with industrial acceptance of the need to work together with regulatory agencies at the early stages of product development, will undoubtedly push these nanopharmaceutics forward [7]. In this manuscript the current challenges and future opportunities of polymer–drug conjugates are reviewed focusing on the above-mentioned four research strategies. Most of the examples that will be reported are in early preclinical–clinical development but it is hoped the progress towards clinically useful medicines will be faster than seen previously for those conjugates currently undergoing late stage clinical trial. There is special discussion of the potential opportunities for personalized medicine in relation to the current industrial pipeline.

Towards new molecular targets Introduction It is 20 years since the term ‘Polymer Therapeutics’ was coined by Prof. Ruth Duncan to define a family of new chemical entities (NCEs) considered the first polymeric nanomedicines [1,2]. In this short period of time, the number of publications in the field has been exponentially growing [3]. Polymer Therapeutics comprise a variety of complex macromolecular systems, their common feature being the presence of a rationally designed covalent chemical bond between a water-soluble polymeric carrier and the bioactive molecule(s) (Box 1). Drug conjugation to a polymer not only enhances its aqueous solubility but also changes drug pharmacokinetics at the whole organism and even subcellular level with the possibility to clearly enhance drug therapeutic value [4,5]. The new research trends in this field fall into four Current Opinion in Biotechnology 2011, 22:894–900

At the beginning, development of polymer–drug conjugates was strongly focused towards cancer therapy. In fact, 15 out of the 16 conjugates currently in clinical trials were designed as anticancer agents. All of them involve conjugation of orthodox chemotherapeutic agents (i.e. doxorubicin (Dox), taxols (PTX), camptothecin (CPT) or platinates (Pt)) [3]. Recent developments emerging from genomics and proteomics research has led to increased understanding of the molecular mechanisms involved in tumor pathogenesis. Therefore, new molecular targets have become available for exploitation in the design of more effective therapies. These new lines mainly rely on the inhibition of specific kinases, activation of apoptosis pathways or angiogenesis modulation [8]. It is expected that the incorporation of drugs directed towards new molecular targets in cancer will result in a significant improvement in polymer conjugate performance. www.sciencedirect.com

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Box 1 Definition of Polymer Therapeutics Polymer Therapeutics can be considered amongst the first polymeric nanomedicines (5–100 nm) [1]. The definition involves rationally designed macromolecular drugs and encompasses polymeric drugs (polymers with inherent activity), polymer–drug conjugates, polymer– protein conjugates, polymeric micelles to which drug is covalently bound, and polyplexes designed as non-viral vectors for gene delivery. From the industrial standpoint, these nanosized medicines are considered NCEs and not conventional pharmaceutical formulations or drug delivery systems that simply physically entrap the drug. Clinical benefit of polymer therapeutics has been already demonstrated with the successful application of polymer–protein conjugates of routine clinical use, and promising results arising from clinical trials with polymer-bound chemotherapy. These facts have laid a firm foundation for more sophisticated second generation constructs.

By direct inhibition, N-(2-hydroxypropylmethacrylamide) (HPMA) copolymer wortmannin conjugates led to an effective blockage of PI-3 kinase [9], and alternatively, geldanamycin conjugates have shown indirect inhibition of several kinases involved in cancer cell survival by inhibition of the chaperone HSP-90 [10]. Therapies focused on the activation of apoptotic pathways are also promising anticancer strategies [11]. The PEGylation of curcumin, a Jab1 inhibitor, and the conjugation of bcl2-inhibitor HA14 to HPMA, are two examples of this proapoptotic approach Box 2 Biological rational behind the design of polymer–drug conjugates Combination of De Duve’s realization that the endocytic pathway might be useful for ‘lysosomotropic drug delivery’ and Ringsdorf’s vision of the idealized polymer chemistry for drug conjugation gave birth to the concept of polymer–drug conjugates in the 70s. Consequently, a collaborative work between Ruth Duncan and Jindrich Kopecek resulted in the clinical evaluation of HPMA copolymer–Dox conjugate (FCE28068, PK1) in 1994, making this conjugate the first synthetic polymer–anticancer drug conjugate to be tested in humans [reviewed in [1,3–6,52]]. Apart from enhancing the aqueous solubility of hydrophobic drugs polymer–drug conjugation promotes passive tumour targeting by the enhanced permeability and retention (EPR) effect and allows for lysosomotropic drug delivery following endocytic capture whilst limiting access to the normal sites of toxicity. After intravenous administration, the ‘leakiness’ of angiogenic tumor vasculature allows selective extravasation of the conjugate in tumor tissue. Additionally, tumor tissue frequently lacks of an effective lymphatic drainage, which subsequently promotes polymer retention. The combination of both factors leads to conjugate accumulation in tumor tissue, a passive targeting phenomenon named by Maeda as EPR effect. EPR-mediated tumor targeting is driven by the plasma concentration of circulating polymer conjugate. Active targeting could be also achieved by the incorporation of additional specific residues within the polymer carrier inducing a receptor-mediated endocytic uptake. It is clear then the crucial role of an adequate polymer–drug(s) linker(s) design. The ideal linker should be stable in blood, but able to release drug at an optimum rate on arrival. As many of the drugs being transported exert their effects via an intracellular pharmacological receptor so it is essential that drug release occurs.

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[12,13]. Anti-angiogenic therapies are already a clinical reality [14,15]. After the first reported anti-angiogenic conjugate, HPMA copolymer-TNP470 by Satchi-Fainaro et al. [16], several conjugates have been developed as single agents or for use as combination therapy (comprehensively reviewed in [17]). Looking at targeting bone metastasis, TNP-470–alendronate and paclitaxel (PTX)– alendronate combination conjugates were described with remarkable preclinical results [16,18,19]. PTX and TNP470 have also been combined with RGD-based peptides, not only owing to their active targeting effect on avb3 integrins but also owing to the inherent anti-angiogenic properties of RGD-peptidomimetics [20]. An even more interesting approach is the growing design of new therapeutic concepts such as the use of coiled-coil systems [21] or conjugates as treatments for other diseases including, infections [22], inflammation [23], rheumatoid arthritis [24] and diabetes [25]. In addition we would particularly like to stress the potential of polymer–drug conjugates for use in regenerative medicine. Advances in understanding of mechanisms for disease-specific targeting and the progressive appearance of new, and more specific carriers should increase the possibility of bringing the active molecule to virtually any biological compartment in the human body. Several preclinical studies have already illustrated potential of conjugates in regenerative medicine, for example, for wound healing [26], ischemia [27] or osteoporosis [28]. More ambitious targets, such as cardiac regeneration or neurodegenerative disorders are the focus of ongoing research projects, and first studies should come to fruition in the near future. This so far, less explored research area constitutes one of the most exciting fields for the future [8].

Polymer-based combination therapy The complex molecular basis of human pathologies often means that the application of single agent therapy is insufficient for effective and sustained therapy. Frequently, more than one drug or type of treatment is used in order to improve therapeutic outcome. Therefore, delivery strategies that simultaneously co-transport the required drugs to the target disease can be advantageous. This is the rationale behind the so-called polymer-based combination therapy (comprehensively reviewed in [29]). Polymer–drug conjugates can be used in combination therapy in four distinct situations. These include, polymer–drug conjugate + free drug (type 1), polymer–drug conjugate + polymer–drug conjugate (type 2), single polymer carrying a combination of drugs (type 3) and, polymer-directed enzyme prodrug therapy (PDEPT, type 4). Although synthesis of a single polymeric carrier bearing a combination of drugs could be more costly than the co-administration of single drugs the clinical benefits resulting from drug synergism could justify the effort [29]. Up until now, only type 1 combinations have been Current Opinion in Biotechnology 2011, 22:894–900

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clinically explored [30,31]. This approach, together with type 2 and 4, has been classically preferred as greater industrial feasibility could be expected. However, type 3 combination conjugates have a significant advantage as this is the only approach that can guarantee simultaneous delivery of both drugs to the same site of action, and with careful design, can enable synergistic drug effects. There is already proof of concept in several studies (reviewed in [29,32]; landmark examples in [33,34]), including the anti-angiogenic combination conjugates discussed above [18–20]. However, it is important to note that there are many fundamental questions that must be addressed in order to optimize polymer-based combinations. First, when selecting drugs for combination, it is important to consider agonistic or synergistic interactions, the best drug ratio, and the capacity of the chosen polymer to achieve the desired drug payload. The linker must be carefully designed to ensure suitable drug release kinetics for each agent. Being that these systems are particularly complex, the physico-chemical characterization, as well as the correlation of in vitro studies with their in vivo behavior are not trivial. The challenges for transfer of these complex nanomedicines into the clinical trial have been the subject of much discussion. Although not with a conjugate, it is important to note that Celator Technologies Inc. have already progressed a liposomal combination therapy into Phase II and this technology was considered by the FDA as a ‘single agent’ in respect of its clinical development path [http://www.celator.ca/new/products.html]. This paves the way for advance of first polymer combination therapy into clinical trials [2,7].

yielded new and more complex but well defined structures [37–39]. However, not only their molecular structure is important, but also their supramolecular conformation adopted in aqueous solution [40]. It is worth pointing out, as an example, the case of polyglycerol dendrons [41]. Among the linear polymers new promising carriers have recently (re-)emerged. This is the case of the pHresponsive polyacetal Fleximer1 [http://mersana.com] or the non-biodegradable POx (poly(2-alkyloxazoline)), a polymer known since 1966 but almost forgotten in the past decades. In addition to its PEG-like properties, POx shows thermosensitive response, can be easily functionalized and it offers the possibility of high drug payload [42]. HESylation (hydroxyethyl starch conjugation) is showing very promising results in preclinical studies, and its implementation for small drugs is also being investigated especially to reduce its natural high polydispersity [43]. It is considered important to emphasize also, the crucial role of the polymer–drug(s) linker design in conjugate development. Apart from the classical cathepsin B responsive linkers, novel enzymatically labile or pH-labile linkers are required to achieve more sophisticated and controlled constructs. In addition, the need for more sophisticated methodology of characterization owing to the increasing complexity of the final structures cannot be neglected (see paragraph below) [44]. The final size and shape of the entire system could affect the biological behavior, at the level of the whole body or at cellular level. Thus, the more globular, larger structures might give a faster internalization but also a faster clearance by the reticulum endoplasmic system (RES). So far the only dendrimer evaluated clinically following systemic administration have been developed for MRI imaging, while the linear polymers have been evaluated most often as drug conjugates [36,40].

Controlled architecture and new carriers A wide variety of natural and synthetic polymers have already been described as carriers for drug delivery. However, very few have been successfully transferred into patients [3] (Figure 1). Unfortunately, there is still a gap between advanced polymer chemistry disciplines and the understanding of the risk–benefit principles required for selection of novel polymer structures that are practical for transfer into the clinic. We are convinced that novel advances in polymer and organic chemistry can provide a new generation of polymeric platforms with more complex and defined architectures to be used in specific applications. However, the total control of crucial parameters such as molecular weight, polydispersity, localization of charge or hydrophobicity–hydrophilicity balance is a must in order to tune the conjugate biodistribution, fate, biological activity and toxicity [35,36]. It is important to remark that, polymer chemists should always base their carrier designs on an adequate biological rational, safety profile and industrial feasibility. New synthetic chemistry approaches, such as click chemistry or controlled polymerization methods, have already Current Opinion in Biotechnology 2011, 22:894–900

We believe that an effective multidisciplinary dialog between polymer, physical and organic chemists, together with biologists and physicians is the real key to fast optimization of polymer conjugate design at the frontiers of medical need. This will accelerate progression to clinical use.

Importance of an exhaustive physicochemical characterization New polymeric platforms have the potential to bring more controlled pharmacokinetics thus resulting in a more efficient drug delivery to the desired cell/compartment. However, the conjugate complexity means they require very careful physico-chemical characterization before biological evaluation. Moreover, uncertainties regarding composition will also lead to regulatory concerns. For a polymer–drug conjugate, conjugate size and molecular weight are not the only key questions to be addressed, and other important factors include polydispersity, Zeta potential and charge distribution, hydrophilic–hydrophobic balance, shape, drug impurities and the drug payload. The fact that these features will all govern www.sciencedirect.com

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Figure 1 Doxorubicin Paclitaxel Platinates Camptothecin

New strategies in cancer Control of angiogenesis Apoptosis Regenerative Medicine

ORTHODOX ANTICANCER DRUGS

NEW TARGETS

COMBINATION THERAPY

POLYMER-DRUG CONJUGATES

FCE 28068, FCE 28069, -

-

REGULATORY ISSUES

ProLindac , Opaxio , NKTR-102........

CLASSICAL POLYMERS

INDUSTRIAL PIPELINES

INDIVIDUALIZED THERAPY

CONTROLLED ARCHITECTURES & NEW CARRIERS

PEG HPMA copolymer PGA

POx, HES RAFT, ATRP, NCA

CHARACTERIZATION Spectroscopy Chromatography Scattering techniques Microscopy Electrical techniques Spectrometry

Current Opinion in Biotechnology

Current state-of the art technology with polymer–drug conjugate relies on strong foundations coming from 30 years of interdisciplinary research. 16 conjugates have already been transferred to the clinics, consequently, a rich industrial pipeline is currently available and exponentially growing. It is accepted that future challenges and opportunities to move this platform technology forward are based on four main strategies: 1) focusing on new molecular targets in cancer as well as other diseases, 2) polymer-based combination therapy, 3) control on polymeric platforms and their conformational behavior in solution and 4) and exhaustive physico-chemical characterization essential to transform a promising conjugate into a candidate for clinical evaluation following regulatory indications. Increased understanding of polymer conjugate features that govern clinical risk– benefit is leading to an appreciation of clinical biomarkers that will open new possibilities for personalized therapy.

clinical risk–benefit means they play an important role determining whether or not a conjugate can progress towards clinical development [7,35]. In addition to the classical methodologies used to characterize polymers, the fast advance of the physical and computational methods is bringing forward a great variety of novel tools that can provide important and complementary information. More sophisticated techniques are currently being used mainly looking at conjugate conformation properties in solution (reviewed in [6,36]). In particular, scattering techniques, such as, Small-Angle Neutron Scattering (SANS) has already showed to be a valuable tool for determining the structure activity relationships in conjugates already in the clinic [45]; and even in complex combination conjugates [33]. Although examples are scarce, the potential of Nuclear Magnetic Resonance (NMR) for the characterization of polymer–drug conjugates has been clearly exemplified for several conjugates including clinical candidates. Special NMR techniques in solution, as pulse-field gradient www.sciencedirect.com

spin-echo NMR experiments, will also help to elucidate the size, morphology and dynamics [6]. In order to reveal the fate of polymer–drug conjugates in vitro and in vivo, sophisticated optical imaging techniques [46,47] are already recognized tools, however with high risk of artifact contamination. Electron Paramagnetic Resonance (EPR) is a very promising alternative [48] as already demonstrated with other type of nanopharmaceutics [49,50] – a direct transfer to polymer–drug conjugates is readily expected. Even though a great number of techniques are available, we still miss universal protocols for characterizing a polymer–drug conjugate, specially coming from recommendations of the regulatory agencies.

Industrial pipeline. Towards individualized therapy Translational research in polymer therapeutics has yielded several compounds into the market, especially polymer–protein conjugates currently in routine clinical Current Opinion in Biotechnology 2011, 22:894–900

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use [3]. In the case of polymer–drug conjugates progression to regulatory approval has been slower. Clinical failures with MAG-CPTTM (HPMA copolymer-CPT conjugate) and PNU166945TM (HPMA copolymerPTX conjugate) owing to a wrong conjugate rational design that yielded to unspecific drug release [51,52], commercial issues in the case of FCE28068 and FCE28069 [51,52] or the lack of activity found in early Phase III trials with OpaxioTM [31] have been some of the issues responsible for this delay. Nevertheless, up to 16 of these compounds are nowadays in advanced clinical trials [3]. The closest to market is OpaxioTM (also known as Xyotax or CT-2103) developed by Cell Therapeutics Inc. [http://www.celltherapeutics.com/opaxio]. This case is of particular interest as clearly shows the importance of understanding conjugate features that govern clinical risk–benefit. After a careful analysis of data from Phase III trials, the therapeutic value of OpaxioTM as anticancer agent turned out to be gender-dependent showing increased survival in women but not in men. A correlation between oestrogen levels and cathepsin B activity, responsible of PTX release, has subsequently been reported and this enzyme is now used as a clinical biomarker guiding ongoing trials that only enroll chemotherapy-naive advanced NSCLC female patients with estradiol levels greater than 25 pg/mL [31]. After the first examples, it is clear now for the pharmaceutical industry that an appreciation of clinical biomarkers will open new possibilities for personalized therapy with these nanopharmaceutics, allowing the selection of patients with more possibilities to benefit from therapy and less probability to suffer side-effects accelerating the progress towards conjugate commercialization. OpaxioTM sister conjugate CT-2106, PGA-CPT, is also in Phase II. Other companies are supporting promising conjugates in Phase II, which will soon become strong candidates to get the certification. One example is ProLindacTM, an HPMA copolymer DACH-platinate from Access Pharmaceuticals that has successfully completed a European Phase II trial in patients with ovarian cancer [52] [http://www.accesspharma.com]. Nektar Therapeutics’ contribution consists of three PEGylated products, NKTR-102 and NKTR-105 for cancer applications and NKTR-118 for opioid-induced constipation (the only one designed for oral administration) [http://Nektar.com]. Mersana is pursuing the commercializing of XMT1001 (Fleximer1-CPT) as its lead candidate but has also a potent anti-angiogenic conjugate XMT1107 (Fleximer1fumagillin) in Phase I. Cerulean Pharma Inc. is currently developing the first conjugate administered as a supramolecular assembled particle of 30 nm, CRLX101 (formerly IT-101) a cyclodextrin-CPT nanoparticle advancing in Phase 2a studies [http://ceruleanrx.com]. Many other small biotech companies are emerging exponentially with time in order to develop novel nanoCurrent Opinion in Biotechnology 2011, 22:894–900

medicines. One example is Serina Therapeutics Inc., a relatively new company with compounds based on a new polymeric carrier, POx, in preclinical early development. Its pipeline includes three anticancer conjugates (Ser201, Ser203 and Ser207) [http://www.serinatherapeutics.com/ pipeline.shtml]. We believe POx, owing to its versatility, controlled polymerization and drug-loading capacity, may become a PEG substitute enlarging the possibilities for new treatments [35,42].

Conclusions Current state of art polymer therapeutics relies on strong foundations coming from 30 years of interdisciplinary research from the bench to the bedside, they can be considered amongst the most successful polymeric nanomedicines. There are a growing number of polymer therapeutics that are products and also entering clinical development as both novel treatments and imaging agents. They are used as Nano-sized Medicines in the form of individual agents or conjugates or as components of complex, self-assembling nanoparticles and micelles. Consequently, an exponentially growing industrial pipeline is currently available in big pharmaceutical companies as well as in small biotechnologies devoted to specific nanoconjugates. It is accepted that future challenges and opportunities to move this platform technology forward are based on new molecular targets in cancer as well as other diseases, polymer-based combination therapy, control on polymeric platforms and their conformational behavior in solution and exhaustive physico-chemical characterization essential to transform a promising conjugate into a candidate for clinical evaluation following regulatory indications. Therefore, there is a need for continued development of validated analytical techniques for characterization of these complex nano-sized medicines and validated methods for establishing preclinical safety. Increased understanding of polymer conjugate features that govern clinical risk–benefit is leading to an appreciation of clinical biomarkers that will open new possibilities for personalized therapy and optimized clinical trial design.

Acknowledgements We thank Prof. Ruth Duncan for critical comments on the manuscript. We acknowledge support by Centro de Investigacio´n Prı´ncipe Felipe and MICINN (EUI2008-03904). MJV is a Ramon y Cajal researcher.

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Current Opinion in Biotechnology 2011, 22:894–900

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