Targeting a rare amyloidotic disease through rationally designed polymer conjugates Inmaculada Conejos–Sánchez a , Isabel Cardoso b , Maria J. Saraiva b,c , María J. Vicent a, ⁎ a Polymer Therapeutics Lab., Centro de Investigación Príncipe Felipe (CIPF), C/Eduardo Primo Yúfera 3, Valencia 46012, Spain b Instituto de Biología Molecular e Celular (IBMC), Rua do Campo Alegre 823, Porto 4150-180, Portugal c Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Largo Prof. Aber Salazar 2, Porto 4099-003, Portugal abstract article info Article history: Received 26 November 2013 Accepted 21 January 2014 Available online 31 January 2014 Keywords: Polymer therapeutics Drug delivery Drug design Nanoconjugates Polymer–drug conjugate Rare diseases Saraiva et al. discovered in 2006 a RAGE-based peptide sequence capable of preventing transthyretin (TTR) aggregate-induced cytotoxicity, hallmark of initial stages of an inherited rare amyloidosis known as Familial Amyloidotic Polyneuropathy (FAP). To allow clinical progression of this peptidic sequence as FAP treatment, a family of polymer conjugates has been designed, synthesised and fully characterised. This approach fulfils the strategies defined in the Polymer Therapeutics area as an exhaustive physico-chemical characterisation fitting ac- tivity output towards a novel molecular target that is described here. RAGE peptide acts extracellularly, therefore, no intracellular drug delivery was necessary. PEG was selected as carrier and polymer–drug linker optimisation was then carried out by means of biodegradable (disulphide) and non-biodegradable (amide) covalent bonds. Conjugate size in solution, stability under in vitro and in vivo scenarios and TTR binding affinity through surface plasmon resonance (SPR) was also performed with all synthesised conjugates. In their in vitro evaluation by mon- itoring the activation of caspase-3 in Schwann cells, peptide derivatives demonstrated retention of peptide activ- ity reducing TTR aggregates (TTRagg) cytotoxicity upon conjugation and a greater plasma stability than the parent free peptide. The results also confirmed that a more stable polymer–peptide linker (amide) is required to secure therapeutic efficiency. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Polymer therapeutics are well established as successful first genera- tion nanomedicines for treatment of infectious diseases and cancer [1]. Polymer–protein, drug and aptamer conjugates are innovative chemical entities capable of improving bioactive compound properties and thus increasing efficacy and decreasing toxicity [2,3]. Design of second gener- ation of conjugates is now focussing on improved polymer structures, polymer–based combination therapy and novel molecular targets with great potential to further progress the clinical importance of these unique technologies [4]. Novel conjugates for the treatment of neuro- pathological disorders are proposed in this study. Amyloidosis is well known in the form of Alzheimer's and Parkinson's disease, but the target disease here is a rarer pathological disorder named familial amyloid polyneuropathy (FAP). FAPs constitute an important group of inherited amyloidosis diseases, and one of the most common FAPs is caused by a mutated protein called transthyretin (TTR), which forms amyloid de- posits, mainly in the peripheral nervous system [5]. The aggregation cascade of this mutated protein, produces a TTR aggregate (TTRagg) able to trigger neurodegeneration through engagement with the receptor-for-advanced-glycation-end-products (RAGE) which is pres- ent on peripheral neurons. RAGE signalling has been defined to be in- volved in many human pathologies such as Alzhehimer's disease, diabetes and ageing, among others. This receptor is also up-regulated in tissues from FAP patients [6]. The secreted RAGE form, named soluble RAGE (sRAGE), acts as a decoy to trap ligands and prevent interaction with cell surface receptors. sRAGE was shown to have important inhib- itory effects in several cell cultures and transgenic mouse models, in which it prevented or reversed full-length RAGE signalling. Saraiva et al. [7] discovered a specific peptidic sequence (named RAGE peptide) that is able to suppress TTRagg-induced cytotoxicity in cell culture. A reduced version of that peptide was proved to maintain the activity and the affinity of the initial peptide. The final peptide (compound A) contains 6aa and responds to the sequence (from N to C terminus): YVRVRY. Although this provides an opportunity to design novel therapeutics for FAP treatment, peptide therapeutics themselves display well known challenges for in vivo use, e.g. low stability, poor pharmacokinetics and potential immunogenicity. Moreover the RAGE peptide demonstrates low solubility in plasma limiting its potential for i.v. administration. Thus the aim of this study was to design a polymer–peptide FAP in- hibitor. PEG was chosen for conjugation as it provides flexibility, water- solubility, and biomolecule spatial availability and it sterically protects Journal of Controlled Release 178 (2014) 95–100 ⁎ Corresponding author at: Therapeutics Lab., Centro de Investigación Príncipe Felipe (CIPF), C/Eduardo Primo Yúfera 3, Valencia 46012, Spain. Tel.: +34 963289680; fax: +34 963289701. E-mail address: mjvicent@cipf.es (M.J. Vicent). 0168-3659/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jconrel.2014.01.019 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel