Polymer Drug Conjugates for the Treatment of Neurodegenerative Disorders

The design of second generation polymer conjugates is directed towards four main research areas: the synthesis of novel polymeric carriers with defined architectures, an exhaustive physico-chemical characterization through leading techniques, the use of combination therapy pursuing synergism and improved treatments and their application on novel molecular targets other than cancer, including the crossing of different biological barriers. These guidelines have been addressed in this thesis in which the main purpose was to build polymer-drug conjugates (PDCs) for the treatment of neurodegenerative disorders. Based on the well established properties of the poly-glutamic acid (PGA) as polymer carrier, a versatile and simple methodology for the preparation of well-defined polyglutamate nanocarriers has been described. For the first time non-nucleophilic tetrafluoroborate anions were used as NCA initiators allowing a multigram scale polyglutamate synthesis with defined molecular weight (up to 1200 units), low polydispersity (<1.2) and controlled end-chain functionality. Systems obtained encompass homopolymers (PGAn), diblocks (DB, PEGm-PGAn, PGAn-PEG-X) and triblock copolymers (TB, PEG-PGAn-PEG-X). The reason behind this design is based on: (i) the availability of points for site-specific conjugation (group X) ( for peptide, protein, antibody, etc binding), (ii) the study of the different cell trafficking and (iii) in vivo fate depending on polymer solution conformation and its effect on the expected biological output. In addition, deprotection methodology of PBLG (poly-benzyl-L-glutamates) was optimised yielding 100% benzyl removal without damaging co-polymers structure as well as maintenance of the adequate stereoselectivity. Then, post-polymerisation modification on the PGA block enabled specific and orthogonal conjugation of different bioactive molecules such as targeting ligands, labelling probes or drugs. Cytotoxicity of the novel nanocarriers was evaluated in HUVEC cells to secure their cell viability. Continuing with the in vitro assays,investigation of the precise mechanism of internalisation in the same cell model was performed aiming to explore possible differences based on the size and shape in solution of the different carriers. In all cases endocytosis was the preferred cell uptake mechanism and a lysosomotropic drug delivery was observed. Historically, many studies with PDCs have been focused on cancer treatment. Once the clinical benefits of these systems have been demonstrated, their use in other diseases has been also encouraged, in particular in the area of tissue regeneration and repair. In the present work, neurodegenerative disorders have been faced up. When damage in the central nervous system (CNS) is involved, drug must surpass a biological obstacle known as blood-brain barrier (BBB). Conjugation of a targeting ligand for blood-brain barrier (BBB) crossing though receptor-mediated endocytosis was the strategy selected for drug delivery into the brain. Ligands towards the transferrin receptor (TfR) (e.g. transferring (Tf), monoclonal antibody OX26 (mAbOX26), iron-mimicking cyclic peptide (cPEP) or Angiopep-2) were covalently linked to our polyglutamates trying to favor the BBB entry by active targeting. Molecular probes for fluorescence optical imaging, contrast agents for MRI or tracers for positron emission tomography (PET) were conjugated to our systems, with and without targeting ligands in order to monitor conjugate biodistribution comparing their BBB crossing capability. In most of the cases, the polymer platform linkage with the ligand was a disulphide bond. The bioreversible union allows the ligands to be released following transcytosis. All of the polyglutamate systems proved to have an appropriate biodistribution, where non-specific accumulation in any organ was observed and body elimination followed renal excretion. Regarding the targeted systems, TB-Tf was found in rat brain after tissue homogenation although in very low percentage. Aiming to increase the percentage, the natural ligand Tf which presents receptor saturation under physiologic conditions was substituted by an antibody able to selectively target this receptor with greater efficiency. mAbOX26 was successfully linked to the carrier but issues related to solubility issues limited a proper detection in brain.Consequently, peptide ligands were used instead. cPEP was directed to TfR and Angiopep-2 to the lipoprotein receptor-related protein-1 (LRP1). This last peptide is the most advanced for BBB crossing in phase II clinical trials. After the results obtained, PGA homopolymer and DB were the carriers selected for further studies. Monitoring of these targeted systems did not show BBB crossing may be due to low availability of the peptide motifs for receptor recognition. Currently other polyglutamates with different structures are being tested to surpass this inconvenience. When CNS is not involved but peripheral nervous system (PNS), brain entering is not a major problem. FAP (familiar amyloidotic polyneuropathy) is a rare amyloidotic disease where PNS is the main organ affected. A mutation in the protein transthyretin (TTR) promotes its aggregation ending up in fibril formation whose deposits promote organ failure. As a first proof of concept, treatment of FAP by means of PDCs was proposed. Continuing with the polymeric platforms exploited in this work, PGA and PEG were selected for conjugation of two bioactive molecules able to disrupt amyloid fibrils (doxycycline) and to suppress cytotoxicity promoted for the early aggregates (RAGE peptide). Two families of conjugates were synthesised following a rational design based on the extracellular target of the compounds and the properties and requirements of each drug. Total drug loading was varied as well as the linkage alternating biodegradable (disulphide) and non-biodegradable (amide) bonds. After in vitro screening in appropriate FAP models, Doxy conjugates showed higher activity than the parent drug at same concentration and RAGE peptide conjugates maintained the original activity. The best candidate of each library (PGA-CONH-Doxy and PEP1-PEG-PEP1) was selected and a full physico-chemical characterisation was performed in order to explain the activity found. Both conjugates proved by LCMS or HPLC techniques to be stable under the in vitro conditions used implying non requirement of drug release to achieve activity while the others not. Furthermore, the conjugates demonstrated stability in plasma and non haemolytic activity confirming their suitability for intravenous administration. Studies on solution conformation by dynamic light scattering (DLS) showed marked differences in the RAGE peptide conjugates where more stable and smaller conjugates (Rh=0.6-3nm) corresponded to the most active ones. Regarding the PGA-Doxy, different ωt% loading did not show major differences in conformation in SANS (small angle neutron scattering) analysis corroborating the similar activity found with the selected conjugates. Previous to in vivo activity evaluation, biodistribution of the conjugates were performed resulting in non-specific organ accumulation and elimination via the kidney. Finally, combination therapy was proposed as a helpful tool due to the target species of the TTR aggregation cascade coexist in the advance stages of the disease. Initially, conjugation of both agents in the same polymer matrix (PGA homopolymer, DB or TB) was suggested but due to the unknown space among respective targets, individual PDCs were first tried and combination of the single conjugates was suggested as alternative. Thus, preliminary in vitro and in vivo studies with both conjugates (PGA-CONH-Doxy and PEP1-PEG-PEP1) were performed. Comparing the obtained results of immunohistochemistry analysis from single conjugates administration and their combination (without control samples) it could be concluded that the combination group showed lower deposition of non-fibrillar TTR. Histological analysis of the organs evidenced normal patterns in morphological tissue structures implying the safety of the administered conjugates. In any case, PGA-Doxy effect is planned to be evaluated in old FAP mice as well as a dose optimisation study should be performed in order to finally proved conjugates effectiveness in FAP.