Aggregation Prone Region analogues: synthetic excipients for protein formulation

In the last few decades, protein therapeutics have become a relevant segment of the pharmaceutical industry. However, clinical use of biotherapeutics can be limited by poor stability of proteins in the different steps of manufacturing, storage and formulation, with subsequent formation of protein aggregates. Aggregation affects not only therapeutics proteins, with detrimental effects on production costs, final product yields and therapeutic efficacy, but also endogenous proteins, as it leads to the formation of large aggregates deposits which have been correlated to different neurodegenerative diseases. Consequently, a range of stabilisers have been developed to increase the stability of biotherapeutics in formulations, or prevent the aggregation of endogenous proteins. Among the different strategies employed, excipients that stabilise proteins through non-covalent interactions have been reported. This thesis focusses on the stabilisation of proteins through hydrophobic interactions: here, the synthesis of short, hydrophobic, stabilisers is reported. These stabilisers were tested on different proteins, to interact with hydrophobic patches on proteins primary strucures, and block these patches from mutual, self-interactions that may lead to proteins aggregation. In the first part of this work, peptides analogous of hen egg lysozyme Aggregation Prone Region (APR), were synthesised, and tested on lysozyme to verify any potential interaction between these synthetic peptides and their homologous sequence on lysozyme, to block its site from self-interactions that lead to aggregation. For improved solubility and to enhance its stabilising effect, the APR peptide fragment was finally copolymerised with monomer N-hydroxyethylacrylamide, to generate a peptide-polyacrylamide copolymer stabiliser. Pleasingly, the copolymer proved to be able to delay the onset of lysozyme aggregation, which was induced in strong basic conditions. Encouraged by these results, in the second experimental chapter this strategy was expanded by developing a library of amphiphilic block copolymers, comprising hydrophobic amino acid-like moieties, potentially able to non-covalently interact with hydrophobic, self-aggregating protein domains, and prevent protein aggregation/denaturation. Three moieties were chosen, indole 3-acetic acid, phenyl acetic acid and methylisobutiric acid, to mimic the side chains of three amino acids, tryptophan, phenylalanine and isoleucine, respectively. The copolymers were tested on two different proteins, hen egg lysozyme, bovine pancreatic insulin, and the antimicrobial peptide IDR 1018. Potential interaction between the proteins and the copolymers was evaluated under stressful conditions, which induced proteins aggregation, measured by turbidity and solubility studies. Promising stabilising effects were shown by some of the indole-contaning copolymers, which proved to be able to prevent the aggregation and to increase the solubility of both insulin and peptide IDR 1018. Hydrophobic Indole-based oligomers were further tested to evaluate their efficacy in encapsulating the antimicrobial peptide IDR 1018. The peptide was first ion paired with the antimicrobial molecule usnic acid, to develop a hydrophobic complex for enhanced IDR 1018 encapsulation and potential co-delivery of two antimicrobial drugs. In the fourth experimental chapter of this thesis, cholanic-polyacrylamides conjugates were synthesised for potential non-covalent protein conjugation. Cholanic acid has been previously investigated for its ability to interact with proteins hydrophobic patches. In particular, a series of PEG-cholanes of different molecular weight were used to efficiently complex two different proteins, the recombinant human growth hormone (rh-GH) and the recombinant human granulocyte colony stimulating factor (rh‐G‐CSF). improving their bioavailability and extending their half-life. Here, cholanic acid was incorporated into a RAFT agent and used to mediate the polymerization of N-hydroxyethylacrylamide, to develop cholanic-polyacrylamides of different length. The polymers were successfully employed as protein complexing agents for two model proteins, bovine serum albumin and bovine pancreatic insulin. Finally, the last chapter is presented in a form of a draft paper, and is part of a collaborative work started by a former PhD student in our group, Joao Madeira do O. A series of linear and 4-arm glycopolymers, were previously prepared by copper azide alkyne cycloaddition (CuAAC) functionalisation of preformed poly(propargyl methacrylate)s with different sugar azides. In this thesis, the reversible, non-covalent interaction between the small hydrophobic molecule Nile Red and linear and 4-arm glycopolymers was evaluated. Results suggest that the interaction occurs between the dye molecule and single polymer chains, suggesting that these glycopolymers do not self-assemble in supramolecular aggregates and act instead as unimolecular micelles

Synthetic macromolecular peptide-mimetics with amino acid substructure residues as protein stabilising excipients

The clinical use of protein and peptide biotherapeutics requires fabrication of stable products. This particularly concerns stability towards aggregation of proteins or peptides. Here, we tested a hypothesis that interactions between a synthetic peptide, which is an aggregation-prone region analogue, and its homologous sequence on a protein of interest, could be exploited to design excipients which stabilise the protein against aggregation. A peptide containing the analogue of lysozyme aggregation-prone region (GILQINSRW) was conjugated to a RAFT agent and used to initiate the polymerisation of N-hydroxyethyl acrylamide, generating a GILQINSRW-HEA90 polymer, which profoundly reduced lysozyme aggregation. Substitution of tryptophan in GILQINSRW with glycine, to form GILQINSRG, revealed that tryptophan is a critical amino acid in the protein stabilisation by GILQINSRW-HEA90. Accordingly, polymeric peptide-mimetics of tryptophan, phenylalanine and isoleucine, which are often present in aggregation-prone regions, were synthesized. These were based on synthetic oligomers of acrylamide derivatives of indole-3 acetic acid (IND), phenylacetic acid (PHEN), or 2-methyl butyric acid (MBA), respectively, conjugated with hydrophilic poly(N-hydroxyethyl acrylamide) blocks to form amphiphilic copolymers denoted as INDm-, PHENm- and MTBm-b-HEAn. These materials were tested as protein stabilisers and it was shown that solution properties and the abilities of these materials to stabilise insulin and the peptide IDR 1018 towards aggregation are dependent on the chemical nature of their side groups. These data suggest a structure–activity relationship, whereby the indole-based INDm-b-HEAn peptide-mimetic displays properties of a potential stabilising excipient for protein formulations

Direct routes to functional RAFT agents from substituted N-alkyl maleimides

N-substituted maleimides have become an indispensable tool for the synthesis of bioconjugates and functional materials. Herein, we present three strategies for the incorporation of N-alkyl substituted maleimides into RAFT agents and show that these maleimide-derived CTAs can be used to easily introduce a range of chemical functionality at the β-position of polymer chains, resulting in α,β,ω-functional RAFT polymers. With both functional maleimides and RAFT agents that are increasingly available on the market, the approach presented in this study could facilitate the synthesis of end-functional macromolecules and will complement well the range of existing synthetic routes, including those utilising N-substituted maleimides, to functional polymeric materials

A Reactive Prodrug Ink Formulation Strategy for Inkjet 3D Printing of Controlled Release Dosage Forms and Implants

We propose a strategy for creating tuneable 3D printed drug delivery devices. 3D printing offers the opportunity for improved compliance and patient treatment outcomes through personalisation, but bottlenecks include finding formulations that provide a choice of drug loading and release rate, are tuneable and avoid the need for surgical removal. Our solution is to exploit 3D inkjet printing freedoms. We use a reactive prodrug that can polymerize into drug-attached macromolecules during 3D printing, and by tuning the hydrophilicity we can facilitate or hinder hydrolysis, which in turn controls the drug release. To demonstrate this approach, we attach ibuprofen to 2-hydroxyethyl acrylate through a cleavable ester bond, formulate it for inkjet 3D printing, and then print to produce a solid dosage form. This allows a much higher loading than is usually achievable-in our case up to 58 wt%. Of equal importance, the 3D inkjet printing freedoms mean that our drug delivery device is highly tuneable: by selection of spacer monomers to adjust the hydrophilicity; through geometry; by spatially varying the components. Consequently, we create bespoke, hierarchical release systems, from the molecular to macro. This approach represents a new paradigm for the formulation of printable inks for drug-loaded medical devices

Control of aggregation temperatures in mixed and blended cytocompatible thermoresponsive block co-polymer nanoparticles

A small library of thermoresponsive amphiphilic copolymers based on polylactide-block-poly((2-(2-methoxyethoxy)ethyl methacrylate)-co-(oligoethylene glycol methacrylate)) (PLA-b-P(DEGMA)-co-(OEGMA)), was synthesised by copper-mediated controlled radical polymerisation (CRP) with increasing ratios of OEGMA:DEGMA. These polymers were combined in two ways to form nanoparticles with controllable thermal transition temperatures as measured by particle aggregation. The first technique involved the blending of two (PLA-b-P(DEGMA)-co-(OEGMA)) polymers together prior to assembling NPs. The second method involved mixing pre-formed nanoparticles of single (PLA-b-P(DEGMA)-co-(OEGMA)) polymers. The observed critical aggregation temperature Tt did not change in a linear relationship with the ratios of each copolymer either in the nanoparticles blended from different copolymers or in the mitures of pre-formed nanoparticles. However, where co-polymer mixtures were based on (OEG)9MA ratios within 5-10 mole% , a linear relationship between (OEG)9MA composition in the blends and Tt was obtained. The data suggest that OEGMA-based copolymers are tunable over a wide temperature range given suitable co-monomer content in the linear polymers or nanoparticles. Moreover, the thermal transitions of the nanoparticles were reversible and repeatable, with the cloud point curves being essentially invariant across at least three heating and cooling cycles, and a selected nanoparticle formulation was found to be readily endocytosed in representative cancer cells and fibroblasts

Aggregation Prone Region analogues: synthetic excipients for protein formulation

In the last few decades, protein therapeutics have become a relevant segment of the pharmaceutical industry. However, clinical use of biotherapeutics can be limited by poor stability of proteins in the different steps of manufacturing, storage and formulation, with subsequent formation of protein aggregates. Aggregation affects not only therapeutics proteins, with detrimental effects on production costs, final product yields and therapeutic efficacy, but also endogenous proteins, as it leads to the formation of large aggregates deposits which have been correlated to different neurodegenerative diseases. Consequently, a range of stabilisers have been developed to increase the stability of biotherapeutics in formulations, or prevent the aggregation of endogenous proteins. Among the different strategies employed, excipients that stabilise proteins through non-covalent interactions have been reported. This thesis focusses on the stabilisation of proteins through hydrophobic interactions: here, the synthesis of short, hydrophobic, stabilisers is reported. These stabilisers were tested on different proteins, to interact with hydrophobic patches on proteins primary strucures, and block these patches from mutual, self-interactions that may lead to proteins aggregation. In the first part of this work, peptides analogous of hen egg lysozyme Aggregation Prone Region (APR), were synthesised, and tested on lysozyme to verify any potential interaction between these synthetic peptides and their homologous sequence on lysozyme, to block its site from self-interactions that lead to aggregation. For improved solubility and to enhance its stabilising effect, the APR peptide fragment was finally copolymerised with monomer N-hydroxyethylacrylamide, to generate a peptide-polyacrylamide copolymer stabiliser. Pleasingly, the copolymer proved to be able to delay the onset of lysozyme aggregation, which was induced in strong basic conditions. Encouraged by these results, in the second experimental chapter this strategy was expanded by developing a library of amphiphilic block copolymers, comprising hydrophobic amino acid-like moieties, potentially able to non-covalently interact with hydrophobic, self-aggregating protein domains, and prevent protein aggregation/denaturation. Three moieties were chosen, indole 3-acetic acid, phenyl acetic acid and methylisobutiric acid, to mimic the side chains of three amino acids, tryptophan, phenylalanine and isoleucine, respectively. The copolymers were tested on two different proteins, hen egg lysozyme, bovine pancreatic insulin, and the antimicrobial peptide IDR 1018. Potential interaction between the proteins and the copolymers was evaluated under stressful conditions, which induced proteins aggregation, measured by turbidity and solubility studies. Promising stabilising effects were shown by some of the indole-contaning copolymers, which proved to be able to prevent the aggregation and to increase the solubility of both insulin and peptide IDR 1018. Hydrophobic Indole-based oligomers were further tested to evaluate their efficacy in encapsulating the antimicrobial peptide IDR 1018. The peptide was first ion paired with the antimicrobial molecule usnic acid, to develop a hydrophobic complex for enhanced IDR 1018 encapsulation and potential co-delivery of two antimicrobial drugs. In the fourth experimental chapter of this thesis, cholanic-polyacrylamides conjugates were synthesised for potential non-covalent protein conjugation. Cholanic acid has been previously investigated for its ability to interact with proteins hydrophobic patches. In particular, a series of PEG-cholanes of different molecular weight were used to efficiently complex two different proteins, the recombinant human growth hormone (rh-GH) and the recombinant human granulocyte colony stimulating factor (rh‐G‐CSF). improving their bioavailability and extending their half-life. Here, cholanic acid was incorporated into a RAFT agent and used to mediate the polymerization of N-hydroxyethylacrylamide, to develop cholanic-polyacrylamides of different length. The polymers were successfully employed as protein complexing agents for two model proteins, bovine serum albumin and bovine pancreatic insulin. Finally, the last chapter is presented in a form of a draft paper, and is part of a collaborative work started by a former PhD student in our group, Joao Madeira do O. A series of linear and 4-arm glycopolymers, were previously prepared by copper azide alkyne cycloaddition (CuAAC) functionalisation of preformed poly(propargyl methacrylate)s with different sugar azides. In this thesis, the reversible, non-covalent interaction between the small hydrophobic molecule Nile Red and linear and 4-arm glycopolymers was evaluated. Results suggest that the interaction occurs between the dye molecule and single polymer chains, suggesting that these glycopolymers do not self-assemble in supramolecular aggregates and act instead as unimolecular micelles

Direct routes to functional RAFT agents from substituted N-alkyl maleimides

N-substituted maleimides have become an indispensable tool for the synthesis of bioconjugates and functional materials. Herein, we present three strategies for the incorporation of N-alkyl substituted maleimides into RAFT agents and show that these maleimide-derived CTAs can be used to easily introduce a range of chemical functionality at the β-position of polymer chains, resulting in α,β,ω-functional RAFT polymers. With both functional maleimides and RAFT agents that are increasingly available on the market, the approach presented in this study could facilitate the synthesis of end-functional macromolecules and will complement well the range of existing synthetic routes, including those utilising N-substituted maleimides, to functional polymeric materials