Polymer synthesis and conjugation strategies for enhancing the stability of oxytocin

The aim of this work was to investigate different methods for the covalent attachment of poly(ethylene glycol) onto the therapeutic peptide oxytocin, a highly important, but thermally unstable therapeutic used globally. This peptide is the WHO recommended therapeutic for prevention of postpartum haemorrhaging, sitting on the WHO list of essential medicines. Tackling the currently unacceptable maternal mortality rate, particularly in developing countries is of paramount importance and is currently one of the WHOs main priorities. Within this project it was speculated that by attachment of PEG to the peptide there would hopefully be an increase in stability, particularly for aqueous formulations at elevated temperatures. Chapter one gives a brief outline of the problem that the world is facing with respect to maternal mortality, and particularly the gap between developed and developing countries and previous strategies that have looked into improving the stability of oxytocin. Additionally the various different site-specific conjugation approaches available for peptide modification are discussed, alongside how these can be implemented with controlled radical polymerisation techniques for the synthesis of alternative polymer architectures. Chapter two discusses two particular targeting chemistries for site-selectively targeting the N-terminal amine (the only amino group on the peptide structure). Some commercially applicable linear PEGylation reagents (such as utilising NHS esters) were utilised for the conjugation of polymers onto oxytocin in this manner. PolyPEGs were synthesised to contain similar α-end group functionality as for the linear polymers and reacted with oxytocin in similar manners. The reversible nature of one of these chemistries was also investigated, and the potential release of the native peptide dependence on conjugate architecture and pH were evaluated. In Chapter 3 the potential for conjugation techniques targeting the sulfhydryl groups arising from a reduction of the disulfide bond were approached in two different manners. Disubstituted maleimide chemistry is particularly useful in this case as it allows the rebridging of the disulfide bond, one of the main degradation sites on the peptide, with a stronger 2-carbon bond. Dithiophenolmaleimide α-end functional polyPEGs were synthesised and conjugated onto the peptide via an in-situ approach alongside traditional conjugations with both polyPEGs and linear PEGs. Another approach was evaluated for the conjugation at the disulfide bond that treats both free cysteine residues for separate conjugations utilising phosphine mediated thiol-ene chemistry. The facile synthesis of ABA block copolymers containing a central ‘oxytocin’ block, however results in a loss of the cyclic structure on the peptide, and likely complete suppression of biological activity. Chapter 4 reports the investigation of the various conjugation strategies raised in chapters 2 & 3 for how the properties of the peptide might have changed post-conjugation comparing linear PEG and polyPEG site selectively added at either position in comparison to the native peptide. This is evaluated for the thermal stability, where oxytocin shows high levels of degradation in aqueous solutions, particularly at elevated temperatures. Also investigated is the potential retention of uterotonic activity, via ex-vivo electrophysiology studies, as well as some previously investigated effects on the inhibition of cell proliferation of the breast cancer cell line MDA-MB231. Chapter 5 focusses on the conjugation to oxytocin, and another small disulfide containing peptide of some different (non-PEG) polymers synthesised in similar manners to those discussed in chapters 2 and 3. These few examples show that there is a large scope within this polymer synthesis and conjugation chemistry for utilising these techniques for a wide range of different monomer classes. Those described include the synthesis of a promising PEG alternative, two different thermoresponsive polymers and polymers containing pendant sugar functionality followed by the subsequent peptide conjugations.