Development and evaluation of delivery strategies for therapeutic peptides

Therapeutic proteins and peptides represent a rapidly expanding class of biologics due to their high target specificity and potent biological activity. Therapeutic peptides are particularly attractive because they can mimic endogenous signaling molecules and selectively interact with receptors regulating key physiological functions. However, their clinical use is often limited by poor physicochemical stability, susceptibility to enzymatic degradation, and short circulation half-life. Therefore, the development of delivery strategies able to improve peptide stability and pharmacokinetic behavior is of major pharmaceutical interest. Among the therapeutic areas in which peptide drugs play an important role, diabetes mellitus represents a major field of application. Diabetes is a chronic metabolic disorder characterized by impaired glucose homeostasis and persistent hyperglycemia, often associated with obesity and other metabolic comorbidities. In this context, peptide-based therapies that reproduce the actions of endogenous hormones have become increasingly important. Pramlintide, an amylin analogue, and semaglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, are two clinically relevant therapeutic peptides that target complementary pathways involved in glucose regulation, gastric emptying, satiety, and body weight control. The aim of this thesis was to develop and evaluate delivery strategies for these two antidiabetic peptides through PEGylation, using different conjugation approaches tailored to their structural features. Pramlintide was modified through a site-selective N-terminal PEGylation strategy based on reductive amination with PEG-CHO 20 kDa, with the objective of improving its pharmacokinetic profile and potentially overcoming its formulation limitations. Semaglutide was instead PEGylated through an enzymatic, site-selective conjugation mediated by microbial transglutaminase (mTGase) using PEG-NH₂ 20 kDa, in order to investigate whether polymer conjugation could represent a complementary delivery-oriented strategy even for an already optimized GLP-1 analogue. The conjugation reactions were monitored by reversed-phase high-performance liquid chromatography (RP-HPLC), and the resulting PEGylated products were purified by ion-exchange chromatography. The isolated conjugates were further characterized by RP-HPLC and SDS-PAGE, which confirmed the successful formation and satisfactory purity of both PEG20kDa-Nter-Pramlintide and PEG20kDa-Gln-Semaglutide. In addition, the pharmacodynamic activity of PEG20kDa-Gln-Semaglutide was evaluated in vivo in diet-induced obese rats following subcutaneous and intratracheal administration. While native semaglutide induced a clear reduction in body weight, the PEGylated conjugate did not show detectable biological activity under the tested conditions, suggesting that the introduction of a large PEG chain may interfere with weight reducing activity of semaglutide. This result can be due to two reasons: 1) the PEG prevents the receptor recognition and compromise peptide functionality, although the selected approach of conjugation yields the polymer coupling far from the area of receptor recognition, or 2) since PEG blocks the blood-brain barrier crossing this will impair the central activity of semaglutide that might be the driving pathway of signalling for this pharmacodynamic effect. Further studies will be directed to evaluate if the peripheric effects of semaglutide are preserved after PEGylation. PEG20kDa-Nter-Pramlintide will be further evaluated in future in vivo studies, which will help clarify whether PEGylation may represent a more suitable strategy for improving the pharmacokinetic and pharmacodynamic properties of amylin analogues. Overall, this work highlights the feasibility of selective PEGylation and the importance of tailoring delivery strategies to each peptide to optimize therapeutic performance.