Development of polypeptide-based therapeutics for topical delivery

Resumen

Topical administration represents the main route to attain local therapeutic activity of bioactive agents in several organs, such as the skin or the heart by means of devices that enhance drug transport through the endothelium acting as a reservoir. Various rational strategies have been employed in an attempt to improve the physico-chemical properties of bioactive agents according to the features of the desired site of action, thereby improving topical delivery and stability. The research carried out in this thesis describes the application of polymer therapeutics, a nanomedicinal approach, to improve the physico-chemical properties and increase both the penetration of bioactive and retention time at the desired site of action. Well-defined polypeptide-based polymer therapeutic approaches offer particular advantages for topical applications such as biodegradability, versatility, multivalence, and high drug loading capacity. The development of new polypeptide-drug conjugates employing stimuli-responsive linking moieties, as well as novel hybrid polypeptide-based carriers to enhance drug delivery through topical applications, can improve the effectiveness of topical treatments. Our work focused on the treatments of skin diseases such as psoriasis, and furthermore, we believe that our newly developed platforms may find wider use, and we also explore polypeptide-drug combinations as an approach to enhance wound healing and treat ischemia/reperfusion injury following myocardial infarction. Psoriasis is a common and chronic inflammatory disease mediated by the immune system with predominantly cutaneous involvement. Approximately 80% of patients are affected by mild to moderate disease, and topical treatment with corticosteroids remains a widely employed therapeutic approach. However, many topical corticosteroids that are currently employed or under assessment in clinical trials lack adequate physico-chemical properties and suffer from local cutaneous and systemic side effects that correlate with the high doses required. Therefore, we propose the implementation of polymer conjugation approaches to overcome these limitations. To this end, we present the development of a biodegradable vehicle, comprising a cross-linked matrix of hyaluronic acid and poly-L-glutamic acid (hyaluronic acid-poly-L-glutamate cross-polymer or HA-CP), for the topical delivery of the advanced therapeutic agents. We exhaustively characterized and biologically evaluated our HA-CP vehicle in vitro, ex vivo, and in vivo revealing multiple advantageous properties regarding the topical administration of therapeutics. Our HA-CP vehicle functioned as a penetration enhancer, using a family of amphiphilic block copolymers of different nature (using PPhe and PBG with different degree of polymerization) and physico-chemical properties, significantly promoting the penetration into the viable epidermis while avoiding the systemic absorption and any associated adverse effects. We also present a straightforward methodology for the synthesis of well-defined polypeptide-based drug conjugates based on poly-L-glutamic acid, or PGA, as the polymeric carrier. We synthesized and characterized a PGA-conjugated corticosteroid (fluocinolone acetonide) via a pH-responsive ester linker. PGA conjugation targeted the corticosteroid to the appropriate skin layer, and the implementation of bioresponsive polymer-drug linking moieties permitted the sustained release of the drug at the required site of action. Both these characteristics promote optimal drug release kinetics and the attainment of a therapeutically relevant concentration of an active agent within the epidermis for the required period of time. We demonstrated that polymer conjugation significantly improved the pharmacological activity of the drug due to greater bioavailability in the required skin layer in vitro, ex vivo, and in vivo. Moreover, the combination of our polypeptide-conjugated corticosteroid within the HA-CP vehicle resulted in synergistic anti-psoriatic activity in vivo, providing a significant reduction in inflammation. Finally, we applied the knowledge acquired from the development of polymer-drug conjugates for the treatment of skin inflammation in other topical applications, as the treatment of skin wound healing, using the PGA-conjugation of the omega-3 polyunsaturated fatty acid didocosahexaenoic acid (diDHA). PGA conjugation enhanced diDHA stability and decreased degradation, which promoted improved therapeutic activity for the conjugate when compared to free diDHA both in skin wound healing and in the treatment of ischemia-reperfusion injury in the mouse heart following myocardial infarction. Overall, our findings highlight the suitability of polymer therapeutic approaches, and polypeptide conjugation in particular, to form drug delivery systems for topical applications. Specifically, PGA-drug conjugates enhance the skin penetration of drugs, while bioresponsive linkers promote the specific release of the drug in the desired skin layer. Moreover, PGA conjugation of fatty acids has also demonstrated the improvement of both safety and effectiveness of the treatment in skin wound healing and in ischemia-reperfusion injury in the heart.