PROJECT SUMMARY Oral delivery of therapeutic peptides, proteins, and oligonucleotides is prevented by a highly effective layer of epithelial cells lining the surface of the gastrointestinal tract. As a result, nearly all biologic therapies are administered via injection or infusion. These methods are costly, painful, inconvenient, and lead to lower rates of patient adherence relative to oral administration. Building on discoveries by Harvard Medical School professor Wayne Lencer, MD, we are developing a lipid-based technology platform that facilitates absorption across the intestinal epithelium, enabling oral delivery of biologics. Previous research demonstrated that glycosphingolipids comprised of a short-acyl chain ceramide are trafficked through epithelial cells in a process known as transcytosis. Additional studies showed that the glycosphingolipids can serve as carriers for conjugated cargo, enabling active and endogenous transcellular transport across the mucosal epithelium in the gut. Because glycosphingolipids are synthetically complex, limiting translation to clinical applications, we have synthesized simplified platforms. One, comprised of just C6-D-erythro-ceramide, is sufficient to enable transcytosis in vitro, but was hindered in vivo by the diminished solubility and amphipathicity of these molecules. Since our last application, we synthesized new ceramide-based vehicles that show enhanced solubility and amphipathicity. Building on these discoveries, we will optimize the new ceramide platform to enable the oral delivery of GLP-1 for the treatment of type II diabetes and obesity. In the studies proposed herein, the ceramide will be chemically modified with different headgroups, each with different physicochemical properties. We will investigate the effects of each headgroup on the permeability, solubility, and activity of the conjugate. Additionally, we will determine the pharmacokinetics and pharmacodynamics of the GLP-1-ceramide conjugates in rodents. We will compare conjugates directly to the only commercially available oral GLP-1 therapy, Rybelsus, and we expect to demonstrate superior bioavailability, half-life, and efficacy for the ceramide fusions. At the completion of these studies, we expect to have identified an optimal orally bioavailable GLP-1 conjugate suitable for preclinical development. Given the need for improved oral treatment options for type II diabetes and obesity, successful completion of these studies will lead to significant impact for the millions of patients suffering with these diseases. Furthermore, validation of the ceramide platform will provide the foundation for developing additional orally bioavailable biologic medicines.

PROJECT NARRATIVE We are developing a ceramide-based platform to enable oral delivery of therapeutic peptides and proteins. Focusing on GLP-1 agonists for the treatment of type II diabetes and obesity, we will optimize the platform specifically around three parameters - permeability, solubility, and activity – and demonstrate its superiority to the only other oral GLP-1 agonist. Once optimized, we will harness these next-generation fusion molecules as therapeutics and begin preclinical studies.