The discovery of glucagon-like peptide-1 (GLP-1), an incretin hormone with important effects on glycemic control and body weight regulation, led to efforts to extend its half-life and make it therapeutically effective in people with type 2 diabetes (T2D). The development of short- and then long-acting GLP-1 receptor agonists (GLP-1RAs) followed. Our article charts the discovery and development of the long-acting GLP-1 analogs liraglutide and, subsequently, semaglutide. We examine the chemistry employed in designing liraglutide and semaglutide, the human and non-human studies used to investigate their cellular targets and pharmacological effects, and ongoing investigations into new applications and formulations of these drugs. Reversible binding to albumin was used for the systemic protraction of liraglutide and semaglutide, with optimal fatty acid and linker combinations identified to maximize albumin binding while maintaining GLP-1 receptor (GLP-1R) potency. GLP-1RAs mediate their effects via this receptor, which is expressed in the pancreas, gastrointestinal tract, heart, lungs, kidneys, and brain. GLP-1Rs in the pancreas and brain have been shown to account for the respective improvements in glycemic control and body weight that are evident with liraglutide and semaglutide. Both liraglutide and semaglutide also positively affect cardiovascular (CV) outcomes in individuals with T2D, although the precise mechanism is still being explored. Significant weight loss, through an effect to reduce energy intake, led to the approval of liraglutide (3.0 mg) for the treatment of obesity, an indication currently under investigation with semaglutide. Other ongoing investigations with semaglutide include the treatment of non-alcoholic fatty liver disease (NASH) and its use in an oral formulation for the treatment of T2D. In summary, rational design has led to the development of two long-acting GLP-1 analogs, liraglutide and semaglutide, that have made a vast contribution to the management of T2D in terms of improvements in glycemic control, body weight, blood pressure, lipids, beta-cell function, and CV outcomes. Furthermore, the development of an oral formulation for semaglutide may provide individuals with additional benefits in relation to treatment adherence. In addition to T2D, liraglutide is used in the treatment of obesity, while semaglutide is currently under investigation for use in obesity and NASH.
Albumin Binding as a Concept for Creating Long-Acting GLP-1 Analogs
Various approaches have since been used to extend the half-life of native GLP-1, several of which have resulted in pharmacological agents that are effective in the treatment of T2D. The first human-based GLP-1RA to be evaluated in clinical trials for OW dosing was taspoglutide (BIM-51077, Aib8,35 GLP-1 [7-36] amide, Roche). The Aib8 protected taspoglutide from DPP-IV degradation. Although a zinc chloride-based formulation of taspoglutide, facilitating subcutaneous precipitation, showed promising results, phase 3 trials were completed, but a submission for approval was discontinued, due to a number of cases of anaphylactic shock. Other approaches have entailed limiting the renal clearance of GLP-1- or exendin-based compounds by covalent fusion of the peptide to a large, stable plasma protein like albumin (albiglutide) or the Fc domain of IgG (dulaglutide).
Human serum albumin (HSA) is among the most stable and abundant of plasma proteins. It comprises three homologous domains assembled into a heart-shaped protein. Approximately 10–15 g is produced by the liver daily, which—owing to its long half-life of several weeks—results in an HSA plasma concentration of 35–50 g/L (~0.6 mM) . The long half-life of HSA is due to its ability to pH-dependently bind the neonatal receptor (FcRn), in the same manner as the Fc domain of antibodies . High-affinity binding of HSA to FcRn recycles the protein back to plasma, thus protecting it from degradation during endocytosis . In the plasma HSA is released due to its low affinity for FcRn at a neutral pH . HSA also plays an important role in achieving homeostasis, by stabilizing plasma pH and denaturation conditions. Furthermore, it is an important antioxidant .
A feature of HSA of particular interest is its ability to bind to a variety of components in plasma, thereby facilitating the solubility and transportation of otherwise insoluble substrates, such as fatty acids and steroids.The fatty acids C10, C12, C14, C16, and C18 have been shown to bind to seven sites on albumin. Several approaches for using albumin as a carrier in drug discovery have been investigated. Various ligands have been identified that bind to albumin, including small molecules, peptides, and proteins. A unique binding epitope on albumin gave a 1:1 binding stoichiometry for a cyclic peptide with the core sequence DICLPRWGCLW, but showed species-specific differences in binding affinity. This albumin-binding peptide was fused to fab fragments, which increased the half-life significantly in mice (37-fold) and rabbits (26-fold) .
In 1998, the first description of this approach was published featuring His7, Arg26 GLP-1(7-37) derivatized with a C8 fatty acid. The compound did not appear to have strong albumin binding and was subject to self-association and physical instability, limiting its pharmaceutical use. Insulin detemir was the first clinically approved protein modified by a fatty acid. Myristic acid, attached to the B29 lysine of desB30 insulin, increased the duration of action of insulin detemir to an extent that made it applicable for once-daily (OD) injection. The underlying mechanism for this increase is primarily subcutaneous deposition after injection, with only a small increase in intravenous half-life due to albumin binding. Insulin degludec is the latest generation of albumin-binding insulin to be based on fatty-acid derivatization. Hexadecandionyl was attached to desB30 human insulin via a L-γ-glutamic acid spacer. Subcutaneous deposition was also an important part of the mechanism of protraction here, facilitated by the fatty acid in insulin degludec leading to self-assembly into multi-hexamers, but insulin degludec also has a longer intravenous half-life than insulin detemir.
A systematic test of derivatives from C12 to C20 fatty acids showed that a C18 di-acid together with a γGlu-2xOEG linker resulted in the highest albumin affinity combined with GLP-1R potency. Structure–activity relations of the derivatives clearly demonstrated that the length of fatty di-acid was important, and that the C18 di-acid used in semaglutide was the optimal choice. There was a clear trend for increasing GLP-1R potency with increasing carbon atoms in di-acids from C12 to C18, but this trend was reversed when longer (>C18) di-acids were used. In a receptor-binding assay the C18 di-acid was also shown to have the highest affinity shift when albumin was added, indicating a strong albumin affinity.
The Pharmacology of Liraglutide and Semaglutide
Liraglutide and semaglutide are both long-acting GLP-1RAs that, despite differing administration intervals and doses, have pharmacodynamic (PD) effects for 24 h/day. Liraglutide was developed for OD administration, and is available as a OD injection of up to 1.8 mg, whereas semaglutide is available as a OW injection of up to 1.0 mg —both for the treatment of T2D. Liraglutide is additionally approved for the treatment of obesity, at a dose of 3.0 mg, while semaglutide is in clinical development as a treatment for obesity (phase 3) and NASH (phase 2). Furthermore, phase 3 clinical trials in T2D are ongoing with semaglutide administered orally (in a co-formulation with an absorption enhancer); mechanistic findings with this formulation are summarized toward the end of this article. The most common side effect of the GLP-1RA class is GI-related adverse events (AE) including nausea, diarrhea, and vomiting which are dose-dependent and typically present in the up-titration phase. Most GI AEs with GLP-1RAs are mild and transient in nature but, in some patients, they can lead to premature treatment discontinuation. Hence, both liraglutide and semaglutide dosing should be titrated initially.
Using fatty acids engineered onto a peptide, thereby facilitating binding to albumin, is a novel concept for drug protraction. Eleven fatty-acid binding sites are described on human albumin and, as the human albumin concentration is ~0.6 mM and the exposure levels of liraglutide and semaglutide are in the 20–40 nM range , there appears to be a large surplus in binding capacity on albumin. Consequently, no apparent safety issues have arisen from this approach. No interactions with other albumin-binding drugs have been shown , likely because most other drugs do not bind to the fatty-acid binding sites. As patients with liver deficiencies can have lower levels of plasma albumin, liraglutide, and semaglutide were evaluated in this population; studies showed no differences in PK and, as such, no dose adjustment is required . Likewise, studies have shown that no dose adjustment of liraglutide or semaglutide is needed in patients with renal deficiencies ; neither drug is cleared via the kidney . Liraglutide, which comprises natural amino acids and a C16 mono-acid, is fully metabolized in the same way as other peptides and fatty acids . Semaglutide, which contains an amino acid described in nature but not in humans (Aib) and a more synthetic component in the spacer region, is still fully metabolized . Indirect comparisons indicate the only difference to be that more semaglutide metabolites are excreted in feces compared with liraglutide .
Clinical efficacy studies evaluated liraglutide and semaglutide with regard to glucose lowering, weight loss and CV risk reduction.