GLP-1 (Glucagon-Like Peptide-1) is an active peptide hormone produced and secreted by intestinal L cells and certain neurons in the brainstem solitary nucleus after eating. In the human body, it exerts various biological functions by binding to GLP-1 receptors (GLP-1R) and activating receptor signaling. These functions include inhibiting glucagon secretion, increasing insulin secretion, slowing gastrointestinal motility, and suppressing appetite, thereby participating in the regulation of blood glucose balance. Additionally, GLP-1 has functions such as delaying gastric emptying, suppressing appetite, and providing cardiovascular
protection.

Figure 1. Effects of GLP1 and GLP1RAs on various tissues. [1]

Despite GLP-1 having many advantages, its most challenging issue in clinical development is its short half-life in the bloodstream. There are two main reasons for this: firstly, within receptors, it undergoes degradation by dipeptidyl peptidase-4 (DPP-4) and neutral endopeptidase (NEP), actions that render GLP-1 inactive. Secondly, GLP-1 is rapidly cleared and degraded by the kidneys in the body, resulting in an approximate half-life of 2 minutes for endogenous GLP-1. Only 10-15% of GLP-1 manages to enter circulation intact, with fasting plasma levels ranging from 0-15 pmol/L.

To address the issue of a short half-life, various methods and strategies have been employed from two perspectives: enhancing tolerance to DPP-4 enzyme and reducing in vivo clearance.

First Genaration Long Acting GLP-1

The first-generation long-acting strategy for GLP-1 involved the modification and alteration of amino acid sequences. Inspiration for this long-acting strategy was drawn from Exendin-4 found in the saliva of the Gila monster (Heloderma suspectum). Although its structure shares only 53% homology with human GLP-1, Exendin-4 demonstrated stronger binding capacity to human GLP receptors compared to GLP-1. This is attributed to the high similarity in the crucial N-terminal amino acids of both.

Compared to GLP-1, Exendin-4 has a Gly substitution for Ala at the second position, making it more resistant to digestion by DPP-4. This increased tolerance results in a half-life of 30 minutes for Exendin-4, and subcutaneous injection can maintain effective concentrations for 2.4 hours. Based on the characteristics of Exendin-4, two drugs, exenatide and liraglutide, were developed through modification and alteration of its sequence.

Exenatide (Byetta), transformed by Amylin Pharmaceutical from Exendin-4, is a polypeptide composed of 36 amino acids. Approved by the FDA in 2005, Exenatide is the world's first GLP-1 RA (glucagon-like peptide-1 receptor agonist) drug and is administered by injection twice daily.

Lixisenatide (Adlyxin, Lyxumia), developed by Sanofi, is used to treat type 2 diabetes with a daily injection frequency. Lixisenatide is a peptide consisting of 44 amino acids, with an amide group at its C-terminus. The first 39 amino acids in its sequence are derived from Exendin-4.

Figure 2. Exenatide and Lixisenatide [2]

Second Genaration Long Acting GLP-1

The second-generation long-acting strategy involves molecular design strategies such as fatty acid conjugate, PEGylation, albumin fusion, Fc proteins fusion, and formulation development strategies like drug delivery systems. It is the application of these strategies that has led to the successful development of weekly formulations of GLP-1.

1. Fatty Acid Conjugate

Covalent attachment of a fatty acid, known as acylation, is used extensively to control the half-life of therapeutic peptides.

Acylation with fatty acids can increase the circulation time of therapeutic peptides by several mechanisms. Initially, these acylated peptides group could self-assemble to supramolecular structures. After subcutaneous injection, they form a depot at the injection site, delaying absorption into the bloodstream. As the modified peptide dissociates from this depot and enters circulation, it binds to serum albumin, protecting against enzymatic degradation and slowing kidney clearance due to the larger peptide-albumin complex. The albumin-bound peptide may also interact with the neonatal Fc receptor (FcRn), increasing circulation time. While FcRn recycling is suggested for albumin-bound peptides, there's currently no experimental proof for acylated GLP-1 RAs.

Figure 3. Half-life extension mechanisms of acylated GLP-1 analogs. [2]

The effectiveness of acylated GLP-1 RAs relies on factors such as acylation site, fatty acid chain characteristics (bulkiness and length), and the spacer used between the peptide and fatty acid. Acylation at the N-terminus, crucial for receptor binding, reduces potency, while C-terminal modifications have minimal impact on potency. Spacers connecting peptide and fatty acid also influence potency. Bulky fatty acids negatively affect potency, and the fatty acid length correlates positively with albumin binding and extended circulation. However, longer fatty acid chains may decrease potency in the presence of serum, requiring a careful balance in selecting acylation site and chain length for optimal in vivo stability and potency.

The most successful example of lipoylation of GLP-1 drugs is Liraglutide, which was approved by the FDA in 2010 under the brand name Victoza®. Liraglutide is derived from natural GLP-1, which binds Lys26 at position 26 to a 16-carbon fatty acid via γ-Glu, while replacing Lys at position 34 with Arg. Upon injection, the fatty acid chain leads to oligomerization of liraglutide and the formation of oligomers localized to the injection site, delaying its uptake and metabolism in the body's circulation. In vitro studies have shown that although liraglutide can be metabolized by DPP-4 and NEP, it is degraded much more slowly than GLP-1, probably due to the spatial site-blocking provided by fatty acid-bound albumin. Also albumin binding prevents renal filtration of liraglutide, and intact liraglutide was not detected in the urine of patients in clinical studies.

Semaglutide is the next-generation GLP-1 drug based on Liraglutide, approved by the FDA in December 2017 under the brand name Ozempic®. Compared to Liraglutide, Semaglutide replaces the eighth alanine (Ala) with α-aminoisobutyric acid (Aib) to reduce the degradative effect of DPP-4. Additionally, Semaglutide's acylated fatty acid changes from C16 to a longer (C18) dienoic fatty acid, and the linker is modified.In comparison to Liraglutide, Semaglutide has higher albumin binding affinity. In clinical studies, Semaglutide exhibits lower enzymatic degradation and reduced renal elimination compared to Liraglutide. Due to its improved design, Semaglutide has a half-life of 7 days, allowing for once-weekly dosing. Despite the reduced dosing frequency, the dosage is lower than that of Liraglutide.

Figure 4. Liraglutide & Semaglutide [2]

2. Albumin fusion

Extending the half-life of drugs by fusing them with albumin is a common technique in the design of peptide drugs. This approach involves modifying recombinant proteins and peptide drugs with human serum albumin (HSA) to increase their half-life, reduce the frequency of administration, and enhance patient compliance.

Figure 5. Application of serum albumin (HSA)

Albumin significantly enhances the half-life of GLP-1 drugs in the body through two main mechanisms: firstly, the fusion of GLP-1 with albumin increases its molecular weight, preventing it from passing through the gaps in the renal glomerular epithelial cells. This escape from renal clearance allows it to continue circulating in the bloodstream and exert its therapeutic effects. Secondly, albumin can bind to the FcRn on endothelial cells, undergo internalization, escape lysosomal degradation, and re-enter the circulatory system.

In the early stages, HSA modification involved directly fusing peptides with HSA without introducing a linker peptide. For instance, GSK developed Albiglutide, which was approved by the FDA on April 15, 2014, for the treatment of type 2 diabetes. It was the first HSA-fused long-acting drug to receive FDA approval. Albiglutide is a recombinant protein composed of two copies of GLP-1 analogs fused to human albumin. The molecule has a Gly8 to Ala substitution in both copies of the GLP-1 analogs to improve resistance to DPP-4 degradation. It has an in vivo half-life of 6–8 days, and is approved for once-weekly administration. However, in 2017, GSK announced the withdrawal of this drug from the global market due to limited prescribing of the medicine. 

Figure 6. Albiglutide [2]

3. Fc fusion

Fc fusion proteins are IgG-based chimeric fusion proteins used to bind peptides or proteins that not only retain the biological activity of the functional protein molecule, but also have some of the properties of an antibody, such as prolonging the half-life by binding to the associated Fc receptor.

Fc-fusion proteins contain the antibody Fc segment, which, while incapable of antigen-antibody reactions, can mediate various biological functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Additionally, these proteins can select fusion partners based on the differences in cytotoxicity mediated by the four subtypes. The prolonged mechanism relying on fusion partners involves extending the half-life of drugs in the plasma and increasing the stability of fusion proteins through the neonatal Fc receptor (FcRn)-mediated recycling pathway.

One highly successful example of using Fc to extend the half-life is Eli Lilly's star drug dulaglutide, which employs multiple long-acting strategies. Firstly, it applies an amino acid substitution strategy by replacing alanine (Ala) at position 8 with glycine (Gly8) to resist DPP-4 degradation, replacing glycine (Gly) at position 22 with glutamic acid (Glu22) to stabilize the secondary structure and enhance potency, and replacing arginine (Arg) at position 36 with glycine (Gly36) to avoid potential T-cell epitopes. Secondly, it selects the Fc region of IgG4 to prolong the half-life, and modifies some sequences to reduce its interaction with Fc receptors, thus avoiding ADCC immune side effects. Finally, it uses an ultra-long linker containing 16 amino acids between GLP-1 analog and the Fc portion to prevent drug inactivation due to fusion with Fc.

Figure 7. Dulaglutide [2]

4. PEGylation

Polyethylene glycol (PEG) is a compound synthesized through the polymerization of ethylene oxide, with a relative molecular weight primarily ranging from 200 to 40000. PEG is a neutral, non-toxic substance characterized by unique physical and chemical properties, as well as excellent biocompatibility. It is also one of the few chemical substances approved by the FDA for use in injectable medications within the body.

PEGylation not only facilitates the long acting of drugs but also alters the physicochemical properties of compounds. This modification allows prodrugs to be activated at specific target sites for therapeutic effects or adjusts their lipophilicity or hydrophilicity to reduce toxicity and more.

PEGylation technology has been applied to various therapeutic modalities including small molecules, aptamers, peptides, and proteins, leading to over 40 PEGylated drugs currently used in the clinic and many investigational PEGylated agents under clinical trials.

Shifting the PEG modification site to the C-terminus (carboxyl group) is an effective modification strategy, such as PEG Loxenatide. In May 2019, the China NMPA approved PEG Loxenatide injection from Hansoh Pharmaceutical through the Priority Review approval process. The drug is the world's first PEGylated, long-acting glucose-lowering drug that requires only one injection per week. The unique PEGylation technology provides loxenatide with a number of advantages, including a longer half-life, less fluctuation in blood glucose while significantly lowering blood glucose, faster onset of action, and a reduced incidence of adverse effects.

Huateng Pharma, as a leading PEG linker supplier, can provide you with both polydispersed PEGs, monodispersed PEGs and multi-arm PEGs for your drug PEGylation need. We are ISO 9001 and EXCiPACT GMP certified, contact us at sales@huatengusa.com for your PEG inquiries.

Figure 8. PEG Loxenatide

5. Sustained-release drug delivery systems

An alternative approach to maintain prolonged plasma levels of GLP-1 receptor agonists (RAs) involves utilizing drug delivery systems. Different systems, such as polymeric hydrogels, nanoparticles, and microparticles, have undergone testing in preclinical studies. However, only implants based on poly(lactic-co-glycolic acid) (PLGA) carrying GLP-1 RAs have received FDA approval to date.

Exenatide Microspheres for Injection (Bydureon®), is an FDA-approved PLGA-based microsphere formulation of exenatide polymer. Although PLGA has a degradation time of more than six months, the Exenatide-PLGA microsphere formulation is only capable of achieving a once-weekly dosing frequency.

Figure 9. Exenatide Microspheres for Injection [2]

A disadvantage of Exenatide microspheres is their inability to release GLP-1 drugs at a steady rate in vivo. In order to achieve a stable and continuous release of the drug, other controlled release devices such as osmotic pumps have been developed. Representative of these is the ITCA 650 developed by Intarcia Therapeutics.The ITCA 650 is a matchstick-sized osmotic pump implanted subcutaneously in the abdomen that continuously releases exenatide microspheres in a zero-stage release.The device is designed to enable twice-yearly dosing of exenatide. Unfortunately, the ITCA 650 was brutally rejected by the FDA twice, in 2017 and 2020, due to safety concerns.

Intarcia continues to advance the ITCA 650 program, which unfortunately was denied approval by the FDA for the third time in September of this year. Most of the 19 voting panelists who participated in the review of the program cited increased nephrotoxicity and cardiovascular adverse events associated with acute kidney injury (AKI) compared to placebo as the main reasons for their negative vote on ITCA 650. Despite the repeated rejections, this clever device design may indeed be useful in other drug areas.

Figure 10. ITCA 650

Third Genaration Long Acting GLP-1

If the second-generation long-acting strategy transformed daily formulations into weekly ones, the third-generation long-acting strategy may potentially advance GLP-1 formulations administered weekly to monthly or even every few months.

1. Antibody conjugates

Fusion proteins are still not an optimal solution for long-lasting mechanisms, peptide-antibody fusions are susceptible to proteolytic hydrolysis, and Regeneron gives new strategies for antibody-conjugated GLP-1 analogs.

On March 17, 2022, Regeneron's patent WO2022056494 for a novel GLP-1R-targeting ADC (Antibody-Drug Conjugate) was disclosed. In this patent, Regeneron describes the combination of antibodies specific to the extracellular domain of GLP-1R or GLP-1 peptide mimetics that functionally activate GLP-1R. This combination forms an antibody-drug conjugate, and Regeneron has coined a new term for this technology: antibody-tethered drug conjugates (ATDCs).

It's worth noting that Regeneron's GLP-1R-targeting ADC differs from traditional ADCs. In this case, the antibody binds to the extracellular region of GLP-1R, while the payload binds to the transmembrane region of GLP-1R. The advantage of this mechanism lies in promoting affinity, enhancing efficacy, stabilizing the GLP-1 peptide, extending its half-life, and reducing clearance rates, ultimately achieving a sustained and stable GLP-1 activation effect.

Compared to GLP-1, this GLP-1R ATDC exhibits stronger agonist activity. In preclinical trials, Regeneron opted for a high-fat diet-induced obese mouse model. Dulaglutide served as a positive control, administered twice a week for 4 weeks, while the three ATDC compounds were administered as a single dose on day 0. The experiment lasted 8 weeks, with each group receiving a dosage of 25 mpk.

The experimental results demonstrate the sustained weight control effects of this new drug: in the dulaglutide group, weight loss was observed only on days 3 and 7, followed by weight rebound. In contrast, mice in the three ATDC experimental groups, despite receiving a single dose, maintained weight loss effects for 4 weeks, 6 weeks, and 8 weeks, respectively. These preclinical results highlight the advantages of ATDC drugs over GLP-1 peptides, suggesting the potential for developing monthly formulations.

Conclusion

The long-acting strategies for GLP-1 drugs have undergone three generations of development, with each generation building upon and optimizing the strategies of the previous one.

Starting from modifications and substitutions in the amino acid sequence, progressing to the introduction of fatty acid chains, and further evolving into strategies involving microspheres, hydrogels, and others, GLP-1 drugs have now reached the stage of once-weekly formulations. However, despite this progress, weekly dosing still falls short in improving patient compliance significantly. This has led to the emergence of strategies such as antibody conjugation. In the near future, there is hope for the development of formulations that require only a few injections per year for glucose-lowering and weight reduction. This would represent a significant breakthrough in the field of GLP-1 therapeutics.

References:
[1] Andersen, A., Lund, A., Knop, F.K. et al. Glucagon-like peptide 1 in health and disease. Nat Rev Endocrinol 14, 390–403 (2018). https://doi.org/10.1038/s41574-018-0016-2
[2] Yu M, Benjamin MM, Srinivasan S, et al. Battle of GLP-1 delivery technologies. Adv Drug Deliv Rev. 2018;130:113-130. doi:10.1016/j.addr.2018.07.009
[3] Harris, J. M. et al. Pegylation: a novel process for modifying pharmacokinetics. Clin. Pharmacokinet. 2001, 40, 539–551.
[4] https://patents.google.com/patent/US20220096648A1/en