Peptides are a class of compounds commonly found in living organisms, and to date, tens of thousands of peptides have been found in various organisms. They are widely involved in and regulate various physiological activities in the organism.

Peptide drugs have the advantages of good specificity, outstanding therapeutic efficacy, high safety, low immunogenicity, good membrane permeability and relatively low cost, etc. However, their disadvantages are also obvious, peptide drugs have poor drug-like properties and are susceptible to enzymatic degradation, renal clearance and receptor-mediated rapid clearance, resulting in a short half-life. In addition, the parenteral administration of peptides causes patient discomfort and inconvenience, which may negatively affect compliance and ultimately limit therapeutic efficacy.

One way to solve these problems is to confer new physicochemical properties on peptide drugs by modifying or reforming their structures to improve their druggability while retaining the drug activity.

Peptide Lipidation

Peptide lipidation involves the coupling of a fatty chain to a suitable moiety of a peptide molecule. In nature, cells use lipidation to control the function and intracellular localization of several proteins. When lipoylation is used for peptide drug modification, it may significantly improve the half-life and cell permeability of peptide molecules, and may even take a big leap towards formulations for non-injectable drug delivery.

Although the more common PEGylation may also achieve these effects, some results have shown that the advantages of lipidation are in some cases superior to PEGylation. Moreover, several lipidated peptide drugs have been approved by the FDA (Table 1), such as semaglutide, liraglutide, and tirzepatide, etc.

Table 1. List of lipidated peptides approved by FDA

1. Tirzepatide

Figure 1. Structure of Tirzepatide

Tirzepatide is a first-in-class and the only dual glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) receptor agonist, that can significantly reduce glycemic levels and improve insulin sensitivity （Trade name: Mounjaro, approved in 2022）, as well as reducing body weight by more than 20% (trade name: Zepbound, approved in 2023) and improving lipid metabolism. Tirzepatide has a C20 fatty-diacid portion attached, used to optimize the uptake and metabolism of the compound. The fatty-diacid section (eicosanedioic acid) is linked via a glutamic acid and two (2-(2-aminoethoxy)ethoxy)acetic acid units to the side chain of the lysine residue, which prolongs its duration of action—leading to a half-life of about five days.

2. Semaglutide

Figure 2. Structure of Semaglutide

Semaglutide is a human GLP-1 analog for the treatment of Type 2 diabetes and obesity.  It boasts structural alterations that enhance its efficacy: a longer (C-18) fatty acid chain linked to Lys26 heightens binding to albumin. Furthermore, the substitution of Ala8 with 2-aminoisobutyric acid fortifies the peptide's resilience against peptidases. Notably, Lys34 is replaced by arginine in its sequence, and additional structural modifications contribute to prolonging its presence in plasma, resulting in an extended half-life of approximately 7 days (165-184 hours). This formulation allows for its administration either through subcutaneous injection once a week (Ozempic®) or orally once a day (Rybelsus®).

3. Liraglutide

Figure 3. Structure of Liraglutide

Liraglutide is a GLP-1R agonist that was approved by the FDA as a once-daily injection for the treatment of T2DM in 2010 and for obesity in 2014. It shares a 97% homology with human GLP-1(7-37) but incorporates two key modifications. Firstly, a Lys-to-Arg amino acid substitution occurs at position 34, and secondly, a 16-carbon acyl moiety is conjugated to the side chain of Lys-26 via a γ-glutamic acid spacer. This attached fatty acid moiety enhances binding to serum albumin, leading to a delay in renal excretion. Consequently, liraglutide exhibits a prolonged half-life of approximately 13 hours in humans.

Advantages of Peptide Lipidation

1. Prolonged Half-life

Lipidation improves drug half-life primarily by enabling albumin binding. Albumin, a 585 amino acid containing monomeric protein with a molecular weight of 66.5 kDa, has a very long half-life (20 days). Albumin's main role is to transport hydrophobic molecules in the bloodstream, including fatty acids. Albumin binding improves half-life by limiting the access of proteases to the drug, greatly reducing renal clearance and leveraging FcRn-mediated recycling.

Since albumin binding is reversible, lipidated peptides can exert their drug effects after unbound. Importantly, under normal physiological conditions, only two out of the seven binding sites on albumin are occupied by fatty acids, thus eliminating the threat of lipidated drugs competing for binding sites with the natural carriers of albumin. Increasing the length of the fatty acids on the lipidated drug usually leads to tighter albumin binding as a way to further increase the half-life. Recent research directions include modification of peptides with longer fatty acids (18 or 20 carbons) and the intervention of dicarboxylic acids.

2. Enhanced drug delivery

Lipidation provides a significant advantage by facilitating improved drug delivery. Firstly, lipidation enables a depot to offer delayed drug absorption after subcutaneous injection. Secondly, lipidation enhances cellular permeabilization and facilitates increased uptake of the drug by cells.

Lipidated peptides and proteins are usually administered subcutaneously and can form reversible non-covalent multimers at the injection site. These noncovalent multimers act like a depot, delaying absorption from the injection site to the bloodstream. For example, the formation of subcutaneous depot almost doubles the half-life of liraglutide compared to intravenous administration. Once the drug molecule enters the bloodstream, binding to albumin becomes the primary mechanism for prolonging the half-life.

Another major contribution of lipidation is to increase the cell membrane permeability of peptide drugs (most of the peptide drug molecules are more hydrophilic and therefore less membrane permeable). The lipid moiety increases affinity for the cell membrane and can be inserted into the cell membrane. Increased affinity for the cell membrane also naturally promotes higher cellular uptake, with potential implications for intracellular delivery. Typically increasing the length of the fatty chain results in increased cellular uptake. The formation of stable non-covalently bound multimers also contributes to the enhancement of membrane permeability.

3. Routes of administration

The increased cell membrane permeability of lipidated peptides has also had a profound impact on the development of routes of administration. Although oral peptide drugs have been on the market for the last century (cyclosporine and desmopressin), lipidation has opened a much wider door for oral peptide formulations. Novo Nordisk's oral peptide formulation for type 2 diabetes, Semaglutide (Rybelsus®, approved for marketing by the FDA in 2019) is a lipidated peptide. 

Semaglutide maintains a long half-life after oral administration (approximately 153-160 hours compared to 168 hours after injection). However, oral delivery significantly reduces its bioavailability, thus requiring higher daily doses. Future lipidated reagents for oral administration of peptides may seek shorter fatty acids (e.g., 12-carbon) than semegluitde (18-carbon), which may increase bioavailability. Lipidated therapeutics that do not create stable multimers in the stomach may exhibit enhanced permeability (and bioavailability) owing to heightened interactions with the membrane. Generally, more efficacious lipidated therapeutics intended for oral delivery have the potential to be designed specifically for that route of administration where higher bioavailability is balanced with improvements in half-life.

Besides, lipidation can be used in combination with permeation enhancers. N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) has been used as an enhancer in Rybelsus®, which together with lipidation has contributed to the introduction of this oral type 2 diabetes drug.

Among non-invasive routes of administration for peptides and proteins, the lungs have a special potential for rapid and complete uptake of lipidated molecules. In particular, the alveolar epithelium is very thin and covered by a small amount of fluid, and typically contains much lower levels of drug-metabolizing enzymes than the skin or gastrointestinal tract. However, the number of inhaled biologics that have gained approval is still very few. The utilization of lipidated peptide drugs in pulmonary delivery has not yet been fully developed, but promising.

4. Increased pharmacological potency

Drug modifications theoretically have an impact on the potency and pharmacological activity of the target therapeutic, for which lipidation is no exception. Factors such as the length of the lipid chain, the lipidation site, and the linker used may all have a critical impact. The effects of lipidation cannot be predicted solely from the chemical structure and require specific analysis.

An increase in the length of the lipid chain may either enhance or reduce the efficacy of the drug. Therefore, the choice of lipid chain needs to be screened without adopting the wrong concept that the longer the better. Although the lipidated structures of semaglutide and tirzepatide appear to be complex, they must have been selected after a rigorous screening process and balancing of various performance indicators.

The site of lipidation is also very specific, and different amino acid modifications may lead to very different effects. Therefore, it is necessary to consider different nucleophilic amino acid residues during the design of drug molecules. In addition to the most commonly used lysine side chain amino groups, including N-terminal amino groups with C-terminal carboxyl groups can also be used as potential lipidation sites.

The structure of the linker used to attach the lipid moiety to the target peptide also affects drug potency. For molecules whose potency decreases as the lipid length increases, the use of a long hydrophilic linker for attachment of longer fatty acids can minimize the potency cost of lipidation. However, how linker selection affects drug potency is often unpredictable. Commonly used linkers include, the combination of γ-Glu with ethylene groups (PEG) repeat structures, such as tirzepatide and semaglutide. These hydrophilic linkers may be useful for synthesis and formulation and could improve solubility problems often encountered in lipidated peptides.

5. Reduced immunogenicity

Lipidation has also been shown to reduce the immunogenicity of potential biologics, which has been a major barrier to the clinical translation of protein therapies. As of 2015, 89% of approved biologics reported some immunogenic reactions, 55% of which had an impact on therapeutic efficacy. These reactions include life-threatening autoimmunity, allergic reactions, and accelerated blood clearance due to the formation of anti-drug antibodies (ADA).

Furthermore, some post-translational modifications intended to increase therapeutic half-life are themselves immunogenic, limiting efficacy. Low levels of anti-drug antibodies have been reported in patients receiving lipidated drugs, but the presence of these antibodies has not been shown to reduce therapeutic efficacy to date. On the other hand, in some cases, the immunogenicity of lipidated peptides is even lower than that of their non-lipidated counterparts.

Low levels of anti-drug antibodies have been reported in patients receiving lipidated drugs, but the presence of these antibodies has not been shown to reduce therapeutic efficacy to date. On the other hand, in some cases, the immunogenicity of lipidated peptides is even lower than that of their non-lipidated counterparts.

Conclusion

Lipidation is a clinically validated chemical modification technique that can be used to optimize the pharmacological properties of peptide and protein drugs. The technique provides a means of extending the inherently short half-life of natural molecules to allow for daily, weekly or even longer dosing intervals.

In addition, lipidation can be used to facilitate the oral delivery of biological drugs and potentially alter their distribution in tissues. Peptide lipidation is safe and effective, it can also be optimized by a variety of medicinal chemistry methods, and it allows for cost-effective mass production. In conclusion, lipidation has been successfully applied to drug engineering and may play an increasingly important role in advancing peptide drug development.

Peptides have become a unique class of therapeutic agents in recent years as a result of their distinct biochemical characteristics and therapeutic potential. Biopharma PEG offers high purity PEG derivatives for PEGylation of peptides which has the potential to improve bioavailability compared to the unmodified molecule.

References:
[1] Kurtzhals, P. et al. Derivatization with fatty acids in peptide and protein drug discovery. Nature Reviews Drug Discovery. 2023, 22, 59–80.
[2] Menacho-Melgar R, Decker JS, Hennigan JN, Lynch MD. A review of lipidation in the development of advanced protein and peptide therapeutics. J Control Release. 2019 Feb 10;295:1-12. doi: 10.1016/j.jconrel.2018.12.032. Epub 2018 Dec 21. PMID: 30579981; PMCID: PMC7520907.
[3] Frias, J. P. Tirzepatide: a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) dual agonist in development for the treatment of type 2 diabetes. Expert Rev Endocrinol Metab. 2020, 15, 379-394
[4] Kapitza, C. et al. Semaglutide, a once-weekly human GLP-1 analog, does not reduce the bioavailability of the combined oral contraceptive, ethinylestradiol/levonorgestrel. Journal of Clinical Pharmacology. 2015, 55, 497–504.
[5] Ahangarpour, M. et al. Photo-induced radical thiol-ene chemistry: a versatile toolbox for peptide-based drug design. Chemical Society Reviews. Royal Society of Chemistry. 2021, 50, 898–944.

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