The Role of Chemical Modifications in the Oral Peptide Development
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The Role of Chemical Modifications in the Oral Peptide Development

For a long time, the primary goals in peptide drug development have been the attainment of high affinity, receptor selectivity, and resistance to enzymatic degradation. However, alongside ensuring biological activity, the delivery of peptide drugs has gradually become a focal point in the industry, especially with the emergence of orally active peptides as a new target. The production of orally active peptides requires a dual optimization process: 1) optimization of affinity and selectivity, and 2) optimization of oral efficacy. Chemical modifications of peptides have a significant impact on achieving the development of oral formulations. It is hoped that through the experiences gained from orally available peptide formulations already on the market or in clinical stages, guidance can be provided for the chemical modification of peptides to simultaneously meet the potential therapeutic activity and oral bioavailability of peptide drugs.

Cyclosporine A

Cyclosporine A (CsA), a fungal metabolite, is an eleven-membered cyclic peptide containing seven N-methylated amino acid residues. CsA, primarily composed of hydrophobic amino acids, has been approved for oral immunosuppressive therapy (marketed as Neoral®). Apart from being used for immunosuppression in transplantation, Neoral® is also indicated for the treatment of conditions such as psoriasis, severe atopic dermatitis, pyoderma gangrenosum, chronic autoimmune urticaria, rheumatoid arthritis, and other related diseases.

Due to its high hydrophobicity, CsA exhibits poor oral bioavailability in pure solution formulations, primarily due to its poor water solubility and metabolism by intestinal epithelial cells. The mechanism behind CsA achieving oral delivery is quite unique. In addition to the inherent advantages brought by its cyclic peptide structure, the conformation of the amide bond between the adjacent residues Me-Leu9 and Me-Leu10 plays a subtle but crucial role. The equilibrium between cis/trans conformation of this structurally unique peptide backbone amide bond is highly dependent on the polarity of the environment surrounding the peptide molecule. In polar solvents or when bound to the receptor cyclophilin, trans conformation predominates. However, in non-polar solvents or crystalline form, cis conformation becomes dominant.

Studies have found that the kinetics of cis/trans interconversion (energy barrier of 19 kcal/mol) are closely related to the binding of CsA to cyclophilin. The formation of the CsA-cyclophilin complex, in turn, affects the activity of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase and a crucial signaling enzyme in T lymphocytes. Since the cis/trans equilibrium of this amide bond in CsA depends on the hydrophilicity of the environment, it is hypothesized that CsA mediates the transformation from cis to trans conformation, favoring oral absorption, while trans conformation favors binding to the receptor. This dual role of CsA in oral absorption and receptor binding perfectly meets the two key requirements for oral peptide drugs. The complexity of CsA's natural mechanism has become a major model for studying the oral bioavailability of peptide formulations. However, replicating the subtle conformational changes in other synthetic peptide analogs is challenging.


Lipid modification has opened up a broad avenue for oral peptide formulations. The success of Novo Nordisk's oral peptide formulation Semaglutide (Rybelsus®) for type 2 diabetes (approved by FDA in 2019) has greatly inspired developers to explore oral peptide formulations, and one of the key successes of Semaglutide is lipid modification of GLP-1 analogs.

After oral administration, Semaglutide maintains a long half-life (approximately 153-160 hours, compared to 168 hours after injection). However, the oral delivery significantly reduces its bioavailability, necessitating higher daily doses. Future lipidation reagents for oral peptide drugs may seek shorter fatty acids than semaglutide (18 carbons), which may increase bioavailability.

Tri-N-methylated Veber-Hirschmann Peptides

Veber-Hirschmann peptide cyclo(PFwKTF) is a cyclized somatostatin analog selective for sst2 and sst5 somatostatin receptors. Its tri-N-methylated analogs show higher bioavailability, effectively crossing Caco-2 cell monolayers. These analogs exhibit resistance to degradation in plasma and achieve an absolute oral bioavailability of 10% in rats. Clearly, the N-methylation of these three solvent-exposed amide bonds increases the peptide's lipophilicity. The peptide backbone forms two βII' and βVI-turn secondary structures and a peptide bond primarily dominated by cis-conformation. Yet, these three modifications maintain the spatial configuration of the active Veber-Hirschmann peptide.


Desmopressin, a peptide analog of the hormone vasopressin, is more resistant to enzymatic degradation than vasopressin. The product DDAVP® is approved for oral administration for the treatment of diabetes insipidus. Nocdurna® (desmopressin) is approved as sublingual tablets for the treatment of nocturia.

Structurally, desmopressin is a hydrophilic cyclic peptide linked by disulfide bonds with extensive hydrogen bonding potential. Oral desmopressin is absorbed into the bloodstream through a paracellular transport mechanism, with poor oral bioavailability (0.08-1%). Nevertheless, due to its high target affinity, desmopressin achieves efficacy at low doses. Because of its potent pharmacological effects and high hydrophilicity, desmopressin, along with alpha-Amanitin, suggests that even with very low oral bioavailability, peptide drugs can still be a choice for oral formulations.


ZYOG1 is an orally available linear peptide containing multiple non-natural amino acids and three Cα-methylated amino acids. ZYOG1, similar to other GLP-1 agonists like exenatide , is a compound derived from exendin-4 and developed for the treatment of diabetes. The N-terminal Val is incorporated into these linear peptide precursors to improve their bioavailability. Although ZYOG1 has relatively low oral bioavailability, it reportedly exhibits similar oral efficacy to exenatide administered subcutaneously. ZYOG1 is currently in clinical trials and requires further development to improve its bioavailability and ultimately prove whether this linear peptide can be used as an oral GLP-1 agonist for diabetes treatment.

Griselimycin Derivatives

Griselimycin derivatives derived from Streptomyces act as highly active antimicrobials by inhibiting DNA polymerase in Mycobacterium tuberculosis. Further enhancement of their depsipeptide moiety (F = 48%) is achieved by incorporating non-natural hydrophobic amino acid residues, resulting in very high oral bioavailability. Griselimycin derivatives with cyclohexyl-substituted Pro (R=Cychexyl) exhibit an 89% oral bioavailability, along with increased biological activity. Compared to other orally active peptides, all three griselimycin derivatives show significantly high bioavailability. The overall increase in lipophilicity leads to higher binding affinity and improved bioavailability (griselimycin derivatives are composed of entirely hydrophobic amino acids).


Common orally available peptides seem to favor hexameric cyclic peptides, inspiring enthusiasm for cyclizing linear hexapeptides. The simplest and most direct approach is the cyclization of all-Ala hexapeptides. Although such peptides lack biological activity, they provide a framework for studying the spatial structure of hexameric peptides and exploring the effects of chemical modifications such as N-methylation on peptide spatial structure to further investigate the new biological features of these structural changes on peptide molecules.

The well-known RGD tripeptide structure (recognized by various integrin receptors) has been incorporated into various hexameric peptides to achieve a range of goals, including enhancing gastrointestinal permeability. However, the presence of hydrophilic amino acids weakens the intestinal permeability of peptide molecules. Therefore, researchers have adopted the so-called LPCM (lipophilic prodrug charge masking) strategy to counteract the adverse effects of high polarity on these RGD cyclic peptides. LPC transforms originally poorly absorbed hydrophilic parent peptides (clog P= -3.3) into peptide prodrugs (clog p=6.6) that enter the bloodstream through transcellular pathways, thereby achieving an oral bioavailability of 44% for Cyclo(N-Me-D-Val-Arg(Hoc)2-Gly-Asp(OMe)-Ala-N-MeAla) peptide. Following oral administration, Cyclo(N-Me-D-Ala-Arg(Hoc)2-Gly-Asp(OMe)-Ala-N-MeAla) is cleaved by serum esterases into its active form, becoming a ligand with high activity and selectivity for integrin αvβ3. This active ligand can enhance VEGF-mediated angiogenesis, making it suitable for combination chemotherapy for cancer treatment.

MyR-c(MyD 4-4)

The cyclic peptide c(MyD 4-4) inhibits the cytoplasmic adaptor protein MyD88 and is developed for the treatment of autoimmune diseases. c(MyD 4-4) disrupts the dimerization of MyD88 and blocks the stimulation of TLR2 and TLR4 in human and murine macrophages. To overcome the low oral bioavailability of c(MyD 4-4) (F=0.84±0.25%), researchers coupled a cardamoyl group (MyR) to an amidine structure to generate the peptide MyR-c(MyD 4-4). The modified peptide exhibits significantly increased oral bioavailability (F=47.6±16.2%) and enhances inhibitor activity. Perhaps due to the introduction of the lipophilic cardamoyl group, the modified peptide alters the spatial structure of the peptide, thereby disrupting the salt bridge between positively charged arginine and negatively charged aspartate.

Endorphin-1 Analogs

The linear opioid peptide endorphin-1, composed of four natural amino acids, undergoes rapid enzymatic degradation, resulting in poor bioavailability and short duration of action. However, glycosylation greatly enhances the bioavailability of endorphin-1, providing a foundation for oral delivery.

Endorphin-1 analogs modified with lactose and succinic acid arms experience a 700-fold increase in membrane permeability and a duration of action lasting up to 2 hours. However, the addition of glucosyl succinic acid units significantly reduces the ligand-peptide binding affinity of mu-opioid receptors (MOR). Despite adverse effects on MOR binding affinity and agonist activity, the modified endomorphin-1 analog still exhibits nanomolar binding affinity. It produces dose-dependent pain relief after oral administration in rats. This example demonstrates that in certain cases, glycosylation helps to improve the bioavailability of peptides while retaining acceptable receptor binding affinity.

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