Cyclic Peptide

What is a cyclic peptide

What is a cyclic peptide?A type of peptide therapeutic in which both ends or other parts of a peptide chain are chemically linked to form a cyclic structure.It is attracting attention as a "middle-molecule modality" that combines the ease of synthesis and cell membrane permeability of small molecules with the high target selectivity and protein-protein interaction (PPI) inhibitory ability of macromolecular antibodies and similar agents. Furthermore, stabilizing the structure through head-to-tail cyclization, disulfide bonds, or thioether cyclization can be expected to improve protease resistance. This also makes it possible to approach "undruggable targets," such as flat PPI interfaces, which were difficult to target with conventional small-molecule drugs.

Representative examples of cyclic peptides

Cyclosporin A

Cyclosporin A is a representative macrocyclic peptide derived from natural products,It is widely used as an immunosuppressant.It suppresses the immune response by binding to cyclophilin, a type of immunophilin, and inhibiting calcineurin. N-methylated amino acids and non-natural amino acids<による強固な構造形成により高い代謝安定性を示し、疎水性と自発的な細胞膜透過性を備えることで、経口投与可能な環状ペプチドの先駆的な例となっています。

Pasireotide

Pasireotide iscyclic hexapeptide designed based on somatostatinIt binds broadly to SSTR1 to 5, showing particularly high affinity for SSTR5. Through the introduction of a cyclic skeleton and non-natural amino acids, it overcomes the metabolic vulnerability of natural somatostatin and represents a typical example of an injectable peptide with enhanced duration of action.

Pegcetacoplan

Pegcetacoplan isPEGylated cyclic peptide dimer targeting complement C3And it is used for the treatment of paroxysmal nocturnal hemoglobinuria and other conditions. It is a representative example of a DDS/PK-modified peptide in which PEG is conjugated to a cyclic peptide that strongly inhibits the protein-protein interaction (PPI) with C3, thereby suppressing renal excretion and significantly improving blood retention and duration of action.

Key challenges in cyclic peptide development

Trade-off between membrane permeability and water solubility, as well as renal excretion and peptidase degradation

For cyclic peptides with a molecular weight of 1,000 to 3,000,Balancing membrane permeability and water solubilityis a major challenge. While increasing hydrophobicity or N-methylation leads to decreased water solubility, aggregation, and biliary excretion, increasing polarity results in rapid renal excretion. Furthermore, identifying metabolites, including ring opening and side-chain cleavage by peptidases in the liver, kidneys, and blood, is difficult, making evaluation at the preclinical stage important.

Divergence between spontaneous conformational change and in vivo PPI inhibitory activity

Cyclic peptides easily change conformation in the liquid phase,Even if it shows high affinity in vitro, it cannot maintain the active conformation under physiological conditions.There are cases where, especially in the presence of albumin or within cells, drug efficacy may be diminished. In addition, because it has a large interaction surface,off-target binding to highly homologous similar proteinsalso becomes a challenge.

Immunogenicity and hepato-renal toxicity caused by non-natural amino acids and special cyclic structures

While unnatural amino acids, D-amino acids, and artificial linkers such as thioethers and triazoles enhance metabolic stability,induces immunogenicity such as the production of anti-drug antibodies (ADA)There is a possibility. Additionally, at high doses, caution is required regarding nephrotoxicity due to accumulation in the renal tubules and hepatotoxicity mediated by hepatic uptake transporters such as OATP.

Nonclinical evaluation required for cyclic peptides

Evaluation 1: Multi-faceted evaluation system for "membrane permeability and metabolic stability" linked with 3D structures and physicochemical properties

In cyclic peptides, not only simple PAMPA and Caco-2, but alsoMultifaceted evaluation based on 3D structure and physical propertiesis important. We analyze conformations using NMR and MD, and evaluate stability in terms of membrane permeability, water solubility, plasma, liver S9, and renal microsomes. We identify ring-opening and cleavage sites using LC-MS/MS and rapidly feed the results back into structural optimization.

Evaluation 2: "Live-cell reality evaluation" demonstrating intracellular targets and PPI inhibition, and Target Occupancy (RO) analysis

Cell-free binding assays alone cannot fully reflect intracellular PPI inhibitory activity. In addition to intracellular target binding and target occupancy (RO) analysis using NanoBRET, we verify intracellular action through CETSA and high-resolution imaging. Furthermore,Integrate intracellular RO and in vivo tissue exposure using a PK/PD model (PBPK), improves the prediction accuracy of the human effective dose.

Rating 3: Evaluation of "multimodality PK/PD" to visualize pharmacokinetics and tissue accumulation specific to medium-sized molecules

Cyclic peptides areUneven distribution in the liver, kidneys, spleen, etc., and accumulation in lysosomesBecause this can occur, precise evaluation of tissue distribution is important. Localization is visualized using MSI and QWBA, and unchanged drugs and metabolites are quantified using LC-MS/MS. By combining both methods, it is verified whether the drug reaches the target cells in the target tissue.

Rating 4: Early screening for "immunogenicity and in vitro safety" to capture risks of non-natural structures

In cyclic peptides containing non-natural amino acids or modifying groups,Early evaluation of immunogenicity and hepatotoxicity/nephrotoxicityis important. We evaluate risks using T cell proliferation and cytokine assays using human PBMCs, MAPPs analysis, and iPS cell-derived kidney and liver Organ-on-a-chip models. By identifying immunogenic epitopes and reflecting them in de-immunization design, we enhance safety from the early stages of development.

Key points when choosing a contract research organization (CRO) for non-clinical studies

In the development of cyclic peptides, PK evaluation alone is not sufficient. It is important to conduct multifaceted evaluations—including membrane permeability, metabolic stability, intracellular target occupancy, tissue distribution, and immunogenicity/safety—and verify their link to drug efficacy. Selecting a CRO capable of seamlessly supporting everything from efficacy and disease models to resistance models is the key to development success.