Peptides and proteins are not orally bioavailable in mammals, although a few peptides are intestinally absorbed in small amounts. Polypeptides are generally too large and polar to passively diffuse through lipid membranes, while most known active transport mechanisms facilitate cell uptake of only very small peptides. Systematic evaluations of peptides with molecular weights above 500 Da are needed to identify parameters that influence oral bioavailability. Here we describe 125 cyclic peptides containing four to thirty-seven amino acids that are orally absorbed by mammals. Cyclization minimizes degradation in the gut, blood, and tissues by removing cleavable N- and C-termini and by shielding components from metabolic enzymes. Cyclization also folds peptides into bioactive conformations that determine exposure of polar atoms to solvation by water and lipids and therefore can influence oral bioavailability. Key chemical properties thought to influence oral absorption and bioavailability are analyzed, including molecular weight, octanol-water partitioning, hydrogen bond donors/acceptors, rotatable bonds, and polar surface area. The cyclic peptides violated to different degrees all of the limits traditionally considered to be important for oral bioavailability of drug-like small molecules, although fewer hydrogen bond donors and reduced flexibility generally favored oral absorption.

The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation.

Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N-methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N-methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N-methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N-methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N-methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog.cyclic peptide | permeability | N-methylation P eptides are potentially valuable compounds for drug development, offering many advantages over other molecular classes (1-4). Specifically, their ability to mimic endogenous bioactive molecules allows them to bind potently and selectively to "difficult" drug targets, including protein-protein interactions that are too challenging for small-molecule therapeutics. However, the widespread use of peptides in the clinic has been slow in coming, in large part because of their generally low stability in vivo, high clearance, and poor oral bioavailability.The low oral bioavailability of peptides is attributed to a disparity between their physicochemical properties and those traditionally expected for "drug-likeness" (5, 6), leading to a perception that peptides are good drug leads but poor drugs. However, this perception is being challenged by a growing number of peptides that seem to be stable (7) and well absorbed within the gastrointestinal tract (8) and examples in which cyclic peptides have shown orally delivered bioactivity in animal disease models, including inflammatory pain (9) and neuropathic pain (10), prompting us to devise new rules for predicting pharmacokinetic properties of this compound class. Arguably the most famous example of a peptide with poor drug-likeness but reasonable oral bioavailability is cyclosporin A, widely used as the immunosuppressant drug cyclosporine (11). Two structural features of cyclosporin A in particular are thought to contribute to its oral bioavailability, namely its macrocyclic architecture and backbone N-methylation.Cyclization imparts increased rigidity to a parent peptide, which not only improves its stability against proteolytic degradation but also directs it into specific conformations that m...

The first total synthesis and three-dimensional solution structure are reported for sanguinamide A, a thiazole-containing cyclic peptide from the sea slug H. sanguineus. Solution phase fragment synthesis, solid phase fragment assembly, and solution macrocyclization were combined to give (1) in 10% yield. Spectral properties were identical for the natural product, requiring revision of its structure from (2) to (1). Intramolecular transannular hydrogen bonds help to bury polar atoms, which enables oral absorption from the gut.