Orally available peptides are desirable to the pharmaceutical industry because they can be self-administered, allow for a wide range of dosage adjustments, and can be quickly terminated if adverse effects occur. However, of the more than 60 peptide drugs, nearly none of them are orally available.

Because peptides are an essential component of food, the gastrointestinal tract contains many enzymes that break them down, thereby rendering peptide-based medications ineffective. Limited oral availability is mainly due to three factors: degradation by proteases, poor crossing of the epithelial layer due to large size and polar surface, and first-pass metabolism in the liver.

Peptides, which are much smaller than proteins and antibodies, are most amenable to oral delivery. Modifications can increase the overall stability of peptides, but the modification process is laborious and often does not produce peptides that have the stability required to survive in the gastrointestinal tract.

In 2018, researchers from the École Polytechnique Fédérale de Lausanne (EPFL) in Switzerland designed a peptide format termed "double-bridged peptides," in which peptides are cyclized by two chemical bridges that provide high stability to the molecule. Cyclic peptides, whose ends are joined together by chemical linkers, are more stable because their backbones are less flexible and thus more difficult for enzymes to reach. Despite the increased stability of these peptides, they still did not withstand degradation in the gastrointestinal tract.

In the current study, the researchers examined a new method to generate peptides that bind a therapeutic target with high affinity and resist proteases in the gastrointestinal tract of a mouse.

First, billions of peptide sequences were cyclized by two chemical bridges that impose conformational constraints to the peptide backbones, creating a library of double-bridged peptides. The peptides were then exposed to protease-resistant fd bacteriophages which allowed for the discovery of target binders after protease digestion. The resistance to protease degradation was tested by incubating phage displays in porcine pancreatin solution. In the last step, the remaining peptides were panned against target proteins (coagulation factor XIa) to determine which peptides have the highest binding affinity.

"It's a bit like searching a needle in a haystack, and this method makes this easy," said Christian Heinis, PhD, professor at EPFL, in a statement.

The researchers generated a peptide inhibitor of the coagulation factor XIa with nanomolar affinity and a gastrointestinal-protease-resistant peptide antagonist for the interleukin-23 receptor (involved in pathogenesis of Crohn's disease and ulcerative colitis).

For the first time, researchers succeeded in engineering target-specific peptides that can resist breakdown in the gastrointestinal tract. The lead peptide from the new method, which inhibits thrombin, was given to mice in an oral gavage. Some of the peptide remained intact long enough to reach the bloodstream and be detected. The researchers indicated that this is a key step toward engineering oral peptide drugs.

"We are focusing on chronic inflammatory diseases of the gastrointestinal tract like Crohn's disease and ulcerative colitis as well as bacterial infections," explained Heinis. "We have already succeeded in generating enzyme-resistant peptides against the interleukin-23 receptor, an important target of these diseases, which affect millions of patients worldwide without any oral drug available."

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