On the Utility of Chemical Strategies to Improve Peptide Gut Stability

Abstract

Inherent susceptibility of peptides to enzymatic degradation in the gastrointestinal tract is a key bottleneck in oral peptide drug development. Here, we present a systematic analysis of (i) the gut stability of disulfide-rich peptide scaffolds, orally administered peptide therapeutics, and well-known neuropeptides and (ii) medicinal chemistry strategies to improve peptide gut stability. Among a broad range of studied peptides, cyclotides were the only scaffold class to resist gastrointestinal degradation, even when grafted with non-native sequences. Backbone cyclization, a frequently applied strategy, failed to improve stability in intestinal fluid, but several site-specific alterations proved efficient. This work furthermore highlights the importance of standardized gut stability test conditions and suggests defined protocols to facilitate cross-study comparison. Together, our results provide a comparative overview and framework for the chemical engineering of gut-stable peptides, which should be valuable for the development of orally administered peptide therapeutics and molecular probes targeting receptors within the gastrointestinal tract.

Introduction

Oral delivery presents one of the greatest challenges in peptide drug development. (1,2) Although preferred by pharmaceutical manufacturers and particularly by patients, less than 10% of current peptide drugs are given orally. (3,4) The unique physiology and physicochemical environment of the gastrointestinal tract render oral administration of peptide and protein therapeutics inherently difficult. (5) The lining of the gut imposes three major barriers on orally ingested peptide drugs: an enzymatic barrier, (6) a mucosal diffusion barrier, (7) and an absorption barrier. (8) In the stomach, parietal cells create a highly acidic environment by secreting hydrochloric acid, which sets the stage for the digestive enzyme pepsin.

This endopeptidase initiates the main digestion process and preferentially cleaves peptide bonds at the site of aromatic and hydrophobic amino acids. (9) The major digestive machinery of the gut is, however, located in the intestine, where a mixture of highly functional peptidases, lipases, and amylases (pancreatic enzymes) degrades nutrients and presents a serious stability hurdle for peptide drugs. Pancreatic peptidases that are secreted into the lumen of the intestine span a broad substrate specificity and include the endopeptidases trypsin (cleavage sites: Arg and Lys), chymotrypsin (cleavage sites: aromatic and hydrophobic residues), and elastase (cleavage sites: small hydrophobic residues) as well as the exopeptidases carboxypeptidase A (cleavage sites: aromatic, neutral, and acidic amino acids) and B (cleavage sites: Arg and Lys). (10) In addition, a series of brush border peptidases, which are located at the surface of the intestinal epithelial lining, add up to the digestive strength of the intestine. (6) Limited permeation through the mucus-covered gut-barrier further contributes to the recognized low oral bioavailability of peptides.

The molecular size (typically >700 Da) and hydrophilic nature (H-bonding capacity) of peptides hinder the diffusion and uptake process into the bloodstream and often require the implementation of specific delivery formulations and technologies. (2,11−13) Recent examples include the permeation enhancer-based oral formulations of the GLP-1 (glucagon-like peptide-1) receptor agonist semaglutide (14) and an engineered insulin analogue (Novo Nordisk’s OI338 and related OI320) (15,16) for the treatment of diabetes. Despite these advances, it remains highly challenging to develop peptide drugs with good systemic oral bioavailability (>20%).

By contrast, druggable receptors accessible within the gut lumen are increasingly recognized targets for orally administered peptide therapeutics because this strategy removes the necessity of crossing the absorption barrier. (3,17−21) Compounds that remain peripherally restricted to the luminal side with no or negligible oral bioavailability are also often safer because of reduced risks of variable absorption and systemic side effects. Conditions that are successfully targeted via local luminal peptide delivery include infections, inflammatory bowel diseases (IBD, including ulcerative colitis and Crohn’s disease), celiac disease, and constipation, with about 10 compounds either on the market or in clinical development. (4,22,23) Potential luminal accessible targets also exist for diabetes, obesity, and abdominal pain. (17,24−26)

Most successfully, orally administered peptides that activate luminal gut GC-C (guanylyl cyclase-C) receptors for the treatment of gastrointestinal disorders have emerged as a novel drug class. (18,23,27−29) Linaclotide, a synthetic 14-mer and three disulfide bond containing GC-C agonist, is available as an oral drug in chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C). (30−32) More recently, close structural analogues of the endogenous GC-C agonist uroguanylin are being considered for the treatment of the same conditions. (33) Plecanatide, a 16-mer with two disulfide bonds, has been approved by the Food and Drug Administration (FDA) for the oral treatment of CIC and IBS-C. (34,35) In view of the gut-specific activity of such compounds, improving peptide stability to maintain sufficient bioactivity in the hostile environment of the gut has become a central aspect in peptide drug development.

Efforts to improve the metabolic stability of peptides have been driven by considerable advances in chemical methodologies available for synthetic peptide modifications. (36−42) To prevent proteolytic cleavage of specific amide bonds, site-directed engineering of the l-α-peptide backbone with unnatural amino acids (e.g., D-α, N_α-alkylated, C_α-substituted, β- and γ-amino acids) (43−47) and amide bond mimetics (e.g., thioamides, (48) azapeptides, (49) 1,4-disubstituted 1,2,3-triazoles (50)) has been developed. (51) More general strategies for molecular peptide stabilization involve polymer conjugation (52,53) and the use of cyclization to engineer rigid structures, (42,54−58) both of which can prevent hydrolysis by hindering protease access to cleavable bonds. N-to-C-terminal backbone cyclization and side-chain stapling via disulfide bonds are also key structural motifs in several natural peptide scaffolds that are proposed as stable templates to engineer drug leads via grafting small epitopes into their framework. (59−63) High thermal, enzymatic, and/or serum stability has been indicated for natural and engineered versions of cyclotides, (64,65) θ-defensins, (66,67) sunflower trypsin inhibitor (SFTI-1), (68,69) conotoxins, (70,71) and chlorotoxin. (72) However, little is known about the utility of these scaffolds and modification strategies to specifically improve gut stability.

Considering the wide implications that gut-stable oral peptide therapeutics would have on peptide drug development and patients with gastrointestinal disorders, we investigated commonly used approaches to study and enhance peptide gut stability. In particular, we (i) evaluated the gut stability of well-known disulfide-rich peptide scaffolds, approved orally administered peptide drugs, and neuropeptides and (ii) assessed several medicinal chemistry strategies to improve gut stability (independent of impact on bioactivity). We also highlighted the importance of using standardized stability test conditions to ensure reliable consistency and comparability across studies.

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Thomas Kremsmayr, Aws Aljnabi, Juan B. Blanco-Canosa, Hue N. T. Tran, Nayara Braga Emidio, and Markus Muttenthaler, Journal of Medicinal Chemistry 2022 65 (8), 6191-6206
DOI: 10.1021/acs.jmedchem.2c00094

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