Abstract
Peptide drugs offer considerable potential for treating a diverse range of diseases. Yet, their clinical application is generally restricted to injectable therapies. The main challenge hindering their broader use through globally accessible, patient-friendly, and non-invasive delivery routes such as oral or buccal, lies in their poor ability to cross biological barriers effectively. Here, we demonstrate that enzymes can be harnessed to transiently reduce these barriers and improve absorption. As a proof of concept, we employ a mucin-specific protease (mucinase) and a phospholipase to increase mucus diffusivity and epithelial cell membrane permeability, respectively. In a canine model, we show that enteric capsules containing both enzymes, and the peptide drug desmopressin achieved a relative bioavailability of 155 % compared to the drug alone. Additionally, a buccal patch loaded with phospholipase and semaglutide displayed a 5-fold higher bioavailability and lower variability (71.5 % reduction in the coefficient of variation) compared to the commercially available oral tablet. These results suggest that enzymatic modulation of biological barriers holds promise as a strategy to improve non-invasive delivery of peptides and potentially other macromolecular drugs.
Introduction
Peptide drugs are highly valued for their specificity and potency, making them excellent therapeutic options for a diverse range of diseases [[1], [2], [3], [4]]. However, their clinical application is often restricted by the necessity of injections, limiting their use to conditions where such interventions are feasible. This can not only impede patient adherence, leading to suboptimal treatment outcomes and higher healthcare costs, but also constrains their availability to regions with developed medical infrastructure [[5], [6], [7]].
In recent years, enormous efforts have been directed towards implementing less invasive, more convenient, and globally accessible delivery systems for peptide drugs [[7], [8], [9], [10], [11]]. Yet, the inherent instability and large molecular size of most therapeutic peptides represent substantial challenges [1,2,4]. While oral dosing is generally regarded as the most desirable administration mode due to its ease of use and cost-effectiveness, it is strongly limited by gastrointestinal (GI) barriers. Upon ingestion, peptides must endure the harsh digestive environment, penetrate the mucus layer, and traverse the intestinal epithelium before being subjected to the hepatic first-pass metabolism [7,9,12].
Mucus is a viscoelastic hydrogel-like substance that protects the underlying epithelium by trapping and clearing pathogens and foreign particles, significantly limiting peptide permeability and facilitating rapid clearance from the delivery site [[13], [14], [15]]. Beyond the mucus layer, peptides encounter the intestinal epithelium, a barrier that is largely impermeable to macromolecules and strictly regulates the passage of molecules into the bloodstream [[16], [17], [18]]. While gastric degradation can be mitigated through the use of enteric formulations that protect drugs until they reach the intestine, the mucus and the intestinal epithelium are absorption barriers that are more difficult to tackle [7,9,12].
Compared to oral delivery, the buccal route is advantageous as its accessibility allows to place the drug directly to the target site,enables the formation of a strong transmucosal concentration gradient, and its unique physiology facilitates direct access of drugs to the systemic circulation, avoiding GI barriers and the hepatic first-pass metabolism [[19], [20], [21]]. However, the epithelial layers of the buccal epithelium effectively prevent the entry of macromolecules [22,23].
In summary, limited mucus penetration, rapid clearance, and inadequate permeability across intestinal or buccal epithelia are significant hurdles that generally lead to low bioavailability (BA) for most peptide drugs (Fig. 1a) [[8], [9], [10],16].
In recent years, a plethora of formulation strategies have been explored to enhance non-invasive peptide delivery via the oral or buccal route. These include chemical permeation enhancers (PE) (e.g. surfactants, bile salts, medium-chain fatty acids, and chelators) [24], mucoadhesives [25], nano- and microparticulate systems [26], and ingestible devices that inject substances directly into the gastric or intestinal mucosa [27,28]. While ingestible devices have shown promising results, achieving BAs in the double-digit range, their translation into the clinic is slowed down by their complexity, reliability issues, uncertain regulatory pathways, and potential safety concerns [[29], [30], [31]]. Despite extensive efforts, clinical success remains limited, with only a few peptide drugs available as non-injectables, underscoring the need for new permeation-enhancing approaches [9,11,32].
Interestingly, bacteria have evolved highly specialized mechanisms to cross biological barriers and infect their hosts [33,34]. Certain bacteria express proteases with a high selectivity for mucins, degrading mucus and allowing the bacterium to penetrate and/or traverse the mucus layer (Fig. 1b,c) [35]. Another approach used by many pathogenic bacteria is phospholipases, enzymes that hydrolyze phospholipids – the major components of cell membranes (Fig. 1d,e) [36,37].
In this work, we investigate the potential of using highly specific and potent bacterial enzymes to improve non-invasive systemic peptide delivery. The central hypothesis is that these bacterial mechanisms can be harnessed to enhance the BA of peptides through oral and buccal routes. As a proof of concept, we employed a O-glycoprotein-specific protease (mucinase) and a phospholipase to specifically, effectively, and transiently reduce mucus and epithelial barriers. StcE, a mucinase from Escherichia coli (E. coli), was selected for its specificity for mucin proteins, enabling the selective degradation of mucin-proteins without affecting other non-mucin glycoproteins, including therapeutic peptides, and its natural evolution for stability and activity in the small intestine, where it targets and degrades GI mucins [38].
The phospholipase C (PLC) from Bacillus cereus was selected for its ability to transiently disrupt epithelial cell membranes by catalyzing the hydrolysis of phospholipids. PLC cleaves the phosphodiester bond between the glycerol backbone and the phosphate group, leading to localized alterations in membrane integrity that may facilitate enhanced peptide absorption [36,37,39]. Additionally, its optimal enzymatic activity occurs at pH 6–8, aligning well with the physiological conditions of the small intestine and buccal environment [40]. These features make StcE and PLC potentially suitable for non-invasive peptide delivery via oral and buccal routes, where overcoming the intestinal mucus and epithelial barriers is critical for enhancing BA. We show that these enzymes can promote peptide absorption, leading to BAs in dogs that can even surpass the oral gold standard commercial formulation of the peptide drug semaglutide (SMG, Rybelsus®). These results showcase the feasibility of using enzymes for non-invasive peptide delivery and lay the groundwork for the development of innovative dosage forms for other macromolecular drugs.
Download the full article as PDF here Enzymatic absorption promoters for non-invasive peptide delivery
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Marilena Bohley Steiger, Angela Steinauer, Daniel Gao, David Klein Cerrejon, Hanna Krupke, Miguel Heussi, Padryk Merkl, Alexander Klipp, Michael Burger, Cristina Martin-Olmos, Jean-Christophe Leroux, Enzymatic absorption promoters for non-invasive peptide delivery, Journal of Controlled Release, Volume 382, 2025, 113675, ISSN 0168-3659, https://doi.org/10.1016/j.jconrel.2025.113675.
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