Mechanistic benchmarking of SNAC, DDM, and LLC reveals enhancer-dependent pathways for intestinal semaglutide delivery

Abstract

The oral delivery of peptide therapeutics remains constrained by poor intestinal permeability and the challenge of enhancing epithelial transport without compromising mucosal integrity. Although sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC) enables gastric absorption of oral semaglutide and has also been explored for small-intestinal absorption of other peptide drugs, its suitability for enteric intestinal delivery of semaglutide remains unexplored. Here, we performed a comparative mechanistic evaluation of three permeation enhancers (SNAC, N-dodecyl-β-D-maltoside, and lauroyl-l-carnitine) for enteric delivery of semaglutide. Mechanisms were screened in epithelial monolayers and in situ intestinal absorption in rats, and the enhancers carried forward were formulated into enteric-coated capsules and evaluated in beagle dogs, with the leading candidate taken in to cynomolgus monkeys.

In differentiated Caco-2 monolayers, all enhancers increased semaglutide transport but exhibited distinct transport mechanisms and barrier-response profiles. SNAC increased permeability without increasing mannitol transport or disrupting ZO-1, indicating a largely non-paracellular, cargo-dependent profile. DDM markedly increased paracellular marker transport and disrupted tight-junction organization, consistent with a primarily paracellular mode of action associated with reduced barrier recoverability. LLC showed a more favorable balance between transport enhancement and barrier recovery, while also increasing intracellular semaglutide-associated fluorescence and FD4 permeability. Rab5 immunostaining and dynasore inhibition indicated a dynamin-sensitive uptake component alongside partial tight-junction modulation. In rats, LLC produced the greatest increase in semaglutide absorption, and this effect was substantially attenuated by dynasore co-treatment. Transmission electron microscopy and tissue immunofluorescence further supported the presence of LLC-associated micelle-like structures, Rab5-related intracellular trafficking and junctional remodelling, with no overt tissue injury on local or systemic histology. For large-animal translation, citric acid and L-arginine improved semaglutide stability in intestinal fluids, and the resulting LLC-containing enteric formulation markedly increased systemic exposure in orally dosed beagle dogs and in cynomolgus monkeys following intraduodenal delivery.

Collectively, these findings identify LLC as a promising permeation enhancer within the present SNAC/DDM/LLC comparison for enteric intestinal delivery of semaglutide and highlight how coordinated uptake-related and barrier-modulating mechanisms can support intestinal semaglutide absorption.

1. Introduction

Oral delivery of peptide therapeutics remains challenging because gastrointestinal absorption is constrained by both luminal instability and poor epithelial permeability [1], [2], [3], [4]. In addition to enzymatic and chemical degradation, large hydrophilic molecules cross the intestinal epithelium inefficiently, resulting in low and often variable systemic exposure [5], [6]. Formulation strategies that improve epithelial transport while preserving barrier integrity therefore remain central to the development of clinically useful oral peptide medicines [7], [8], [9], [10].

Permeation enhancers (PEs) are now being used in approved and clinically advanced oral peptide formulations. The most prominent example is sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), the PE used in Rybelsus, the first approved oral semaglutide tablet. Each tablet contains 300 mg SNAC with 3, 7, or 14 mg semaglutide, yet absolute oral bioavailability is reported to be only 0.4–1% [11], [12]. The PIONEER 1 trial demonstrated glycaemic efficacy of oral semaglutide monotherapy versus placebo in patients with type 2 diabetes [13]. Rybelsus is designed to promote absorption primarily in the stomach, where SNAC contributes to local pH modulation, limits proteolysis, and enhances semaglutide permeation [12]. However, SNAC activity is not confined to this gastric context or to a strictly transcellular-only mechanism. Its use has been investigated with other peptide and macromolecular cargoes, including vitamin B12, low-molecular-weight heparin, calcitonin, and intraduodenal hPTH(1–34)/teriparatide, and SNAC-associated changes in tight-junction proteins have also been reported in some epithelial and in vivo models [7], [9], [14], [15], [16]. SNAC behavior is therefore likely cargo-, formulation-, concentration-, tissue- and model-dependent. The in vivo pig hPTH(1–34) study used a different peptide, direct duodenal instillation, and combined permeation enhancement with protease inhibition, and therefore, does not directly establish the suitability of SNAC for enteric intestinal semaglutide delivery [15].

Whether SNAC retains useful activity when semaglutide is instead released in the small intestine remains unresolved. As intestinal delivery strategies continue to be explored for peptides and other macromolecules, there is a need to identify enhancers whose activity is compatible with the enteric intestinal epithelium.
The selection of an intestinal PE is complicated by the mechanistic diversity of this class. Increased apparent permeability may arise from tight-junction modulation, membrane perturbation, altered intracellular trafficking, peptide–enhancer association, or nonspecific epithelial injury [9], [17], [18]. These mechanisms are not interchangeable, because they differ in reversibility, safety margin, cargo dependence, and suitability for different gastrointestinal release sites. Sodium caprate (C10), one of the most extensively studied intestinal PEs, for example, has provided an important benchmark for tight-junction and membrane-associated enhancement in epithelial and intestinal models [7], [9], [19], [20]. However, while SNAC and C10 have already been compared extensively under matched Caco-2 and isolated intestinal mucosal conditions, these studies have not been extended to other PEs [19], [20]. This present study focuses on less directly benchmarked enhancer classes that may offer distinct mechanistic profiles for intestinal semaglutide delivery. These mechanistic differences highlight the need for matched comparisons when selecting PEs for intestinal peptide delivery.

N-dodecyl-β-D-maltoside (DDM) and lauroyl-l-carnitine (LLC) were selected together with SNAC to provide a cross-mechanism comparison. DDM is a non-ionic alkylmaltoside enhancer that has been investigated for transmucosal delivery and is commonly associated with membrane interaction and epithelial barrier modulation [21], [22], [23], [24]. Acylcarnitines have long been investigated as gastrointestinal absorption enhancers [25]. LLC is an acylcarnitine-based enhancer with a different amphiphilic structure and has clinical-stage precedent within Enteris BioPharma’s Peptelligence platform, where acylcarnitine-containing formulations have been combined with citric acid for oral peptide delivery, including leuprolide formulations that have advanced to clinical evaluation [7], [9], [26], [27]. Thus, SNAC, DDM, and LLC were chosen not as an exhaustive screen of all available PEs, but as mechanistically and translationally relevant representatives: SNAC as the clinically validated benchmark for oral semaglutide, DDM as an alkylmaltoside enhancer with barrier-modulating activity, and LLC as an acylcarnitine enhancer with clinical-stage oral peptide delivery precedent. This design allows the study to ask whether maltoside- or carnitine-based enhancers provide a more suitable intestinal enhancement profile than SNAC under enteric delivery conditions, while recognizing that C10 has already been addressed extensively in prior head-to-head studies.

Here, we compared SNAC, DDM, and LLC mechanistically for enteric intestinal semaglutide delivery across an escalating sequence of models. Transport mechanisms were resolved in Caco-2 monolayers and tested by in situ intestinal absorption in rats, and the enhancers carried forward were formulated into enteric-coated capsules for pharmacokinetic evaluation in beagle dogs and cynomolgus monkeys. As such, the study addresses how route-specific enhancer behavior should inform formulation design for oral peptide delivery.

2. Materials and methods

2.1. Chemicals and reagents

Semaglutide was obtained from Neuftech (Jiangsu, China). Salcaprozate sodium (SNAC) was supplied by Leyan (Shanghai, China). N-Dodecyl-β-D-maltoside (DDM) and lauroyl-l-carnitine (LLC) were purchased from Neuftech (Jiangsu, China) and Glpbio (USA), respectively. L-Arginine was acquired from Sigma (Japan). d-Mannitol was sourced from Adamas Life (Shanghai, China). Fluorescein isothiocyanate–dextran 4 kDa (FITC-dextran) was obtained from Sigma-Aldrich (Sweden). Ready-to-use enteric-coated capsules (EUDRACAP® Enteric, size 2) were obtained from Evonik (Shanghai, China). Citric acid monohydrate, and all excipients used for granulation were of pharmaceutical grade and were purchased from Adamas Life (Shanghai, China). The dynamin inhibitor Dynasore and Cell Counting Kit-8 (CCK-8) were purchased from Sigma-Aldrich (USA) and Dojindo Laboratories (Japan), respectively. iFluor™ 647 phalloidin was supplied by Yeasen (Shanghai, China).

Primary antibodies against ZO-1, Claudin-4, Occludin, Rab5, PEPT1, and SLC22A5 were procured from Sigma-Aldrich (USA). Species-specific fluorescent secondary antibodies were also obtained from Sigma-Aldrich (USA). All cell culture media and supplements, including Minimum Essential Medium (MEM), fetal bovine serum, and penicillin-streptomycin, were purchased from Gibco (USA). All other chemicals and solvents were of analytical grade or higher and used as received.

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Banghao Yuan, Yusheng Sui, Hanxiang Li, Jinyan Huang, Ye Chan Oh, Yajie Zhao, Xueyu Li, Yuheng Sun, Liu Dou, Abdul W. Basit, Yang Mai, Mechanistic benchmarking of SNAC, DDM, and LLC reveals enhancer-dependent pathways for intestinal semaglutide delivery, Journal of Controlled Release, 2026, 115455, ISSN 0168-3659,
https://doi.org/10.1016/j.jconrel.2026.115455.