Salcaprozate-based ionic liquids for GLP-1 gastric delivery: A mechanistic understanding of in vivo performance

Abstract

Oral delivery of peptides requires formulations with high concentrations of permeation enhancer (PE) to promote absorption, and often necessitates fasting time between dosing and food ingestion. Improved formulations promoting a more rapid absorption would increase convenience of use but requires a faster onset of action. We have developed a salcaprozate-based ionic liquid (IL) formulation, namely choline salcaprozate (CHONAC), for oral delivery of a glucagon-like peptide-1 (GLP-1) analogue via gastric absorption. In vitro studies confirmed the higher amount of PE accommodated in the same volume of dosage form as well as faster release of the active pharmaceutical ingredient (API) and PE compared to the tablet reference. Storage stability of the CHONAC formulation was demonstrated for up to 3 weeks at 4 °C. The peptide absorption efficacy of the IL formulation was first evaluated in vivo in rats and anesthetized dogs, showing a faster absorption compared to the reference formulations. In awake dogs, while the CHONAC formulation still enabled earlier API absorption, its overall exposure was inferior to the tablet reference. This was attributed mostly to the gastric physiology, causing formulation dilution in the presence of additional fluid as well as fast transit of liquids into the duodenum, where peptides liable to proteolytic degradation such as the one used in this study showed a negligible absorption, potentially also due to a lower permeation-enhancing capability of CHONAC in the duodenal region. Exploring these issues, an in vivo study in anesthetized dogs involving repeated dosing of a liquid salcaprozate-based formulation in the stomach revealed the potential to sustain peptide absorption throughout the dosing period with a constant absorption rate. In conclusion, combining the advantages of high PE amounts and fast onset of action provided by the IL formulation, and ensuring a prolonged interaction of peptide and PE at a relevant concentration with the stomach epithelium, are necessary to enhance oral peptide bioavailability via gastric delivery.

Introduction

Oral formulations of biomacromolecules, such as proteins and peptides, can provide a more convenient and less invasive alternative to intravenous or subcutaneous administration. However, there are several significant challenges associated with oral delivery of biomacromolecules, including their enzymatic degradation in the gastrointestinal tract and their low permeability across the absorptive epithelium due to their size and polarity, thereby resulting in poor bioavailability [[1], [2], [3]]. In addition, the absorption rate and extent of oral biomacromolecules can be influenced by multiple factors, including food intake, gastrointestinal motility, and interactions with other drugs or excipients, leading to variability in pharmacokinetics [4,5].

To overcome these obstacles, new strategies for oral delivery of biomacromolecules have been developed, such as the use of permeation enhancers (PEs), protease inhibitors and nanoparticle-based delivery systems [6,7]. Among the PEs, sodium salcaprozate (SNAC) has gained attention due to the success in the clinical trials of oral semaglutide [8,9], a glucagon-like peptide-1 (GLP-1) analogue. SNAC enables gastric absorption of semaglutide by fluidizing the epithelial membrane and creating a microenvironment of higher pH which protects the peptide and inactivates the surrounding enzymes [10]. Nevertheless, PE-based tablet formulations are still suboptimal as oral dosage forms, and the achieved bioavailability is not yet close to that of small molecules. Moreover, they often require high doses of PE per dosage form to be effective. For instance, the currently marketed oral semaglutide tablets contain 300 mg of SNAC, whereas oral insulin tablets tested in clinical trials comprise 550 mg of sodium caprate, another PE [[11], [12], [13]]. The dosing conditions are also more restricted compared to standard solid oral formulations, as food intake typically leads to dilution and degradation of biomacromolecules [14]. Therefore, it is important to ensure that most active pharmaceutical ingredient (API) and PE are available for absorption as fast as possible to minimize the time between dosing and food ingestion [15].

In the case of tablet formulations, a fast tablet disintegration is required to increase the rate of dissolution of API and PE [16,17]; however, it is difficult to achieve without the addition of several excipients considering the high doses and hydrophobic nature of the PEs. Alternatively, liquid formulations can provide a faster onset of action by bypassing the disintegration and dissolution steps. Nevertheless, PE-based aqueous formulations require very high volumes to accommodate the large amount of PE per dosage unit, considering the limited PE water solubility in low-to-mid pH environment [18]. Moreover, ensuring the stability of API and PE, especially at high concentrations in aqueous media, is challenging. Therefore, novel liquid vehicles are needed for oral delivery of peptides.

Ionic liquids (ILs) have been recently emerging as a drug delivery system due to their unique physicochemical properties [19,20]. In fact, attempts have been made to use ILs as delivery systems for peptides, such as the choline-geranic acid (CAGE) system used for oral delivery of insulin in rats [21]. ILs are salts where the composing ions are poorly coordinated, resulting in a liquid state below 100 °C, or even at room temperature [21,22]. ILs can dissolve a variety of compounds, including polar and non-polar substances. PEs such as SNAC contain an organic anion, which could lead to the formation of a poorly coordinated salt, i.e., IL, by replacing the sodium cation with an appropriate one. In doing so, the permeation enhancing capabilities would be retained, as the organic moiety is responsible for interacting with the epithelial membrane, while the cation would affect the physical form of the salt, leading to a liquid state which would enable high concentrations of PE per dosage unit.

Fig. 1. (A) Chemical structure of the low potency GLP-1 analogue used in this study (B) CHONAC formation reaction, choline bicarbonate is mixed with the acid form of SNAC (HNAC) in 1:1 M ratio, forming carbon dioxide and water as byproducts (C) CHONAC ionic liquid, and (D) Scheme of CHONAC manufacturing process. HNAC is added as powder to an 80 % w/w solution of choline bicarbonate; the neutralization reaction takes place immediately, being tracked by the formation of CO2 bubbles; the reaction is accelerated by increasing the temperature to 60 °C; in this step any thermostable solid can be incorporated to the formulation; after its completion, the formed CHONAC is dried by rotary evaporation and vacuum heating to remove water from the choline bicarbonate initial solution and formed during the neutralization reaction; the solvent content of the dried CHONAC base can be adjusted then to the desired value; any thermosensitive solids as the API used in this work can be incorporated in this final step.

In this work, we developed a salcaprozate-based IL formulation containing a fatty acid protracted, low potency, long elimination half-life GLP-1 analogue API (Fig. 1A) by using the organic anion N-(8-(2-hydroxybenzoyl)amino)caprylate (NAC) of SNAC and choline as counter ion. We assessed the in vitro release of API from the IL and corresponding tablet formulations. Moreover, we investigated an array of formulation parameters, such as ionic strength, viscosity and API loading, potentially impacting the in vivo behavior. Ultimately, a series of IL formulations were evaluated in vivo in dogs, to gain a mechanistic understanding of such systems and elucidate their potential to serve as oral delivery vehicle for enhancing peptide absorption and bioavailability.

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Materials

The API and SNAC were supplied by Novo Nordisk. NaOH 2 N and HCl 2 N were purchased from Merck (Darmstadt, Germany). Ultrapure water (MQ, 18 MΩ) was prepared in the laboratory with a Millipore-Q system (Darmstadt, Germany) for all aqueous solutions. NaOH 1 N, HCl 1 N and Choline bicarbonate 80 % w/w were purchased from Merck (Darmstadt, Germany). Deionized water (18 MΩ) was prepared in the laboratory with a Millipore-Q system (Darmstadt, Germany). Hard shell gelatin-based capsules (Capsugel®) were obtained from Lonza (Basel, Switzerland).

Analytical grade KH2PO4, NaOH pellets, NaCl, Titrisol ampoules 1 N NaOH and HCl, HPLC grade acetonitrile were all purchased from Merck (Darmstadt, Germany). HPLC grade trifluoroacetic acid (TFA) was purchased from Fisher Scientific (Loughborough, UK). Canine powder for fasted-state simulated gastric fluid (FaSSGF) was purchased from Biorelevant (London, UK). HPLC grade maleic acid was purchased from Sigma-Aldrich (St. Louis, MO, US).

René Rebollo, Zhigao Niu, Lasse Blaabjerg, Damiano La Zara, Trine Juel, Henrik Duelund Pedersen, Vincent Andersson, Michaela Benova, Camilla Krogh, Raphaël Pons, Tobias Palle Holm, Per-Olof Wahlund, Li Fan, Zhuoran Wang, Adam Kennedy, Rune Ehrenreich Kuhre, Philip Christophersen, Pierre-Louis Bardonnet, Philip Jonas Sassene,
Salcaprozate-based ionic liquids for GLP-1 gastric delivery: A mechanistic understanding of in vivo performance,
Journal of Controlled Release, Volume 377, 2025, Pages 267-276, ISSN 0168-3659, https://doi.org/10.1016/j.jconrel.2024.11.036.