To meet unmet medical needs, middle-to-large molecules, including peptides and oligonucleotides, have emerged as new therapeutic modalities. Owing to their middle-to-large molecular sizes, middle-to-large molecules are not suitable for oral absorption, but there are high expectations around orally bioavailable macromolecular drugs, since oral administration is the most convenient dosing route. Therefore, extensive efforts have been made to create bioavailable middle-to-large molecules or develop absorption enhancement technology, from which some successes have recently been reported. For example, Rybelsus^® tablets and Mycapssa^® capsules, both of which contain absorption enhancers, were approved as oral medications for type 2 diabetes and acromegaly, respectively. The oral administration of Rybelsus and Mycapssa exposes their pharmacologically active peptides with molecular weights greater than 1000, namely, semaglutide and octreotide, respectively, into systemic circulation. Although these two medications represent major achievements in the development of orally absorbable peptide formulations, the oral bioavailability of peptides after taking Rybelsus and Mycapssa is still only around 1%. In this article, we review the approaches and recent advances of orally bioavailable middle-to-large molecules and discuss challenges for improving their oral absorption.

Intestinal absorption is a complex process involving the permeability of the epithelial barrier, efflux transporter activity, and intestinal metabolism. Identifying the key factors that govern intestinal absorption for each investigational drug is crucial. To assess and predict intestinal absorption in humans, it is necessary to leverage appropriate in vitro systems. Traditionally, Caco-2 monolayer systems and intestinal Ussing chamber studies have been considered the ‘gold standard’ for studying intestinal absorption. However, these methods have limitations that hinder their universal use in drug discovery and development. Recently, there has been an increasing number of reports on complex in vitro models (CIVMs) using human intestinal organoids derived from intestinal tissue specimens or iPSC-derived enterocytes plated on 2D or 3D in microphysiological systems. These CIVMs provide a more physiologically relevant representation of key ADME-related proteins compared to conventional in vitro methods. They hold great promise for use in drug discovery and development due to their ability to replicate the expressions and functions of these proteins. This review highlights recent advances in gut CIVMs employing intestinal organoid model systems compared to conventional methods. It is important to note that each CIVM should be tailored to the investigational drug properties and research questions at hand.

Inflammatory bowel disease is a set of chronic inflammatory diseases that mainly develop in the gastrointestinal mucosa, including ulcerative colitis and Crohn’s disease. Gastrointestinal membrane permeability is an important factor influencing the pharmacological effects of pharmaceuticals administered orally for treating inflammatory bowel disease and other diseases. Understanding the presence or absence of changes in pharmacokinetic properties under a disease state facilitates effective pharmacotherapy. In this paper, we reviewed the gastrointestinal membrane function in ulcerative colitis and Crohn’s disease from the perspective of in vitro membrane permeability and electrophysiological parameters. Information on in vivo permeability in humans is summarized. We also overviewed the inflammatory bowel disease research using gut-on-a-chip, in which some advances have recently been achieved. It is expected that these findings will be exploited for the development of therapeutic drugs for inflammatory bowel disease and the optimization of treatment options and regimens.

Protein-tyrosine phosphatase 1B (PTP1B) inhibitory activity of the 2'-O-(2-methoxy)ethyl (2'- MOE)-modified gapmer antisense oligonucleotide, ISIS113715, was previously reported. This antisense oligonucleotide increases insulin sensitivity and normalizes plasma glucose levels in diabetic ob/ob and db/db mice. In the present study, the isosequential 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified oligonucleotide, ENA-1, was synthesized, and its ability to further improve the downregulation of PTP1B in db/db mice was examined. We demonstrated that, compared with ISIS113715, intraperitoneal and subcutaneous administration of ENA-1 more effectively decreased the plasma glucose levels in db/db mice. Moreover, ENA-1 decreased expression of PTP1B in the liver and fat of db/db mice more effectively than ISIS113715. We describe for the first time the functional comparison of 2'-MOE- and ENA-modified antisense oligonucleotides. Our data indicate that the enhancement of the efficacy of antisense oligonucleotides by ENA modifications is superior to that of second-generation 2'-MOE modifications in certain aspects.

Intestinal organoid-derived monolayers have emerged as a novel in vitro model for investigating drug absorption and metabolism. Air-liquid interface (ALI) culture method enhances physiologically relevance of epithelial cells. In this study, we applied ALI culture to tissue stem cell-derived human intestinal organoids with the aim of achieving further improvement of their pharmacokinetic function. We established human duodenal and jejunal tissue-derived organoid monolayers under ALI culture using commercially available ready-to-use media and evaluated their utility for their pharmacokinetic application in comparison with conventional submerged monolayers. Although transepithelial electrical resistance values were significantly lower and more stable in ALI monolayers, monolayers under both conditions maintained barrier integrity as indicated by low apparent permeability of fluorescein-isothiocyanate dextran 4000, a paracellular transport marker. ALI monolayers exhibited markedly higher expression of CYP3A4 along with enhanced CYP3A activity than submerged monolayers. Correspondingly, an estimated intestinal availability value of midazolam, a CYP3A substrate, in ALI monolayers closely matched a reported human in vivo value, outperforming submerged monolayers. Activities of phase II metabolic enzymes (uridine 5'-diphospho-glucuronosyltransferase and sulfotransferases) and P-glycoprotein, as well as CYP3A4 and P-gp inducibility, were comparable between the culture conditions. Our findings demonstrate that ALI-cultured human intestinal organoid-derived monolayers provide a robust in vitro platform for investigating intestinal first-pass metabolism of CYP3A substrate drugs. SIGNIFICANCE STATEMENT: This study provides a detailed comparison of tissue-derived human intestinal organoid monolayers cultured under air-liquid interface (ALI) and conventional submerged conditions, focusing on their applicability in pharmacokinetic studies. Our findings highlight the enhanced capability of ALI monolayers to recapitulate CYP3A-mediated intestinal first-pass metabolism. Leveraging commercially available ready-to-use organoid culture media, the ALI model offers a practical and efficient platform for drug metabolism studies, facilitating broader adoption depending on specific research needs.