PERSpECTIVES

that have been investigated in clinical trials. In particular, the recent FDA approval of Advances in oral peptide therapeutics an oral formulation of the glucagon-like peptide 1 (GLP1) receptor agonist Daniel J. Drucker semaglutide for the treatment of type 2 diabetes (T2D) is a landmark in the field Abstract | Protein and peptide therapeutics require parenteral administration, that could energize the development of oral which can be a deterrent to medication adherence. For this reason, there have peptide therapeutics. been extensive efforts to develop alternative delivery strategies, particularly for peptides such as insulin that are used to treat endocrine disorders. Oral delivery is Barriers to oral peptide delivery especially desirable, but it faces substantial barriers related to the structural Peptide transit across the intestinal epithelium, followed by secretion into organization and physiological function of the gastrointestinal tract. This article the lymphatic system or bloodstream of a highlights strategies designed to overcome these barriers, including permeation substantially non-degraded therapeutic, enhancers, inhibitors of gut enzymes, and mucus-penetrating and cell-penetrating requires the circumvention of multiple peptides. It then focuses on the experience with oral peptides that have reached structural and functional barriers, which are clinical trials, including insulin, calcitonin, parathyroid hormone and vasopressin, summarized briefly here and shown in Fig. 1. with an emphasis on the advances that have recently led to the landmark approval Readers are referred to other, comprehensive reviews for more extensive discussion of of an oral formulation of the glucagon-like peptide 1 receptor agonist semaglutide these barriers4,8,9. for the treatment of type 2 diabetes. Protein-degrading enzymes. Ingested peptide The medicinal utility of biologic well as restricting the access of toxins and formulations first encounter digestive therapeutics — encompassing large microbial pathogens, which is achieved by enzymes, including amylase and lipase, in proteins, antibodies, hybrid fusion proteins, cellular and mucosal barriers. the saliva. Upon entry into the stomach, the antibody–drug conjugates and therapeutic This article focuses on developments ingested peptides encounter a low pH and peptides — continues to expand. Such in oral peptide therapeutics — arbitrarily enzymes such as pepsin and cathepsin that therapies are typically administered by defined as smaller proteins with a are highly efficient at proteolysis. Additional parenteral injection because of their molecular mass of <9,000 Da4 — as these proteolytic enzymes are present in the poor oral bioavailability. Although this provide more feasible opportunities for lumen of the small intestine, including those is less of a barrier in the context of acute oral delivery than do larger proteins5. emanating from pancreatic secretions, as administration for limited time periods, Although there have been efforts to well as brush border membrane peptidases daily injections of therapeutics such as develop approaches to optimizing expressed by enterocytes. These include insulin for decades may pose a challenge the pharmaceutical characteristics of trypsin, chymotrypsin, carboxypeptidase for medication adherence. Commonly peptides — including in silico models that and numerous dipeptidases and voiced issues include aversion to injections, incorporate size and molecular features aminopeptidases10. concerns about needle size or discomfort such as hydrogen-bonding capacity, The cell or mucosal models commonly at the injection site1, and persistence with lipophilicity, cyclization and polar surface used to examine the efficiency of drug injections remains suboptimal, even area5,6 — currently no validated framework transport across epithelial cells11 may with once-weekly therapies2. can be generally applied to the rational not fully mimic the complex enzymatic Consequently, extensive efforts have development of an oral formulation of environment, including differences in explored the feasibility of delivering proteins a given peptide. Furthermore, the oral regional pH, within the gut lumen, and via alternative routes, including formulations delivery technologies for which success has thus lead to overestimation of peptide suitable for oral, nasal, ophthalmic, been reported in preclinical models have bioavailability. Wang and colleagues pulmonary, buccal and transdermal often failed to translate into sufficient oral assessed the stability and enzymatic administration3. The widespread use and bioavailability in clinical trials. The aim degradation of 17 different peptide-based convenience of oral drug delivery makes of this article is not to comprehensively drugs using gastric and small-intestinal this route particularly attractive for patients. discuss such technologies, and readers are fluids from pigs and humans, as well However, the substantial challenges of oral referred to other recent reviews for further as enzyme-supplemented (pepsin and biologic delivery become evident when analysis of oral delivery technologies3,4,7. pancreatin) simulated fluids. Several of considering the anatomical structure and Rather, after briefly discussing the barriers the peptides examined (somatostatin, function of the gastrointestinal tract (Fig. 1). to oral peptide delivery and selected calcitonin, glucagon, secretin and insulin) These include the enzymatic breakdown of approaches to addressing them, this article were at least 12 amino acids or longer12. ingested complex nutrients such as proteins focuses on the experiences with oral Small peptides, including those with cyclic into easily absorbed smaller molecules, as formulations of peptides such as insulin structures, were generally more stable,

Nature Reviews | Drug Discovery Perspectives

Peptide as well as mucus pore size, which in turn Enteral microbiota influence mobility and permeation through Gastrointestinal and pathogens experimental mucus systems18. However, enzymes pH the extent to which studies of peptide drug Mucus permeation through experimental mucus layer systems modelled ex vivo have recapitulated Enterocyte the complexity of the mucous layer in vivo Protein Intracellular remains uncertain. Tight luminal export environment junction or degradation Paracellular or transcellular routes. The Transcellular passage of orally ingested peptide drugs across transport Paracellular transport the gut epithelium involves either navigation of the intercellular spaces surrounding and between cells (the paracellular route) or drug transit through cells (the transcellular route), so as to exit intact into the vascular space Vascular access adjacent to the basolateral surface of the gut epithelium (Fig. 1). Transit through the paracellular | Fig. 1 schematic representation of intestinal structure and the associated challenges to route is challenging because of a complex efficient oral absorption of peptides. The challenges of oral peptide delivery become evident when series of molecular barriers, including considering the normal physiological roles of the gastrointestinal tract. Ingested proteins are effi- ciently broken down, via a range of pH-sensitive proteases, into amino acids, which are then actively tight junctions (zonula occludens), transported across the mucosa via a complex family of transporter proteins. Similar digestive pro- adherens junctions (zonula adherens) 19 cesses ensure the enzymatic breakdown of complex carbohydrates and lipids into smaller, easily and desmosomes (macula adherens) . absorbed molecules. A secondary and simultaneous function of the gut epithelium is the recognition Collectively, these junctions encircle and exclusion of toxins, bacteria, viruses and related microbial pathogens. Hence, the lining of the gastro­ epithelial cells and maintain epithelial intestinal tract has evolved a sophisticated set of cellular and mucus barriers to restrict the access of structure and integrity. Junctions permit foreign and noxious agents. Various strategies have been pursued to overcome these barriers (high- the necessary flux of water, ions and lighted in orange) to enable peptide drug transit across the intestinal epithelium and secretion into solutes, while selectively excluding toxins, the lymphatic system or bloodstream, which are summarized in Fig. 2. Paracellular approaches are macromolecules and microorganisms20. designed to enable the passage of a peptide drug through transient disruption of cellular junctions, Occludens, claudins and junctional adhesion whereas transcellular approaches facilitate the passage of peptide drugs through the cytoplasm of enterocytes, followed by secretion into the lymphatic or systemic circulation. Image adapted with molecules form highly regulated and permission from ref.54, Elsevier. dynamic complexes, directly linked to the actin–myosin cytoskeleton21. While transient disruption of junctional complexes whereas larger peptides were more rapidly disulfide bonds to form macromolecules facilitates peptide absorption from the gut degraded. Although this is less extensively that are highly glycosylated, which enables lumen, sustained functional impairment of characterized, the local intestinal microbiota stabilization of the mucin complex and tight junction complexes by drugs, toxins are known to influence drug metabolism, protection against enzymatic degradation. or infections has been linked to systemic and may also liberate enzymes contributing The size and structure of mucus, together endotoxaemia and gastrointestinal to peptide degradation13. with the presence of intermolecular covalent inflammation22. and non-covalent interactions, dictate the The passage of drugs through the The gastrointestinal mucus layer. A complex diffusion of peptides and larger molecules transcellular pathway presents a different mucus layer, composed of cell-associated through the mucus layer. Mucus also serves set of challenges. Absorptive enterocytes mucins and glycoproteins, lies adjacent to as a reservoir for antibacterial defensins and have a specialized system of molecular absorptive enterocytes within the epithelial physically traps microorganisms in outer carriers to transport amino acids, sugars, mucosa of the stomach and bowel, serving superficial mucus layers16, preventing the fatty acids, bile acids and related essential as both a lubricant for ingested nutrients direct access of most bacterial species, as micronutrients from the lumen to the and a physicochemical barrier that traps well as large molecules, to the enterocyte basolateral surface and circulation23,24. larger molecules and pathogens14. Within surface. Some mucins, exemplified by However, a series of intracellular pathways the stomach, the pH of the mucus layer MUC2, also control the extent of luminal simultaneously promote efficient targeting ranges from below 2 at the luminal surface, bacterial colonization through modulation of foreign intracellular proteins to to a neutral pH approximating the epithelial of dendritic cell activity, leading to the intracellular lysosomal pathways25, leading surface15, and mucin turnover is highly suppression of inflammatory responses and to degradation. Alternatively, foreign dynamic, reflecting degradation in the the augmentation of tolerogenic responses intracellular proteins may be rerouted back acidic and enzyme-rich environment, in the gut mucosa17. to the mucosal surface for luminal, rather balanced by constant resynthesis of new Extensive studies of biosimilar and than basolateral, secretion. Additional mucus15. The ~20 different mucin proteins naturally occurring mucus in both challenges for oral peptide absorption may be either secreted or cell-associated cell systems and cell-free models have include a lack of receptors for peptides on molecules with short cytoplasmic tails that established the principles determining the luminal surface of enterocytes and the enable intracellular signal transduction15. hydrophobic drug–mucus interactions and absence of pathways facilitating the uptake Secreted mucins are linked together via the importance of peptide size and charge, of luminal proteins.

www.nature.com/nrd Perspectives

Inter-individual variability. Another major Enhancers must achieve sufficient thought to enhance transcellular and consideration for development of an oral yet transient disruption of the intestinal paracellular permeability, but recent studies peptide is the tremendous inter-individual epithelium to enable meaningful peptide have highlighted a predominant transcellular variability in the physiology of the absorption, while maintaining an acceptable mode of transit through the gastric gastrointestinal tract26, including the extent safety profile and minimal local or systemic epithelium for semaglutide co-formulated of mucus and enzyme production and toxicity. Screening for agents that enhance with SNAC38, which is discussed in greater control of gastric emptying and gut motility. permeability generally employs isolated depth below. 8-(N-2-hydroxy-5-chloro- The rate of gastrointestinal transit is a key gut mucosal segments studied ex vivo, benzoyl)-amino-caprylate (5-CNAC) is factor determining epithelial exposure to or gut epithelial cell lines, analysed using another acylated amino acid that has been an ingested peptide and may be influenced Ussing chambers. This approach enables tested in clinical trials — for example, with by the size, composition and timing of meal the measurement of drug transport across calcitonin (see below). It is rapidly absorbed ingestion, as well as by ageing. Variability cell layers, as well as indirect assessment and metabolized in humans, with renal in gut motility and differential rates of of cell and tight junction integrity through excretion representing the predominant absorption are particularly relevant to an analysis of transepithelial electrical mode of metabolite clearance39. the development of therapeutics such resistance31. For example, Whitehead and Ideally, permeation enhancers comprise as insulin for diabetes; individuals with colleagues analysed the properties of several simple, easy-to-manufacture and safe mild dysglycaemia or diabetes may have dozen permeability enhancers in assays ingredients that can be utilized, using a dysregulation of gut motility27, and even using intestinal epithelial Caco-2 cells and range of formulations, to enhance the suboptimal glycaemic control leads to found that the chemical structure of specific absorption of multiple proteins, without impaired gastric emptying in individuals compounds correlated in a general manner substantial need for alteration of the with type 1 diabetes (T1D) or T2D28. with their mechanisms of action; fatty core components of clinically validated Moreover, inter-individual differences in the esters predominantly enhanced paracellular enhancers36. Multiple theoretical concerns luminal epithelial environment, including permeability, whereas zwitterionic and include the potential for enhanced the relative expression of digestive enzymes, cationic surfactants augmented transcellular peptidases and relevant transporters, permeability32. Permeation enhancers with potentially contribute to the timing and different molecular mechanisms of action extent of transmucosal drug absorption29. may be combined and used together at • Permeation lower doses, to minimize cell toxicity while enhancers Approaches to oral peptide delivery • Enzyme inhibitors achieving synergistic enhancement of • Acid-stable Key considerations in evaluating the permeation efficiency33. The development of coating suitability of oral peptide formulations reconstituted human intestinal tissue or gut • Patches • Milliposts include the extent of absorption across organoids with functional tight junctions34 the intestinal mucosa, the achievement of holds promise for the rapid screening of Stomach detectable pharmacodynamic activity and permeability enhancers that safely and systemic bioavailability. If bioavailability is transiently modify epithelial permeability. • Permeation too low, the cost of the manufactured drug While numerous permeation enhancers enhancers product may be excessive, and commercial have exhibited efficacy in preclinical • Mucolytics development may not be viable. studies, only a few enhancers have shown • Hydrogels • Patches Multiple strategies to facilitate oral sufficient safety and efficacy to progress into • Microneedle peptide delivery are being pursued, often clinical trials35. Of these, two permeation injector devices Small intestine in combination, including permeation enhancers — sodium caprate (C10; also enhancers, methods to combat enzymatic known as decanoic acid) and sodium degradation, nanoparticle carriers, intestinal N-[8-(2-hydroxybenzoyl)amino] caprylate patches and microneedle delivery devices (SNAC; also known as salcaprozate sodium) (Fig. 2). These approaches have been — have been used in proprietary delivery • Targeted 3 nanoparticles reviewed recently , so this section focuses platforms for many years and have been • Permeation on representative technologies, with an tested more extensively in humans than enhancers 36 • Cell-penetrating emphasis on those that have reached clinical any other enhancers . Sodium caprate, a peptides trials or have strong promise to move into medium-chain fatty acid that is approved clinical evaluation. as a food additive, exists in an ionized Colon soluble form with detergent capacity at Permeation enhancers. Permeation pH values that typically occur in the small Fig. 2 | Technologies that have been applied enhancers4 can target either the intestine. Its mode of action is thought to for gastrointestinal peptide absorption in dif- transcellular route, by facilitating the have both transcellular aspects, based on ferent regions of the gastrointestinal tract. passage of non-degraded peptides through epithelial plasma membrane interactions, Examples of potential approaches enabling regional targeting of orally administered peptide epithelial cells, or the paracellular route, and paracellular aspects, based on effects 36 drugs are shown in the boxes next to the stom- via interference with the function of on junctional proteins . SNAC, which was ach, small intestine and bowel. Among these intercellular junctional and adhesion identified on the basis of observations that strategies, permeation enhancers have been the proteins — for example, with chelators that acylated amino acids formed microspheres most extensively developed, and a permeation sequester calcium30, which is simultaneously that facilitated the oral absorption of small enhancer is a component of the recently approved needed for E-cadherin function and as a peptides37, has ‘generally recognized as safe’ oral formulation of semaglutide, summarized cofactor for some proteases. (GRAS) status. SNAC was also originally in Fig. 3.

Nature Reviews | Drug Discovery Perspectives

absorption of noxious agents (bacteria, co-formulated together with peptide cargo chloride-coated PLGA nanoparticles fungi, viruses and toxins), development of to provide localized protection of the containing cell-penetrating peptide immune responses or alteration of the gut peptide at the site of mucosal absorption45. (CPP)–insulin conjugates52. While microbiome through the use of permeability The most clinically advanced example of mucus-penetrating strategies continue enhancers. However, such toxicities have direct enzyme inhibition as a component to be studied extensively, their safety and not yet emerged as problems in routine of an oral peptide formulation may be efficacy have not yet been validated in large monitoring of adverse events in clinical an oral insulin formulation developed clinical trials53. trials4,40. For example, in the largest trial by Oramed, known as ORMD-0801 (see to date examining the safety of an oral below)46. This formulation includes soybean Cell-penetrating peptides. CPPs may be insulin, while more subjects treated with trypsin inhibitor as well as a chelating peptide sequences derived from viruses an insulin analogue encapsulated with agent that scavenges calcium, which is a that are normally efficient at membrane sodium caprate, known as I338, developed cofactor for many proteases. However, the translocation or cell entry, non-viral human anti-insulin and cross-reacting utility and safety of enzyme inhibition as a proteins or smaller molecules such as antibodies (18 versus 5 in the placebo general strategy for enhancing oral peptide octa-arginine54,55. CPPs may interact group), the small numbers of patients with absorption is uncertain. with membrane glycosaminoglycans, antibodies and a lack of associated adverse traversing endocytic pathways and events precluded meaningful conclusions Peptide cyclization. Cyclization strategies ultimately delivering their protein cargo about the clinical significance of these remove exposed N and C termini from to the systemic circulation via exocytosis. antibodies41. Similarly, no imbalance of peptides, which are particularly susceptible Alternatively, CPPs may facilitate cell entry serious adverse events was detected in the to enzymatic cleavage. A cyclic structure by traversing membrane lipid bilayers largest trial of oral semaglutide, PIONEER is a feature of many naturally occurring via energy-independent mechanisms54. 6, which was designed to examine the small peptides, including two that have A large number of energy-dependent long-term cardiovascular safety of oral been successfully developed for oral and energy-independent pathways and semaglutide co-formulated with SNAC administration: the immunomodulatory mechanisms have been identified as (see below)42. The success and relative safety drug cyclosporine47, for which an oral candidates facilitating the entry of CPPs of several permeation enhancers in the microemulsion branded Neoral was and their cargo, and the efficiency of the clinic has fostered great interest in exploring approved in the 1990s, and desmopressin, pathways engaged may be highly CPP, cargo opportunities for further enhancing the an analogue of the peptide hormone and cell-type specific54. CPPs have been efficiency of transmucosal passage of vasopressin that has greater resistance explored for the delivery of anticancer and protein therapeutics. to enzymatic degradation than does antimicrobial therapies and as imaging vasopressin48 (see below). The extent agents, but their use for enhancing the oral Modulation of pH. Pepsin in the acidic to which cyclization is a meaningful absorption of peptide therapies has not yet environment of the stomach readily cleaves and generalizable strategy to enhance been validated in the clinic. multiple peptides. Tablets containing oral peptide absorption is not yet clear. peptides for oral delivery can be coated Nielsen and colleagues recently reported Intestinal patches. Intestinal patches with an acid-stable enteric coat to prevent the physicochemical parameters and oral for oral drug delivery physically protect their dissolution in the stomach. Once a absorption properties of 125 cyclic peptides, a small reservoir of drug from local tablet leaves the stomach and reaches the noting that the majority of these small degradation while positioning the drug upper intestine, the elevation in pH results peptides had limited oral bioavailability, close to the absorptive epithelium. For in dissolution of the enteric coat and release and most had been examined in only example, a mucoadhesive patch was of the tablet contents, as is illustrated for a small number of preclinical and even created by compressing a polymeric an oral formulation of calcitonin that has fewer clinical studies49. matrix containing carbopol, pectin, been tested in clinical trials43 (see below). sodium carboxymethylcellulose and Intestinal and pancreatic enzymes are also Mucus-penetrating agents. Several strategies salmon calcitonin and coating this matrix capable of rapidly degrading peptides. have co-formulated peptides with mucolytic with ethyl cellulose on all but one side. The optimal pH for these gastrointestinal agents or mucus-penetrating agents to The patch adhered to the small intestine enzymes is neutral to basic; the inclusion enhance the rate of passage of peptides mucosa in pigs and rats and released of citric acid in the tablet results in a local, across the mucus barrier. In preclinical salmon calcitonin that was detectable in transient decrease in pH, resulting in studies, the addition of hydrophilic the systemic circulation and was associated inhibition of the resident peptidases. Indeed, polymers such as polyethylene glycol (PEG) with a reduction in blood calcium in rats56. co-administration of citric acid together chains enhances mucus penetration, as More recently, a mucoadhesive patch that with oral salmon calcitonin is presumed to does co-formulation of peptides such as exploits iontophoresis to disrupt intestinal enhance bioavailability in part by reducing insulin within nanoparticles containing a tight junctions and facilitate paracellular the activity of local tryptic enzymes, hydrophilic coating of 2-hydroxypropyl drug transport has been reported, which resulting in enhanced absorption of oral methacrylamide copolymer (pHPMA) delivered insulin into the systemic salmon calcitonin in beagles44. derivatives50. Self-nanoemulsifying drug circulation of rats following a brief period delivery systems (SNEDDS) have also of electric current, without evidence of Direct enzyme inhibition. Another been tested that contain mixtures of oil, major structural impairment of the local strategy to circumvent gut enzyme activity surfactant and cosurfactant51. Insulin gut mucosa57. While progress continues is direct enzyme inhibition. Enzyme delivery in preclinical studies in rats has in patch technology for oral drug delivery, inhibitors including aprotinin, soybean also been achieved through the development clinical proof of concept has not yet trypsin inhibitor and leupeptin have been of mucoadhesive N-trimethyl chitosan been forthcoming.

www.nature.com/nrd Perspectives

Hydrogels. Multiple hydrogel formulations of intestinal perforation62. Abramson and to degradation by vasopressinase64,66. have been explored for enhancing intestinal colleagues also described an orally ingested In clinical trials, oral desmopressin rapidly absorption of peptides from both the small self-orienting millimetre-scale applicator reduced urine volumes in a dose-dependent and the large bowels (for example, refs58–60). (SOMA) that adheres to gastric mucosa manner. Several-fold higher doses of Hydrogels contain water, a crosslinked and delivers, via injection, pharmaceutical oral desmopressin relative to intranasal polymer and a protein cargo, and potentially products such as insulin, without puncturing dosing, and ~200-fold higher relative to also mucoadhesive polymers, to facilitate the outer layer of the stomach. A compressed subcutaneous dosing, were required in prolonged retention and enable a prolonged mixture containing insulin and polyethylene order to achieve effective antidiuresis67. Oral peptide residency time within specific gut oxide was delivered from the SOMA device desmopressin is approved for the treatment regions, while simultaneously resisting via milliposts containing biodegradable of central diabetes insipidus and primary enzymatic degradation. So far, however, polymers, resulting in detectable levels of nocturnal enuresis, whereas injectable hydrogels for therapeutic peptide delivery insulin associated with progressive reduction desmopressin has been favoured for use have not made meaningful progress towards of blood glucose in non-diabetic swine, in individuals with clotting disorders, due the clinic. without histological or functional evidence to its actions of increasing the circulating of gastrointestinal injury63. The extent to levels and activity of the clotting proteins Microneedle devices and milliposts. The which LUMI or SOMA devices can be safely factor VIII and von Willebrand factor68. inherent attractiveness of device-based and reproducibly targeted to the appropriate However, in the context of oral peptides in delivery technologies reflects their regions of the gastrointestinal epithelium general, desmopressin can be viewed as an generalized suitability for delivering a following oral ingestion requires further exception, as its cyclic nature is atypical, broad range of peptides and proteins, with investigation. and its exceptional potency means that a fewer limitations based on protein size. bioavailability of only 0.17% for the oral Rani Therapeutics is developing a small Oral peptides assessed in humans formulation, Minirin, is still viable36. enteric-coated capsule for the delivery of So far, endocrine disorders have been biological therapeutics, including peptides. a strong focus of efforts to deliver Insulin. Insulin (Mr = 5,808), the most The capsule undergoes pH-dependent oral peptide therapies. Analogues widely used injectable peptide therapeutic, dissolution in the small intestine, with of vasopressin, calcitonin, insulin, is currently available as rapid-acting generation of carbon dioxide inflating somatostatin, parathyroid hormone (PTH), formulations, as well as longer-acting a small balloon that in turn positions a thyroid hormone-releasing hormone, basal formulations suitable for once-daily microneedle-based device adjacent to uroguanylin and GLP1 have all been administration. As insulin is used by the epithelium for mucosal injection of formulated for oral administration35. millions of people with diabetes worldwide, therapeutic cargo61. Preclinical studies have Progress in the development of oral peptide it has received considerable attention as a demonstrated that the capsule can deliver therapies that have been tested in humans is candidate peptide for oral delivery. As well up to 3,000 μg of drug, equivalent to highlighted in this section. as reducing the burden of injections, oral ~80 units of insulin, within 30 min of capsule delivery could theoretically mimic a more 61 deployment within the gut . The company Vasopressin. A nonapeptide (Mr = 1,069) with physiological route of insulin delivery to has reported that the safety of the capsule six amino acids in a ring structure, joined by the liver via the portal system, although the device (known as the RaniPill), without a disulfide bridge and a three-amino-acid long-term benefit or safety of liver-targeted active drug substance, has been successfully tail, vasopressin was among the first peptide insulin remains unknown46. However, tested in humans following administration hormones developed for oral administration. the development of a rapid-acting oral in either the fasted or fed state, and clinical Originally described as a hormone with insulin formulation is highly challenging, trials using octreotide have been planned vasoconstrictor activity, vasopressin, also given inter-individual and intra-individual (see the Rani Therapeutics press release in known as antidiuretic hormone, is made variability in rates of gastric emptying28 and the Related links). in the hypothalamus, is released from the timing of food ingestion, as well as the Preclinical studies with two innovative the posterior pituitary and functions as a risk of differences in pharmacodynamic devices have been reported recently by potent regulator of water absorption in the responses, potentially enhancing risks Abramson and colleagues62,63. An orally kidney, thereby decreasing urine output64. of hypoglycaemia69. Furthermore, some dosed microneedle delivery device, Individuals with loss of central vasopressin individuals with obesity and/or diabetes designated a luminal unfolding microneedle secretion (central diabetes insipidus) or have impaired intestinal expression of injector (LUMI), contains multiple with nocturnal enuresis are candidates for tight junction proteins, defective intestinal drug-loaded microneedles encapsulated vasopressin therapy. barrier function and a ‘leaky gut’70,71, within a poly(methacrylic acid-co-ethyl Desmopressin, a modified analogue of suggesting that inter-individual differences acrylate) and PEG coating, and is designed vasopressin with deamination of the in gastrointestinal permeability may also to dissolve at pH levels encountered in the first amino acid and substitution of influence the delivery of oral peptides. Oral small intestine. Following delivery of the eighth amino acid, l-arginine, with delivery of a long-acting basal insulin may the device into the swine gut, the capsule d-arginine, was developed in tablet form be more feasible. dissolved, liberating spring-enabled for the treatment of diabetes insipidus in Several hundred studies have described biodegradable microneedles loaded with the 1980s. Desmopressin has much greater chemical and technical approaches to insulin, which penetrated the gut mucosa. antidiuretic activity than native vasopressin, the oral delivery of insulin, targeting the Plasma insulin levels increased and glucose and very little vasoconstrictive activity65,66. stomach, small intestine or colon, using decreased within 15–30 min, with a systemic Desmopressin also has a prolonged a wide range of drug delivery systems bioavailability of co-formulated insulin of antidiuretic action relative to native (reviewed in7,72). The majority of the studies more than 10% without histological evidence vasopressin, due in part to its resistance have demonstrated glucose-lowering

Nature Reviews | Drug Discovery Perspectives

effects of an externally administered insulin a chelator46 that has been assessed in geranate enhanced the paracellular transport preparation in animals, often with only a subjects with either T1D77 or T2D78. In an of insulin within an ionic liquid formulation single dose assessed. Comparatively few open-label study of eight individuals with while simultaneously reducing enzymatic studies have examined the feasibility of T1D, ORMD-0801 three times daily for insulin degradation and decreasing the chronic oral insulin dosing in diabetic 10 days reduced the mean 24-h glucose area thickness of the gut mucus layer, to augment animals for weeks to months, and only a under the curve (AUC) by 17%, while rates exposure of insulin at the surface of the handful of technologies have progressed of hypoglycaemia trended higher than mucosal epithelium83. Co-formulation of beyond phase I human testing. Selected pretreatment baseline data77. In a phase II insulin with deoxycholic acid and chitosan agents that have reached clinical trials are dose-ranging study, 31 adult patients with conjugate-coated nano-sized liposomes discussed below, as well as selected emerging T2D treated with ORMD-0801 showed enables resistance to degradation and preclinical technologies. improvements in mean placebo-subtracted enhanced mucosal absorption by targeting Insulin has been formulated with a AUC glucose ranging from −7.65 mg dl−1, the apical sodium-dependent bile acid number of permeation enhancers for oral for once-daily dosing, to −9.91 mg dl−1, for transporter84. Finally, although most oral delivery. Among the enhancers highlighted three-times-daily dosing78. No difference insulin programmes target the stomach above, SNAC has not been pursued, because in rates of hypoglycaemia was noted when and small intestine as sites of protein higher concentrations of SNAC attenuate comparing placebo with ORMD-801- absorption, the colon has also been viewed the glucose-lowering response of insulin73, treated subjects, and no serious adverse as a favourable region, due to reduced but sodium caprate has been extensively events were reported78. ORMD-0801 transit times, a more neutral pH, lower studied in the clinic. For example, Halberg continues to be evaluated in separate clinical levels of degradative enzyme activity and and colleagues compared a long-acting trials of subjects with T1D and T2D. The susceptibility to permeation enhancers. basal insulin analogue formulated in a bioavailability of ORMD-0801 was estimated Among recently described strategies for tablet with sodium caprate developed at 5–8% from studies in beagle dogs46. colonic peptide targeting, insulin was by Novo Nordisk, known as I338, for Diasome has reported clinical testing of co-formulated with a number of modified once-daily administration against once-daily a liver-targeted insulin, formulated for both nanoparticles, together with amphipathic subcutaneous injections of insulin glargine subcutaneous and oral administration79. chitosan derivatives and a series of CPPs over 8 weeks in a phase II trial involving The oral formulation consists of vesicles such as Tat, to facilitate transmucosal 50 individuals with T2D41. Reductions in that carry insulin and a proprietary passage via the transcellular route85. fasting glucose, the primary trial end point, hepatocyte-targeting molecule in the Overall, despite the attractiveness of oral were similar in both treatment arms, and phospholipid bilayer, which act to protect insulin delivery, the poor bioavailability no imbalance in rates of adverse events was insulin from proteolytic degradation. The of most oral insulin delivery systems, detected between groups41. Despite these oral HDV-1 insulin formulation reduced coupled with the high cost of manufacturing promising results, the clinical development postprandial glucose in human subjects with recombinant insulin, means that marketing of I338 was discontinued, largely due to T1D, but appeared to be less effective than an oral insulin at a competitive price could low bioavailability, which was estimated at the subcutaneous formulation. Oral HDV-1 be challenging, particularly given the 1.5–2%. Intriguingly, higher insulin antibody insulin also reduced postprandial glucose in intense scrutiny of the rising costs of new titres were detected in subjects treated with subjects with T2D but did not show a clear insulins86. The discontinuation of the clinical I338 versus those receiving insulin glargine, dose–response relationship, perhaps because development of I338 despite its promising without evidence of associated adverse of the precise timing of dosing in relation to clinical effects illustrates the economic events or reduced clinical efficacy41. meal administration80. challenges of oral insulin development. Biocon has designed insulin tregopil, Extensive efforts continue to be devoted a human insulin analogue with a to optimizing oral insulin absorption, Glucagon-like peptide 1. GLP1 is a gut peptide, methoxy-triethylene-glycol-propionyl through the use of permeation enhancers originally described as an incretin hormone moiety linked to the Lys-β29 amino group and protein delivery strategies, and a few that potentiates meal-stimulated insulin and formulated with sodium caprate, as a examples that have recently demonstrated release (see ref.87 for a review). Subsequent fast-acting agent to reduce postprandial glucose lowering in preclinical models studies have demonstrated that GLP1 hyperglycaemia74. Dose-ranging studies are highlighted here. Insulin-containing inhibits appetite and promotes weight loss, have demonstrated dose-proportional nanoparticles showed improved epithelial decelerates gastric emptying and attenuates increases in plasma insulin levels coupled uptake, reduced lysosomal targeting of glucagon secretion — properties collectively with corresponding reductions of blood intracellular degradation and enhanced useful for the treatment of T2D87. glucose following acute administration transmucosal passage of insulin relative Native GLP1 is cleaved at the N of 10–30-mg tablets75. Additional studies to native, non-modified insulin alone81. terminus by dipeptidyl peptidase-4 (DPP4), testing 30 or 45 mg of insulin tregopil Self-assembling ‘bubble’ carriers composed necessitating pharmaceutical strategies that versus insulin aspart in subjects with T2D of diethylene triamine pentaacetic circumvent the inactivation of GLP1 (such have been completed (ClinicalTrials.gov acid (DTPA) dianhydride and sodium as DPP4 inhibitors and degradation-resistant registration number NCT03430856), but bicarbonate can incorporate insulin within peptides) to prolong its half-life. Exenatide the study results have not yet been reported. the water film of the bubbles. Sodium (also known as exendin-4), a 39-amino-acid The bioavailability of insulin tregopil was dodecyl sulfate (SDS) in the bubble peptide isolated from Heloderma suspectum estimated to range from 0.82% to 0.85% in carriers enhances the dispersion of insulin venom, was the first GLP1 receptor agonist dog studies76. molecules, stabilizes the bubble carriers approved (in 2005) for the treatment of ORMD-0801 is a formulation of native and acts as both a protease inhibitor and T2D88. Exenatide contains a position 2 insulin with a non-disclosed permeation a permeation enhancer82. An oral insulin glycine, rendering it more resistant to DPP4 enhancer, soybean trypsin inhibitor and preparation formulated with choline and than GLP1, and exhibits a longer half-life

www.nature.com/nrd Perspectives

in vivo. Exenatide is used clinically as a Gastric mucosa twice-daily injection, or as a once-weekly Semaglutide long-acting microsphere preparation SNAC injected subcutaneously89. Multiple short-acting and long-acting GLP1 receptor agonists have been approved for the treatment of T2D, including lixisenatide, a short-acting exenatide analogue suitable Transcellular transport for once-daily delivery90, and liraglutide, an acylated long-acting human GLP1 analogue administered once daily91, which is also approved at a higher daily dose for Stomach the treatment of obesity92. Dulaglutide93, a GLP1–IgGFc fusion protein, and semaglutide94, an acylated DPP4-resistant Vascular access human GLP1 peptide analogue95, are approved for once-weekly dosing. GLP1 may be well-suited for oral Fig. 3 | Absorption of oral semaglutide. The available evidence, derived from studies based on scin- absorption, particularly when engineered tigraphy , immunohistochemistry and analysis of drug within the intestinal microcirculation, suggests that oral semaglutide, together with sodium N-[8-(2-hydroxybenzoyl)amino] caprylate (SNAC), is pre- for resistance to enzymatic degradation in dominantly absorbed in the stomach, via the transcellular route38. Both SNAC and semaglutide were combination with non-covalent protein detected within 30 min of oral ingestion, even in studies in which the stomach was ligated to prevent binding to enable a prolonged half-life. passage of the SNAC–semaglutide formulation into the intestine38. Unlike insulin, the glucose-dependent mechanisms of GLP1 action minimize the risk of hypoglycaemia. Many strategies have Interestingly, there seems to be a critical forming oligomers to a predominantly mono­ been pursued for the development of orally range of concentrations for co-formulated meric state, which might aid semaglutide available GLP1 receptor agonists, most SNAC to optimize semaglutide absorption, absorption. SNAC also probably attenuates frequently using exenatide (for example, as 600 mg of SNAC resulted in lower levels the enzymatic digestion of semaglutide by refs96–100) and semaglutide. Efforts with of systemic semaglutide than did the 300-mg increasing the local gastric pH specifically semaglutide have recently culminated dose38. Furthermore, small differences in the proximity of the semaglutide–SNAC in the regulatory approval of an oral in either the peptide or the enhancer formulation, and absorption of semaglutide formulation developed by Novo Nordisk for have substantial effects on absorption was anatomically restricted to the area the treatment of T2D, based on the largest efficacy; oral co-administration of SNAC of the semaglutide–SNAC tablet within phase III trial programme conducted to and the closely related GLP1 receptor the stomach38. date for an oral peptide. The mechanistic agonist liraglutide produced negligible First-in-human studies have examined characteristics of this formulation have also circulating levels of liraglutide in rats, and a single doses (2–20 mg) of oral semaglutide, been thoroughly studied and reported, so structural orthoisomer of SNAC, α-SNAC, co-formulated with 150–600 mg SNAC, the remainder of this section focuses on was markedly less efficient at enhancing in healthy human male subjects in the this agent. semaglutide absorption than SNAC38. fasting state102. Systemic semaglutide

Semaglutide (Mr = 4,113) was first As the authors highlighted, this indicates exposure was highest when mixed with developed and approved as a once-weekly that a tailored approach is needed to identify 300 mg SNAC. Within-subject variability injectable GLP1 receptor agonist, and this suitable combinations of peptides and in 24-h AUC semaglutide exposure profiles formulation has a circulating half-life of permeation enhancers for oral delivery, ranged from 19.7% to 34.9% at steady state, 165 h in humans with T2D101. Semaglutide which may explain the limited success of whereas the inter-individual variability in was developed for oral delivery through many efforts in the field in general38. semaglutide levels was considerably greater. formulation in a tablet containing the Several lines of evidence support a pre- The importance of fasting for semaglutide permeation enhancer SNAC38. Although dominant transcellular route for semaglutide absorption is highlighted by findings of no SNAC-co-formulated peptides were absorption (Fig. 3), including the detection of meaningful detectable systemic semaglutide originally thought to be absorbed in the substantial levels of semaglutide within cell exposure when the tablet was dosed in the small intestine36, more recent research has monolayers following exposure to SNAC, fed state38. Oral semaglutide is therefore established the stomach as the major site whereas EDTA (a modulator of tight junction administered with water in the morning of oral semaglutide absorption in dogs and function) did not modify the transport of in the fasted state, at least 30 min before humans38. Scintigraphic studies showed semaglutide38. Semaglutide immunoreactivity breakfast. The extent to which the strict complete tablet dissolution within the was detected in the basal cytoplasm of mucus requirement for dosing in the fasting stomach of humans within 60–140 min of cells but not in the extracellular spaces state could limit the adherence to and ingestion of a tablet containing 10 mg between the mucus cells under junctional effectiveness of oral semaglutide is unclear. of semaglutide and 300 mg of SNAC, and complexes following oral dosing in rats and Phase II dose-ranging trials of oral consistent with the scintigraphic data, dogs38. At the molecular level, differential semaglutide in subjects with T2D indicated pyloric ligation in dogs did not alter the scanning calorimetry revealed physical promising effects on blood glucose103. systemic appearance of semaglutide38. interactions between SNAC and the cellular Levels of semaglutide were not affected The bioavailability of oral semaglutide was lipid membrane. SNAC appeared to shift by impaired liver104 or kidney function102, estimated at 1.22% in dogs38. the proportion of semaglutide molecules and no significant concerns were raised in

Nature Reviews | Drug Discovery Perspectives

studies of drug–drug interactions105. The not statistically significant. The rates of enhancer 5-CNAC, known as SMC021 overall safety profile of oral semaglutide cardiovascular death and all-cause mortality (0.8 mg calcitonin plus 200 mg 5-CNAC), was consistent with findings across the were also lower with semaglutide, and both which has a reported bioavailability of GLP1 class. Rates of premature treatment HbA1c reduction (−1.0% versus −0.3%) ~1%116 and a calculated elimination half-life discontinuation, predominantly reflecting and body weight loss (−4.2 kg versus 0.8 kg) of 1.5 h39. In a placebo-controlled phase gastrointestinal adverse events, were greater were greater with semaglutide than with III study involving 4,665 postmenopausal with the higher doses of semaglutide, placebo42. More subjects discontinued women with osteoporosis, treatment and were >20% with 20-mg and 40-mg semaglutide than placebo (11.6% versus with SMC021 resulted in small increases doses in phase II studies103. The doses of 6.5%), predominantly due to gastrointestinal in lumbar spine bone density, but no oral semaglutide selected for the phase III adverse events. differences in vertebral fracture rates were programme — 3, 7 and 14 mg once daily — Collectively, the results of the PIONEER detected between the treatment groups117. were selected to enable reasonable efficacy clinical trial programme (Table 1) supported Nausea, gastrointestinal complaints and hot without unduly high rates of adverse events. the filing of new drug applications with flashes were more commonly reported in The efficacy and safety of once-daily oral regulatory authorities. In September 2019, subjects treated with SMC021, and more semaglutide for the treatment of T2D were the FDA became the first of these authorities patients discontinued study participation examined in the Peptide Innovation for Early to approve oral semaglutide to improve in the SMC021 cohort relative to placebo Diabetes Treatment (PIONEER) programme the control of blood sugar in adult patients (17% and 11%, respectively). Unexpectedly, of eight phase III trials (Table 1), including with T2D. plasma drug levels were lower in this study head-to-head studies versus oral agents such than were the levels detected in phase I/II as the SGLT2 inhibitor empagliflozin and the Calcitonin. Calcitonin, an amidated testing. The clinical development of DPP4 inhibitor sitagliptin, as well as versus 32-amino-acid peptide produced SMC021 was halted due to failure to injectable liraglutide106–112. The PIONEER predominantly in the C cells of the thyroid, meet the clinical primary end point of trials were carried out in subjects generally suppresses bone resorption by osteoclasts. fracture reduction117, perhaps reflecting the representative of the T2D population, with Pharmacological administration of suboptimal bioavailability of oral calcitonin a substantial proportion of study subjects calcitonin lowers blood calcium, but the over the 3-year study period. who were overweight or obese, either on importance of endogenous calcitonin for Overall, despite some evidence for metformin therapy alone or metformin plus physiological control of calcium homeostasis clinical anti-resorptive activity of oral additional oral antidiabetic agents (Table 1). is uncertain113. The anti-resorptive calcitonin preparations, the lack of reduction PIONEER 7 examined the efficacy of oral properties of calcitonin supported its use as in rates of fracture, concerns about safety semaglutide in subjects with T2D treated a therapy for acute hypercalcaemia, whereas and the rapid development of competing with insulin. sustained calcitonin administration has been anabolic and anti-resorptive agents marketed Mean reductions in glycated developed for the treatment of osteoporosis. for osteoporosis118 have diminished haemoglobin (HbA1c), from a starting Salmon calcitonin shares 50% amino acid enthusiasm for the commercialization of oral baseline HbA1c generally over 8%, ranged identity with human calcitonin, yet it is calcitonin formulations. from 0.8% to 1.3% and 1.1% to 1.5%, for the more biologically potent in humans in vivo 7-mg and 14-mg doses of oral semaglutide, and was approved as a parenteral injection Parathyroid hormone. PTH is an respectively (Table 1), over 26–78 weeks. for the treatment of osteoporosis in 1985, 84-amino-acid peptide that controls Weight loss was consistently observed across and later as a nasal spray in 1995. The nasal bone resorption and bone formation. The the programme, ranging from just over 2 kg formulation exhibits a bioavailability of full-length PTH(1–84) molecule is marketed to 5 kg. The proportion of subjects achieving 1–3% and was approved for the treatment of as a once-daily injection for the treatment an HbA1c of <7% on the 14-mg once-daily osteoporosis on the basis of a 30% reduction of hypoparathyroidism119, whereas the dose ranged from 59% to 80% (Table 1). of vertebral fractures114, spurring efforts to PTH(1–34) molecule, commercially Nausea and gastrointestinal complaints were develop oral formulations. developed in the form of teriperatide, retains the most common adverse events, leading to Several oral formulations of salmon bioactivity and is marketed as an anabolic premature trial discontinuation in ~10–15% calcitonin have been investigated in therapy for osteoporosis120. Attempts to of subjects. Collectively, the efficacy and clinical trials115. Salmon calcitonin (200 mg develop oral human PTH have utilized safety of oral semaglutide is consistent with formulated in an enteric coating resistant to shorter forms of the molecule, including the range of reported responses for injectable acid digestion, together with citric acid to PTH(1–34) and PTH(1–31), to facilitate GLP1 receptor agonists. enhance protease resistance and paracellular transmucosal passage. PIONEER 6, the largest and longest transport) was studied in a phase III trial EnteraBio has developed an oral trial in the programme (with a 15.9-month involving 565 postmenopausal women formulation of PTH(1–34), which has been median time in the trial), examined the with osteoporosis43. Treatment with this tested in phase I trials. Administration of safety of oral semaglutide in 3,183 subjects oral formulation resulted in improvements 1.5 mg PTH(1–34) produced a total AUC with T2D at high risk for cardiovascular in bone mineral density at the lumbar similar to that achieved following 20 μg disease, who were randomized to once-daily spine after 48 weeks of treatment that of teriperatide injection, with a twofold oral semaglutide or placebo, with 82% of the were superior to those obtained with a lower Cmax for oral PTH (see the Entera subjects being randomized to semaglutide commercial nasal formulation or placebo, Bio press release in the Related links). The maintained at 14 mg once daily by the end and the oral formulation was safe and as well permeation enhancer 5-CNAC has also of the study42. Fewer cardiovascular events tolerated as the nasal spray or placebo43. been used to formulate teriperatide for oral were reported in the subjects randomized Another oral salmon calcitonin agent delivery. In a pharmacokinetic study of to semaglutide (3.8%) than with placebo that has reached phase III trials is a several dose and enhancer combinations (4.8%), although the difference was co-formulation with the permeability in healthy postmenopausal women121,

www.nature.com/nrd Perspectives

Table 1 | The phase iii PioNeer programme for semaglutide Patient population intervention and results on primary results on secondary safety findings comparator end point end points PIONEER 1 Subjects with T2D, previously Oral semaglutide at Semaglutide produced Placebo-subtracted Heart rate and lipase managed with diet and doses of 3, 7 and 14 mg greater reductions in differences in body weight elevations were observed to a exercise, prior duration of once daily for 26 weeks HbA1c than placebo with oral semaglutide were greater extent with the higher diabetes of 3.5 years, mean versus placebo (0.6%, 0.9% and 1.1% 0.1, 0.9 and 2.3 kg doses of semaglutide age of 55 years, 51% male, across the three doses) baseline HbA1c of 8% at 26 weeks Treatment discontinuation was more common with the 7-mg and 14-mg doses than with placebo, predominantly due to gastrointestinal AEs PIONEER 2 Subjects with T2D for Oral semaglutide Semaglutide produced Body weight losses were Rates of common and SAEs ≥90 days, age ≥ 18 years, initiated at 3 mg once greater reductions in similar (4.2 versus 3.8 kg) were similar ; gastrointestinal stable dose of metformin for daily , escalated to 7 mg at HbA1c (1.4% versus at 26 weeks, semaglutide AEs were more common with ≥90 days, HbA1c of 7.0–10.5% week 4, and to 14 mg 0.9%) at 26 weeks versus empagliflozin, semaglutide at week 8 versus 25 mg respectively empagliflozin for 26–52 Trial discontinuation was weeks more common with oral semaglutide than with empagliflozin (10.7% versus 4.4%) PIONEER 3 Subjects with T2D, mean Three doses (3, 7 and Semaglutide produced Body weight reductions More subjects on 14 mg age of 58 years, duration 14 mg once daily) of oral a greater reduction in were greater with oral semaglutide discontinued of diabetes of 8.6 years, semaglutide studied, HbA1c than sitagliptin semaglutide than with trial participation relative BMI of 32.5, stable dose of dose escalation at (1.3% for the 14-mg dose sitagliptin: 3.3 versus to sitagliptin therapy (11.6% metformin ± SU for ≥90 days, 4-week intervals, versus versus 0.8%) at 26 weeks 0.7 kg at 26 weeks, and 3.5 versus 5.2%, respectively) entry HbA1c of 7.0–10.5% 100 mg sitagliptin daily versus 1.1 kg at 78 weeks, respectively Two subjects were positive for anti-semaglutide antibodies at week 26; none at weeks 52–78 PIONEER 4 Subjects with T2D, mean Oral semaglutide (14 mg Greater reduction Weight loss was greater AEs were more common age of 56 years, 48% female, once daily) versus of HbA1c with oral with semaglutide than with with semaglutide baseline HbA1c of 7–9.5%, on liraglutide (1.8 mg daily) semaglutide than with liraglutide at 26 and a background of metformin over 52 weeks liraglutide at 52 weeks 52 weeks (4.7 versus 3.2 kg 11% versus 9% versus 4% of therapy , with or without a (1.2% versus 0.9%, and 5.0 versus 3.1 kg, participants discontinued SGLT2 inhibitor respectively) respectively), from a treatment early due to AEs, for baseline weight of 94 kg semaglutide versus liraglutide versus placebo, respectively PIONEER 5 Individuals with T2D and Oral semaglutide titrated Semaglutide was Weight loss was greater Gastrointestinal AEs reduced eGFR of 30–59 ml/ to 14 mg once daily superior to placebo with semaglutide than with were more common with min/1.73 m², duration of versus placebo for with respect to HbA1c placebo (3.7 versus 1.1 kg) semaglutide diabetes of ~14 years, mean 26 weeks reduction (1.1% versus age of 70 years, 52% female, 0.1%) at 26 weeks Changes in renal function baseline HbA1c of 7–9.5% were not different on a background therapy of More subjects discontinued ±metformin, ±SU, ±insulin study drug on semaglutide than on placebo (15% versus 5%, respectively) PIONEER 6 Subjects with T2D, age Oral semaglutide titrated Rates of major Rates of cardiovascular Gastrointestinal AEs leading >50 years with established to 14 mg once daily , cardiovascular events death and all-cause to discontinuation of study cardiovascular or kidney versus placebo and were not significantly mortality were reduced on drug were more common with disease, or age >60 years with usual care; event driven, different for semaglutide semaglutide oral semaglutide (11.6% versus cardiovascular risk factors median time on drug versus placebo (hazard 6.5%, respectively) 15.9 months ratio, 0.79; 95% Reductions in HbA1c confidence interval, (1% versus 0.3%) and 0.57–1.11; P < 0.001 for body weight (4.2 versus non-inferiority) 0.8 kg) were greater with semaglutide than with placebo, respectively

Nature Reviews | Drug Discovery Perspectives

Table 1 (cont.) | The phase iii PioNeer programme for semaglutide Patient population intervention and results on primary results on secondary safety findings comparator end point end points PIONEER 7 Subjects with T2D, Randomization to oral From a baseline HbA1c Weight loss was AEs, predominantly age >18 years, HbA1c of semaglutide with flexible of 8.3%, more subjects greater with oral gastrointestinal, were more 7.5–9.5%, inadequately dose adjustments to on semaglutide than on semaglutide than with common with semaglutide controlled on stable daily 3, 7 or 14 mg once daily , sitagliptin achieved a sitagliptin (2.9 versus 0.8 kg, doses of one or two oral or sitagliptin 100 mg HbA1c of 7% or less (63% respectively) Drug therapy discontinuation glucose-lowering drugs once daily over 52 weeks versus 28%, respectively) was more common in subjects allocated to semaglutide PIONEER 8 Subjects with T2D for 3, 7 or 14 mg oral Placebo-subtracted Placebo-subtracted Rates of premature study ≥90 days, age ≥18 years, semaglutide once daily HbA1c reduction of reductions in body weight discontinuation were greater HbA1c of 7.0–9.5%, stable versus placebo over 0.6%, 1% and 1.4% of 1, 2 and 3.3 kg were with oral semaglutide 14 mg dose of either basal, 26–52 weeks were found with measured at 52 weeks than with placebo (13.3% basal-bolus or premixed 3, 7 and 14 mg of versus 2.7%, respectively) insulin for ≥90 days oral semaglutide, metformin respectively , at 26 weeks Insulin dose reduction was Rates of severe or ± greater with semaglutide symptomatic hypoglycaemia than with placebo were not significantly different (26.5% versus 29.3% for 14 mg of semaglutide versus placebo, respectively) AEs, adverse events; BMI, body mass index; eGFR , estimated glomerular filtration rate; HbA1c, glycated haemoglobin; SAEs, severe adverse events; SGLT2, sodium–glucose transport protein 2; SU, sulfonylurea; T2D, type 2 diabetes. the pharmacokinetic profiles of the 2.5-mg and lysine-linked deoxycholic acid growth factor 1 (IGF1) and growth hormone and 5-mg oral doses, in combination with showed anabolic bone activity124. Whether levels in the 7-month core treatment period, 200 mg of 5-CNAC, most closely resembled further progress will enable sufficient and 88 of 102 subjects who completed the those obtained with injectable teriperatide, bioavailability in humans to support core treatment (86%) elected to enrol in a and the bioavailability of oral PTH was the commercial viability of oral PTH 6-month extension128. The top-line results estimated to be ~1%121. formulations remains unclear. of a phase III placebo-controlled trial of An enteric-coated tablet formulation oral octreotide for maintenance treatment of recombinant human PTH(1–31)NH2 Somatostatin. Octreotide is an octapeptide of adults with acromegaly have recently containing citric acid has also been tested somatostatin analogue (Mr = 1,019) been reported, with Chiasma announcing in humans. In pharmacokinetic studies, with high affinity for the somatostatin 2 that 58% of patients on octreotide capsules the formulation resulted in pulsatile receptor (SSTR2) and moderate affinity for maintained their IGF1 response, compared plasma levels of oral PTH(1–31), similar SSTR5, which has been developed as an with 19% of the patients on placebo (see the to or greater than those achieved with injectable therapy (with both short-acting Chiasma press release in the Related links). subcutaneous administration of 20 μg and longer-acting formulations) for the of recombinant PTH(1–34), although treatment of acromegaly and endocrine Summary and perspectives greater intra-individual variability in tumours125. Chiasma has developed an oral Oral peptide delivery is an active and highly pharmacokinetics was observed with the formulation of octreotide by encapsulating promising area, yet formidable challenges oral formulation122. In an open-label phase octreotide with medium-chain free fatty remain. Several peptides have been tested in II trial in postmenopausal women with acids and sodium caprylate in order phase III trials, yet only oral desmopressin osteoporosis, both the Cmax and the AUC for to transiently enhance paracellular is widely used in the clinic at present, and PTH levels with this oral PTH formulation permeability through disruption of it is an exception, as noted above. Insulin were similar to or greater than those for intestinal tight junction proteins126. has been studied particularly extensively teriperatide, but the increase in bone density In pharmacokinetic studies in monkeys for oral administration. The development of at the lumbar spine for oral recombinant and humans, the pharmacokinetic profile oral insulins is facilitated by the availability human PTH(1–31) was lower than that for of oral octreotide at a 20-mg dose was of species-specific insulin enzyme-linked teriparatide (2.2% compared with 5.1%), not substantially different from that immunosorbent assays (ELISAs), and a and teriperatide resulted in greater and obtained with subcutaneous injection of simple end point, blood glucose, as an more sustained effects on markers of bone 0.1 mg octreotide126,127, and oral octreotide immediate readout of pharmacodynamic resorption than did oral PTH123. administration strongly suppressed activity. Nevertheless, progress towards The low bioavailability obtained with secretagogue-stimulated growth hormone meaningful efficacy in the clinic with oral oral PTH formulations remains a challenge secretion in healthy human volunteers127. insulin technologies has been slow and for commercial development. Greater A subsequent phase III trial examined disappointing, with the majority of the bioavailability (17% following intrajejunal twice-daily oral octreotide in 155 subjects vast oral insulin literature reporting on administration) has been reported in a with acromegaly previously controlled with non-clinical data129. Moreover, the clinical preclinical study in ovariectomized rats, in injectable somatostatin receptor agonists128. trial data to date, while demonstrating which enteric microcapsules containing an Of the enrolled population, 65% achieved efficacy, continue to show suboptimal ionic nanocomplex between PTH(1–34) the primary end point, based on insulin-like bioavailability, which is a challenge for

www.nature.com/nrd Perspectives

cost-effective commercialization of oral Nevertheless, the phase III data suggest 9. Groschwitz, K. R. & Hogan, S. P. Intestinal barrier function: molecular regulation and disease 41,129 insulin formulations . that oral semaglutide is competitive with pathogenesis. J. Allergy Clin. Immunol. 124, 3–20; While they are exciting from a or better than (in terms of HbA1c and quiz 21–22 (2009). 10. Lundquist, P. & Artursson, P. Oral absorption of technological and conceptual viewpoint, weight reduction) all currently available peptides and nanoparticles across the human intestine: oral peptide delivery technologies come oral glucose-lowering agents (all of which opportunities, limitations and studies in human tissues. Adv. Drug Deliv. Rev. 106, 256–276 (2016). with a unique set of safety challenges. Some are less expensive) used to treat T2D. 11. Kisser, B. et al. The Ussing chamber assay to study novel delivery devices may potentially An ongoing larger cardiovascular safety drug metabolism and transport in the human intestine. Curr. Protoc. Pharmacol. 77, 7.17.1–7.17.19 (2017). be associated with partial intestinal study for oral semaglutide will provide 12. Wang, J., Yadav, V., Smart, A. L., Tajiri, S. & Basit, A. W. obstruction, infection or perforation. further data on the long-term effects and Toward oral delivery of biopharmaceuticals: an assessment of the gastrointestinal stability of 17 Controlling for rates of gastrointestinal value of oral semaglutide administration. peptide drugs. Mol. Pharm. 12, 966–973 (2015). transit and variable pharmacokinetics Competing technologies are also in 13. Zimmermann, M., Zimmermann-Kogadeeva, M., Wegmann, R. & Goodman, A. L. Separating host and arising secondary to unpredictable patterns development, such as allosteric agonists microbiome contributions to drug pharmacokinetics of drug absorption may be concerning for and peptide mimetics with properties and toxicity. Science 363, eaat9931 (2019). 14. Johansson, M. E., Sjovall, H. & Hansson, G. C. The proteins with narrow therapeutic and safety suitable for oral drug delivery, as well as gastrointestinal mucus system in health and disease. windows. Understanding the potential small-molecule agonists targeting the GLP1 Nat. Rev. Gastroenterol. Hepatol. 10, 352–361 (2013). 15. Ensign, L. M., Cone, R. & Hanes, J. Oral drug delivery pharmacodynamic differences arising from receptor, such as PF-06882961, which is with polymeric nanoparticles: the gastrointestinal drug absorption via the gut, with more rapid undergoing clinical assessment in subjects mucus barriers. Adv. Drug Deliv. Rev. 64, 557–570 (2012). initial hepatic exposure relative to injected with T2D. 16. Antoni, L. et al. Human colonic mucus is a reservoir for therapeutics, may be relevant for drugs The apparent success of the oral antimicrobial peptides. J. Crohns Colitis 7, e652–e664 (2013). such as insulin that exert important actions semaglutide development programme is 17. Shan, M. et al. Mucus enhances gut homeostasis and on both peripheral tissues and the liver130. likely to stimulate more interest in new oral tolerance by delivering immunoregulatory signals. Science 342, 447–453 (2013). Whether sustained gastrointestinal exposure technologies designed to deliver peptide 18. Boegh, M., Garcia-Diaz, M., Mullertz, A. & to high concentrations of active drug therapeutics and larger proteins via the Nielsen, H. M. Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion substance or permeation enhancers will gastrointestinal tract. To date, the pace and peptide permeation. Eur. J. Pharm. Biopharm. produce clinically relevant changes in the of translating the basic science of enteral 95, 136–143 (2015). 19. Lemmer, H. J. & Hamman, J. H. Paracellular drug gut microbiome is not known. Similarly, protein delivery into clinical progress absorption enhancement through tight junction the extent to which therapeutic proteins has been slow, with the promise of most modulation. Expert Opin. Drug Deliv. 10, 103–114 (2013). delivered via the gastrointestinal tract, often novel technologies that have exhibited 20. Garcia, M. A., Nelson, W. J. & Chavez, N. Cell–cell co-formulated with unique chemical entities, preclinical efficacy remaining unfulfilled in junctions organize structural and signaling networks. Cold Spring Harb. Perspect. Biol. 10, a029181 (2018). will engender unanticipated immune the clinic. However, the science of protein 21. Fanning, A. S., Van Itallie, C. M. & Anderson, J. M. responses requires careful investigation. chemistry, formulation and drug delivery is Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized The clinical development of oral advancing rapidly and is likely to accelerate, epithelia. Mol. Biol. Cell 23, 577–590 (2012). semaglutide represents the largest reflecting the opportunities posed by 22. Han, X., Fink, M. P., Yang, R. & Delude, R. L. Increased iNOS activity is essential for intestinal epithelial tight phase III programme conducted to date the increasing number of biologics and junction dysfunction in endotoxemic mice. Shock 21, for an oral peptide. While most doses of therapeutic peptides that currently require 261–270 (2004). 23. Hamman, J. H., Demana, P. H. & Olivier, E. I. Targeting oral semaglutide assessed in the phase II parenteral administration. receptors, transporters and site of absorption to programme were not as efficacious as Daniel J. Drucker improve oral drug delivery. Drug Target Insights 2, once-weekly injected semaglutide103, 71–81 (2007). Department of Medicine and Lunenfeld-Tanenbaum 24. Terada, T. & Hira, D. Intestinal and hepatic drug oral semaglutide was more effective on Research Institute, Mount Sinai Hospital, University of transporters: pharmacokinetic, pathophysiological, HbA1c and weight reduction than was Toronto, Toronto, ON, Canada. and pharmacogenetic roles. J. Gastroenterol. 50, 508–519 (2015). once-daily injectable liraglutide in a e-mail: [email protected] 25. Bissa, B., Beedle, A. M. & Govindarajan, R. Lysosomal head-to-head phase III trial109. Several https://doi.org/10.1038/s41573-019-0053-0 solute carrier transporters gain momentum in research. Clin. Pharmacol. Therap. 100, 431–436 (2016). questions surround the ultimate utility and Published online xx xx xxxx 26. Tyagi, P., Pechenov, S. & Anand Subramony, J. Oral performance of oral semaglutide in the peptide delivery: translational challenges due to 1. Spain, C. V., Wright, J. J., Hahn, R. M., Wivel, A. & physiological effects. J. Control. Release 287, marketplace, including price, long-term Martin, A. A. Self-reported barriers to adherence and 167–176 (2018). tolerability and adherence in the real persistence to treatment with injectable medications 27. Boronikolos, G. C. et al. Upper gastrointestinal motility for type 2 diabetes. Clin. Ther. 38, 1653–1664.e1 and symptoms in individuals with diabetes, prediabetes world, where patients may not always take (2016). and normal glucose tolerance. Diabetologia 58, the medication as instructed on an empty 2. Holman, R. R. et al. Effects of once-weekly exenatide 1175–1182 (2015). on cardiovascular outcomes in type 2 diabetes. 28. Bharucha, A. E., Kudva, Y. C. & Prichard, D. O. Diabetic stomach and wait 30 min before meal N. Engl. J. Med. 377, 1228–1239 (2017). gastroparesis. Endocrine Rev. 40, 1318–1352 (2019). ingestion. Preliminary indications suggest 3. Anselmo, A. C., Gokarn, Y. & Mitragotri, S. 29. Sugihara, M. et al. Analysis of intra- and intersubject Non-invasive delivery strategies for biologics. variability in oral drug absorption in human that despite the much lower bioavailability Nat. Rev. Drug Discov. 18, 19–40 (2019). bioequivalence studies of 113 generic products. of oral semaglutide (for which the dose is 4. Maher, S., Mrsny, R. J. & Brayden, D. J. Intestinal Mol. Pharm. 12, 4405–4413 (2015). permeation enhancers for oral peptide delivery. 30. Artursson, P. & Magnusson, C. Epithelial transport of 14 mg daily, equivalent to 98 mg weekly), Adv. Drug Deliv. Rev. 106, 277–319 (2016). drugs in cell culture. II: effect of extracellular calcium it will be priced similarly to injectable 5. Di, L. Strategic approaches to optimizing peptide concentration on the paracellular transport of drugs of ADME properties. AAPS J. 17, 134–143 (2015). different lipophilicities across monolayers of intestinal semaglutide (for which the dose is 1 mg 6. Doak, B. C., Over, B., Giordanetto, F. & Kihlberg, J. epithelial (Caco-2) cells. J. Pharm. Sci. 79, 595–600 once weekly). Notably, the once-weekly Oral druggable space beyond the rule of 5: insights (1990). from drugs and clinical candidates. Chem. Biol. 21, 31. Whitehead, K., Karr, N. & Mitragotri, S. Safe and injectable 1-mg dose appears slightly more 1115–1142 (2014). effective permeation enhancers for oral drug delivery. effective in reducing HbA1c and body 7. Wong, C. Y., Al-Salami, H. & Dass, C. R. Microparticles, Pharm. Res. 25, 1782–1788 (2008). 103 microcapsules and microspheres: a review of recent 32. Whitehead, K. & Mitragotri, S. Mechanistic analysis of weight, as inferred from phase II studies . developments and prospects for oral delivery of chemical permeation enhancers for oral drug delivery. Hence, it will be instructive to see how the insulin. Int. J. Pharm. 537, 223–244 (2018). Pharm. Res. 25, 1412–1419 (2008). 8. Viggiano, D. et al. Gut barrier in health and disease: 33. Whitehead, K., Karr, N. & Mitragotri, S. Discovery value proposition for oral semaglutide is focus on childhood. Eur. Rev. Med. Pharmacol. Sci. of synergistic permeation enhancers for oral drug perceived and plays out in key markets. 19, 1077–1085 (2015). delivery. J. Control. Release 128, 128–133 (2008).

Nature Reviews | Drug Discovery Perspectives

34. Madden, L. R. et al. Bioprinted 3D primary human for oral delivery of insulin. Biol. Pharm. Bull. 41, improving payload stability and regulating the intestinal tissues model aspects of native physiology 811–814 (2018). transcytosis pathway. ACS Appl. Mater. Interfaces 10, and ADME/Tox functions. iScience 2, 156–167 59. Kamei, N. et al. Complexation hydrogels for intestinal 34039–34049 (2018). (2018). delivery of interferon beta and calcitonin. J. Control. 82. Lin, P. Y. et al. Safety and efficacy of self-assembling 35. Aguirre, T. A. et al. Current status of selected oral Release 134, 98–102 (2009). bubble carriers stabilized with sodium dodecyl sulfate peptide technologies in advanced preclinical 60. Ahmad, N., Mohd Amin, M. C., Ismail, I. & Buang, F. for oral delivery of therapeutic proteins. J. Control. development and in clinical trials. Adv. Drug Deliv. Rev. Enhancement of oral insulin bioavailability: in vitro and Release 259, 168–175 (2017). 106, 223–241 (2016). in vivo assessment of nanoporous stimuli-responsive 83. Banerjee, A. et al. Ionic liquids for oral insulin 36. Twarog, C. et al. Intestinal permeation enhancers for hydrogel microparticles. Expert Opin. Drug Deliv. 13, delivery. Proc. Natl Acad. Sci. USA 115, 7296–7301 oral delivery of macromolecules: a comparison 621–632 (2016). (2018). between salcaprozate sodium (SNAC) and sodium 61. Hashim, M. et al. Jejunal wall delivery of insulin 84. Wu, S. et al. A delivery system for oral administration caprate (C10). Pharmaceutics 11, E78 (2019). via an ingestible capsule in anesthetized swine—a of proteins/peptides through bile acid transport 37. Leone-Bay, A. et al. N-acylated alpha-amino acids as pharmacokinetic and pharmacodynamic study. channels. J. Pharm. Sci. 108, 2143–2152 (2019). novel oral delivery agents for proteins. J. Med. Chem. Pharmacol. Res. Perspect. 7, e00522 (2019). 85. Guo, F. et al. Enhanced oral absorption of insulin 38, 4263–4269 (1995). 62. Abramson, A. et al. A luminal unfolding microneedle using colon-specific nanoparticles co-modified with 38. Buckley, S. T. et al. Transcellular stomach absorption injector for oral delivery of macromolecules. Nat. Med. amphiphilic chitosan derivatives and cell-penetrating of a derivatized glucagon-like peptide-1 receptor 25, 1512–1518 (2019). peptides. Biomater. Sci. 7, 1493–1506 (2019). agonist. Sci. Transl. Med. 10, eaar7047 (2018). 63. Abramson, A. et al. An ingestible self-orienting system 86. Cefalu, W. T. et al. Insulin Access and Affordability 39. Gschwind, H. P. et al. Metabolism and disposition for oral delivery of macromolecules. Science 363, Working Group: conclusions and recommendations. of the oral absorption enhancer 14C-radiolabeled 611–615 (2019). Diabetes Care 41, 1299–1311 (2018). 8-(N-2-hydroxy-5-chlorobenzoyl)-amino-caprylic acid 64. Qureshi, S., Galiveeti, S., Bichet, D. G. & Roth, J. 87. Drucker, D. J., Habener, J. F. & Holst, J. J. Discovery, (5-CNAC) in healthy postmenopausal women and Diabetes insipidus: celebrating a century of vasopressin characterization, and clinical development of the supplementary investigations in vitro. Eur. J. Pharm. therapy. Endocrinology 155, 4605–4621 (2014). glucagon-like peptides. J. Clin. Investig. 127, Sci. 47, 44–55 (2012). 65. Manning, M., Balaspiri, L., Moehring, J., Haldar, J. & 4217–4227 (2017). 40. McCartney, F., Gleeson, J. P. & Brayden, D. J. Safety Sawyer, W. H. Synthesis and some pharmacological 88. Eng, J., Kleinman, W. A., Singh, L., Singh, G. & concerns over the use of intestinal permeation properties of deamino(4-threonine,8-D-arginine) Raufman, J. P. Isolation and characterization of enhancers: a mini-review. Tissue Barriers 4, e1176822 vasopressin and deamino(8-D-arginine)vasopressin, exendin-4, an exendin-3 analogue, from Heloderma (2016). highly potent and specific antidiuretic peptides, and suspectum venom. Further evidence for an exendin 41. Halberg, I. B. et al. Efficacy and safety of oral basal (8-D-arginine)vasopressin and deamino-arginine- receptor on dispersed acini from guinea pig pancreas. insulin versus subcutaneous insulin glargine in type 2 vasopressin. J. Med. Chem. 19, 842–845 (1976). J. Biol. Chem. 267, 7402–7405 (1992). diabetes: a randomised, double-blind, phase 2 trial. 66. Vavra, I. et al. Effect of a synthetic analogue of 89. Drucker, D. J. et al. Exenatide once weekly versus Lancet Diabetes Endocrinol. 7, 179–188 (2019). vasopressin in animals and in patients with diabetes twice daily for the treatment of type 2 diabetes: 42. Husain, M. et al. Oral semaglutide and cardiovascular insipidus. Lancet 1, 948–952 (1968). a randomised, open-label, non-inferiority study. Lancet outcomes in patients with type 2 diabetes. N. Engl. 67. Hammer, M. & Vilhardt, H. Peroral treatment of 372, 1240–1250 (2008). J. Med. 381, 841–851 (2019). diabetes insipidus with a polypeptide hormone 90. Meier, J. J. et al. Contrasting effects of lixisenatide 43. Binkley, N. et al. A phase 3 trial of the efficacy and analog, desmopressin. J. Pharmacol. Exp. Ther. 234, and liraglutide on postprandial glycemic control, safety of oral recombinant calcitonin: the Oral 754–760 (1985). gastric emptying, and safety parameters in patients Calcitonin in Postmenopausal Osteoporosis (ORACAL) 68. Mannucci, P. M. Desmopressin (DDAVP) in the with type 2 diabetes on optimized insulin glargine with trial. J. Bone Miner. Res. 27, 1821–1829 (2012). treatment of bleeding disorders: the first 20 years. or without metformin: a randomized, open-label trial. 44. Lee, Y. H. et al. Impact of regional intestinal pH Blood 90, 2515–2521 (1997). Diabetes Care 38, 1263–1273 (2015). modulation on absorption of peptide drugs: oral 69. Heinemann, L. & Jacques, Y. Oral insulin and buccal 91. Drucker, D. J., Dritselis, A. & Kirkpatrick, P. absorption studies of salmon calcitonin in beagle insulin: a critical reappraisal. J. Diabetes Sci. Technol. Liraglutide. Nat. Rev. Drug Discov. 9, 267–268 dogs. Pharm. Res. 16, 1233–1239 (1999). 3, 568–584 (2009). (2010). 45. Liu, H., Tang, R., Pan, W. S., Zhang, Y. & Liu, H. 70. Genser, L. et al. Increased jejunal permeability in 92. Pi-Sunyer, X. et al. A randomized, controlled trial of Potential utility of various protease inhibitors for human obesity is revealed by a lipid challenge and is 3.0 mg of liraglutide in weight management. N. Engl. improving the intestinal absorption of insulin in rats. linked to inflammation and type 2 diabetes. J. Pathol. J. Med. 373, 11–22 (2015). J. Pharm. Pharmacol. 55, 1523–1529 (2003). 246, 217–230 (2018). 93. Glaesner, W. et al. Engineering and characterization 46. Arbit, E. & Kidron, M. Oral insulin delivery in a 71. Thaiss, C. A. et al. Hyperglycemia drives intestinal of the long-acting glucagon-like peptide-1 analogue physiologic context: review. J. Diabetes Sci. Technol. barrier dysfunction and risk for enteric infection. LY2189265, an Fc fusion protein. Diabetes Metab. 11, 825–832 (2017). Science 359, 1376–1383 (2018). Res. Rev. 26, 287–296 (2010). 47. Barone, G. et al. The pharmacokinetics of a 72. Gedawy, A., Martinez, J., Al-Salami, H. & Dass, C. R. 94. Lau, J. et al. Discovery of the once-weekly glucagon-like microemulsion formulation of cyclosporine in primary Oral insulin delivery: existing barriers and current peptide-1 (GLP-1) analogue semaglutide. J. Med. Chem. renal allograft recipients. The Neoral Study Group. counter-strategies. J. Pharm. Pharmacol. 70, 58, 7370–7380 (2015). Transplantation 61, 875–880 (1996). 197–213 (2018). 95. Pratley, R. E. et al. Semaglutide versus dulaglutide once 48. Matsui, K. et al. Resistance of 1-deamino- 73. Kidron, M. et al. A novel per-oral insulin formulation: weekly in patients with type 2 diabetes (SUSTAIN 7): [8-D-arginine]-vasopressin to in vitro degradation as proof of concept study in non-diabetic subjects. a randomised, open-label, phase 3b trial. Lancet compared with arginine vasopressin. Endocrinol. Jpn Diabet. Med. 21, 354–357 (2004). Diabetes Endocrinol. 6, 275–286 (2018). 32, 547–557 (1985). 74. Khedkar, A. et al. Impact of insulin tregopil and its 96. Suzuki, K., Kim, K. S. & Bae, Y. H. Long-term oral 49. Nielsen, D. S. et al. Orally absorbed cyclic peptides. permeation enhancer on pharmacokinetics of administration of exendin-4 to control type 2 diabetes Chem. Rev. 117, 8094–8128 (2017). metformin in healthy volunteers: randomized, in a rat model. J. Control. Release 294, 259–267 50. Shan, W. et al. Overcoming the diffusion barrier open-label, placebo-controlled, crossover study. (2019). of mucus and absorption barrier of epithelium by Clin. Transl. Sci. 12, 276–282 (2019). 97. Xu, Y. et al. Novel strategy for oral peptide delivery self-assembled nanoparticles for oral delivery of 75. Khedkar, A. et al. A dose range finding study of novel in incretin-based diabetes treatment. Gut https:// insulin. ACS Nano 9, 2345–2356 (2015). oral insulin (IN-105) under fed conditions in type 2 doi.org/10.1136/gutjnl-2019-319146 (2019). 51. Karamanidou, T. et al. Effective incorporation of diabetes mellitus subjects. Diabetes Obes. Metab. 12, 98. Song, Y. et al. Synthesis of CSK-DEX-PLGA insulin in mucus permeating self-nanoemulsifying 659–664 (2010). nanoparticles for the oral delivery of exenatide to drug delivery systems. Eur. J. Pharm. Biopharm. 97, 76. Gregory, J. M. et al. Enterically delivered insulin improve its mucus penetration and intestinal 223–229 (2015). tregopil exhibits rapid absorption characteristics and absorption. Mol. Pharm. 16, 518–532 (2019). 52. Sheng, J. et al. Enhancing insulin oral absorption a pharmacodynamic effect similar to human insulin in 99. Zhang, L. et al. The use of low molecular weight by using mucoadhesive nanoparticles loaded with conscious dogs. Diabetes Obes. Metab. 21, 160–169 protamine to enhance oral absorption of exenatide. LMWP-linked insulin conjugates. J. Control. Release (2019). Int. J. Pharm. 547, 265–273 (2018). 233, 181–190 (2016). 77. Eldor, R., Arbit, E., Corcos, A. & Kidron, M. 100. Soudry-Kochavi, L., Naraykin, N., Nassar, T. & 53. Boegh, M. & Nielsen, H. M. Mucus as a barrier to Glucose-reducing effect of the ORMD-0801 oral Benita, S. Improved oral absorption of exenatide using drug delivery—understanding and mimicking the insulin preparation in patients with uncontrolled an original nanoencapsulation and microencapsulation barrier properties. Basic Clin. Pharmacol. Toxicol. type 1 diabetes: a pilot study. PLOS ONE 8, e59524 approach. J. Control. Release 217, 202–210 (2015). 116, 179–186 (2015). (2013). 101. Kapitza, C. et al. Semaglutide, a once-weekly human 54. Rehmani, S. & Dixon, J. E. Oral delivery of anti-diabetes 78. Eldor, R., Neutel, J., Homer, K. & Kidron, M. Multiple GLP-1 analog, does not reduce the bioavailability of therapeutics using cell penetrating and transcytosing oral insulin (ORMD-0801) doses elicit a cumulative the combined oral contraceptive, ethinylestradiol/ peptide strategies. Peptides 100, 24–35 (2018). effect on glucose control in T2DM patients. Diabetes levonorgestrel. J. Clin. Pharmacol. 55, 497–504 55. Niu, Z. et al. Rational design of polyarginine 67 (Suppl. 1), 982-P (2018). (2015). nanocapsules intended to help peptides overcoming 79. Geho, W. B., Geho, H. C., Lau, J. R. & Gana, T. J. 102. Granhall, C., Sondergaard, F. L., Thomsen, M. & intestinal barriers. J. Control. Release 263, 4–17 Hepatic-directed vesicle insulin: a review of formulation Anderson, T. W. Pharmacokinetics, safety and (2017). development and preclinical evaluation. J. Diabetes tolerability of oral semaglutide in subjects with renal 56. Gupta, V. et al. Mucoadhesive intestinal devices for Sci. Technol. 3, 1451–1459 (2009). impairment. Clin. Pharmacokinet. 57, 1571–1580 oral delivery of salmon calcitonin. J. Control. Release 80. Geho, W. B., Rosenberg, L. N., Schwartz, S. L., (2018). 172, 753–762 (2013). Lau, J. R. & Gana, T. J. A single-blind, placebo- 103. Davies, M. et al. Effect of oral semaglutide compared 57. Banerjee, A., Chen, R., Arafin, S. & Mitragotri, S. controlled, dose-ranging trial of oral hepatic-directed with placebo and subcutaneous semaglutide on Intestinal iontophoresis from mucoadhesive patches: vesicle insulin add-on to oral antidiabetic treatment glycemic control in patients with type 2 diabetes: a a strategy for oral delivery. J. Control. Release 297, in patients with type 2 diabetes mellitus. J. Diabetes randomized clinical trial. J. Am. Med. Assoc. 318, 71–78 (2019). Sci. Technol. 8, 551–559 (2014). 1460–1470 (2017). 58. Fukuoka, Y. et al. Combination strategy with 81. Zheng, Y. et al. Multifunctional nanoparticles enable 104. Baekdal, T. A., Thomsen, M., Kupcova, V., Hansen, C. W. complexation hydrogels and cell-penetrating peptides efficient oral delivery of biomacromolecules via & Anderson, T. W. Pharmacokinetics, safety, and

www.nature.com/nrd Perspectives

tolerability of oral semaglutide in subjects with salmon calcitonin in the treatment of osteoporosis. 127. Tuvia, S. et al. Oral octreotide absorption in human hepatic impairment. J. Clin. Pharmacol. 58, Expert Opin. Drug Metab. Toxicol. 12, 681–689 subjects: comparable pharmacokinetics to parenteral 1314–1323 (2018). (2016). octreotide and effective growth hormone suppression. 105. Baekdal, T. A., Borregaard, J., Hansen, C. W., 116. Buclin, T., Cosma Rochat, M., Burckhardt, P., Azria, M. J. Clin. Endocrinol. Metab. 97, 2362–2369 (2012). Thomsen, M. & Anderson, T. W. Effect of oral & Attinger, M. Bioavailability and biological efficacy of 128. Melmed, S. et al. Safety and efficacy of oral octreotide semaglutide on the pharmacokinetics of lisinopril, a new oral formulation of salmon calcitonin in healthy in acromegaly: results of a multicenter phase III trial. warfarin, digoxin, and metformin in healthy subjects. volunteers. J. Bone Miner. Res. 17, 1478–1485 J. Clin. Endocrinol. Metab. 100, 1699–1708 (2015). Clin. Pharmacokinet. 58, 1193–1203 (2019). (2002). 129. Zijlstra, E., Heinemann, L. & Plum-Morschel, L. Oral 106. Aroda, V. R. et al. PIONEER 1: randomized clinical 117. Henriksen, K. et al. A randomized, double-blind, insulin reloaded: a structured approach. J. Diabetes trial of the efficacy and safety of oral semaglutide multicenter, placebo-controlled study to evaluate Sci. Technol. 8, 458–465 (2014). monotherapy in comparison with placebo in patients the efficacy and safety of oral salmon calcitonin in the 130. Buse, J. B. et al. Randomized clinical trial comparing with type 2 diabetes. Diabetes Care 42, 1724–1732 treatment of osteoporosis in postmenopausal women basal insulin peglispro and insulin glargine in patients (2019). taking calcium and vitamin D. Bone 91, 122–129 with type 2 diabetes previously treated with basal 107. Montanya, E. et al. 54-OR: oral semaglutide vs. (2016). insulin: IMAGINE 5. Diabetes Care 39, 92–100 (2016). empagliflozin added on to metformin monotherapy 118. Compston, J. E., McClung, M. R. & Leslie, W. D. in uncontrolled type 2 diabetes: PIONEER 2. Diabetes Osteoporosis. Lancet 393, 364–376 (2019). Acknowledgements 68 (Suppl. 1), 54-OR (2019). 119. Mannstadt, M. et al. Efficacy and safety of D.J.D. is supported in part by a Banting and Best Diabetes 108. Mosenzon, O. et al. Efficacy and safety of oral recombinant human parathyroid hormone (1–84) Centre Novo Nordisk Chair in Incretin Biology, by CIHR semaglutide in patients with type 2 diabetes and in hypoparathyroidism (REPLACE): a double-blind, Foundation Grant 154321 and by investigator-initiated moderate renal impairment (PIONEER 5): a placebo-controlled, randomised, phase 3 operating grants for preclinical GLP1 science from Novo placebo-controlled, randomised, phase 3a trial. study. Lancet Diabetes Endocrinol. 1, 275–283 Nordisk. Lancet Diabetes Endocrinol. 7, 515–527 (2019). (2013). 109. Pratley, R. et al. Oral semaglutide versus 120. Hodsman, A. B. et al. Parathyroid hormone and Competing interests subcutaneous liraglutide and placebo in type 2 teriparatide for the treatment of osteoporosis: D.J.D. has served as an adviser or consultant or speaker diabetes (PIONEER 4): a randomised, double-blind, a review of the evidence and suggested guidelines for within the past 12 months to Forkhead Biotherapeutics, phase 3a trial. Lancet 394, 39–50 (2019). its use. Endocr. Rev. 26, 688–703 (2005). Heliome Inc., Intarcia Therapeutics, Kallyope, Merck Research 110. Rosenstock, J. et al. Effect of additional oral 121. Hammerle, S. P. et al. The single dose pharmacokinetic Laboratories, Novo Nordisk Inc., Pfizer Inc. and Sanofi Inc. semaglutide vs sitagliptin on glycated hemoglobin profile of a novel oral human parathyroid hormone Neither D.J.D. nor his family members hold stock directly or in adults with type 2 diabetes uncontrolled with formulation in healthy postmenopausal women. Bone indirectly in any of these companies. GLP2 is the subject of a metformin alone or with sulfonylurea: the PIONEER 3 50, 965–973 (2012). patent licence agreement between Shire Inc. and the randomized clinical trial. J. Am. Med. Assoc. 321, 122. Sturmer, A. et al. Pharmacokinetics of oral recombinant University of Toronto, Toronto General Hospital (UHN) and 1466–1480 (2019). human parathyroid hormone [rhPTH(1–31)NH(2)] in D.J.D. 111. Zinman, B. et al. Efficacy, safety and tolerability of postmenopausal women with osteoporosis. Clin. oral semaglutide versus placebo added to insulin ± Pharmacokinet. 52, 995–1004 (2013). Publisher’s note metformin in patients with type 2 diabetes: the 123. Henriksen, K. et al. Evaluation of the efficacy, safety Springer Nature remains neutral with regard to jurisdictional PIONEER 8 trial. Diabetes Care 42, 2262–2271 and pharmacokinetic profile of oral recombinant claims in published maps and institutional affiliations. (2019). human parathyroid hormone [rhPTH(1–31)NH(2)] in 112. Rodbard, H. W. et al. Oral semaglutide versus postmenopausal women with osteoporosis. Bone 53, empagliflozin in patients with type 2 diabetes 160–166 (2013). Related links uncontrolled on metformin: the PIONEER 2 trial. 124. Hwang, S. R., Seo, D. H., Byun, Y. & Park, J. W. chiasma press release: http://ir.chiasmapharma.com/node/ Diabetes Care 42, 2272–2281 (2019). Preparation and in vivo evaluation of an orally 8156/pdf 113. Naot, D., Musson, D. S. & Cornish, J. The activity of available enteric-microencapsulated parathyroid entera Bio press release: https://www.globenewswire.com/ peptides of the calcitonin family in bone. Physiol. Rev. hormone (1–34)-deoxycholic acid nanocomplex. Int. J. news-release/2019/09/23/1919104/0/en/Entera-Bio-Reports- 99, 781–805 (2019). Nanomed. 11, 4231–4246 (2016). Positive-Results-from-a-Phase-2-PK-PD-Study-of-Oral-PTH- 114. Chesnut, C. H. III et al. A randomized trial of nasal 125. Lamberts, S. W., Van der Lely, A. J., De Herder, W. W. 1-34-in-Patients-with-Hypoparathyroidism.html spray salmon calcitonin in postmenopausal women & Hofland, L. J. Octreotide. N. Engl. J. Med. 334, rani therapeutics press release: https://res.cloudinary.com/ with established osteoporosis: the prevent recurrence 246–254 (1996). vwp/v1551478356/First_Human_Study_of_RaniPill_Capsule_ of osteoporotic fractures study. PROOF Study Group. 126. Tuvia, S. et al. A novel suspension formulation to_Replace_Injections_Announced_by_Rani_Therapeutics_ Am. J. Med. 109, 267–276 (2000). enhances intestinal absorption of macromolecules pbzlhb.pdf. 115. Bandeira, L., Lewiecki, E. M. & Bilezikian, J. P. via transient and reversible transport mechanisms. Pharmacodynamics and pharmacokinetics of oral Pharm. Res. 31, 2010–2021 (2014). © Springer Nature Limited 2019

Nature Reviews | Drug Discovery