A two-step method preparation of semaglutide through solid-phase synthesis and inclusion body expression.

Semaglutide is currently the most promising antidiabetic drug, especially for the treatment of type 2 diabetes mellitus, due to its excellent efficacy in glycemic control and weight loss. However, the production of semaglutide remains high cost, and high yield, low cost, and high purity still remains a challenge. Herein, we reported a convenient and high-yield strategy for the preparation of semaglutide through fragmented condensation coupling, involving solid-phase peptide synthesis of tetrapeptide and on-column refolding and on-column enzyme cleavage based inclusion body expression of Lys26Arg34GLP-1 (11-37) with fused protein tags in an X-Y-D4K-G pattern. The optimized N-terminal protein tag significantly boosts inclusion body expression level, while on-column refolding and on-column enzyme cleavage avoid precipitation, enhancing efficiency and yield together with one-step purification. The successful preparation of semaglutide is expected to achieve large-scale industrial production with low cost, high yield and high purity.

The Synthesis of SNAC Phenolate Salts and the Effect on Oral Bioavailability of Semaglutide.

PURPOSE: Sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) is a well-known penetration enhancer widely used in commercial applications. This study aims to broaden its properties through a novel strategy of converting it into its phenolate salts. The objective is to investigate the synthesis of SNAC phenolate salts, specifically SNAC-choline (SNAC-CH), SNAC-sodium (SNAC-Na), and SNAC-phosphatidylcholine (SNAC-PC), and to explore their potential application in improving the oral absorption of semaglutide. METHODS: The synthesis of SNAC phenolate salts was confirmed through 1H-NMR, FTIR, and an elemental analysis of C, H, N, and O. In vivo testing was conducted to assess the oral delivery of semaglutide using these synthesized SNAC phenolate salts. Pharmacokinetic (PK) values were measured to evaluate the impact on drug absorption. RESULTS: The synthesis of SNAC phenolate salts (SNAC-CH, SNAC-Na, and SNAC-PC) was successfully achieved under appropriate conditions, and their structures were confirmed using analytical techniques such as IR, NMR, and CHN elemental analysis. The paradigm of their use was evaluated through an oral pharmacokinetic (PK) in vivo study using SNAC phenolate salts, which did not impair the original SNAC PK values. This suggests that this strategy holds promise as a potential new effective enhancer for oral absorption. CONCLUSIONS: The utilization of SNAC phenolate salts presents a novel and promising strategy for extending the verity of penetration enhancers' molecules and properties. Synthesizing phenolate salts represents a new chemical strategy that may open new avenues in molecular development. This approach holds future potential to enhance the oral delivery of peptide drugs like semaglutide without compromising therapeutic efficacy. Overall, it offers significant advancements in the field by providing a potential alternative to injectable peptides through oral delivery systems.

Semaglutide, delayed gastric emptying, and intraoperative pulmonary aspiration: a case report.

PURPOSE: We report a case in which the use of semaglutide for weight loss was associated with delayed gastric emptying and intraoperative pulmonary aspiration of gastric contents. CLINICAL FEATURES: A 42-yr-old patient with Barrett's esophagus underwent repeat upper gastrointestinal endoscopy and ablation of dysplastic mucosa. Two months earlier, the patient had started weekly injections of semaglutide for weight loss. Despite having fasted for 18 hr, and differing from the findings of prior procedures, endoscopy revealed substantial gastric content, which was suctioned before endotracheal intubation. Food remains were removed from the trachea and bronchi using bronchoscopy. The patient was extubated four hours later and remained asymptomatic. CONCLUSION: Patients using semaglutide and other glucagon-like peptide 1 agonists for weight management may require specific precautions during induction of anesthesia to prevent pulmonary aspiration of gastric contents.

RéSUMé: OBJECTIF: Nous rapportons un cas dans lequel l'utilisation de sémaglutide à des fins de perte de poids a été associée à un retard de vidange gastrique et à une aspiration pulmonaire peropératoire du contenu gastrique. CARACTéRISTIQUES CLINIQUES: Un patient de 42 ans souffrant d'un œsophage de Barrett a subi une cinquième endoscopie gastro-intestinale supérieure avec ablation de la muqueuse dysplasique. Deux mois plus tôt, le patient avait commencé à recevoir des injections hebdomadaires de sémaglutide pour perdre du poids. Bien qu'à jeun depuis 18 heures et à la différence des évaluations lors des interventions antérieures, l'endoscopie a révélé un contenu gastrique important, qui a été aspiré avant l'intubation endotrachéale. Les restes de nourriture ont été retirés de la trachée et des bronches par bronchoscopie. Le patient a été extubé quatre heures plus tard et est demeuré asymptomatique. CONCLUSION: Les patients utilisant du sémaglutide et d'autres agonistes du peptide analog au glucagon-1 pour la gestion du poids pourraient nécessiter des précautions spécifiques lors de l'induction de l'anesthésie pour empêcher l'aspiration pulmonaire du contenu gastrique.

Human glucose-dependent insulinotropic polypeptide (GIP) is an antimicrobial adjuvant re-sensitising multidrug-resistant Gram-negative bacteria.

Increasing antibiotic resistance in Gram-negative bacteria has mandated the development of both novel antibiotics and alternative therapeutic strategies. Evidence of interplay between several gastrointestinal peptides and the gut microbiota led us to investigate potential and broad-spectrum roles for the incretin hormone, human glucose-dependent insulinotropic polypeptide (GIP) against the Enterobacteriaceae bacteria, Escherichia coli and Erwinia amylovora. GIP had a potent disruptive action on drug efflux pumps of the multidrug resistant bacteria E. coli TG1 and E. amylovora 1189 strains. The effect was comparable to bacterial mutants lacking the inner and outer membrane efflux pump factor proteins AcrB and TolC. While GIP was devoid of direct antimicrobial activity, it has a potent membrane depolarizing effect, and at low concentrations, it significantly potentiated the activity of eight antibiotics and bile salt by reducing MICs by 4-8-fold in E. coli TG1 and 4-20-fold in E. amylovora 1189. GIP can thus be regarded as an antimicrobial adjuvant with potential for augmenting the available antibiotic arsenal.

Oral Semaglutide, the First Ingestible Glucagon-Like Peptide-1 Receptor Agonist: Could It Be a Magic Bullet for Type 2 Diabetes?

The gastrointestinal tract secretes gut hormones in response to food consumption, and some of these stimulate insulin secretion. Glucagon-like peptide-1 (GLP-1) is an incretin peptide hormone released from the lower digestive tract that stimulates insulin secretion, suppresses glucagon secretion, and decreases hunger. GLP-1 receptor agonist (GLP-1RA) mimics the action of endogenous GLP-1, consequently reversing hyperglycemia and causing weight reduction, demonstrating its efficacy as an antidiabetic and antiobesity agent. Previously restricted to injection only, the invention of the absorption enhancer sodium N-(8-[2-hydroxybenzoyl]amino) caprylate resulted in the development of oral semaglutide, the first ingestible GLP-1RA. Oral semaglutide demonstrated its efficacy in glycemic management and body weight loss with a low risk of hypoglycemia as a monotherapy and in combination with other hypoglycemic medications in its clinical trial programs named Peptide Innovation for Early Diabetes Treatment. Consistent with other injectable GLP-1RAs, gastrointestinal side effects were often reported. Additionally, cardiovascular safety was established by demonstrating that oral semaglutide was not inferior to a placebo in terms of cardiovascular outcomes. Thus, oral semaglutide represents a novel treatment option that is particularly well-suited for patients with type 2 diabetes and/or obesity.

Engineering of a Biologically Active Glycosylated Glucagon-Like Peptide-1 Analogue.

Glucagon-like peptide receptor (GLP-1R) agonists (e.g., semaglutide, liraglutide, etc.) are efficient treatment options for people with type 2 diabetes and obesity. The manufacturing method to produce semaglutide, a blockbuster GLP-1 drug on the market, involves multistep synthesis. The large peptide has a hydrophobic fatty acid side chain that makes it sparingly soluble, and its handling, purification, and large-scale production difficult. The growing demand for semaglutide that the manufacturer is not capable of addressing immediately triggered a worldwide shortage. Thus, we have developed a potential alternative analogue to semaglutide by replacing the hydrophobic fatty acid with a hydrophilic human complex-type biantennary oligosaccharide. Our novel glycoGLP-1 analogue was isolated in an ∼10-fold higher yield compared with semaglutide. Importantly, our glycoGLP-1 analogue possessed a similar GLP-1R activation potency to semaglutide and was biologically active in vivo in reducing glucose levels to a similar degree as semaglutide.

Functional association of the N-terminal residues with the central region in glucagon-related peptides.

GLP-1 is an incretin peptide involved in the regulation of glucose metabolism and the glucose-dependent stimulation of insulin secretion. Ex-4 is a paralog of GLP-1 that has comparable GLP-1R potency but extended physiological action. GLP-1 and Ex-4 are helical peptides that share ∼50% sequence homology but differ at several residues, notably the second amino acid which controls susceptibility to DPP-IV cleavage. This single amino acid difference yields divergent receptor potency when studied in the context of the two hormone sequences. Ex-4 uniquely tolerates Gly2 through select amino acid differences in the middle region of the peptide that are absent in GLP-1. We report that substitution of Ex-4 amino acids Glu16, Leu21, and Glu24 to the GLP-1 sequence enabled Gly2 tolerance. The coordination of the N-terminus with these central residues shows an interaction of substantial importance not only to DPP-IV stability but also to receptor activation. Extension of this observation to glucagon-based co-agonist peptides showed different structural requirements for effective communication between the N-terminus and the mid-section of these peptides in achieving high potency agonism at the GLP-1 and GCGRs.

Mutational Landscape of the Proglucagon-Derived Peptides.

Strong efforts have been placed on understanding the physiological roles and therapeutic potential of the proglucagon peptide hormones including glucagon, GLP-1 and GLP-2. However, little is known about the extent and magnitude of variability in the amino acid composition of the proglucagon precursor and its mature peptides. Here, we identified 184 unique missense variants in the human proglucagon gene GCG obtained from exome and whole-genome sequencing of more than 450,000 individuals across diverse sub-populations. This provides an unprecedented source of population-wide genetic variation data on missense mutations and insights into the evolutionary constraint spectrum of proglucagon-derived peptides. We show that the stereotypical peptides glucagon, GLP-1 and GLP-2 display fewer evolutionary alterations and are more likely to be functionally affected by genetic variation compared to the rest of the gene products. Elucidating the spectrum of genetic variations and estimating the impact of how a peptide variant may influence human physiology and pathophysiology through changes in ligand binding and/or receptor signalling, are vital and serve as the first important step in understanding variability in glucose homeostasis, amino acid metabolism, intestinal epithelial growth, bone strength, appetite regulation, and other key physiological parameters controlled by these hormones.

Structure and dynamics of semaglutide- and taspoglutide-bound GLP-1R-Gs complexes.

The glucagon-like peptide-1 receptor (GLP-1R) regulates insulin secretion, carbohydrate metabolism, and appetite and is an important target for treatment of type 2 diabetes and obesity. Multiple GLP-1R agonists have entered into clinical trials, with some, such as semaglutide, progressing to approval. Others, including taspoglutide, failed due to the high incidence of side effects or insufficient efficacy. GLP-1R agonists have a broad spectrum of signaling profiles, but molecular understanding is limited by a lack of structural information on how different agonists engage with the GLP-1R. Here, we report cryoelectron microscopy (cryo-EM) structures and cryo-EM 3D variability analysis of semaglutide- and taspoglutide-bound GLP-1R-Gs protein complexes. These reveal similar peptide interactions to GLP-1 but different motions within the receptor and bound peptides, providing insights into the molecular determinants of GLP-1R peptide engagement.

Total Synthesis of Semaglutide Based on a Soluble Hydrophobic-Support-Assisted Liquid-Phase Synthetic Method.

Considering the high cost of the production of semaglutide, which is currently the most promising antidiabetic drug especially for the treatment of type 2 diabetes mellitus, a new synthetic route of semaglutide production that possesses excellent yield and high purity is of vital importance. Herein, we reported a newly developed synthetic route of semaglutide that is simple and efficient, based on a soluble hydrophobic-support-assisted liquid-phase synthetic method by applying Alloc-chemistry to the synthesis of the main chain peptide and side chain peptide of semaglutide. With careful optimization of the reaction conditions and innovative strategy of post-synthetic treatments, the total yield and purity of the crude semaglutide was improved satisfactorily.

Profile of semaglutide in the management of type 2 diabetes: design, development, and place in therapy.

Type 2 diabetes mellitus (T2DM) has become one of the leading causes of morbidity and mortality in developed countries. Low efficacy, weight gain, and hypoglycemia are the main pitfalls of previous treatments for T2DM. New therapies have been designed with the aim of improving the results in efficacy and quality of life. Glucagon-like peptide 1 (GLP-1) receptor agonists (GLP-1 RA) increase glucose-dependent insulin secretion, decrease gastric emptying, and reduce postprandial glucagon secretion. The last GLP-1 RA approved by the US Food and Drug Administration and European Medicines Agency was semaglutide. This review describes its pharmacology, core clinical data coming from the randomized controlled trials included in the development program, proven cardiovascular benefits, safety issues, and precautions for the use of semaglutide in special populations. Additionally, an overview of the positioning of semaglutide in T2DM therapy and practical issues regarding semaglutide initiation are offered.

The development of an oral GLP-1 receptor agonist for the management of type 2 diabetes: evidence to date.

Glucagon-like peptide 1 receptor agonists (GLP1-RA) are prominent agents in the therapeutics of type 2 diabetes mellitus due to their exemplary efficacy in both preprandial and postprandial glycemia, their safety, low risk of hypoglycemia, their multilevel pathophysiological superiority, weight loss and importantly the observed benefits in cardiovascular disease reduction. Their major drawback is the subcutaneous route of administration, constituting a barrier to adoption and reason for treatment discontinuation. Thus, the development of an oral GLP1-RA agent would promote medication adherence and quality of life, further consolidating its beneficial effects in real-life clinical practice. However, this task is hampered by suboptimal gastrointestinal protein absorption. Yet, the introduction of oral semaglutide, a modified form of semaglutide with the addition of a carrier sodium N-(8-[2-hydroxybenzoyl] amino) caprylate, may have provided a safe and effective way to reach systemic circulation while other molecules are in development. Whether this molecule still has the impressive cardiovascular effects demonstrated with the use of its precursor remains to be explored. However, to date, its efficacy and safety have already been showcased in a randomized trial. More research is warranted in order to further consolidate these findings across different type 2 diabetes mellitus (T2DM) subpopulations, and adequately powered studies with a longer follow-up that would allow the exploration of microvascular and macrovascular complications are needed. Finally, studies comparing oral semaglutide and similar molecules with other currently established antidiabetic agents to evaluate the relative efficacy, the cost-effectiveness and further understand its place in T2DM therapeutic algorithm are needed. This review focuses on the development of oral GLP1-RA agents and summarizes the challenges, milestones and expected benefits associated with a successful introduction.

Subcutaneous semaglutide (NN9535) for the treatment of type 2 diabetes.

INTRODUCTION: It is critical for individuals with type 2 diabetes mellitus (T2DM) to maintain optimal glycemia while avoiding hypoglycemia, control body weight, and reduce cardiovascular risk. The GLP-1 receptor agonists stimulate glucose-dependent insulin release (low risk of hypoglycemia), inhibit glucagon secretion, slow gastric emptying and suppress appetite (weight loss). The new members of the class are available as once daily or weekly injections. Additionally, some members of the class have demonstrated reduced cardiovascular risk. Areas covered: This manuscript describes semaglutide - a new investigational long acting GLP-1 receptor agonist. The key trials from the clinical development process are reviewed and important end-points highlighted. Expert opinion: Once-weekly semaglutide has shown superiority in reducing glycosylated hemoglobin and body weight in comparison with placebo and active comparators when used as monotherapy or in combination treatment. In addition, semaglutide improved markers of ß-cell function and have shown cardiovascular risk reduction similar to once daily liraglutide. Although, overall semaglutide safety was comparable to other GLP-1 receptor agonists (low risk of hypoglycemia and high frequency of gastrointestinal side effects), increase in retinopathy complications requires further investigation.

Effect of fatty acid chain length on suppression of ghrelin and stimulation of PYY, GLP-2 and PP secretion in healthy men.

We have evaluated the effects of fatty acid chain length on ghrelin, peptide YY (PYY), glucagon-like peptide-2 (GLP-2) and pancreatic polypeptide (PP) secretion and hypothesized that intraduodenal administration of dodecanoic ("C12"), but not decanoic ("C10"), acid would decrease plasma ghrelin and increase PYY, GLP-2 and PP concentrations. Plasma hormone concentrations were measured in seven healthy men during 90-min intraduodenal infusions of: (i) C12, (ii) C10 or (iii) control (rate: 2 ml/min, 0.375 kcal/min for C12/C10) and after a buffet-meal consumed following the infusion. C12 markedly suppressed plasma ghrelin and increased both PYY and GLP-2 (all P < 0.05) compared with control and C10, while C10 had no effect. Both C10 and C12 increased PP concentrations slightly (P < 0.05). We conclude that the effects of intraduodenal fatty acids on ghrelin, PYY and GLP-2 secretion are dependent on their chain length.

Conserved and non-conserved loci of the glucagon gene in old world ruminating ungulates.

The homology and diversification of genomic sequence encoding glucagon gene among native Egyptian buffalos, camel and sheep were tested using cattle as model. Oligodeoxynucleotide primers designed from the available GenBank data were used for PCR probing of the glucagon gene encoding sequence at different loci. The DNA oligomer probes were constructed to flank either the whole gene encoding sequence or different intra-gene encoding sequences. The PCR products were visualized using agarose gel electrophoresis. All species showed a same size band of prepro-glucagon when PCR was used to amplify the whole gene encoding sequence. In contrary, amplifications of different intra-gene loci failed to give the same results. The results indicated variable degrees of diversity among old world ruminating ungulates in the glucagon gene encoding sequence. Compared with other ruminants, the variation appears predominantly in camel. Surprisingly, the similarity in size between both amplification products of whole gene encoding sequence and the proposed size of glucagon cDNA definitely excludes the possibility of large intervening introns spanning the genomic sequence of the glucagon gene in these species. This indicates that, in contrast to other tested mammals, the glucagon gene includes an essentially full-length copy of glucagon mRNA. The study revealed a possible new aspect of glucagon gene evolution in order to correlate its corresponding protein function among different ruminant species.

Oxyntomodulin and related peptides control somatostatin secretion in RIN T3 cells.

We studied the effects of oxyntomodulin (OXM), of its C-terminal (19-37) fragment (OXM (19-37)) and of glucagon (GLU) on somatostatin release, cyclic AMP accumulation and inositol phosphate turnover in somatostatin-secreting RIN T3 cells in culture. Rapid changes in cellular free Ca2+ were also measured using fura-2. Carbachol was used as a control test agent for the parameters involving the inositol phosphate/Ca2+ cascade. OXM, GLU and OXM (19-37) were all able to stimulate somatostatin release with relative ED50 of approx. 1, 22 and 45, respectively. OXM and GLU stimulated cyclic AMP levels with relative ED50 of approx. 1 and 30, respectively, whereas OXM (19-37) was totally ineffective on this parameter. In contrast to carbachol, none of the peptides significantly modified the inositol phosphate turnover or induced rapid changes in cellular free Ca2+. We conclude that the RIN T3 cells contain a receptor-cyclic AMP system similar to that found in gastric mucosa and that this system is linked to somatostatin release. Another receptor-second messenger mechanism linked to somatostatin release is triggered by the (19-37) fragment. This mechanism is not the inositol phosphate/Ca2+ cascade triggered in the same cells by cholinergic agents.

Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide.

Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analogue that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analogue by increasing albumin affinity and secure full stability against metabolic degradation. The fatty acid moiety and the linking chemistry to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analogue. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib(8), Arg(34)) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clinical testing.

Oxyntomodulin-like immunoreactivity: diurnal profile of a new potential enterogastrone.

The biological specificity of oxyntomodulin toward the gastric mucosa results from its C-terminal octapeptide. A RIA using a specific antibody raised against this region permitted quantification of the whole set of proglucagon-derived peptides that interact with the oxyntomodulin recognition systems, corresponding to the new concept of oxyntomodulin-like-immunoreactivity (OLI). The present report describes the physiological 24-h OLI profile in human plasma (eight men and eight women; mean age, 45 yr; range, 20-77 yr). Blood was withdrawn every hour from 0700-1900 h and every 2 h from 2100-0500 h. A meal-dependent profile was found for circulating OLI, with basal values (60 +/- 7 ng/L) at 0500 h and rises elicited by each food intake. The highest value (136 +/- 21 ng/L) was obtained at 2100 h. Plasma concentrations and diurnal variations of OLI were similar to those of the other intestinal peptides known to exert an endocrine function. The mean circulating OLI values increased with age, whereas no change was noticed according to sex. The inhibitory effect exerted by the peptides of the OLI family on gastric acid secretion, the meal dependence of their plasma concentrations, and the observed synchronism of their diurnal profile with that previously described for somatostatin make them candidates for an enterogastrone action.

H-Lys-Arg-Asn-Lys-Asn-Asn-OH is the minimal active structure of oxyntomodulin.

Oxyntomodulin inhibits gastric acid secretion via its C-terminal octapeptide. Its minimal active structure was delineated by testing, on histamine-stimulated gastric acid secretion in the conscious rat, the inhibitory effect of octapeptide analogues, shortened either or both on their N- or C- terminus. The octapeptide may be simplified by deleting the two C-terminal amino acids while keeping its efficacy and the slope of the dose-response curve. Suppressing the first N-terminal amino acid dramatically decreased the activity. The nonprotected peptides are metabolized by aminopeptidases and endopeptidases. The increased potency of the N-acetylated forms is related, at least in part, with their protection against aminopeptidases.

Self-assembling glucagon-like peptide 1-mimetic peptide amphiphiles for enhanced activity and proliferation of insulin-secreting cells.

Current treatment for type 1 diabetes mellitus requires daily insulin injections that fail to produce physiological glycemic control. Islet cell transplantation has been proposed as a permanent cure but is limited by loss of ß-cell viability and function. These limitations could potentially be overcome by relying on the activity of glucagon-like peptide 1 (GLP-1), which acts on ß-cells to promote insulin release, proliferation and survival. We have developed a peptide amphiphile (PA) molecule incorporating a peptide mimetic for GLP-1. This GLP-1-mimetic PA self-assembles into one-dimensional nanofibers that stabilize the active secondary structure of GLP-1 and can be cross-linked by calcium ions to form a macroscopic gel capable of cell encapsulation and three-dimensional culture. The GLP-1-mimetic PA nanofibers were found to stimulate insulin secretion from rat insulinoma (RINm5f) cells to a significantly greater extent than the mimetic peptide alone and to a level equivalent to that of the clinically used agonist exendin-4. The activity of the GLP-1-mimetic PA is glucose-dependent, lipid-raft dependent and partially PKA-dependent consistent with native GLP-1. The GLP-1-mimetic PA also completely abrogates inflammatory cytokine-induced cell death to the level of untreated controls. When used as a PA gel to encapsulate RINm5f cells, the GLP-1-mimetic PA stimulates insulin secretion and proliferation in a cytokine-resistant manner that is significantly greater than a non-bioactive PA gel containing exendin-4. Due to its self-assembling property and bioactivity, the GLP-1-mimetic PA can be incorporated into previously developed islet cell transplantation protocols with the potential for significant enhancement of ß-cell viability and function.