The Discovery and Development of Liraglutide and Semaglutide

Abstract

The discovery of glucagon-like peptide-1 (GLP-1), an incretin hormone with important effects on glycemic control and body weight regulation, led to efforts to extend its half-life and make it therapeutically effective in people with type 2 diabetes (T2D). The development of short- and then long-acting GLP-1 receptor agonists (GLP-1RAs) followed. Our article charts the discovery and development of the long-acting GLP-1 analogs liraglutide and, subsequently, semaglutide. We examine the chemistry employed in designing liraglutide and semaglutide, the human and non-human studies used to investigate their cellular targets and pharmacological effects, and ongoing investigations into new applications and formulations of these drugs. Reversible binding to albumin was used for the systemic protraction of liraglutide and semaglutide, with optimal fatty acid and linker combinations identified to maximize albumin binding while maintaining GLP-1 receptor (GLP-1R) potency. GLP-1RAs mediate their effects via this receptor, which is expressed in the pancreas, gastrointestinal tract, heart, lungs, kidneys, and brain. GLP-1Rs in the pancreas and brain have been shown to account for the respective improvements in glycemic control and body weight that are evident with liraglutide and semaglutide. Both liraglutide and semaglutide also positively affect cardiovascular (CV) outcomes in individuals with T2D, although the precise mechanism is still being explored. Significant weight loss, through an effect to reduce energy intake, led to the approval of liraglutide (3.0 mg) for the treatment of obesity, an indication currently under investigation with semaglutide. Other ongoing investigations with semaglutide include the treatment of non-alcoholic fatty liver disease (NASH) and its use in an oral formulation for the treatment of T2D. In summary, rational design has led to the development of two long-acting GLP-1 analogs, liraglutide and semaglutide, that have made a vast contribution to the management of T2D in terms of improvements in glycemic control, body weight, blood pressure, lipids, beta-cell function, and CV outcomes. Furthermore, the development of an oral formulation for semaglutide may provide individuals with additional benefits in relation to treatment adherence. In addition to T2D, liraglutide is used in the treatment of obesity, while semaglutide is currently under investigation for use in obesity and NASH.

Keywords: GLP-1; albumin; liraglutide; obesity; once-weekly; semaglutide; type 2 diabetes.

Figure 1

Structure of human serum albumin, representing domains and fatty-acid binding sites. Green, subdomain I; blue, subdomain II; gray, subdomain III; yellow/red, fatty acids. The PDB file has code 1E7E resolved to 2.5 Å resolution. Reprinted from Bhattacharya et al. (10). Copyright 2018, with permission from Elsevier.

Figure 2

Chemical structures of liraglutide and semaglutide. Adapted with permission from Lau et al. (53). Copyright 2018, American Chemical Society.

Figure 3

Albumin affinity of fatty-acid derivatized peptides vs. native GLP-1. (A) Derivatives of Aib8, Arg34 GLP-1(7-37) with C12 to C22 di-acids and attached to Lys26 using γGlu- and OEG-based linkers. GLP-1R binding at 0 and 2% HSA using baby BHK cells expressing the human GLP-1R, and in vitro potency measured in BHK cells that express both the human GLP-1R and a luciferase reporter system; (B) HSA binding of GLP-1 derivatives, assessed using analytical ultracentrifugation; (C) In vivo protraction in rats following intravenous administration of 32 (5.5 nmol/kg), semaglutide (4.2 nmol/kg), 19 (3.3 nmol/kg), 16 (5.5 nmol/kg), and 15 (5.3 nmol/kg). BHK, baby hamster kidney; EC₅₀, effective concentration; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; HSA, human serum albumin. Adapted with permission from Lau et al. (53). Copyright 2018, American Chemical Society.

Figure 4

Crystal structure of the semaglutide peptide backbone (gray) in complex with the GLP-1 receptor extracellular domain (golden surface). Individual residues are shown as sticks, with nitrogen and oxygen atoms colored blue and red, respectively. (A) Overall structure with boxed area magnified in (B,C). (B) The structure of the C-terminus of the semaglutide peptide backbone. Hydrogen bond interactions are illustrated as dotted lines. (C) Arg36 closes the hydrophobic ligand-receptor interface by aligning with Trp31 and Glu68. A water molecule is coordinated by Glu27 and Arg34. With permission from Lau et al. (53). Copyright 2018, American Chemical Society.

Figure 5

GLP-1 receptor immunoreactivity using monoclonal antibody 3F52 on monkey and human pancreas. (A) GLP-1R IHC with antibody MAb 3F52 on paraffin-embedded monkey pancreas (A–G) and ISLB with ¹²⁵I-GLP-1 on a frozen section from monkey pancreas (H–J). (B,C) Magnifications of solid-line boxed areas in (A,B), respectively. (D) High magnification of the dashed-line boxed area in (B). (I) High magnification of the boxed area in (H). (A–D,F) GLP-1R immunoreactivity using a DAB IHC protocol. (E,G) Images from a double-labeling GLP-1R insulin IHC fluorescence protocol. In (E), double labeling for GLP-1R (green) and insulin (red) shows complete co-localization, demonstrating that all GLP-1R–positive cells are beta-cells. The ductal epithelium is negative for GLP-1R (D). In (F), weakly GLP-1R–immunopositive acinar cells (arrows) can be seen adjacent to strongly staining islets (left). (G) High-magnification image of the main duct area of a cynomolgus monkey pancreas double-immunostained for GLP-1R (green) and insulin (red). (J) Image from the section adjacent to (I) and incubated with ¹²⁵I-GLP-1 plus an excess of unlabeled GLP-1. Scale bars correspond to 1 mm (A) and 100 μm (B–J). (B) A normal (A–C,G–J) and a diabetic (D–F) human pancreas double labeled for GLP-1R (green) and insulin (red). (B,E) High magnification images of the dashed-line boxes in (A,D), respectively. (C,F) High-magnification images of the solid-line boxes in (A,D), respectively. Note the complete co-localization of signals for GLP-1R and insulin in islets in (A,B,D,E) and absence of staining of ductal epithelium in (C,F). G-I, GLP-1R (green)/cytokeratin-19 (red) double staining in sample of normal human pancreas containing part of the main duct with PDGs. (G) Low-magnification overview. H, High magnification of area (dashed-line box in G) containing PDGs and showing no GLP-1R immunoreactivity. I, High magnification of area (solid line box in G) containing an islet with GLP-1R immunoreactive beta-cells. (J) Two GLP-1R/insulin-positive cells are located within the ductal epithelium. Scale bars correspond to 0.5 mm (A,D,G), 100 μm (B,C,E,F,H,J), and 50 μm (I). DAB, diaminobenzidine; GLP-1R, glucagon-like peptide 1 receptor; IHC, immunohistochemistry; ISBL, in situ ligand binding; MAb, monoclonal antibody; PDGs, pancreatic duct glands. Reproduced with permission from Pyke et al. (73). By permission of Oxford University Press on behalf of the Endocrine Society, available at: https://academic.oup.com/endo/article/155/4/1280/2423090?searchresult=1. This figure is not included under the CC-BY license of this publication. For permissions, please contact journals.permissions@oup.com.

Figure 6

GLP-1 receptor immunoreactivity in non-human primate smooth muscle cells. (A) GLP-1R immunoreactivity in non-human primate smooth muscle cells of a vas afferens (va) arteriole at the vascular pole. Note the absence of signal in the macula densa (md), tubuli (t), glomerulus (^*), and endothelial cells within an arteriole (arrow). (B,C) Near-adjacent sections showing double labeling for GLP-1R (green)/renin (red) and GLP-1R (green)/SMA (red), respectively. (B′,C′) Same as (B,C), respectively, with red fluorescence digitally removed to highlight GLP-1R immunoreactivity. (B″,C″) Same as (B,C), respectively, with green fluorescence digitally removed to highlight renin and SMA immunoreactivity, respectively. Scale bars correspond to 50 μm.GLP-1R, glucagon-like peptide 1 receptor; SMA, smooth muscle actin. Reproduced with permission from Pyke et al. (73). By permission of Oxford University Press on behalf of the Endocrine Society, available at: https://academic.oup.com/endo/article/155/4/1280/2423090?searchresult=1. This figure is not included under the CC-BY license of this publication. For permissions, please contact journals.permissions@oup.com.

Figure 7

Distribution of fluorescently labeled liraglutide in the mouse brain. (A) Representative whole brain images viewed in the (A) dorsoventral or (B) sagittal plane from C57BL/6J mice administered with liraglutide⁷⁵⁰. The brain tissue was scanned at 620 and 710 nm, representing both autofluorescence from the tissue (gray) and specific signal (green). The red regions in (C,F,I) are shown at higher magnification in (D,G,J), respectively. Images in (D,E,G,H,J,K) show high-magnification views of a single section from (D,G,J) C57BL/6J or (E,H,K) Glp1r^−/−mice administered liraglutide⁷⁵⁰. Liraglutide⁷⁵⁰ was detectable in (C,D) PVN, (F,G) ME and ARC, and (I,J) AP. (E,H,K) In mice lacking a functional GLP-1R, no liraglutide⁷⁵⁰ signal could be detected in any of these regions. Scale bars: 200 μm (A,B,C,F,I); 50 μm (D,E); 100 μm (G,H,J,K). (B) The brain tissue was scanned at 620 and 710 nm, representing both autofluorescence from the tissue (gray) and specific signal (green). The red regions in (A,D,G,J) are shown at higher magnification in (B,E,H,K), respectively (unspecific staining has been removed). Images in the middle and right columns represent enlargements of a single section from (B,E,H,K) C57BL/6J or (C,F,I,L) Glp1r^−/− mice administered with liraglutide⁷⁵⁰. Liraglutide⁷⁵⁰ was detectable in (A,B) organum vasculosum of the lamina terminalis (D,E), subfornical organ (G,H), supraoptic nucleus and supraoptic decussation, and (J,K) ChP. (C,F,I,L). In mice lacking a functional GLP-1R, no liraglutide⁷⁵⁰ signal could be detected in any of these regions except from ChP. Scale bars: 200 μm (A,D,G,J–L); 100 μm (B,C,E,F,H,I). AP, area postrema; ARC, arcuate nucelus; ChP, choroid plexus; ME, median eminence; NTS, nucleus of the solitary tract; OVLT, organum vasculosum of lamina terminalis; PVN, paraventricular nucleus; SFO, subfornical organ; SO, supraoptic nucleus; SOD, supraoptic decussation. Republished with permission of American Society For Clinical Investigation, from Secher et al. (117), Copyright 2018; permission conveyed through Copyright Clearance Center, Inc.

Figure 8

Liraglutide and the mouse brain. Distribution of liraglutide⁵⁹⁴ or exendin(9-39)⁵⁹⁴ in brain. (A,B) liraglutide⁵⁹⁴ had access to ARC, in which it bound the GLP-1R and internalized. (B) High-magnification image showed that liraglutide⁵⁹⁴ was internalized and the fluorescent signal was located in the cytoplasm. Staining, Hoechst nuclear stain (blue) and liraglutide⁵⁹⁴/exendin(9-39)⁵⁹⁴(red). Scale bars: 100 μm. Liraglutide treatment regulates ARC gene expression and ARC neuronal activity. (C) Liraglutide treatment for 28 days in DIO rats significantly increased mean Cart mRNA levels in the ARC (^*p < 0.001 liraglutide vs. vehicle and vs. weight matched), whereas Pomc expression was unaffected. (D) Npy and Agrp mRNA levels were significantly increased in weight-matched rats–but not following treatment with liraglutide (†p < 0.05 weight matched vs. vehicle and vs. liraglutide). Data are mean ± SEM, and statistical analyses were performed using 1-way ANOVA, with Fisher's post hoc test. Images (E,F) show neuronal accumulation and activity following GLP-1R simulation. Specifically, panel (E) shows CART- and liraglutide⁵⁹⁴-positive cells shown as mean ± SEM. Staining, Hoechst nuclear strain (blue), liraglutide594 (red) and CART (green). Scale bar: 25 μm. Panel (F) shows the effects of GLP-1(7-36)amide on firing rate of spontaneous action potentials in POMC/CART neurons. (G) Proposed regulation of neuronal activation by liraglutide. Summary diagram demonstrating the suggested regulatory pathway of GLP-1 on ARC NPY and POMC neurons. GLP-1 stimulates POMC neurons directly through the GLP-1R and is suggested to indirectly inhibit ARC-NPY neurons through a local inhibitory GABA neuron. ANOVA, analysis of variance; ARC, arcuate nucleus; CART, cocaine- and amphetamine-regulated transcript; DIO, diabetes induced obesity; GABA, gamma-amino butyric acid; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; NYP, neuropeptide Y; POMC, proopiomelanocortin; SEM, standard error of the mean; veh, vehicle. Republished with permission of American Society For Clinical Investigation, from Secher et al. (117), Copyright 2018; permission conveyed through Copyright Clearance Center, Inc.

Figure 9

Liraglutide, cFOS, and the hindbrain. Brain region with liraglutide^VT750 access. Bar graph (A) shows the mean fold change and standard deviation (SD) of the total fluorescence signal in selected brain regions comparing liraglutide^VT750 and vehicle (n = 5). An asterisk marks significant difference between treatments when analyzed in individual brain regions using a false discovery rate value of 5% to correct for multiple comparisons. Note the split y-axis when interpreting result and standard deviation. Neural activation following liraglutide administration. Bar graph (B) shows the mean fold change SD to total c-Fos heat map signal in selected brain regions comparing liraglutide- and vehicle-dosed animals. Regions were selected as having either liraglutide^VT750 access, GLP-1R expression, or c-Fos response. An asterisk marks significant difference between treatments when analyzed in individual brain regions using a false discovery rate value of 20% to correct for multiple comparisons. (C) Liraglutide-specific c-Fos increase overlaid with the average liraglutide^VT750 distribution from bar graph (B). (D) Connectivity maps visualized by horizontal maximum intensity projection overlaid with the average c-Fos increase following liraglutide administration from (C). ACB, nucleus accumbens; AOB, accessory olfactory bulb; AP, area postrema; ARH, arcuate hypothalamic nucleus; AUDd, dorsal auditory area; BLA, basolateral amygdalar nucleus; BMA, basomedial amygdalar nucleus; BST, bed nuclei of the stria terminalis; CA (1, 2, and 3), field CA(1, 2, and 3); CeA, central amygdalar nucleus; COA, cortical amygdalar area; CU, cuneate nucleus; DG, dentate gyrus; DMH, dorsomedial nucleus of the hypothalamus; DMX, dorsal motor nucleus of the vagus nerve; DR, dorsal nucleus raphe; ECT, ectorhinal area; ECU, external cuneate nucleus; GENd, geniculate group, dorsal thalamus; IC, inferior colliculus; LC, locus ceruleus; LH, lateral habenula; LHA, lateral hypothalamic area; LPO, lateral preoptic area; LRNm, lateral reticular nucleus, magnocellular part; LS, lateral septal nucleus; ME, median eminence; MEPO, median preoptic nucleus; MH, medial habenula; MM, medial mammillary nucleus; MPT, medial pretectal area; MRN, midbrain reticular nucleus; MTN, midline group of the dorsal thalamus; NI, nucleus incertus; NLL, nucleus of the lateral lemniscus; NTS, nucleus of the solitary tract; OV, vascular organ of the lamina terminalis; PAG, periaqueductal gray; PB, parabrachial nucleus; PCG, pontine central gray; PG, pontine gray; PH, posterior hypothalamic nucleus; PSTN, parasubthalamic nucleus; PVH, paraventricular hypothalamic nucleus; PVp, periventricular hypothalamic nucleus, posterior part; RHP, retrohippocampal region; SCm, superior colliculus, motor related; SCH, suprachiasmatic nucleus; SF, septofimbrial nucleus; SFO, subfornical organ; SNc, substantia nigra, compact part; SNr, substantia nigra, reticular part; SO, supraoptic nucleus; TTd, taenia tecta, dorsal part; TU, tuberal nucleus; VL, lateral ventricle; VMH, ventromedial hypothalamic nucleus; VTA, ventral tegmental area; ZI, zona incerta; V4, fourth ventricle. Reproduced from Salinas et al. (200). Creative common license available at: http://creativecommons.org/licenses/by/4.0/.

Figure 10

Plaque lesion development with semaglutide and liraglutide in apolipoprotein E and low-density lipoprotein receptor knockout mice aorta, and effect on atherosclerosis. (A) Semaglutide significantly decreased WD-induced plaque lesion development at all dose levels in ApoE^−/− mice: ^*p = 0.0266, ^**p = 0.0046, ^****p < 0.0001 vs. vehicle WD, and (B) LDLr^−/− mice: ^****p < 0.0001 vs. vehicle WD. (C) WD-induced increases in body weight were significantly lowered by liraglutide and in the weight-matched comparator: ^****p < 0.0001 vs. vehicle, WD. (D) Liraglutide significantly attenuated WD-induced plaque lesion development, whereas the weight-matched comparator group did not: ^*p = 0.0448, ^****p < 0.0001 vs. vehicle, WD; liraglutide, WD vs. weight-matched, WD, p = 0.06). ApoE, apolipoprotein E; LDLr, low-density lipoprotein receptor; WD, Western diet. Reprinted from Rakipovski et al. (111), Copyright 2018, with permission from Elsevier.

Figure 11

Effects of GLP-1RAs on atherosclerosis is hypothesized to occur via inflammation. (A) Gene expression changes by WD and semaglutide (compared with vehicle-dosed chow-fed animals), exemplifying genes that represent pathways with well-described relevance to plaque formation and the pathophysiology of atherosclerosis, Benjamini–Hochberg corrected p-values; ^****p < 0.0001; ^***p < 0.001; ^**p < 0.01; ^*p < 0.05 vs. LDLr^−/−, WD; ^####p < 0.0001, ^###p < 0.001; ^##p < 0.01; ^#p < 0.05 vs. ApoE^−/−, WD. (B) Subcutaneous administration of semaglutide in an LPS inflammation model reduced plasma levels of TNF-alfa, ^**p = 0.0024 vs. vehicle at 1 h and p = 0.048 vs. vehicle at 4 h. (C) Proposed model illustrating how long-acting GLP-1RAs could reduce atherosclerotic burden. ABCA 1, ATP-binding cassette transporter 1; ApoE, apolipoprotein E; BW, body weight; CCL2, chemokine [C-C motif] ligand 2; CD, cluster of differentiation; GLP-1RA, glucagon-like peptide-1 receptor agonist; IEL, intestinal intraepithelial lymphocyte; IL, interleukin; LDL, low-density lipoprotein; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; OPN, osteopontin; PTGIS, prostaglandin I2 synthase; SELE, selectin E; TG, triglyceride; TNF-alfa, tumor necrosis factor-alfa; VCAM 1, vascular adhesion molecule 1; WD, Western diet. Reprinted from Rakipovski et al. (111), Copyright 2018, with permission from Elsevier.

Figure 12

Anatomical site of absorption of oral semaglutide. (A) Gamma scintigraphic imaging of tablet erosion in the stomach from 2 to 140 min after a single dose of oral semaglutide (10 mg) in a representative healthy individual. White line outlines the stomach; colors within the stomach (red/yellow/green/blue) represent the tablet core and released radioactivity. (B) Illustration of the splenic vein, which drains the gastric cavity, and the portal vein, which drains the gastrointestinal system. Mean semaglutide plasma concentration–time profiles in the splenic and portal veins after a single dose of oral semaglutide (10 mg) in beagle dogs (n = 15). R. gastric, right gastric; L. gastric, left gastric; R. gastroepiploic, right gastroepiploic; L. gastroepiploic, left gastroepiploic. The ratio and 95% CI of the splenic vs. portal veins for AUC_0−30min were calculated [1.94 (1.15 to 2.74)], and statistical significance was determined on the basis of a null hypothesis value of 1 (p < 0.05). The horizontal dashed line (right) represents similar semaglutide plasma concentrations in the splenic and portal veins. Error bars show ±SEM calculated on the original scale or calculated on a log-scale and back-transformed to the original scale. From Buckley et al. (262). Reprinted with permission from the American Association for the Advancement of Science (AAAS).

Figure 13

Absorption and localization of semaglutide in dog gastric mucosa. (A) Local 3F15 (semaglutide) immunofluorescence reactivity (red) under the tablet. Nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 2 mm. (B) 3F15 reactivity (red) restricted to the neck region. The bulk of H₊/K₊ ATPase (green)–positive parietal cells reside in deeper layers, but a few scattered parietal cells can be found in the neck region exposed to luminal semaglutide. Scale bar, 200 μm. (C) 3F15 (red), β-catenin (purple), ZO1 (green), and DAPI (blue). Sloughing of the uppermost region of the epithelium is marked by white asterisks; semaglutide is also detected in deeper, intact layers (white box). Scale bar, 200 μm. (D) Higher magnification of the boxed area in (C). Intact tight junctions labeled with apical ZO1 (green) in direct contact with luminal semaglutide. Scale bar, 40 μm. (E) Same region as (D) without β-catenin. Intracellular 3F15 reactivity (red) is observed in mucosal cells (marked by white arrows). 3F15 also detects semaglutide in capillaries under the epithelium (marked by white asterisks). Scale bar, 40 μm. (F) Maximum projection image from a 63 × confocal 11-μm z stack, showing semaglutide (red) associated with a blood vessel (marked by white arrows) labeled with smooth muscle actin (green). Scale bar, 40 μm. From Buckley et al. (262). Reprinted with permission from the American Association for the Advancement of Science (AAAS).