GLP-1; glucagon receptor/GLP-1 receptor (GCGR/GLP-1R)

Survodutide (BI-456906) is a potent, selective, GCGR/GLP-1R dual agonist in vitro [1]
To establish the effect of peptide modifications on the binding of Survodutide (BI-456906) to GCGRs and GLP-1Rs, we applied a series of functional assays to characterize BI 456906 in vitro. The functional potency (EC50) of BI 456906 in CHO-K1 cells was 0.33 and 0.52 nM for GLP-1R and GCGR, respectively, showing an approximately 10-fold lower potency compared with the native ligands GLP-1 (60 pM) and glucagon (20 pM) (Figure 1B). The EC50 for the endogenous mouse GLP-1R was assessed in the insulinoma cell line MIN6 (Figure 1B); the EC50 was 0.36 nM for BI 456906 and 60 pM for GLP-1. The ability of BI 456906 to activate the endogenous GCGR was determined in primary mouse, rat, cynomolgus monkey, and human hepatocytes, based on cumulative measurements of cAMP and functional inhibition of glycogen synthesis in primary rat hepatocytes (Figure 1B).
The impact of the albumin binding half-life extension on the receptor potencies for Survodutide (BI-456906) was characterized in the presence of 0.5% or 100% human or mouse plasma and compared with endogenous receptor ligands and other incretin agonists (Figure 1C). In 0.5% human and mouse plasma, BI 456906 showed a similar potency to that of endogenous GLP-1. For the GCGR, in 0.5% human and mouse plasma, BI 456906 was ∼6-fold less potent (0.29 and 0.17 nM, respectively) in relation to endogenous glucagon (47 and 30 pM, respectively) (Figure 1C). When the potency was assessed in the presence of 100% human and mouse plasma, BI 456906 showed a potency of 1.0 and 1.6 nM for the human GLP-1R and 8.3 and 16 nM for the human GCGR, respectively. The shift in potency was higher in 100% mouse plasma compared with 100% human plasma, suggesting a stronger affinity of BI 456906 to mouse plasma proteins compared with human. The increased affinity to mouse plasma proteins is supported by the pharmacokinetic profiles of BI 456906 and the long-acting GLP-1R agonist semaglutide in mouse plasma, with a longer terminal half-life seen for BI 456906 compared with semaglutide (Table S1).
The functional relevance of the effects of BI 456906 was confirmed for potentiating GSIS in mouse and rat pancreatic islets, which was similar to native GLP-1 and the long-acting GLP-1R agonist liraglutide (Figures 1D, E, S1a and b). In perifused human islets, BI 456906 showed a first- and second-phase insulin-secretory response that was similar to that of liraglutide (Figure 1F).

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Survodutide (BI-456906) drives transcriptional changes in hepatocytes, providing novel mechanistic insights into glucagon receptor agonism [1]
The livers from the study represented in Figure 3 were subjected to bulk mRNA sequencing to identify genes that were dose-dependently regulated by Survodutide (BI-456906), but not semaglutide, and therefore not considered secondary to the degree of weight loss. In total, 93 mRNAs fulfilled these criteria (Figures 5A and S7). When grouped by their functional and biochemical relevance, transcriptional changes were seen for mRNAs associated with the methionine cycle (Figure 5B and C), G protein-coupled receptor (GPCR) signaling (Figure 5D–F), oxidative phosphorylation (Figure 5G and H), cholesterol metabolism (Figure 5I–K), and cell cycle and differentiation (Figure 5L–N).
GCGR activation alters plasma amino acid concentrations due to its catabolic effects. We therefore measured plasma amino acid concentrations in samples from GLP-1R KO mice (Figure 2C) and the subchronic bodyweight-lowering study (Figure 3). Treatment with Survodutide (BI-456906) in both studies resulted in a dose-dependent reduction of plasma amino acid levels compared with vehicle and semaglutide (Figure 6A–G and S8), suggesting GCGR engagement. This effect was significant for alanine, citrulline, glutamine, ornithine, serine, threonine, and tyrosine. In addition, transcripts for amino acid-metabolizing enzymes were dose-dependently changed with BI 456906 treatment (downregulated Ddah1, upregulated Asns, Ass1, Gls2, Got1, and Sds; Figure 6H–M). We correlated the transcriptional changes for the genes Sds, Gls2, and Got1 with the plasma concentrations of serine and glutamine (Figure 6N–P), and for the 93 gene set with the liver cholesterol and triglyceride concentrations (Figure S9). These analyses demonstrated a significant negative correlation, which was not observed for semaglutide, strengthening the conclusion that changes in plasma amino acids are attributed to GCGR agonistic activity of BI 456906 in the liver (Figure 6N–P).

Survodutide (BI-456906) engages the glucagon-like peptide-1 receptor in vivo upon single dosing to reduce food intake, improve glucose tolerance, and inhibit gastric emptying [1]
The acute potency of BI 456906 in engaging the GLP-1R was tested in lean mice after single dosing; the ability of BI 456906 to reduce food intake, improve glucose tolerance, and inhibit gastric emptying was compared with semaglutide. Acute food intake was dose-dependently reduced by BI 456906 (Figure 2A) and semaglutide (Figure 2B) in WT but not in GLP-1R KO mice (Figure 2C), showing a lower potency for BI 456906 (Figure 2I). Similarly, both BI 456906 and semaglutide improved intraperitoneal and oral glucose tolerance in WT (Figures 2D, E, and S2) but not in GLP-1R KO mice (intraperitoneal, Figures 2F, S2c; oral, Figure S2h). Reduced acetaminophen excursion, a measure for inhibition of gastric emptying, was observed for both BI456906 (Figure 2G) and semaglutide (Figure 2H), with a higher potency for semaglutide compared to BI 456906, which was effective only at higher doses of 100 and 300 nmol/kg.
Overall, the efficacy of BI 456906 to reduce food intake, improve glucose tolerance, and delay gastric emptying was similar to that of semaglutide, but with an approximately 10- to 20-fold lower potency at the GLP-1R (Figure 2I). This is in concordance with the lower in vitro potency of BI 456906 compared with semaglutide determined for the human GLP-1R and GCGR in CRE-Luc cells in 100% mouse plasma (1.6 nM compared with 0.098 nM; Figure 1C), suggesting that plasma protein binding is notably higher for BI 456906 in mouse plasma.

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Survodutide (BI-456906) achieves a greater bodyweight-lowering efficacy in diet-induced obese mice compared with maximally effective doses of semaglutide [1]
Upon repeated QD SC administration of BI 456906 or semaglutide to DIO mice, BI 456906 dose-dependently reduced bodyweight from baseline by up to 32% at Day 28 at a dose of 30 nmol/kg (Figure 3A). The efficacy of 30 nmol/kg BI 456906 to reduce bodyweight was greater than semaglutide at maximally effective doses of 20 nmol/kg (25%) and 100 nmol/kg (27%), the difference reaching significance compared with the 20 nmol/kg dose but not 100 nmol/kg. The marked reductions in bodyweight upon treatment with BI 456906 and semaglutide were reflected by an immediate suppression of food intake compared with vehicle-treated mice, with a maximal anorectic effect seen between Days 1 and 2 (Figure 3B). However, the acute food intake reduction of BI 456906 was less pronounced compared with semaglutide, despite equimolar doses, suggesting an additional mechanism driving the superior weight loss efficacy after 28 days of treatment. Food intake gradually returned to baseline levels and began to normalize around Day 15 for both BI 456906 and semaglutide.

Survodutide (BI-456906) and semaglutide dose-dependently decreased fat mass, with significant reductions seen with doses of 10, 20, and 30 nmol/kg and 20 and 100 nmol/kg for the two compounds, respectively (Figure S3a). Lean mass was significantly reduced by BI 456906 at doses of 20 and 30 nmol/kg and semaglutide at doses of 20 nmol/kg, respectively (Figure S3b). The dose-dependent lowering of liver triglycerides and both liver and plasma cholesterol by BI 456906 (versus vehicle) was similar to the effects seen with semaglutide (Figure 3C–E). Plasma triglycerides were significantly reduced by 30 nmol/kg BI 456906 versus vehicle (Figure 3F). Relative to vehicle, concentrations of ALT were significantly reduced by BI 456906 and semaglutide at all doses investigated, and AST concentrations were significantly reduced by 30 nmol/kg BI 456906 and 20 nmol/kg and 100 nmol/kg semaglutide (Figure 3G and H). Despite lesser reductions in bodyweight compared with 20 nmol/kg semaglutide, doses of 3 and 10 nmol/kg BI 456906 significantly reduced liver triglycerides, plasma cholesterol, and ALT levels (Figure 3C, E, and g). Furthermore, BI 456906 dose-dependently and significantly decreased plasma insulin and leptin versus vehicle (Figure 3I and J), similar to semaglutide. At the end of the study, when food intake had returned to levels in vehicle-treated animals, 100 nmol/kg semaglutide-treated mice showed significantly increased plasma levels of the orexigenic hormone ghrelin versus vehicle, a non-significant trend that was also observed with increasing BI 456906 doses (Figure 3K).
Survodutide (BI-456906) dose-dependently reduced plasma glucagon, an effect that was not observed with semaglutide (Figure 3L). Engagement of the GCGR by BI 456906 was supported by dose-dependent increases in plasma FGF-21 (Figure 3M).
Upon termination of the study, an ex vivo bioactivity assay was carried out on plasma samples to determine drug exposures and the relative bioactivity for the human GLP-1R and GCGR (Figure 3N; Table S2) to support the interpretation of the pharmacokinetic and pharmacodynamic profiles of the two peptides (Table S1). Diluted plasma samples were used to determine the nM activity and relative bioactive fraction (EC50) for BI 456906 and semaglutide at the GLP-1R (Figure S4) and the GCGR (Figure S5). As illustrated in Figure 3O, the ex vivo bioactivity allowed the determination of the bioactivities for the respective receptors in relation to the treatment exposure (Table S2); the relative bioactivity of BI 456906 at the GLP-1R was lower compared with semaglutide (Figure 3O). The ex vivo activity of semaglutide in plasma exceeded its EC50 in 100% mouse plasma more than 1000-fold, suggesting full engagement of the GLP-1R at both doses investigated (Figure 3N and O). BI 456906, at its maximal dose of 30 nmol/kg, showed a bioequivalence of less than 1000-fold and 100-fold for the GLP-1R and GCGR, respectively (Figure 3N). Taken together, these analyses support the conclusion that, unlike semaglutide, the bodyweight-lowering efficacy of BI 456906 is related to engagement of both receptors.

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Survodutide (BI-456906) lowers bodyweight while maintaining glycemic control and increasing energy expenditure in mice with diet-induced obesity [1]
To demonstrate that weight-lowering effects of BI 456906 in DIO mice can be achieved without compromising glycemic control, a subchronic dosing study was performed comparing BI 456906, semaglutide, and LA-GCG at doses expected to yield similar weight reductions after 10 days of treatment. BI 456906 decreased bodyweight from baseline versus vehicle (Figure 4A) to a similar level as semaglutide (Figure 4A), while LA-GCG achieved significant (Figure 4A), yet less extensive, bodyweight reductions versus vehicle. Both BI 456906 and semaglutide significantly decreased blood glucose at Days 1 and 3 versus vehicle, whereas LA-GCG significantly increased blood glucose at Day 5 (Figure 4B). Lowering of plasma glucagon was observed with BI 456906 and LA-GCG up to Day 8 compared with vehicle while semaglutide led to an initial drop in glucagon, returning to vehicle levels after subchronic dosing (Figure 4C). Plasma acyl ghrelin levels were unchanged by treatment with LA-GCG but were significantly increased versus vehicle by BI 456906 and semaglutide at Days 3 and 5, respectively (Figure 4D).
Survodutide (BI-456906) engages the glucagon receptor in vivo upon single dosing, increases liver nicotinamide N-methyltransferase mRNA, and reduces plasma serine and glutamine [1]
Hepatic expression of the Nnmt gene was dose-dependently and specifically upregulated by BI 456906, but not semaglutide, implying GCGR agonism in the liver (Figure 5B). Consistent with these findings, single dosing of lean mice with BI 456906, but not semaglutide, dose-dependently increased Nnmt mRNA expression in the liver (Figure 7A), reaching significance at 100 and 300 nmol/kg BI 456906 versus vehicle. In addition, plasma amino acids were measured as circulating biomarkers for target engagement of the GCGR; single dosing of mice with BI 456906 significantly reduced plasma glutamine and serine levels compared with control and semaglutide-treated groups at a dose of 10 nmol/kg (Figure 7B and C).

In vitro functional receptor potencies [1]
The functional potencies for of Survodutide (BI-456906) were determined in Chinese hamster ovary (CHO)-K1 cells expressing human GLP-1R and GCGR cDNAs, mouse insulinoma MIN6 cells, and primary human, cynomolgus monkey, mouse, and rat hepatocytes. These assays were applied to support the structure–activity relationship and respective potencies to stimulate cyclic AMP generation, which were determined in the presence of 0.1% BSA. Cells were seeded in 96-well microtiter plates in 100 μL growth medium; growth medium was removed after 24 h (4 h for primary hepatocytes) and the cells were washed with Krebs-Ringer Bicarbonate HEPES buffer (KRBH, 200 μL). The buffer was removed, and the cells were incubated for 15 min at room temperature in 10 μL KRBH (KRBH + 10 mM HEPES, 5 mM NaHCO3, 0.1% W/V bovine serum albumin [BSA]) with 0.1 mM 3-isobutyl-1-methylxanthine. The reaction was stopped by the addition of lysis buffer (0.1% W/V BSA, 5 mM HEPES, 0.3% V/V Tween®20) for 10 min at room temperature. Lysates were transferred to 384-well plates and incubated with 10 μL of acceptor/donor bead mixture for 1 h at room temperature in the dark, then the cAMP content was determined using the AlphaScreen™ cAMP Functional Assay Kit according to the manufacturer’s instructions.
To characterize the impact of plasma protein binding for fatty-acid protracted peptides, the cAMP responsive element (CRE)-induced luciferase activity assay was applied, and receptor potencies were determined in the presence of low (0.5%) and high (100%) levels of mouse and human plasma. Human HEK293 CRE-luc2P cells expressing recombinant GLP-1R and GCGR were cultivated in Dulbecco’s Modified Eagle Medium (with high glucose/l-glutamine) supplemented with 10% fetal bovine serum, 50 μg/mL hygromycin, and 400 μg/mL Geneticin™. For the assays, cells were resuspended either in KRBH with 0.5% human or mouse plasma or in 100% human or mouse plasma. Cells were treated for 4 h at 37 °C with the different peptides (all n = 3; all tested peptides were produced in house, handled as 1 mM stock solution in dimethyl sulfoxide, and tested at a final concentration range between 0.2 pM and 1 μM). In vitro potency was assessed using the Bright-Glo™ Luciferase Assay System, measuring the production of cAMP through a CRE-controlled luciferase. The potency (EC50), plasma shift, and GLP-1R/GCGR ratio was calculated for each plasma condition.

Inhibition of glycogen synthesis in rat hepatocytes and glucose-stimulated insulin secretion from primary islets in vitro [1]
For determining glycogen synthesis inhibition in primary rat hepatocytes, cells were plated in 24-well plates in medium (Williams E Medium containing 200 mM glutamine, 1 mg/mL gentamicin, dexamethasone 0.1 μM, Insulin-Transferrin-Sodium-Selenite-Supplement 0.017 μM) at 37 °C and 5% CO2. After 24 h, the medium was removed, cells were washed once with PBS, and 180 μL KRBH (134 mM NaCl, 3.5 mM KCl, 1.2 mM KH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 5 mM NaHCO3, 10 mM HEPES, pH 7.4) containing 0.1% BSA, 22.5 mM glucose, the respective peptide concentration, and 40 μCi/mL D-[U-14C]-glucose was added. After 180 min, the incubation buffer was aspirated; the cells were washed once with ice-cold PBS and lysed for 30 min at room temperature with 1 mol/l NaOH. Lysates were transferred to 96-well filter plates and glycogen was precipitated by incubation for 120 min at 4 °C followed by washing four times with ice-cold ethanol (70%). The precipitates were filtered to dryness and the synthesized glycogen determined by counting the amount of radioactivity (incorporated 14C-glucose) on the filter in a topcount scintillation counter. Each cell plate contained wells with vehicle controls (0.1% DMSO in KRBH) as reference for non-inhibited glycogen synthesis (100% CTL) and wells without D-[U-14C]-glucose as control for non-specific background, which was subtracted from all values.
The potencies to increase glucose-stimulated insulin secretion (GSIS) were assessed in mouse and rat islets under static conditions as previously described. Rat and mouse islets were treated with GLP-1, Survodutide (BI-456906), or liraglutide. Untreated cells were exposed to 1.0 mM glucose and compared with those kept under treatment conditions. Glucose was added (8.3 mM for rat islets, 16.7 mM for mouse islets), and insulin secretion was measured and expressed as a percentage of total insulin.
The Perifusion System was used to determine the dynamic insulin secretion of human islets after application of 10 nM Survodutide (BI-456906), 30 nM liraglutide, or buffer (KRBH with 0.1% BSA). Six chambers were used, each containing 50 islet cells. Insulin secretion (ng/mL) was measured over 120 min, with 1 mM glucose applied after 20 min, 8.3 mM glucose after 35 min, 1 mM glucose after 75 min, and 60 mM KCl after 105 min.

Pharmacokinetic properties of Survodutide (BI-456906) [1]
For pharmacokinetic analysis, male C57BL/6NRj mice were given a single intravenous (IV) or subcutaneous (SC) Survodutide (BI-456906) dose (20 nmol/kg; N = 3 per route). In addition, male Beagle dogs received an IV or SC dose of 5 nmol/kg or 10 nmol/kg (N = 3) Survodutide (BI-456906), respectively. Plasma samples were generated at different time points post dosing and stored at −20 °C until further analysis. Plasma concentrations of Survodutide (BI-456906) were measured using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with a calibration range of 0.5–1000 nM on a QTRAP 6500+ . Samples were pre-treated with ethanol for protein precipitation before the analysis.

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Inhibition of acute food intake in NMRI outbred mice [1]
Three-week-old, male, lean NMRI outbred mice (wild-type [WT] and GLP-1R knockout [KO]) were obtained from Janvier Labs (Le Genest-Saint-Isle, France) and were group-housed four mice per cage on a 12 h/12 h dark/light cycle (lights off at 15:00). The room temperature was controlled at 21 °C ± 1 °C, with 60% ± 20% humidity. Animals had ad libitum access to regular rodent chow and tap water. Six-week-old male mice were randomized into treatment groups based on food intake and bodyweight (n = 8–10 per group). Mice were fasted for 6 h before randomization and SC dosing of Survodutide (BI-456906), semaglutide, or vehicle 1 h before the dark phase. Food intake was measured for 24 h using a fully automated Herdsman-2 food-intake monitoring system. Blood was drawn at 24 h post dosing, and amino acid levels were measured (see later). ED50 values were calculated in GraphPad Prism 9 using the nonlinear fit tool.

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Acute glucose tolerance tests in lean mice [1]
The effect of Survodutide (BI-456906) on acute glucose tolerance was assessed by intraperitoneal and oral glucose tolerance tests. Male WT and GLP-1R KO lean C57BL6/J mice, 10–12 weeks old, were randomized (n = 7 per group) and fasted for 12 h prior to study initiation. Pre-treatment blood glucose was measured at −5 h. Mice were administered SC Survodutide (BI-456906) or semaglutide at −4 h. Baseline blood glucose was measured at −1 h, and then an intraperitoneal bolus of 2 g/kg glucose was applied at 0 h. Blood glucose was measured at 0, 15, 30, 60, and 120 min, and compound exposure was measured at 140 min.
Male WT and GLP-1R KO lean C57BL6/J mice, 6–7 weeks old (n = 6–7 per group), were administered SC vehicle, Survodutide (BI-456906), or semaglutide at −24 h. Mice were then fasted from −10 h, before bolus ingestion of glucose (2 g/kg) at 0 h. Blood glucose was measured at 0, 15, 30, 60, and 120 min, and compound exposure was measured at 120 min. In both experiments, glucose was measured in whole blood using a commercially available glucometer with test strips. Data are represented as mean ± standard error of mean (SEM) and were compared using GraphPad Prism 9 using two-way ANOVA, followed by Dunnett’s method for multiple comparisons versus vehicle. Significant differences were identified at p < 0.05.

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Acute gastric emptying in lean mice [1]
Lean, male, 8–10-week-old B6J mice were fasted for 12 h before study initiation, with SC compound dosing of vehicle, Survodutide (BI-456906), or semaglutide at −4 h. At 0 h, mice had a bolus ingestion of acetaminophen (100 mg/kg)–glucose (2 g/kg), and the acetaminophen concentration was measured by plasma sampling via the vena facialis at 10, 30, and 60 min. After 60 min, ex vivo assays assessing acetaminophen, blood glucose, and terminal exposure were carried out. Acetaminophen was measured using an automated analyzer. AUC was calculated from acetaminophen concentrations measured between 0 and 60 min. ED50 values were calculated in GraphPad Prism 9 using the nonlinear fit tool.
Nicotinamide N-methyltransferase gene expression in liver [1]
Male WT C57BL6/J mice, 6–7 weeks old (n = 5 per group), were administered SC vehicle, semaglutide, or Survodutide (BI-456906) at −18 h. Mice were then fasted from −12 h until 0 h, when they were euthanized, a lobe of the liver was preserved in RNAlater™ Stabilization Solution at 4 °C, and nicotinamide N-methyltransferase (NNMT) mRNA expression in the liver was measured by real-time PCR using a TaqMan gene-expression assay.

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Imaging study in cAMP response element-luciferase transgenic mice [1]
Male cAMP response element (CRE)-luciferase (Luc) transgenic mice were obtained at 12–13 weeks old. Animals were fed irradiated Teklad 2918.15 Rodent Diet and water ad libitum. Mice were sorted into study groups (n = 5 per group) based on bodyweight and baseline images obtained on Day −1. All mice were dosed according to individual bodyweight with SC vehicle, semaglutide, Survodutide (BI-456906), or a long-acting glucagon (LA-GCG) analog (each 100 nmol/kg) or dosed with isoproterenol by intraperitoneal injection (10 mg/kg). In vivo bioluminescence imaging was carried out at Day −1 (pre-dose) and Day 0 (4, 8, and 12 h post dose). Bioluminescence imaging was performed using an IVIS Spectrum under 1–2% isoflurane gas anesthesia. Mice were injected subcutaneously at the base of the neck with 250 mg/kg (25 mg/mL) D-luciferin and imaged in the prone, supine, and left lateral positions 10 min after injection. Images were analyzed using Living Image software. At 12 h post dose following the last imaging time point, all mice were euthanized via overexposure to CO2 for blood and tissue collection. Whole blood was collected via cardiac puncture and used to generate plasma. For dose–response effects of respective compounds, luciferase activity was measured ex vivo. Here, mice were dosed according to individual bodyweight with SC vehicle, semaglutide, Survodutide (BI-456906), or LA-GCG. After 4 h, liver and pancreas were harvested and homogenized. Luciferase activity was measured using the luciferase assay system.

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Biomarker study in mice with diet-induced obesity [1]
Male C57BL6/J mice pre-fed with a 60% high-fat diet (HFD) were obtained from The Jackson Laboratories at >16 weeks of age (n = 11 per group). Animals were housed with a 10:00–22:00 dark/light cycle throughout. For 10 days, the mice were given chronic repeated SC compound dosing at 08:00 with vehicle (twice daily [BID]), Survodutide (BI-456906) (7.5 nmol/kg once daily [QD]), semaglutide (10 nmol/kg QD), or LA-GCG (30 nmol/kg BID), with bodyweight and food intake measured every day. On treatment Days 1, 3, 5, 8, and 10, blood was drawn at the same time each day, and blood glucose (n = 11) and plasma active ghrelin and glucagon (n = 6) were measured. Data are represented as mean ± SEM and were compared using GraphPad Prism 9 using two-way ANOVA followed by Dunnett’s method for multiple comparisons versus vehicle. Significant differences were identified at p < 0.05.

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Energy expenditure study in mice with diet-induced obesity [1]
Male C57BL/6J mice were obtained from Charles River Laboratories at age 6–7 weeks. Animals were housed in groups of five in individually ventilated cages with a 12 h light/dark cycle (06:00–18:00) and were acclimatized for 1 week with ad libitum feeding. Animals were then fed a 45% HFD for 18 or 30 weeks, before randomization to groups with similar average bodyweight at Day −1. Animals were dosed QD with SC vehicle (25 mM phosphate-buffered saline) or Survodutide (BI-456906) (5, 10, or 20 nmol/kg) for 9 days, with energy metabolism, activity, and core body temperature measured each day. On Day 10, mice were sacrificed by cervical dislocation after a final bleeding by puncture of the retrobulbar venous plexus.

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Subchronic, repeated dosing study in diet-induced obese mice [1]
Male C57BL6/J mice pre-fed with a 60% HFD were obtained from The Jackson Laboratories at >16 weeks of age. Upon arrival, mice were single-housed to obtain accurate and individual food intake measurements for each animal. During the entire study, animals had ad libitum access to food and tap water. Before the start of the study, animals were randomized based on their bodyweight measured 1 week prior to the start of treatment (n = 11 per group). At study start, the age of the mice was 22 weeks. Mice were administered chronic repeated SC dosing of vehicle, Survodutide (BI-456906) (3, 10, 20, or 30 nmol/kg), or semaglutide (20 or 100 nmol/kg) daily for 30 days, with bodyweight and food intake measured every day. At Day 29, EchoMRI™ 4in1-900 was carried out. At study termination (Day 30), exposure was measured at 7 h (Cmax) and 24 h (Cmin) post dose, along with multiple biochemistry markers. Blood was drawn, and plasma triglycerides, cholesterol, free fatty acids, aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucagon, fibroblast growth factor-21 (FGF-21), amino acids, and ex vivo bioactivity were measured. The liver was homogenized, and concentrations of triglycerides and cholesterol were measured. Plasma and liver triglycerides, cholesterol, plasma amylase, and lipase were measured using an automated analyzer. Plasma glucagon and FGF-21 were measured using an enzyme-linked immunosorbent assay and colorimetric kits. Insulin, leptin, and active ghrelin were measured using a MILLIPLEX® Mouse Metabolic Hormone Multiplex Assay. An enzyme-linked immunosorbent and colorimetric assay kit was used to measure free fatty acids in EDTA plasma.
To determine the activity of semaglutide and Survodutide (BI-456906), heparin plasma was derived from euthanized animals. The plasma was diluted in 100% mouse plasma, and the respective concentrations to induce luciferase activities were determined in vitro using human embryonic kidney cells expressing recombinant human GLP-1R and human GCGR and the CRE-inducible luciferase. A standard curve in 100% mouse plasma was generated with human GLP-1-(7–36) amide (containing 1 μM linagliptin) and human glucagon. The bioactivity of semaglutide and Survodutide (BI-456906) ex vivo was calculated for the GLP-1R and GCGR for each individual sample and animal based on the standard curves for GLP-1 and glucagon, calculated as relative activity over the actual potency (EC50) for each molecule and dose and displayed in relation to the exposure of the molecule. For Survodutide (BI-456906), a relative potency ratio ex vivo for the GLP-1R and GCGR was derived from the activity of endogenous GLP-1 and glucagon observed in vitro.

AUC0-∞ and Cmax of survodutide were similar in participants with Child-Pugh class A, B, or C cirrhosis compared with healthy participants, with 90% CIs for the adjusted geometric mean ratios spanning 1 (Table 2). Values for other pharmacokinetic parameters were also similar (Table S2). Drug-related adverse events occurred in 25.0% of healthy participants and 0%, 25.0%, and 0% of Child-Pugh A, B, or C participants, respectively. No adverse events were serious, fatal, or led to trial discontinuation, and no cases of hepatic injury occurred (Table 3).[2]

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In the single-dose cohorts (n = 41), mean AUC0-∞ and Cmax were similar in those with cirrhosis compared with healthy individuals (90% CIs for adjusted geometric mean ratios spanned 1). Drug-related adverse events occurred in 25.0% of healthy individuals and ≤25.0% of those with cirrhosis after single doses, and 82.4% and 87.5%, respectively, of the multiple-dose cohorts (n = 41) over 28 weeks. Liver fat content, liver stiffness, liver volume, body weight, and other hepatic and metabolic disease markers were generally reduced after 28 weeks of survodutide treatment. Conclusions: Survodutide is generally tolerable in people with compensated or decompensated cirrhosis, does not require pharmacokinetic-related dose adjustment, and may improve liver-related non-invasive tests, supporting its investigation for MASH-related cirrhosis. Impact and implications: Survodutide is a glucagon receptor/glucagon-like peptide-1 receptor dual agonist in development for treatment of metabolic dysfunction-associated steatohepatitis (MASH), which causes cirrhosis in ∼20% of cases. This trial delineates the pharmacokinetic and safety profile of survodutide in people with compensated or decompensated cirrhosis, and revealed associated reductions in liver fat content, markers of liver fibrosis and body weight. These findings have potential relevance for people with MASH-including those with decompensated cirrhosis, who are usually excluded from clinical trials of investigational drugs. Based on this study, further investigation of survodutide for MASH-related cirrhosis is warranted.[2]

Tolerability, safety and efficacy of open-label therapy in cirrhosis [2]
During the 28 weeks of survodutide treatment (24 weeks of up-titration from 0.3 mg to 6.0 mg then 4 weeks at maximal doses) dose reductions occurred in 3/8 participants without cirrhosis who did not prematurely discontinue trial drug, 2/11 with Child-Pugh A, and 0/6 with Child-Pugh B cirrhosis. Drug-related adverse events occurred in 14/17 (82.4%), 14/16 (87.5%), and 7/8 (87.5%) participants without cirrhosis or with Child-Pugh A or B cirrhosis, respectively (Table 3). Serious adverse events occurred in 0, five (31.3%), and three (37.5%) participants, respectively (Table S3). None were fatal or drug-related except hepatic encephalopathy, which occurred in one participant; this event was minimal hepatic encephalopathy and did not cause discontinuation of survodutide or prevent the participant completing the trial (as summarized in the supplementary data). Adverse events leading to discontinuation of trial drug occurred in eight (47.1%), three (18.8%), and two (25.0%) participants without cirrhosis or with Child-Pugh A or B cirrhosis, respectively – mostly due to gastrointestinal disorders such as nausea or vomiting, which were also the most common type of adverse event in all three cohorts. No cases of acute kidney injury occurred in participants with vomiting or diarrhea.
Mean change in heart rate at 28 weeks was +4.1, +8.5, and +7.6 beats per minute in those without cirrhosis, and Child-Pugh A and B participants, respectively. No participants had onset of QTcF >500 ms or change from baseline of >60 ms, and no safety signals suggestive of increased risk of arrhythmias or QTcF prolongation were identified.
All three cohorts exhibited reductions at week 28 in liver fat content by MRI-PDFF (–43.29%, –51.59%, –0.48% in those without cirrhosis, Child-Pugh A, Child-Pugh B, respectively), liver stiffness by magnetic resonance elastography (–0.13 kPa, –1.12 kPa, –1.02 kPa, respectively), ELF scores (–0.149, –0.369, –0.522, respectively), and plasma Pro-C3 levels (–35.79%, –22.59%, –22.67%, respectively) (Fig. 3). Similarly, there were generally reductions at week 28 in FibroScan-measured liver fat content (–17.9%, –18.9%, 9.9% in those without cirrhosis, Child-Pugh A, Child-Pugh B, respectively) and stiffness (3.85%, –11.62%, –2.63%, respectively), liver volume by MRI (–15.24%, –17.36%, –22.00%, respectively), and body weight (–14.80%, –10.40%, –8.69%, respectively) (Table 4). Liver enzymes were generally reduced across cohorts, with minor less consistent changes in bilirubin and international normalized ratio.

[1]. BI 456906: Discovery and preclinical pharmacology of a novel GCGR/GLP-1R dual agonist with robust anti-obesity efficacy. Mol Metab. 2022 Dec:66:101633.

[2]. Efficacy, tolerability and pharmacokinetics of survodutide, a glucagon/glucagon-like peptide-1 receptor dual agonist, in cirrhosis. J Hepatol. 2024 Nov;81(5):837-846.

Objective: Obesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906. Methods: BI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy. Results: BI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906's superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay. Conclusions: BI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.[1]

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In summary, BI 456906 is a potent, appropriately balanced agonist of both the GCGR and the GLP-1R that produces bodyweight reductions through GLP-1R and GCGR dual agonism by the inhibition of food intake, gastric emptying, and increase in energy expenditure, while maintaining normoglycemia in obese, insulin-resistant animals. The preclinical profiling of BI 456906 and its mode of action supports its clinical investigation in Phase II trials in people with overweight/obesity (NCT04667377), diabetes (NCT04153929), and NASH (NCT041771273).[1]

C192H288N47O61NA

4261

Survodutide;2805997-46-8

White to off-white solid powder

Survodutide; BI-456906; 2805997-46-8; BI 456906 sodium

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Please refer to the "Guidelines for Dissolving Peptides" section in the 4th page of the "Instructions for use" file (upper-right section of this webpage) for how to dissolve peptides.

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H2O :≥ 100 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)