Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract Disclosure: S. Erlendsson: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. A. Kulakova: Employee; Self; Novo Nordisk. S. Poulsen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. P. Thygesen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. Growth hormone (GH) and GH analogues are characterized by their ability to bind and activate the GH receptor (GHR). Binding affinity and functional activity are pivotal parameters for development and characterization of new GH analogues for treatment of growth disorders. The GHR is a member of the class I cytokine family and obtains a tripartite structure consisting of two extracellular fibronectin type III domains (ECD), a single-pass transmembrane domain (TMD), and a disordered intracellular domain (ICD). These receptors lack intrinsic kinase activity but rely on recruitment of intracellular signalling kinases and regulatory proteins. Activation of the downstream JAK-STAT signalling cascade is initiated by formation of an asymmetric GHR dimer induced by binding of GH to the GHR ECDs via two partially overlapping binding sites designated site I and II. The structural rearrangement in the ECDs propagates through the TMD and results in a separation of the ICDs which leads to activation through cross-phosphorylation of the Janus kinases 2 (JAK2). Here we utilize isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and small angle x-ray scattering (SAXS) to characterize the binding of native GH and GH analogues including somapacitan and pegvisomant to the monomeric GHR as well as an artificial soluble stabilized dimeric GHR. The GH analogues with good binding properties were subsequently tested in vitro for biological activity using a STAT3-luciferase reporter gene assay (RGA) and a proliferation assay with BAF3 cells expressing the human GHR. Both assays were established in two formats enabling testing of both receptor agonists and antagonists. Using SPR we show that the binding of all tested compounds is preserved between the monomeric and dimeric GHR with affinities ranging from near-picomolar to lower micromolar and we are subsequently able to confirm the expected stoichiometry by utilizing a combination of ITC and SAXS. In the RGA and the proliferation assay we demonstrate that a 1:2 stoichiometry is critical for receptor activation regardless of the affinity and therefore we propose high throughput screening of receptor stoichiometry as key in discovery of new growth disorder therapeutics. Presentation: Thursday, June 15, 2023

INTRODUCTION:Immunogenicity causing development of anti-drug antibodies (ADAs) are major challenges in the treatment of haemophilia, as well as other diseases where proteins are used for treatment. Furthermore, it is a complication for preclinical testing of such therapies in animal models.AIM:To investigate if the immunosuppressive drug CTLA4 immunoglobulin (CTLA4-Ig) can induce tolerance in haemophilia A (HA) rats receiving recombinant human coagulation factor VIII (rhFVIII) treatment.METHODS:Two different prophylactic rhFVIII compounds were given intravenously to HA rats for 4 weeks. Both rhFVIII compounds were co-administered with commercially available CTLA4-Ig or human IgG subclass 4 (hIgG4) as control, and blood samples were collected. To functionally test if pharmacological efficacy was retained, rats were subjected to a bleeding experiment under anaesthesia at end of study.RESULTS:The mean inhibitory level after 4 weeks in rats receiving rhFVIII and hIgG4 was 85.7 BU for octocog alfa and 37.4 BU for rurioctocog alfa pegol. In contrast, co-administration with CTLA4-Ig during rhFVIII therapy prevented the formation of ADAs (both binding and inhibitory) in 14/14 rats receiving octocog alfa and in 7/7 rats receiving rurioctocog alfa pegol. Moreover, we were able to show that the pharmacological efficacy of rhFVIII was preserved.CONCLUSION:In a rat model with spontaneous bleeding, co-administration of CTLA4-Ig with rhFVIII prevented antibody formation. No FVIII antibodies were detected, demonstrating that CTLA4-Ig co-administration can be applicable as a method to prevent immunogenicity, when evaluating human proteins in preclinical systems permitting continuous pharmacokinetic and pharmacodynamic assessment.

Somapacitan is a growth hormone derivative approved for once-weekly treatment of growth hormone deficiency in adults and currently in clinical development for once-weekly dosing in children. The purpose of this study was to obtain non-clinical data from rats to support the safety evaluation of the most abundant metabolites of somapacitan in humans. The aims were to identify somapacitan metabolites and their relative proportions in rat plasma, identify the structure of abundant metabolites and measure the systemic metabolite exposure at the no observed-adverse-effect level in the rat. After a single dose of radiolabelled somapacitan and analysis by high-performance liquid chromatography with radiochemical detection, seven somapacitan-related metabolites were detected in plasma from male rats, of which six were seen in plasma from female rats. The three most abundant metabolites (M1, M2 and M3) were structurally identified from liquid chromatography and mass spectrometry data, and a fourth metabolite (P1) was characterised from its specific retention time (lacking retention to the stationary phase) in plasma analysis with reversed-phase liquid chromatography and radiochemical detection. The metabolites were products from proteolysis of the peptide backbone in somapacitan. A deamidation product of the M1 metabolite (M1B) was also identified. Following multiple, twice-weekly dosing for 4 weeks, somapacitan was the principal plasma component up to 36 h after dosing. After 36 h, metabolites M1+M1B were the most abundant plasma components. Pharmacokinetic models were developed for somapacitan and metabolite P1 and used for steady-state assessment in the rat. Comparison of our data generated from rats with data from the parallel human study demonstrated that the most abundant metabolites were present in rats at higher levels than in humans. This study has provided non clinical safety data that contribute to an overall safety assessment of somapacitan.

Hemophilia A is a bleeding disorder resulting from deficient factor VIII (FVIII), which normally functions as a cofactor to activated factor IX (FIXa) that facilitates activation of factor X (FX). To mimic this property in a bispecific antibody format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with equilibrium dissociation constant values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by 4 orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation, with potencies 13 and 18 times higher than a sequence-identical analogue of emicizumab. A similar potency difference was observed in a tail vein transection model in hemophilia A mice, whereas reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetic parameters of Mim8 were investigated and a half-life of 14 days shown in cynomolgus monkeys. In conclusion, Mim8 is an activated FVIII mimetic with a potent and efficacious hemostatic effect based on preclinical data.

Blocking the proteolytic capacity of urokinase-type plasminogen activator (uPA) with a monoclonal antibody (mAb) reduces arthritis progression in the collagen-induced mouse arthritis model to an extent that is on par with the effect of blocking tumor necrosis factor-alpha by etanercept. Seeking to develop a novel therapy for rheumatoid arthritis, a humanized mAb, NNC0266-0043, was selected for its dual inhibition of both the zymogen activation and the proteolytic capacity of human uPA. The antibody revealed nonlinear elimination kinetics in cynomolgus monkeys consistent with binding to and turnover of endogenous uPA. At a dose level of 20.6 mg kg−1, the antibody had a plasma half-life of 210 h. Plasma uPA activity, a pharmacodynamic marker of anti-uPA therapy, was reduced to below the detection limit during treatment, indicating that an efficacious plasma concentration was reached. Pharmacokinetic modeling predicted that sufficient antibody levels can be sustained in arthritis patients dosed subcutaneously once weekly. The anti-uPA mAb was also well tolerated in cynomolgus monkeys at weekly doses up to 200 mg kg−1 over 4 weeks. The data from cynomolgus monkeys and from human material presented here indicates that anti-uPA mAb NNC0266-0043 is suitable for clinical testing as a novel therapeutic for rheumatic diseases.

Somapacitan is a long-acting, once-weekly, albumin-binding growth hormone (GH) derivative. The reversible albumin-binding properties leads to prolonged circulation half-life. Here, we investigated and compared somapacitan with human GH on downstream receptor signaling in primary hepatocytes and hepatocellular models and using isothermal titration calorimetry to characterize receptor binding of somapacitan in the presence or absence of human serum albumin (HSA). With non-invasive fluorescence imaging we quantitatively visualize and compare the temporal distribution and examine the tissue-specific growth hormone receptor (GHR) activation at distribution sites. We found that signaling kinetics were slightly more rapid and intense for GH compared with somapacitan. Receptor binding isotherms were characterized by a high and a low affinity interaction site with or without HSA. Using in vivo optical imaging we found prolonged systemically biodistribution of somapacitan compared with GH, which correlated with plasma pharmacokinetics. Ex vivo mouse organ analysis revealed that the temporal fluorescent intensity in livers dosed with somapacitan was significantly increased compared with GH-dosed livers and correlated with the degree of downstream GHR activation. Finally, we show that fluorescent-labeled analogs distributed to the hypertrophic zone in the epiphysis of proximal tibia of hypophysectomized rats and that somapacitan and GH activate the GHR signaling in epiphyseal tissues.

Somapacitan, a human growth hormone derivative that binds reversibly to albumin, was investigated for human serum albumin (HSA) and HSA domain binding. Isothermal titration calorimetry (ITC) binding profiles showed high-affinity binding (∼100-1000 nM) of one somapacitan molecule and low-affinity binding (∼1000-10000 nM) of one to two somapacitan molecules to HSA. The high-affinity site was identified in HSA domain III using size exclusion chromatography (SEC) and ITC. SEC studies showed that the neonatal Fc receptor shields one binding site for somapacitan, indicating its position in domain III. A crystal structure of somapacitan in complex with HSA optimized for neonatal Fc receptor binding, having four amino acid residue replacements, identified a low-affinity site in fatty acid-binding site 6 (domain II). Surface plasmon resonance (SPR) showed these replacements affect the kinetics of the high-affinity binding site. Furthermore, small-angle X-ray scattering and SPR brace two somapacitan-binding sites on HSA.

The treatment of hemophilia A (HA) is primarily based on replacement of factor VIII (FVIII), and in people with HA with inhibitors (HAwI) on the use of by-passing agents. Recently, a FVIII mimetic bispecific antibody emicizumab (Hemlibra®) was approved for treatment of HA and HAwI, offering a subcutaneous, prophylactic treatment opportunity with potential for significantly reducing the treatment burden. We describe the development and pre-clinical characterization of Mim8, a novel, next-generation FVIII mimetic human bispecific antibody. Mim8 is a highly potent molecule bridging factor IXa (FIXa) and factor X (FX) in development for subcutaneous treatment of people with HA and HAwI. Development of Mim8 utilized the Duobody® platform to initially screen for compatible anti-FIXa and anti-FX antibodies followed by several iterations of systematic mutational optimization. In total, more than 30,000 bispecific antibodies were analyzed. The optimization process aimed for efficient Mim8-mediated activation of FX by FIXa in the presence of procoagulant membrane, low target binding in solution, low immunogenicity risk, and for desirable biophysical parameters such as low viscosity. In vitro characterization demonstrated that Mim8 efficiently localizes FIXa and FX to the phospholipid surface and enhances FXa activation. The monovalent anti-FIXa arm alone stimulates the proteolytic activity of FIXa in the range of 15,000-fold and is an important contributor to the activity of the bispecific antibody. The dissociation constants (Kd) of Mim8 for FIXa and FX is in the micromolar range, minimizing target binding in the blood. Using thrombin generation assay in congenital HA plasma and thrombelastography (TEG) in whole blood from healthy volunteers spiked with anti-FVIII antibodies, Mim8 was capable of normalizing thrombin generation and blood clot formation, respectively, with approximately 15 times greater potency than emicizumab (Figure 1). A similar potency improvement was demonstrated in a tail vein transection bleeding model in FVIII-deficient mice co-dosed with human FIX and FX to circumvent lack of Mim8 cross reactivity to murine FIX and FX. The terminal half-life of Mim8 was estimated to 14 days (range 10-17 days) in cynomolgus monkeys and the subcutaneous bioavailability to 97%. In conclusion, Mim8 is a novel, next-generation FVIII mimetic bispecific antibody with anti-FIXa and anti-FX arms that potently stimulates FX activation resulting in efficacious haemostasis in vitro and in vivo. Mim8 has a high potency allowing for administration of small volumes in a pen device, good PK parameters, minimal target binding in the blood, and good biophysical properties. Collectively, these properties support clinical development of Mim8 as a potentially improved next-generation FVIII-mimetic prophylactic treatment option for persons with hemophilia A regardless of inhibitor status. Figure 1: Left: FXI-triggered thrombin generation assay in congenital HA plasma (mean and SD of n = 5). Right: thromboelastography in whole blood from healthy donors spiked with polyclonal anti-FVIII antibody (mean and SD of n = 3). Coagulation was triggered with low concentration (∼30 fM) of tissue factor (Innovin® 1:200,000). Shaded areas: standard deviation of controls. Blue circles: Mim8. Grey squares: a sequence identical analogue (SIA) to emicizumab (comparable data were obtained with a commercially available batch of Hemlibra®). Disclosures Kjellev: Novo Nordisk A/S: Employment, Equity Ownership. Østergaard:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Greisen:Novo Nordisk A/S: Equity Ownership, Patents & Royalties: Patents. Hermit:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Thorn:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hansen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Zhou:Novo Nordisk A/S: Equity Ownership, Other: Previous employment, Patents & Royalties: Patents. Bjelke:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Kjalke:Novo Nordisk A/S: Employment, Honoraria. Lund:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Holm:Novo Nordisk A/S: Equity Ownership, Other: Previous employment. Ley:Novo Nordisk A/S: Employment, Equity Ownership. Elenius:Novo Nordisk A/S: Equity Ownership, Other: Previous employment; Leo Pharma A/S: Employment, Equity Ownership. Thygesen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Loftager:Novo Nordisk A/S: Employment, Equity Ownership. Rasch:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lorenzen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Gandhi:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lamberth:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Egebjerg:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lund:Novo Nordisk A/S: Employment, Equity Ownership. Henriksen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Rahbek-Nielsen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. 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Background: Adult onset growth hormone (GH) deficiency (AGDH) is a potentially underdiagnosed condition, caused by damage to the pituitary gland. AGHD is treated with growth hormone replacement therapy. A large variety of clinical symptoms and changes in the metabolic homeostasis can be observed and quantified. New large animal models are needed for future drug development. New method: In this study, we evaluate methods for a new large non-primate animal model of GH deficiency in post pubertal Göttingen Minipigs (minipig). Lesions in the pituitary gland were made by stereotaxic monopolar thermo-coagulation guided by magnetic resonance imaging (MRI), and pituitary function was evaluated using insulin tolerance test (ITT) with measurements of growth hormone secretion induced by hypoglycemia. Results: Lesions were successfully applied to the pituitary gland without any damage to surrounding tissue including the hypothalamus, which was confirmed by post-operative MRI and post mortem histology. Plasma levels of GH during ITT showed no decrease in secreted levels one week after surgery compared to levels obtained before surgery. Comparison with existing methods: Compared to other GH insufficiency models, eloquent brain tissue is spared. Furthermore, alternatively to rodent models, a large animal model would allow the use of human intended equipment to evaluate disease. Using the minipig avoids social, economical and ethical issues, compared with primates. Conclusion: The lesions did not remove all GH production, but proof of concept is demonstrated. In addition, the ITT is presented as a safe and efficient method to diagnose GH deficiency in minipigs.

Genetic absence of the urokinase-type plasminogen activator (uPA) reduces arthritis progression in the collagen-induced arthritis (CIA) mouse model to an extent just shy of disease abrogation, but this remarkable observation has not been translated into therapeutic intervention. Our aim was to test the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes mouse uPA significantly reduced arthritis progression in the CIA and delayed-type hypersensitivity arthritis models. In the CIA model, the impact of anti-uPA treatment was on par with the effect of blocking TNF-α by etanercept. A pharmacokinetics evaluation of the therapeutic Ab revealed target-mediated drug disposition consistent with a high turnover of endogenous uPA. The cellular expression patterns of uPA and uPAR were characterized by double immunofluorescence in the inflamed synovium from patients with rheumatoid arthritis and compared with synovium from healthy donors. The arthritic synovium showed expression of uPA and uPAR in neutrophils, macrophages, and a fraction of endothelial cells, whereas there was little or no expression in synovium from healthy donors. The data from animal models and human material provide preclinical proof-of-principle that validates uPA as a novel therapeutic target in rheumatic diseases.

The present work describes a series of human growth hormone (hGH) albumin binder conjugates with an extended in vivo half-life. A broad range of different conjugates were studied by varying the albumin binder structure and conjugation site. Conjugates were conveniently obtained by reductive alkylation or by alkylation to introduced cysteines using functionalized albumin-binding side chains. In vitro and in vivo profiling provided the basis for identification of position L101C in human growth hormone as the most optimal position for conjugation, where both a sufficient level of receptor binding and a suitably long half-life could yield a molecule with potential for a once-weekly dosing regimen.

Growth hormone (GH) resistance may develop as a consequence of inflammation during conditions such as inflammatory bowel disease, encompassing ulcerative colitis (UC). However, the specific role of the GH–insulin growth factor (IGF)-1-axis and/or the functional consequences of GH resistance in this condition are unclear. In situ hybridization targeting the GH receptor (GHR) and relevant transcriptional analyses were performed in patients with UC and in IL-10 knock-out mice with piroxicam accelerated colitis (PAC). Using cultured primary epithelial cells, the effects of inflammation on the molecular mechanisms governing GH resistance was verified. Also, the therapeutic potential of GH on mucosal healing was tested in the PAC model. Inflammation induced intestinal GH resistance in UC and experimental colitis by down-regulating GHR expression and up-regulating suppressor of cytokine signalling (SOCS) proteins. These effects are driven by pro-inflammatory mediators (tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6) as confirmed using primary epithelial cells. Treatment of experimental colitis with GH increased IGF-1 and body weight of the mice, but had no effects on colonic inflammation or mucosal healing. The high transcriptional similarity between UC and experimental colitis accentuates the formation of intestinal GH resistance during inflammation. Inflammation-induced GH resistance not only impairs general growth but induces a state of local resistance, which potentially impairs the actions of GH on mucosal healing during colitis when using long-acting GH therapy.

OBJECTIVE:Somapacitan is an albumin-binding growth hormone derivative intended for once weekly administration, currently in clinical development for treatment of adult as well as juvenile GH deficiency. Nonclinical in vivo pharmacological characterisation of somapacitan was performed to support the clinical trials. Here we present the pharmacokinetic and pharmacodynamic effects of somapacitan in rats, minipigs, and cynomolgus monkeys.METHODS:Pharmacokinetic studies investigating exposure, absorption, clearance, and bioavailability after single intravenous (i.v.) and subcutaneous (s.c.) administration were performed in all species. A dose-response study with five dose levels and a multiple dose pharmacodynamic study with four once weekly doses was performed in hypophysectomised rats to evaluate the effect of somapacitan on growth and IGF-I production.RESULTS:Pharmacokinetic profiles indicated first order absorption from the subcutaneous tissue after s.c. injections for somapacitan in all three species. Apparent terminal half-lives were 5-6h in rats, 10-12h in minipigs, and 17-20h in monkeys. Somapacitan induced a dose-dependent growth in hypophysectomised rats (p<0.001) and an increase in plasma IGF-I levels in rats (p<0.01), minipigs (p<0.01), and cynomolgus monkeys (p<0.05) after single dose administration. Multiple once weekly dosing of somapacitan in hypophysectomised rats induced a step-wise increase in body weight with an initial linear phase the first 3-4days in each dosing interval (p<0.001).CONCLUSION:The nonclinical pharmacokinetic and pharmacodynamic studies of somapacitan showed similar pharmacokinetic properties, with no absorption-limited elimination, increased clearance and increased and sustained levels of IGF-I in plasma for up to 10days after a single dose administration in all three species. Somapacitan induced a dose-dependent increase in body weight and IGF-I levels in hypophysectomised rats. Multiple dosing of somapacitan in hypophysectomised rats suggested a linear growth for the first 3-4days in each weekly dosing interval, whereas daily hGH dosing showed linear growth for approximately two weeks before reaching a plateau level.

BACKGROUND AND PURPOSEWe aimed to develop a mechanistic mixed-effects pharmacokinetic (PK)-pharmacodynamic (PD) (PKPD) model for recombinant human growth hormone (rhGH) in hypophysectomized rats and to predict the human PKPD relationship.EXPERIMENTAL APPROACHA non-linear mixed-effects model was developed from experimental PKPD studies of rhGH and effects of long-term treatment as measured by insulin-like growth factor 1 (IGF-1) and bodyweight gain in rats. Modelled parameter values were scaled to human values using the allometric approach with fixed exponents for PKs and unscaled for PDs and validated through simulations relative to patient data.KEY RESULTSThe final model described rhGH PK as a two compartmental model with parallel linear and non-linear elimination terms, parallel first-order absorption with a total s.c. bioavailability of 87% in rats. Induction of IGF-1 was described by an indirect response model with stimulation of kin and related to rhGH exposure through an E-max relationship. Increase in bodyweight was directly linked to individual concentrations of IGF-1 by a linear relation. The scaled model provided robust predictions of human systemic PK of rhGH, but exposure following s.c. administration was over predicted. After correction of the human s.c. absorption model, the induction model for IGF-1 well described the human PKPD data.CONCLUSIONSA translational mechanistic PKPD model for rhGH was successfully developed from experimental rat data. The model links a clinically relevant biomarker, IGF-1, to a primary clinical end-point, growth/bodyweight gain. Scaling of the model parameters provided robust predictions of the human PKPD in growth hormone-deficient patients including variability.

The kidneys are thought to play an important role in the clearance of recombinant human growth hormone (rhGH), but the relative importance is not clear. Obtaining knowledge of clearance pathway is an important prerequisite for the development of new long acting growth hormone analogues targeted at treatment of patients with growth hormone disorders. The purpose of this study was to investigate the relative importance of the kidneys in the clearance of rhGH. The study employed a newly validated nephrectomy rat model and a population based pharmacokinetic approach to assess renal clearance of rhGH in non-anesthetized rats, anesthetized rats and in nephrectomized anesthetized rats. Clearance in non-anesthetized rats was 290 ml/h/kg. This was reduced to 185 ml/h/kg by anesthesia and further reduced to 18 ml/h/kg by nephrectomy. As nephrectomy was able to reduce clearance with 90%, we conclude that renal clearance plays a pivotal role in the elimination of rhGH in rats.

Most countries have some form of biosafety regulation covering genetically modified organisms (GMOs) and genetically modified products including food. I lead the Office of the Gene Technology Regulator (OGTR) in administering the Australian regime (under the Gene Technology Act 2000) to protect human health and safety and the environment by identifying and managing risks posed by GMOs.

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.