Development of a Long-Acting and Stapled Dual Amylin and Calcitonin Receptor Agonist as Monotherapy and Combination with GLP-1R Agonists for the Treatment of Obesity.

Amylin treatment improves body weight and glucose control, although it is limited by a short action and need for high doses. Dual amylin and calcitonin receptor agonists (DACRAs) are dual amylin and calcitonin receptor agonists with beneficial effects beyond those of amylin. However, to what extent the additional benefits reside in their higher potency or their targeting of the calcitonin receptor remains unclear. Here we deconstruct the receptors involved in the effects of a DACRA, KBP-088, by comparing it to rat amylin (rAMY), rat calcitonin (rCT), and their combination in obese high-fat diet (HFD) and diabetic Zucker diabetic fatty (ZDF) rats. HFD-fed Sprague-Dawley rats and ZDF rats were treated for 4 weeks with KBP-088 (5 µg/kg per day), rAMY (300 µg/kg per day), rCT (300 µg/kg per day), and the combination of rAMY and rCT (300+300 µg/kg per day) using infusion pumps. Body weight, food intake, fasting glycemia, glycated hemoglobin type A1c levels, and glucose tolerance were assessed. In obese HFD-fed rats, KBP-088, rAMY, and the combination of rAMY and rCT significantly reduced body weight and improved glucose tolerance, whereas rCT alone had no effect. In diabetic ZDF rats, rCT was efficient in lowering fasting glycemia similar to rAMY, whereas dual activation by KBP-088 and the combination of rAMY and rCT were superior to activating either receptor alone. In conclusion, calcitonin therapy regulates fasting blood glucose in a diabetic rat model, thereby underscoring the importance of calcitonin receptor activation as well as the known role of amylin receptor agonism in the potent metabolic benefits of this group of peptides. SIGNIFICANCE STATEMENT: We deconstruct the receptors activated by dual amylin and calcitonin receptor agonist (DACRA) therapy to elucidate through which receptor the beneficial metabolic effects of the DACRAs are mediated. We show that calcitonin receptor activation is important for blood glucose regulation in diabetes. This is in addition to the known metabolic beneficial role of amylin receptor activation. These data help in understanding the potent metabolic benefits of the DACRAs and underline the potential of DACRAs as treatment for diabetes and obesity.

Amylin analogs have been shown to significantly reduced body weight and dosage of insulin. Dual amylin and calcitonin receptor agonists (DACRAs) are the most potent amylin receptor agonists. We developed a series of novel long-acting amylin agonists to assess the potency on both calcitonin (CTR) and amylin 3 (AMY3) receptor activation on body weight and other metabolic parameters. We report herein the results from an evaluation of these compounds in rodent models.In vitro screening consisted of measurement of ligand-induced β-arrestin recruitment and assessment of cAMP production on human CTR and AMY3 receptors as an indicator of receptor activity, followed by in vivo evaluation. Acute food intake and pharmacokinetic evaluations in lean rats were determined following single subcutaneous administration of selected analogs. Diet-induced obese (DIO) mice were treated with subcutaneous injections of vehicle or one of a series of amylin agonists for 24 days. Cagrilintide was used as positive control. Cohorts were assessed for changes in BW, food intake and other markers.A series of amylin agonists reduced food intake in rats in the range of 56-93%, compared to vehicle, in the period from 0-72 hr post-dose. In comparison, cagrilintide was able to reduce food intake mostly from 0-48 hr and up to 72 hr only at 30 nmol/kg. Durable appetite suppression was further confirmed in a pharmacokinetic study. Treatment with selected novel amylin agonists resulted in reductions to BW in rats up to 10% compared to vehicle. In DIO mice, BW loss effects were comparable to those observed in cagrilintide-treated animals. Improvements in other metabolic markers were also observed. A novel series of amylin analogs produced significant reductions in BW in rodents, with effect sizes comparable to those observed in an active control group. Dual agonism of CTR and AMY3 receptors represents a promising therapeutic approach to metabolic disorders such as obesity and diabetes. Further evaluation of these compounds is ongoing. Disclosure K. Yagiz: None.

Introduction and Objective: Amylin, a peptide hormone co-secreted with insulin by pancreatic beta cells in response to food intake, suppresses appetite and delays gastric emptying, making it an attractive target for obesity therapy. Cagrilintide, a long-acting dual amylin and calcitonin receptor agonist (DACRA) peptide, has demonstrated promising efficacy and safety profile in clinical trials, but an oral small molecule DACRA with good bioavailability and stability could provide a more convenient and accessible alternative for obesity treatment. This study evaluates the in vitro and in vivo pharmacological properties of ACCG-2671 for obesity treatment. Methods: The in vitro binding affinity and functional potency of ACCG-2671 were assessed for the calcitonin receptor (CTR) and amylin receptor (AMY3R). Chronic weight-loss effects were evaluated in diet-induced obese (DIO) rats by oral daily dosing of ACCG-2671 as single treatments or in combination treatments with semaglutide using two treatment regimens: add-on and concurrent combination. Results: ACCG-2671 demonstrated high binding affinity and balanced potency in human CTR and AMY3R functional assays. In DIO rats, oral administration of ACCG-2671 resulted in significant, dose-dependent body weight reductions. Combination treatments (add-on and concurrent) resulted in superior weight loss compared to single treatments. Conclusion: ACCG-2671 is a novel oral small molecule DACRA that significantly reduces body weight in obese rats. Its pharmacological profile and preclinical efficacy support its advancement as a development candidate, alone and in combination for obesity treatment, potentially offering a novel, convenient, and accessible therapeutic option. Disclosure X. Fang: Employee; Structure Therapeutics, Inc. Z. Zhang: Employee; Structure Therapeutics, Inc. H. Zhang: Employee; Structure Therapeutics, Inc. C. Lu: None. D. Xue: Employee; Structure Therapeutics, Inc. Z. Wang: Employee; Structure Therapeutics, Inc. S. Zhan: Employee; Structure Therapeutics, Inc. X. Wang: Employee; Structure Therapeutics, Inc. W. Liang: Employee; Structure Therapeutics, Inc. T. Ma: Employee; Structure Therapeutics, Inc. N. Hu: Employee; Structure Therapeutics, Inc. J.J. Zhang: None. L. Chen: None. X. Lai: None. D. He: None. C. Li: Employee; Structure Therapeutics, Inc. Z. Gao: Employee; Structure Therapeutics, Inc. X. Lin: None. X. Jiang: Employee; Structure Therapeutics, Inc. H. Lei: Employee; Structure Therapeutics, Inc. F. Zhang: Stock/Shareholder; Eli Lilly and Company, Novo Nordisk A/S. Employee; Structure Therapeutics, Inc.

Parallel activation of the calcitonin receptor (CTR) and amylin receptor (AMYR) is considered a more effective weight-loss strategy. Although the novel dual amylin and calcitonin receptor agonist (DACRA), petrelintide, is currently undergoing phase II clinical trials, its agonistic activity remains insufficient compared with natural agonists. Further optimization of the agonistic capabilities of petrelintide is an attractive strategy for developing DACRAs. Due to the lack of structure-activity relationship (SAR) and target binding information, a step-by-step process involving three rounds of modifications was performed guided by structure-based drug design and molecular dynamics (MD) simulations. Two successful methylation strategies led to the identification of the more efficient novel DACRA, BGM1812, with excellent performance in terms of half-life, stability, and solubility. In both in vivo and in vitro studies, BGM1812 showed significantly enhanced efficacy. This finding provides valuable insights into the SAR of petrelintide and highlights the potential of BGM1812 as a promising obesity drug candidate.

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.

The present study investigated a novel oral dual amylin and calcitonin receptor agonist (DACRA), KBP-042, in head-to-head comparison with salmon calcitonin (sCT) with regard to in vitro receptor pharmacology, ex vivo pancreatic islet studies, and in vivo proof of concept studies in diet-induced obese (DIO) and Zucker diabetic fatty (ZDF) rats. In vitro, KBP-042 demonstrated superior binding affinity and activation of amylin and calcitonin receptors, and ex vivo, KBP-042 exerted inhibitory action on stimulated insulin and glucagon release from isolated islets. In vivo, KBP-042 induced a superior and pronounced reduction in food intake in conjunction with a sustained pair-fed corrected weight loss in DIO rats. Concomitantly, KBP-042 improved glucose homeostasis and reduced hyperinsulinemia and hyperleptinemia in conjunction with enhanced insulin sensitivity. In ZDF rats, KBP-042 induced a superior attenuation of diabetic hyperglycemia and alleviated impaired glucose and insulin tolerance. Concomitantly, KBP-042 preserved insulinotropic and induced glucagonostatic action, ultimately preserving pancreatic insulin and glucagon content. In conclusion, oral KBP-042 is a novel DACRA, which exerts antiobesity and antidiabetic efficacy by dual modulation of insulin sensitivity and directly decelerating stress on the pancreatic α- and β-cells. These results could provide the basis for oral KBP-042 as a novel therapeutic agent in type 2 diabetes.

BackgroundWeight loss therapy is becoming more and more important, and two classes of molecules, namely amylin receptor and GLP-1 receptor agonists, have shown promise in this regard. Interestingly, these molecules have several overlapping pharmacological effects, such as suppression of gastric emptying, reduction of glucagon secretion and weight loss in common; however, they also have distinct effects on prandial insulin secretion. Hence, a combination of these two mechanisms is of significant interest.MethodsIn this study, we investigated the add-on potential of the dual amylin and calcitonin receptor agonist (DACRA) KBP-089 in combination with the GLP-1 receptor agonist liraglutide as obesity treatment in high-fat diet (HFD) fed rats.ResultsIncreasing doses of KBP-089 and liraglutide alone and in combination were studied with respect to their effects on body weight, food intake and glucose metabolism during a 9-week intervention study conducted in HFD rats. Further, the gastric emptying rate during an oral glucose tolerance was assessed. Treatment with KBP-089 and liraglutide dose-dependently lowered body weight 15% (at 2.5 μg/kg/day) and 7% (at 400 μg/kg/day) in HFD rats, respectively, while the combination resulted in a 21% body weight reduction, which was mirrored by reduction in fat depot sizes. Gastric emptying and glucose metabolism were improved, primarily by KBP-089, although liraglutide led to a reduction in fasting plasma glucagon.ConclusionDACRAs complement GLP-1 on food intake, body weight, and glucose tolerance indicating the potential for an add-on therapy.

Introduction and Objective: Amylin analogues are being investigated as potential obesity treatments. Amylin receptors (AMYRs) belong to the calcitonin receptor (CTR) family and consist of heterodimers of the calcitonin receptor and receptor activity modifying proteins (RAMPs). CTR and AMYRs exhibit distinct physiological roles with CTR activation being important for calcium homeostasis, while AMYRs are involved in appetite regulation and body weight. It is however uncertain what the clinical role is of different receptor profiles. In this study we compare three amylin analogues in clinical development (cagrilintide, eloralintide and NN1213) on CTR and AMYRs activation as well as calcium lowering ability in rats to evaluate AMYR selectivity and furthermore address the efficacy on food intake and body weight reduction. Methods: Receptor potency was measured in vitro. Receptor selectivity was further investigated in rats, by measuring Ca2+ plasma levels after a single subcutaneous injection of vehicle, cagrilintide (10 nmol/kg), eloralintide (10 or 30 nmol/kg) or NN1213 (10 or 30 nmol/kg). In addition, the effect of NN1213 (10 nmol/kg, qd, sc.) on body weight and food intake was investigated in diet-induced obese rats during 17 days of dosing. Body weight and Ca2+ levels and food intake were analysed by a mixed effect model or 2-way repeated measure ANOVA, respectively, followed by Dunnett’s multiple comparison test. Results: In vitro, cagrilintide and eloralintide activated both rat and human AMYRs and CTR whereas NN1213 was more selective for AMYRs. In rats, cagrilintide and eloralintide induced a prolonged decrease in Ca2+ plasma levels in rats while NN1213 did not. Furthermore, NN1213 decreased body weight and food intake in diet-induced obese rats. Conclusion: Cagrilintide and eloralintide are both AMYR and CTR agonists whereas NN1213 is a selective AMYRs agonist that also decreases body weight in rats. Disclosure D. Ipsen: Employee; Novo Nordisk A/S. B. Ballarin-Gonzalez: Employee; Novo Nordisk. Stock/Shareholder; Novo Nordisk. E. Moo: Employee; Novo Nordisk. A. Secher: Employee; Novo Nordisk A/S. Stock/Shareholder; Novo Nordisk A/S. K. Raun: Employee; Novo Nordisk A/S. Stock/Shareholder; Novo Nordisk A/S.

Pharmacological treatment with dual amylin and calcitonin receptor agonists (DACRAs) cause significant weight loss and improvement of glucose homeostasis. In this study, the maximally efficacious dose of the novel DACRA, KeyBiosciencePeptide (KBP)-066, was investigated. Two different rat models were used: high-fat diet (HFD)-fed male Sprague-Dawley rats and male Zucker diabetic fatty (ZDF, fa/fa) rats to determine the maximum weight loss and glucose homeostatic effect, respectively. One acute study and one chronic study was performed in HFD rats. Two chronic studies were performed in ZDF rats: a preventive and an interventive. All studies covered a dose range of 5, 50, and 500 µg/kg KBP-066 delivered by subcutaneous injection. Treatment with KBP-066 resulted in a significant weight reduction of 13%-16% and improved glucose tolerance in HFD rats, which was independent of dose concentration. Dosing with 50 and 500 µg/kg led to a transient but significant increase in blood glucose, both in the acute and the chronic study in HFD rats. All doses of KBP-066 significantly improved glucose homeostasis in ZDF rats, both in the preventive and interventive study. Moreover, dosing with 50 and 500 µg/kg preserved insulin secretion to a greater extent than 5 µg/kg when compared with ZDF vehicle rats. Taken together, these results show that maximum weight loss is achieved with 5 µg/kg, which is within the range of previously reported DACRA dosing, whereas increasing dosing concentration to 50 and 500 µg/kg may further improve preservation of insulin secretion compared with 5 µg/kg in diabetic ZDF rats. SIGNIFICANCE STATEMENT: Here we show that KeyBiosciencePeptide (KBP)-066 induces an equally potent body weight loss across a broad dose range in obese rats. However, higher dosing of KBP-066 may improve insulin action in diabetic rats both as preventive and interventive treatment.

Amylin receptors (AMYRs), heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins, are promising obesity targets. A hallmark of AMYR activation by Amy is the formation of a 'bypass' secondary structural motif (residues S19-P25). This study explored potential tuning of peptide selectivity through modification to residues 19-22, resulting in a selective AMYR agonist, San385, as well as nonselective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, and San45 bound to AMY3R or CTR. San45, via its conjugated lipid at position 21, was anchored at the edge of the receptor bundle, enabling a stable, alternative binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. Targeted lipid modification may provide a single intervention strategy for design of long-acting, nonselective, Amy-based DACRAs with potential anti-obesity effects.

Improved metabolic efficacy of a dual amylin and calcitonin receptor agonist when combined with semaglutide or empagliflozin

Does receptor balance matter? – Comparing the efficacies of the dual amylin and calcitonin receptor agonists cagrilintide and KBP-336 on metabolic parameters in preclinical models

Obesity and associated co-morbidities, such as type 2 diabetes and non-alcoholic fatty liver disease, are major health challenges. Hence, there is an important need to develop weight loss therapies with the ability to reduce the co-morbidities. The effect of the dual amylin and calcitonin receptor agonist (DACRA), KBP-089, on body weight, glucose homeostasis and fatty acid accumulation in liver and muscle tissue and on food preference was investigated. Furthermore, we elucidated weight-independent effects of KBP-089 using a weight-matched group. Rats fed a high-fat diet were treated, s.c., with KBP-089 0.625, 1.25, 2.5μg·kg-1 or vehicle. KB-089 induced in a dose-dependent and sustained weight loss (~17% by 2.5μg·kg-1 ). Moreover, KBP-089 reduced fat depot size and reduced lipid accumulation in muscle and liver. In Zucker Diabetic Fatty rats, KBP-089 improved glucose homeostasis through improved insulin action. To obtain a weight-matched group, significantly less food was offered (9% less than in the KBP-089 group). Weight matching led to improved glucose homeostasis by reducing plasma insulin; however, these effect were inferior compared to those of KBP-089. In the food preference test, rats fed a normal diet obtained 74% of their calories from chocolate. KBP-089 reduced total caloric intake and induced a relative increase in chow consumption while drastically reducing chocolate consumption compared with vehicle. The novel DACRA, KBP-089, induces a sustained weight loss, leading to improved metabolic parameters including food preference, and these are beyond those observed simply by diet-induced weight loss.

This study aims to elucidate the mechanism behind the potent weight loss induced by dual amylin and calcitonin receptor agonists (DACRA) through comparison of the novel DACRA KBP-088 with the amylinomimetic davalintide with regard to in vitro receptor pharmacology and in vivo efficacy on food intake and body weight. KBP-088 and davalintide were tested for their ability to activate the amylin and calcitonin receptors as function of dose and time. Two doses of KBP-088 (1.67 and 5.0 μg/kg) were compared with similar davalintide doses in high-fat diet (HFD)-fed rats receiving subcutaneous dosing once daily for 62 days. Glucose tolerance was assessed after 3 and 7 wk of treatment. KBP-088 demonstrated activation of amylin and calcitonin receptors and prolonged receptor activation compared with davalintide as well as a potent reduction of acute food intake. KBP-088 transiently reduced food intake and induced and notably sustained a significant ∼16% vehicle-corrected weight loss without significant weight loss in the calorie-restricted control groups. Additionally, KBP-088 reduced white adipose tissues and adipocyte hypertrophy. Finally, KBP-088 alleviated hyperinsulinemia and improved oral glucose tolerance even with significantly lower insulin levels after 3 and 7 wk of treatment. KBP-088 is a potent amylin and calcitonin receptor agonist with prolonged receptor activation compared with davalintide. Moreover, KBP-088 induced and sustained significant weight loss and reduced overall adiposity and adipocyte hypertrophy in HFD rats. Finally, KBP-088 improved oral glucose tolerance and alleviated hyperinsulinemia, underscoring the potential of KBP-088 as an antiobesity agent with benefits on glucose control.

Targeting the root cause of obesity-related comorbidities through weight independent and dependent actions on insulin sensitivity: Benefits of dual amylin and calcitonin receptor agonists.