Ligand-Dependent Modulation of G Protein Conformation Alters Drug Efficacy.

G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a molecular level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the molecular basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism. VIDEO ABSTRACT.

Structural insight into selectivity of amylin and calcitonin receptor agonists.

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.

Dual amylin and calcitonin receptor agonist treatment reduces biomarkers associated with kidney fibrosis in diabetic rats.

Dual amylin and calcitonin receptor agonists (DACRAs) are effective treatments for obesity and type 2 diabetes (T2D). They provide beneficial effects on body weight, glucose control, and insulin action. However, whether DACRAs protect against diabetes-related kidney damage remains unknown. We characterize the potential of long-acting DACRAs (KBP-A, Key Bioscience Peptide-A) as a treatment for T2D-related pathological alterations of the kidney extracellular matrix (ECM) in Zucker diabetic fatty rats (ZDF). We examined levels of endotrophin (profibrotic signaling molecule reflecting collagen type VI formation) and tumstatin (matrikine derived from collagen type IVα3) in serum and evaluated kidney morphology and collagen deposition in the kidneys. We included a study in obese Sprague-Dawley rats to further investigate the impact of KBP-A on ECM biomarkers. In ZDF vehicles, levels of endotrophin and tumstatin increased, suggesting disease progression along with an increase in blood glucose levels. These rats also displayed damage to their kidneys, which was evident from the presence of collagen formation in the medullary region of the kidney. Interestingly, KBP-A treatment attenuated these increases, resulting in significantly lower levels of endotrophin and tumstatin than the vehicle. Levels of endotrophin and tumstatin were unchanged in obese Sprague-Dawley rats, supporting the relation to diabetes-related kidney complications. Furthermore, KBP-A treatment normalized collagen deposition in the kidney while improving glucose control. These studies confirm the beneficial effects of DACRAs on biomarkers associated with kidney fibrosis. Moreover, these antifibrotic effects are likely associated with improved glucose control, highlighting KBP-A as a promising treatment of T2D and its related late complications.NEW & NOTEWORTHY These studies describe the beneficial effects of using a dual amylin and calcitonin receptor agonist (DACRA) for diabetes-related kidney complications. DACRA treatment reduced levels of serological biomarkers associated with kidney fibrosis. These reductions were further reflected by reduced collagen expression in diabetic kidneys. In general, these results validate the use of serological biomarkers while demonstrating the potential effect of DACRAs in treating diabetes-related long-term complications.

Phase-plate cryo-EM structure of a class B GPCR-G-protein complex.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαsßγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gß subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.

The long-acting amylin/calcitonin receptor agonist ZP5461 suppresses food intake and body weight in male rats.

The peptide hormone amylin reduces food intake and body weight and is an attractive candidate target for novel pharmacotherapies to treat obesity. However, the short half-life of native amylin and amylin analogs like pramlintide limits these compounds' potential utility in promoting sustained negative energy balance. Here, we evaluate the ability of the novel long-acting amylin/calcitonin receptor agonist ZP5461 to reduce feeding and body weight in rats, and also test the role of calcitonin receptors (CTRs) in the dorsal vagal complex (DVC) of the hindbrain in the energy balance effects of chronic ZP5461 administration. Acute dose-response studies indicate that systemic ZP5461 (0.5-3 nmol/kg) robustly suppresses energy intake and body weight gain in chow- and high-fat diet (HFD)-fed rats. When HFD-fed rats received chronic systemic administration of ZP5461 (1-2 nmol/kg), the compound initially produced reductions in energy intake and weight gain but failed to produce sustained suppression of intake and body weight. Using virally mediated knockdown of DVC CTRs, the ability of chronic systemic ZP5461 to promote early reductions in intake and body weight gain was determined to be mediated in part by activation of DVC CTRs, implicating the DVC as a central site of action for ZP5461. Future studies should address other dosing regimens of ZP5461 to determine whether an alternative dose/frequency of administration would produce more sustained body weight suppression.

Safety, tolerability and pharmacokinetic characterisation of DACRA KBP-042 in healthy male subjects.

There is a need for antidiabetic agents successfully targeting insulin sensitivity and treating obesity control at the same time. The aim of this first-in-human study was (a) to evaluate safety and tolerability, (b) to evaluate pharmacokinetics and (c) to assess indications of receptor engagement of single ascending doses of KBP-042, a dual amylin and calcitonin receptor agonist (DACRA) that has shown promising preclinical data, with superior activity in terms of typical amylin-induced responses including reduction of food intake, weight loss and gluco-regulatory capacities. A randomised double-blind placebo-controlled single ascending dose study was performed with six dose levels of KBP-042 (5, 7.5, 10, 20, 20 (evening), 40 ug) in healthy male adults. KBP-042 or placebo was administered as a single dose after an overnight fast, followed by a standardized lunch after 4 hours. KBP-042 was associated with dose-dependent complaints of nausea and vomiting, with a lack of tolerability at doses of 20 µg and above. Doses of 5-40 µg KBP-042 behaved according to a linear pharmacokinetic profile. Indications of target receptor engagement were observed at the level of glucose control and lowering of bone resorption, compared to placebo. The results of this study showed that doses up to 40 µg were safe, although tolerability was not present at the highest doses. The study confirmed target receptor engagement at the studied doses.

The dual amylin and calcitonin receptor agonist KBP-089 and the GLP-1 receptor agonist liraglutide act complimentarily on body weight reduction and metabolic profile.

BACKGROUND: Weight 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. METHODS: In 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. RESULTS: Increasing 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. CONCLUSION: DACRAs complement GLP-1 on food intake, body weight, and glucose tolerance indicating the potential for an add-on therapy.

The Dual Amylin and Calcitonin Receptor Agonist, KBP-066, Induces an Equally Potent Weight Loss Across a Broad Dose Range While Higher Doses May Further Improve Insulin Action.

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.

Dose Frequency Optimization of the Dual Amylin and Calcitonin Receptor Agonist KBP-088: Long-Lasting Improvement in Food Preference and Body Weight Loss.

Dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for treatment of type 2 diabetes and obesity because of their beneficial effects on body weight, blood glucose, insulin sensitivity, and food preference, at least short-term. DACRAs activate the receptors for a prolonged time period, resulting in metabolic effects superior to those of amylin. Because of the prolonged receptor activation, different dosing intervals and, hence, less frequent receptor activation might change the efficacy of DACRA treatment in terms of weight loss and food preference. In this study, we compared daily dosing to dosing every other day with the aim of understanding the optimal balance between efficacy and tolerability. Obese and lean male Sprague-Dawley rats were treated with the DACRA KBP-088, applying two different dosing intervals (1.5 nmol/kg once daily and 3 nmol/kg every other day) to assess the effect on body weight, food intake, glucose tolerance, and food preference when given the choice between chow (13% fat) and a high-fat diet (60% fat). Treatment with KBP-088 induced significant weight loss, reduction in adiposity, improvement in glucose control, and altered food preference toward food that is less calorie-dense. KBP-088 dosed every other day (3 nmol/kg) was superior to KBP-088 once daily (1.5 nmol/kg) in terms of weight loss and improvement of food preference. The beneficial effects were evident in both lean and obese rats. Hence, dosing KBP-088 every other day positively affects overall efficacy on metabolic parameters regardless of the lean/obese state, suggesting that less-frequent dosing with KBP-088 could be feasible. SIGNIFICANCE STATEMENT: Here, we show that food preference can be altered chronically toward choices that are less calorie-dense by pharmacological treatment. Further, pharmacological dosing regimens affect the efficacy differently, as dosing every other day improved body weight loss and alterations in food preference compared with daily dosing. This suggest that alterations of the dosing regimens could be feasible in the treatment of obesity.

The Calcitonin Receptor Plays a Major Role in Glucose Regulation as a Function of Dual Amylin and Calcitonin Receptor Agonist Therapy.

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.

Cell active and functionally-relevant small-molecule agonists of calcitonin receptor.

The natural calcitonin (CT) receptor and its peptide agonists are considered validated targets for drug discovery. A small molecule agonist, SUN-B8155, has previously been shown to efficiently activate cellular CTR. Herein, we report the synthesis of a series of compounds (S8155 1-9) derived from SUN-B8155, and investigate the structural-functional relationship, bias properties and their cellular activity profile. We discover that the N-hydroxyl group from the pyridone ring is required for G protein activity and its affinity to the CT receptor. Among the compounds studied, S8155-7 exhibits improved G protein activity while S8155-4 displays a significant ß-arrestin-2 signaling bias. Finally, we show that both S8155-4 and S8155-7 inhibit tumour cell invasion through CTR activation. These two compounds are anticipated to find extensive applications in chemical biology research as well drug development efforts targeting CT receptor.

The Dual Amylin and Calcitonin Receptor Agonist KBP-088 Induces Weight Loss and Improves Insulin Sensitivity Superior to Chronic Amylin Therapy.

KBP-088 (KeyBiosciencePeptide 088) is a potent dual amylin and calcitonin receptor agonist (DACRA). DACRAs are known to elicit potent activity in terms of typical amylin-induced responses, such as reducing food intake and body weight. However, to what extent amylin infusion can mimic the effects of the dual agonist KBP-088 is unknown. We studied the effect of acute dosing with KBP-088 (5 µg/kg) and rat amylin (100, 300, and 1000 µg/kg) and subsequently compared the chronic effect of KBP-088 (5 µg/kg per day) to increasing doses of rat amylin (100, 300, and 1000 µg/kg per day) delivered by continuous subcutaneous infusion, in high-fat diet (HFD) fed Long-Evans rats. Furthermore, acute amylin sensitivity was investigated. Single dose KBP-088 (5 µg/kg) potently reduced acute food intake for a prolonged period compared with amylin (100, 300, and 1000 µg/kg), confirming the difference in potency. Independent of dose, chronic amylin administration (100, 300, and 1000 µg/kg per day) was less effective than KBP-088 (5 µg/kg per day) in inducing body weight loss (15% with KBP-088, and 5%, 9%, and 8% with amylin, vehicle corrected) and reducing overall adiposity in HFD rats. Moreover, KBP-088 improved oral glucose tolerance with significantly reduced insulin levels (80% reduction) that were better than all doses of amylin (68%, 53%, and 7% reduction). Acute amylin sensitivity was independent of the chronic treatment. Dual activation of amylin and calcitonin receptors by KBP-088 is superior to amylin in reducing body weight and improving glucose tolerance, indicating a role for the calcitonin receptor.

A dual amylin and calcitonin receptor agonist inhibits pain behavior and reduces cartilage pathology in an osteoarthritis rat model.

OBJECTIVES: Pain and disability are the main clinical manifestations of osteoarthritis, for which only symptomatic therapies are available. Hence, there is a need for therapies that can simultaneously alter disease progression and provide pain relief. KBP is a dual amylin- and calcitonin-receptor agonist with antiresorptive and chondroprotective properties. In this study we investigated the effect of KBP in a rat model of osteoarthritis. METHODS: Medial meniscectomy (MNX) was performed in 39 rats, while 10 underwent sham surgery. Rats were treated with KBP and/or naproxen. Nociception was assessed by mechanical and cold allodynia, weight bearing asymmetry, and burrowing behavior. Blood samples were collected for biomarker measurements, and knees for histology. Cartilage histopathology was evaluated according to the advanced Osteoarthritis Research International (OARSI) score and KBPs in vitro antiresorptive effects were assessed using human osteoclasts cultured on bone. RESULTS: The MNX animals displayed an increased nociceptive behavior. Treatment with KBP attenuated the MNX-induced osteoarthritis-associated joint pain. The cartilage histopathology was significantly lower in rats treated with KBP than in MNX animals. Bone and cartilage degradation, assessed by CTX-I and CTX-II plasma levels, were decreased in all KBP-treated groups and KBP potently inhibited bone resorption in vitro. CONCLUSIONS: Our study demonstrates the effectiveness of KBP in ameliorating osteoarthritis-associated joint pain and in protecting the articular cartilage, suggesting KBP as a potential drug candidate for osteoarthritis.

Rodent models of leptin receptor deficiency are less sensitive to amylin.

The pancreatic hormone amylin is released from beta cells following nutrient ingestion and contributes to the control of body weight and glucose homeostasis. Amylin reduces food intake by activating neurons in the area postrema (AP). Amylin was also shown to synergize with the adipokine leptin, with combination therapy producing greater weight loss and food intake reduction than either hormone alone. Although amylin and leptin were initially thought to interact downstream of the AP in the hypothalamus, recent findings show that the two hormones can act on the same AP neurons, suggesting a more direct relationship. The objective of this study was to determine whether amylin action depends on functional leptin signaling. We tested the ability of amylin to induce satiation and to activate its primary target neurons in the AP in two rodent models of LepR deficiency, the db/db mouse and the Zucker diabetic fatty (ZDF) rat. When compared with wild-type (WT) mice, db/db mice exhibited reduced amylin-induced satiation, reduced amylin-induced Fos in the AP, and a lower expression of calcitonin receptor (CTR) protein, the core component of all amylin receptors. ZDF rats also showed no reduction in food intake following amylin treatment; however, unlike the db/db mice, levels of amylin-induced Fos and CTR in the AP were no different than WT rats. Our results suggest that LepR expression is required for the full anorexic effect of amylin; however, the neuronal activation in the AP seems to depend on the type of LepR mutation.

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI- cells, which yield core N-glycans (Man5GlcNAc2). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Optimization of tolerability and efficacy of the novel dual amylin and calcitonin receptor agonist KBP-089 through dose escalation and combination with a GLP-1 analog.

Amylin and GLP-1 agonism induce a well-known anorexic effect at dose initiation, which is managed by dose escalation. In this study we investigated how to optimize tolerability while maintaining efficacy of a novel, highly potent dual amylin and calcitonin receptor agonist (DACRA), KBP-089. Furthermore, we tested the GLP-1 add-on potential of KBP-089 in high-fat diet (HFD)-fed rats. KBP-089 potently activated both the amylin and calcitonin receptors in vitro and demonstrated a prolonged receptor activation as well as a potent reduction of acute food intake. HFD rats dosed every day or every second day obtained equal weight loss at study end, albeit with an uneven reduction in both food intake and body weight in rats dosed every second day. In a 4-fold dose escalation, KBP-089 induced a transient reduction in food intake at every escalation step, with reducing magnitude over time, and the following treatment with 2.5, 10, and 40 µg/kg resulted in an ~15% vehicle-corrected weight loss, a corresponding reduction in adipose tissue (AT), and, in all treatment groups, improved oral glucose tolerance (P < 0.01). Twofold and linear escalations suppressed body weight evenly with no significant reduction in food intake at either escalation step. KBP-089 (1.25 µg/kg) and liraglutide (50 µg/kg) reduced 24-h food intake by 29% and 37% compared with vehicle, respectively; however, when they were combined, 24-h food intake was reduced by 87%. Chronically, KBP-089 (1.25 µg/kg) and liraglutide (50 µg/kg) lowered body weight 8% and 2% in HFD rats, respectively, whereas the combination resulted in a 12% body weight reduction. Moreover, the combination improved glucose tolerance (P < 0.05). In conclusion, DACRAs act complementarily with GLP-1 on food intake and body weight. Furthermore, on escalation, KBP-089 was well tolerated and induced and sustained a significant weight loss and a reduction in AT in lean and HFD rats, underscoring the potential of KBP-089 as an anti-obesity agent.

The Dual Amylin- and Calcitonin-Receptor Agonist KBP-042 Works as Adjunct to Metformin on Fasting Hyperglycemia and HbA1c in a Rat Model of Type 2 Diabetes.

KBP-042 is a dual amylin and calcitonin receptor agonist that increases glucose tolerance and insulin action and reduces body weight in rat models of obesity and prediabetes. The objective of the present study was to 1) evaluate KBP-042 as a treatment of late-stage type 2 diabetes in a rat model and 2) assess the value of adding KBP-042 to the standard of care, metformin, to consider KBP-042 as a relevant drug for treating patients with type 2 diabetes. Two studies were included: an intervention study and a prevention study. In the intervention study, treatment with 5 µg/kg KBP-042 was initiated in 11-week-old Zucker diabetic fatty (ZDF) rats, in which glucose tolerance, fasting glycemia, and glycated hemoglobin were assessed after 4 weeks. In the prevention study, either metformin (400 mg/kg), KBP-042 (5 µg/kg), or a combination of both were administered to ZDF rats for a total of 9 weeks. Glycemia, glucose tolerance, and insulin tolerance were tested. Furthermore, fasting plasma insulin and glucagon levels were evaluated. Finally, pancreatic content of insulin was assessed as a surrogate marker of beta-cell mass. It was found that KBP-042 was efficient in lowering fasting plasma glucose as well as improving glucose tolerance, both as prevention and intervention of disease progression. Furthermore, KBP-042 was efficient in combination with metformin and had additional effects compared with either therapy alone. In conclusion, KBP-042 is a highly relevant therapeutic candidate against type 2 diabetes, effective both as an add-on therapy to metformin and as a stand-alone therapy.

KBP-088, a novel DACRA with prolonged receptor activation, is superior to davalintide in terms of efficacy on body weight.

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.

Are insulin sensitizers the new strategy to treat Type 1 diabetes? A long-acting dual amylin and calcitonin receptor agonist improves insulin-mediated glycaemic control and controls body weight.

BACKGROUND AND PURPOSE: Insulin therapies for Type 1 diabetes (T1D) have limitations, such as glucose fluctuations, hypoglycaemia, and weight gain. Only pramlintide is approved with insulin. However, its short half-life limits efficacy, requiring multiple daily injections and increasing hypoglycaemia risk. New strategies are needed to improve glycaemic control. Dual amylin and calcitonin receptor agonists are potent insulin sensitizers developed for Type 2 diabetes (T2D) as they improve glucose control, reduce body weight, and attenuate hyperglucagonemia. However, it is uncertain if they could be used to treat T1D. EXPERIMENTAL APPROACH: Sprague Dawley rats received a single intravenous injection of streptozotocin (STZ) (50 mg·kg-1) to induce T1D. Humulin (1 U/200 g·day-1 or 2 U/200 g·day-1) was continuously infused, while half of the rats received additional KBP-336 (4.5 nmol·kg-1 Q3D) treatment. Bodyweight, food intake, and blood glucose were monitored throughout the study. An oral glucose tolerance test was performed during the study. KEY RESULTS: Treatment with Humulin or Humulin + KBP-336 improved the health of STZ rats. Humulin increased body weight in STZ rats, but KBP-336 attenuated these increases and maintained a significant weight loss. The combination exhibited greater blood glucose reductions than Humulin-treated rats alone, reflected by improved HbA1c levels and glucose control. The combination prevented hyperglucagonemia, reduced amylin levels, and increased pancreatic insulin content, indicating improved insulin sensitivity and beta-cell preservation. CONCLUSION AND IMPLICATIONS: The insulin sensitizer KBP-336 lowered glucagon secretion while attenuating insulin-induced weight gain. Additionally, KBP-336 may prevent hypoglycaemia and improve insulin resistance, which could be a significant advantage for individuals with T1D seeking therapeutic benefits.

Dual amylin and calcitonin receptor agonist treatment improves insulin sensitivity and increases muscle-specific glucose uptake independent of weight loss.

Dual amylin and calcitonin receptor agonists (DACRAs) are known to induce significant weight loss as well as improve glucose tolerance, glucose control, and insulin action in rats. However, to what extent DACRAs affect insulin sensitivity beyond that induced by weight loss and if DACRAs affect glucose turnover including tissue-specific glucose uptake is still unknown. Hyperinsulinemic glucose clamp studies were carried out in pre-diabetic ZDSD and diabetic ZDF rats treated with either the DACRA KBP or the long-acting DACRA KBP-A for 12 days. The glucose rate of disappearance was assessed using 3-3H glucose and tissue-specific glucose uptake was evaluated using 14C-2-deoxy-D-glucose (14C-2DG). In diabetic ZDF rats, KBP treatment significantly reduced fasting blood glucose and improved insulin sensitivity independent of weight loss. Furthermore, KBP increased the rate of glucose clearance, likely by increasing glucose storage, but without altering the endogenous glucose production. This was confirmed in pre-diabetic ZDSD rats. Direct assessment of tissue-specific glucose uptake showed, that both KBP and KBP-A significantly increased glucose uptake in muscles. In summary, KBP treatment significantly improved insulin sensitivity in diabetic rats and markedly increased glucose uptake in muscles. Importantly, in addition to their well-established weight loss potential, the KBPs have an insulin-sensitizing effect independent of weight loss, highlighting DACRAs as promising agents for the treatment of type 2 diabetes and obesity.