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 &lt; 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 &lt; 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. </jats:p

Prolonged ligand dissociation kinetics on membranes preparations and live cells.

<p>A) Total binding of ¹²⁵I-sCT and ¹²⁵I-hCT ligand bound to U2OS CALCR cells at different time points (0, 2, 4, 8, 24, 48 and 72 hours). B₀ is total ¹²⁵I-ligand bound to cells at t = 0 and B is ¹²⁵I-ligand bound. B) Specific activity of ¹²⁵I-sCT and ¹²⁵I-hCT ligand released into the supernatant by cells in (B) at different time points (0, 2, 4, 8, 24, 48 and 72 hours). C) ¹²⁵I-sCT and ¹²⁵I-hCT ligand dissociation was investigated using isolated U2OS CALCR membrane preparations. The dissociation from the membranes was measured after 1, 2, 4, 8, 24, 48 and 72 hours with or without the presence of 20 μM GTPγS to determine G-protein dependency. B₀ is total ¹²⁵I-ligand bound to membranes at t = 0 and B is ¹²⁵I-ligand bound.B₀ is total ¹²⁵I-ligand bound to cells at t = 0 and S is ¹²⁵I-ligand in the medium. Live cell assays were conducted at 37°C and membrane assays were conducted at room temperature.</p

Acid wash decrease, but do not attenuate sCT mediated prolonged cAMP and β-arrestin signaling.

<p>Effects of acid wash on cAMP production (A) and β-arrestin recruitment (B). U2OS CALCR cells were initially stimulated for 60 min with sCT or hCT using U2OS CALCR cells, then acid washed for 2×2 min and cultured for another 3, 6, 24 or 48 hours in fresh ligand free culture medium, and then assayed at end incubation. In parallel, standard assay groups of sCT and hCT were included as controls. C) Internalization rate of ¹²⁵I-sCT and ¹²⁵I-hCT stimulated CT_(a) receptors in U2OS CALCR. Cells were pre-stimulated for 45 min at 4°C with 0.25 nM ¹²⁵I-sCT or 0.25 nM ¹²⁵I-hCT for ensure receptor occupancy, then incubating for an additional 0, 15, 30, 60 and 120 min at 37°C to assess internalization. Asterisk (*) indicates significant difference between AUC sCT, Not Washed and AUC sCT, Acid Wash. Asterisk (#) indicates significant difference between AUC hCT, Not Washed and AUC hCT, Acid Wash. p&lt;0.05 was considered to be significant. *  = p&lt;0.05, **  = p&lt;0.01, ***  = p&lt;0.001. cAMP data are shown as mean ± SEM and representative of three individual conducted experiments with five replicates. β-arrestin data are shown as mean ± SEM of 3 experiments performed with five replicates. Internalization data are shown as mean ± SEM and representative of two individual conducted experiments in triplicates.</p

Prolonged ERK activation/phosphorylation in Cos-7 CT_(a)R cells by sCT, but not hCT.

<p>Cos-7 CT_(a)R Cells were stimulated for 5 min, 10 min, 30 min, 120 min, 24 hours or 48 hours with either Vehicle (A), 100 nM hCT (B) or 100 nM sCT (C). At each time point, the cells were lysed and protein content extracted. The protein extracts was then analyzed by western blotting investigating ligand-mediated phospho-ERK1/2 activation. Total ERK1/2 was used as a protein loading control.</p

Difference in CT_(a)R -mediated cAMP response by prolonged continuous hCT and sCT stimulation.

<p>A) cAMP production as a function of ligand concentration as U2OS CALCR cells were stimulated with sCT or hCT at a dose range of 10 pM to 10 nM for a prolonged period of time, ranging from 1, 4, 24, 48 to 72 hours. Ligands and medium were only added during the initiation of the experiment. Assays were conducted without IBMX in the medium to assess time dependent cAMP production. B) Single dose of 10 nM sCT, 10 nM hCT and Vehicle shown in (A) plotted as a function of time. Asterisk (*) indicate significant difference between AUC sCT and AUC hCT, p&lt;0.05 was considered to be significant. *  = p&lt;0.05, **  = p&lt;0.01, ***  = p&lt;0.001. Data are shown as mean ± SEM and representative of three individual conducted experiments using five replicates.</p

Difference in CT_(a)R-mediated β-arrestin recruitment by prolonged continuous hCT and sCT stimulation.

<p>A) Data show β-arrestin recruitment as a function of ligand concentration. U2OS CALCR cells were stimulated with sCT or hCT at a dose range of 0.1 nM to 100 nM for a prolonged period of time, ranging from 1, 4, 24, 48 to 72 hours. Ligands and medium were only added at the experiment initiation. B) Single dose of 100 nM sCT, 100 nM hCT and Vehicle shown in (A) plotted as a function of time. Asterisk (*) indicate significant difference between AUC sCT and AUC hCT, p&lt;0.05 was considered to be significant. *  = p&lt;0.05, **  = p&lt;0.01, ***  = p&lt;0.001. Data are shown as mean ± SEM of 3 experiments performed with five replicates.</p

Continuous stimulation attenuates additional prolonged responses in inverse proportion to ligand concentration.

<p>U2OS CALCR cells were stimulated with sCT for 4, 24, 48, 72 or 96(A) or hCT for 4, 24, 48 or 72 hours (B) in a similar prolonged setup as demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092042#pone-0092042-g002" target="_blank">Figure 2</a>. In parallel, one set of cells was stimulated for 96h (sCT) or 72h (hCT), following which the culture medium was replaced with fresh medium with ligands and incubated for another 4, 24 or 48 hours. Ligand concentration sCT or hCT: 10 nM, 1 nM, 0.1 nM or vehicle. The vertical arrow indicates time of medium-ligand replacement.</p