The Inhibitory Effect of Salmon Calcitonin on Tri-Iodothyronine Induction of Early Hypertrophy in Articular Cartilage

Salmon calcitonin has chondroprotective effect both in vitro and in vivo, and is therefore being tested as a candidate drug for cartilage degenerative diseases. Recent studies have indicated that different chondrocyte phenotypes may express the calcitonin receptor (CTR) differentially. We tested for the presence of the CTR in chondrocytes from tri-iodothyronin (T3)-induced bovine articular cartilage explants. Moreover, investigated the effects of human and salmon calcitonin on the explants.Early chondrocyte hypertrophy was induced in bovine articular cartilage explants by stimulation over four days with 20 ng/mL T3. The degree of hypertrophy was investigated by molecular markers of hypertrophy (ALP, IHH, COLX and MMP13), by biochemical markers of cartilage turnover (C2M, P2NP and AGNxII) and histology. The expression of the CTR was detected by qPCR and immunohistochemistry. T3-induced explants were treated with salmon or human calcitonin. Calcitonin down-stream signaling was measured by levels of cAMP, and by the molecular markers.Compared with untreated control explants, T3 induction increased expression of the hypertrophic markers (p<0.05), of cartilage turnover (p<0.05), and of CTR (p<0.01). Salmon, but not human, calcitonin induced cAMP release (p<0.001). Salmon calcitonin also inhibited expression of markers of hypertrophy and cartilage turnover (p<0.05).T3 induced early hypertrophy of chondrocytes, which showed an elevated expression of the CTR and was thus a target for salmon calcitonin. Molecular marker levels indicated salmon, but not human, calcitonin protected the cartilage from hypertrophy. These results confirm that salmon calcitonin is able to modulate the CTR and thus have chondroprotective effects

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

Fold expression (mean(95%CI)) of pre-hypertrophic marker in response to 1 and 10 nM sCT stimulation of T3-induced explants compared with T3-induced alone (T3).

§<p>P-value of ANOVA test,</p>#<p>P-value of Dunnett’s post-test.</p

Immunolocalization of the calcitonin receptor (CTR) in cartilage explants induced with or without T3 for 4 days.

<p>First panel shows the superficial layer of the cartilage, second panel the mid layer of the cartilage and the third panel the deep layer of the cartilage. Treatment scheme is indicated at the top of the pictures. The pictures are representative of 3 replicates from the same experiment. Positive staining is visualized by brown color.</p

Expression and induction of the calcitonin receptor (CTR) as a result of T3 stimulation.

<p>The level of cAMP produced by the chondrocytes two hours after application of A) salmon calcitonin (sCT) or B) human calcitonin (hCT). The measurements were taken at day 0, 4 and 8 in explants with or without T3. The measurements were normalized to vehicle control for each time point, giving the fold induction. Significance level; *p<0.05, and ***p<0.001, data shown as geometric mean with 95%-CI (N = 2, n = 10).</p

The effect of co-stimulation with salmon calcitonin (sCT) on cartilage turnover.

<p>A) Cell viability, measured by Alamar blue, of the cartilage explants. B) Induction of alkaline phosphatase (ALP) activity in the conditioned medium. C) Measurement of matrix metalloproteinase (MMP)-derived type II collagen degradation fragments (C2M) released to the conditioned medium. D) Measurement of type II procollagen (P2NP) released to the conditioned medium. E) Measurement of MMP-derived aggrecan collagen degradation fragments (AGNx-II) released to the conditioned medium. Comparisons were performed by one-way ANOVA with Dunnetts post test. Significance levels; *p<0.05 and **p<0.01, data shown as mean with CI-95% n = 6.</p