Salmon Calcitonin Research Articles

Salmon Calcitonin Exerts an Antidepressant Effect by Activating Amylin Receptors.

Depressive disorder is defined as a psychiatric disease characterized by the core symptoms of anhedonia and learned helplessness. Currently, the treatment of depression still calls for medications with high effectiveness, rapid action, and few side effects, although many drugs, including fluoxetine and ketamine, have been approved for clinical usage by the Food and Drug Administration (FDA). In this study, we focused on calcitonin as an amylin receptor polypeptide, of which the antidepressant effect has not been reported, even if calcitonin gene-related peptides have been previously demonstrated to improve depressive-like behaviors in rodents. Here, the antidepressant potential of salmon calcitonin (sCT) was first evaluated in a chronic restraint stress (CRS) mouse model of depression. We observed that the immobility duration in CRS mice was significantly increased during the tail suspension test and forced swimming test. Furthermore, a single administration of sCT was found to successfully rescue depressive-like behaviors in CRS mice. Lastly, AC187 as a potent amylin receptor antagonist was applied to investigate the roles of amylin receptors in depression. We found that AC187 significantly eliminated the antidepressant effects of sCT. Taken together, our data revealed that sCT could ameliorate a depressive-like phenotype probably via the amylin signaling pathway. sCT should be considered as a potential therapeutic candidate for depressive disorder in the future.

Examination of Effective Buccal Absorption of Salmon Calcitonin Using Cell-Penetrating Peptide-Conjugated Liposomal Drug Delivery System.

IntroductionThe buccal route has been considered an attractive alternative delivery route for injectable formulations. Cell-penetrating peptides (CPPs) are gaining increased attention for their cellular uptake and tissue permeation effects. This study was aimed to evaluate the in vitro and ex vivo permeation-enhancing effect of penetratin-conjugated liposomes for salmon calcitonin (sCT) in TR146 human buccal cells and porcine buccal tissues.MethodsPenetratin was conjugated to phospholipids through a maleimide-thiol reaction. Liposomes were prepared and sCT was encapsulated using a thin-film hydration method. Physical properties such as particle size, zeta potential, encapsulation efficiency, and morphological images via transmission electron microscopy were obtained. Cellular uptake studies were conducted using flow cytometry (FACS) and confocal laser scanning microscopy (CLSM). A cell permeation study was performed using a Transwell® assay, and permeation through porcine buccal tissue was evaluated. The amount of sCT permeated was quantified using an ELISA kit and was optically observed using CLSM.ResultsThe particle size of penetratin-conjugated liposomes was approximately 123.0 nm, their zeta potential was +29.6 mV, and their calcitonin encapsulation efficiency was 18.0%. In the cellular uptake study using FACS and CLSM, stronger fluorescence was observed in penetratin-conjugated liposomes compared with the solution containing free sCT and control liposomes. Likewise, the amount of sCT permeated from penetratin-conjugated liposomes was higher than that from the free sCT solution and control liposomes by 5.8-fold across TR146 cells and 91.5-fold across porcine buccal tissues.ConclusionPenetratin-conjugated liposomes are considered a good drug delivery strategy for sCT via the buccal route.

Hypophagia induced by salmon calcitonin, but not by amylin, is partially driven by malaise and is mediated by CGRP neurons

ObjectiveThe behavioral mechanisms and the neuronal pathways mediated by amylin and its long-acting analog sCT (salmon calcitonin) are not fully understood and it is unclear to what extent sCT and amylin engage overlapping or distinct neuronal subpopulations to reduce food intake. We here hypothesize that amylin and sCT recruit different neuronal population to mediate their anorectic effects. MethodsViral approaches were used to inhibit calcitonin gene-related peptide (CGRP) lateral parabrachial nucleus (LPBN) neurons and assess their role in amylin’s and sCT’s ability to decrease food intake in mice. In addition, to test the involvement of LPBN CGRP neuropeptidergic signaling in the mediation of amylin and sCT’s effects, a LPBN site-specific knockdown was performed in rats. To deeper investigate whether the greater anorectic effect of sCT compared to amylin is due do the recruitment of additional neuronal pathways related to malaise multiple and distinct animal models tested whether amylin and sCT induce conditioned avoidance, nausea, emesis, and conditioned affective taste aversion. ResultsOur results indicate that permanent or transient inhibition of CGRP neurons in LPBN blunts sCT-, but not amylin-induced anorexia and neuronal activation. Importantly, sCT but not amylin induces behaviors indicative of malaise including conditioned affective aversion, nausea, emesis, and conditioned avoidance; the latter mediated by CGRPLPBN neurons. ConclusionsTogether, the present study highlights that although amylin and sCT comparably decrease food intake, sCT is distinctive from amylin in the activation of anorectic neuronal pathways associated with malaise.

Involvement of nitric oxide pathway in the acute anticonvulsant effect of salmon calcitonin in rats

Epilepsy is a chronic neurological disease that is thought to affect up to 1% of the world’s population. Evidence suggests that salmon calcitonin (sCT) has positive effects on epileptic seizures and epileptogenesis. However, it remains unknown that whether nitric oxide (NO) pathway contributed to this antiepileptic effect of sCT. We have used the pentylenetetrazole (PTZ)-induced seizure rat model to identify the effect of sCT on seizure score, seizure-induced cognitive deficit, and the NO pathway in the brain. We found that sCT increases the first myoclonic jerk (FMJ), decreased Racine’s convulsion scale (RCS), and abates seizure-induced cognitive impairment. We further demonstrated that sCT attenuated the abnormal increase of NO in the brain. These results revealed that sCT exerts an antiepileptic effect by modulating the NO pathway in the brain.

Neurotoxic amyloid prefibrillar oligomers: Do salmon calcitonin and amyloid β1-42 wear the same “outfit”?

We were impressed by the similarity and complementarity between experimental results concerning neurotoxicity induced by prefibrillar oligomers (PFOs) of two different proteins belonging to the “amyloid” family: salmon Calcitonin (sCT) and Amyloid-β1-42 (Aβ1-42).

Osteoprotective Effect of Enamel Matrix Derivatives on the Regeneration of Mandibular Defects in Experimentally Glucocorticoid-Induced Osteoporosis.

Purpose Osteoporosis is a progressive systematic skeletal illness characterized by low bone mineral density and susceptibility to fracture caused by bone resorption. Aim of the Study. This study intended to evaluate the possible role of emdogain in combination with calcitonin on the healing of surgically induced mandibular defects performed on osteoporotic rats. Materials and Methods Forty healthy female white albino rats were included in this study and divided into four groups. In group I (negative control), 10 rats received a vehicle injection after which a unilateral mandibular defect was created in each rat of all groups. Three groups were subjected to induction of osteoporosis by subcutaneous injection of 0.1 mg/kg/day dexamethasone for 60 days. In group II, rats were kept without treatment. In group III, rats were treated with daily intramuscular injection of 2.5 IU/kg of synthetic salmon calcitonin. In group IV, rats were handled as group III, and the created cavity was filled with emdogain. Rats were euthanized at 2nd and 4th week postsurgically. Hematoxylin and eosin, Masson's trichrome, NF-κB (nuclear factor of activated B cells), and immunohistochemical stains were used, followed by statistical analysis. Results Group I showed normal stages of bone defects healing. Group II revealed the formation of granulation tissue with dilated blood vessels, while groups III and IV showed enhanced bone healing and proper collagen fibers. The percentage area of newly formed collagen fibers was significantly higher in group IV at 2nd week (13.96 ± 0.020%) and 4th week (16.95 ± 0.024%) than in group II (8.75 ± 0.015% and 10.29 ± 0.015%, respectively) and group III (12.93 ± 0.015% and 14.61 ± 0.021%, respectively), but was lower than that in group I (15.75 ± 0.015% and 17.49 ± 0.015%, respectively). Conclusion The local application of emdogain combined with systemically injected calcitonin improves bone healing in surgically induced bone defects in osteoporotic rats.

Development of High Affinity Calcitonin Analog Fragments Targeting Extracellular Domains of Calcitonin Family Receptors.

The calcitonin and amylin receptors (CTR and AMY receptors) are the drug targets for osteoporosis and diabetes treatment, respectively. Salmon calcitonin (sCT) and pramlintide were developed as peptide drugs that activate these receptors. However, next-generation drugs with improved receptor binding profiles are desirable for more effective pharmacotherapy. The extracellular domain (ECD) of CTR was reported as the critical binding site for the C-terminal half of sCT. For the screening of high-affinity sCT analog fragments, purified CTR ECD was used for fluorescence polarization/anisotropy peptide binding assay. When three mutations (N26D, S29P, and P32HYP) were introduced to the sCT(22–32) fragment, sCT(22–32) affinity for the CTR ECD was increased by 21-fold. CTR was reported to form a complex with receptor activity-modifying protein (RAMP), and the CTR:RAMP complexes function as amylin receptors with increased binding for the peptide hormone amylin. All three types of functional AMY receptor ECDs were prepared and tested for the binding of the mutated sCT(22–32). Interestingly, the mutated sCT(22–32) also retained its high affinity for all three types of the AMY receptor ECDs. In summary, the mutated sCT(22–32) showing high affinity for CTR and AMY receptor ECDs could be considered for developing the next-generation peptide agonists.

Dopamine-Conjugated Carbon Dots Inhibit Human Calcitonin Fibrillation.

The development of biocompatible nanomaterials has become a new trend in the treatment and prevention of human amyloidosis. Human calcitonin (hCT), a hormone peptide secreted from parafollicular cells, plays a major role in calcium–phosphorus metabolism. Moreover, it can be used in the treatment of osteoporosis and Paget’s disease. Unfortunately, it tends to form amyloid fibrils irreversibly in an aqueous solution, resulting in a reduction of its bioavailability and therapeutic activity. Salmon calcitonin is the replacement of hCT as a widely therapeutic agent due to its lower propensity in aggregation and better bioactivity. Herein, we used citric acid to synthesize carbon dots (CDs) and modified their surface properties by a variety of chemical conjugations to provide different functionalized CDs. It was found that dopamine-conjugated CDs can effectively inhibit hCT aggregation especially in the fibril growth phase and dissociate preformed hCT amyloids. Although the decomposition mechanism of dopamine-conjugated CDs is not clear, it seems to be specific to hCT amyloids. In addition, we also tested dopamine-conjugated mesoporous silica nanoparticles in preventing hCT fibrillization. They also can work as inhibitors but are much less effective than CDs. Our studies emphasized the importance of the size and surface functionalization of core materials in the development of nanomaterials as emerging treatments for amyloidosis. On the other hand, proper functionalized CDs would be useful in hCT formulation.

Evaluation of the efficacy of salmon calcitonin nasal spray on bone healing following open reduction and internal fixation of mandibular fractures — A randomized controlled trial

The objective of this study was to assess the efficacy of calcitonin spray on bone healing following open reduction internal fixation (ORIF) of mandibular fractures. Fourteen patients were subdivided into a study group and a control group. A standardized surgical protocol for ORIF was followed. Postoperatively, salmon calcitonin nasal spray was administered to only the study group. The outcome parameters assessed were serum osteocalcin, pain, and radiographic bone healing. Serum osteocalcin was assessed pre- and postoperatively. Postoperative pain was documented using a visual analogue scale (VAS) on the 7th, 14th, 23rd, and 30th days. An orthopantomogram was used to score fracture healing at four time intervals, as follows: 1 — absence of callus; 2 — presence of minimal callus; 3 — considerable callus; and 4 — complete fusion of fracture. Pain scores were lower for the study group, with no pain from the fifth day, while the control group produced a mean score for day 5 of 2.43 ± 0.98 (p = 0.001). Mean postoperative serum osteocalcin levels were higher for the study group (67.82 ± 8.89) compared with the control group (57.69 ± 6.22; p = 0.029). Bone healing at 12 weeks postoperatively was level 4 for 28.6% of patients in the study group and level 3 for 71.4%. In comparison, 85.7% in the control group demonstrated level 3 healing, while 14.3% remained at level 2 (p = 0.462). Within the limitations of the study, it can be concluded that intranasal salmon calcitonin spray reduces postoperative pain and facilitates fracture healing, although its economic efficiency is still to be proven.

A serological type II collagen neoepitope biomarker reflects cartilage breakdown in patients with osteoarthritis

ObjectivesThere is an unmet medical need for biomarkers in OA which can be applied in clinical drug development trials. The present study describes the development of a specific and robust assay measuring type II collagen degradation (T2CM) and discusses its potential as a noninvasive translational biomarker. MethodsA type II collagen specific neoepitope (T2CM) was identified by mass spectrometry and monoclonal antibodies were raised towards the epitope, employed in a chemiluminescence immunoassay. T2CM was assessed in bovine cartilage explants with or without MMP-13 inhibitor, and explant supernatants were analyzed by Western blot. T2CM was measured in plasma samples from one study (n ​= ​48 patients) where OA patients were referred to total knee replacement (TKR). Additionally, T2CM was quantified in serum from OA patients receiving salmon calcitonin treatment (sCT) (n ​= ​50) compared to placebo (n ​= ​57). ResultsThe T2CM assay was technically robust (13/4 ​% inter/intra-variation) and specific for the type II collagen fragment cleaved by MMP-1 and -13. The MMP-13 inhibitor reduced the T2CM release from bovine cartilage explants receiving catabolic treatment. These results were confirmed by Western blot. In human end-stage OA patients (scheduled for TKR), the T2CM levels were elevated compared to moderate OA (p<0.004). The OA patients receiving sCT had lower levels of T2CM compared to placebo group after 1, 6, and 24 months of treatment (p ​= ​0.0285, p ​= ​0.0484, p ​= ​0.0035). ConclusionsTo our knowledge, T2CM is the first technically robust serological biomarker assay which has shown biological relevance in ex vivo models and OA cohorts. This suggests that T2CM may have potential as a translational biomarker for cartilage degradation.

The effect of salmon calcitonin against glutamate-induced cytotoxicity in the C6 cell line and the roles the inflammatory and nitric oxide pathways play.

Recent evidence has shown that salmon calcitonin (sCT) has positive effects on the nervous system. However, its effect and mechanisms on glutamate-induced cytotoxicity are still unclear. The current experiment was designed to examine the effect of sCT on glutamate-induced cytotoxicity in C6 cells, involving the inflammatory and nitric oxide stress pathways. The study used the C6 glioma cell line. Four cell groups were prepared to evaluate the effect of sCT on glutamate-induced cytotoxicity. The control group was without any treatment. Cells in the glutamate group were treated with 10mM glutamate for 24h. Cells in the sCT group were treated with various concentrations (3, 6, 12, 25, and 50µg/mL) of sCT for 24h. Cells in the sCT + glutamate group were pre-treated with various concentrations of sCT for 1h and then exposed to glutamate for 24h. The cell viability was evaluated with an XTT assay. Nuclear factor kappa b (NF-kB), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), neuronal nitric oxide synthase (nNOS), nitric oxide (NO), cyclic guanosine monophosphate (cGMP), caspase-3, and caspase-9 levels in the cells were measured by ELISA kits. Apoptosis was detected by flow cytometry method. sCT at all concentrations significantly improved the cell viability in C6 cells after glutamate-induced cytotoxicity (p < 0.001). Moreover, sCT significantly reduced the levels of NF-kB (p < 0.001), TNF-α, and IL-6 levels (p < 0.001). sCT also decreased nNOS, NO, and cGMP levels (P < 0.001). Furthermore, it decreased the apoptosis rate and increased the live-cell rate in the flow cytometry (P < 0.001). In conclusion, sCT has protective effects on glutamate-induced cytotoxicity in C6 glial cells by inhibiting inflammatory and nitric oxide pathways. sCT could be a useful supportive agent for people with neurodegenerative symptoms.

A selective role for receptor activity-modifying proteins in subchronic action of the amylin selective receptor agonist NN1213 compared with salmon calcitonin on body weight and food intake in male mice.

The role of receptor activity‐modifying proteins (RAMPs) in modulating the pharmacological effects of an amylin receptor selective agonist (NN1213) or the dual amylin–calcitonin receptor agonist (DACRA), salmon calcitonin (sCT), was tested in three RAMP KO mouse models, RAMP1, RAMP3 and RAMP1/3 KO. Male wild‐type (WT) and knockout (KO) littermate mice were fed a 45% high‐fat diet for 20 weeks prior to the 3‐week treatment period. A decrease in body weight after NN1213 was observed in all WT mice, whereas sCT had no effect. The absence of RAMP1 had no significant effect on NN1213 efficacy, and sCT was still inactive. However, the absence of RAMP3 impeded NN1213 efficacy but improved sCT efficacy. Similar results were observed in RAMP1/3 KO suggesting that the amylin receptor 3 (AMY3 = CTR + RAMP3) is necessary for NN1213's maximal action on body weight and food intake and that the lack of AMY3 allowed sCT to be active. These results suggest that the chronic use of DACRA such as sCT can have unfavourable effect on body weight loss in mice (which differs from the situation in rats), whereas the use of the amylin receptor selective agonist does not. AMY3 seems to play a crucial role in modulating the action of these two compounds, but in opposite directions. The assessment of a long‐term effect of amylin and DACRA in different rodent models is necessary to understand potential physiological beneficial and unfavourable effects on weight loss before its transition to clinical trials.

Skin and bone development in Atlantic salmon (Salmo salar) influenced by hatchery environment

Recirculating aquaculture systems (RAS) are receiving greater attention because they have potential to reduce the use of water, enable better control of environmental conditions and give greater biosecurity. However, little is known about the effects of conditions in RAS on early development. Here we compare the survival, development and gene expression of Atlantic salmon grown in a RAS and flow through (FT) system from egg until before they start to feed (at around 875°d). The epidermis of RAS fish was significantly thinner and contained larger numbers of mucosal cells, which were positioned more toward the skin surface, and less mineralisation of the vertebral body, than FT fish. Genes known to be involved in bone development and mineralisation were significantly up-regulated (inner ear specific collagen, transforming growth factor-beta-inducible early growth response protein 3 and salmon calcitonin) or down-regulated (metalloproteinase inhibitor 2-like, 25-hydroxyvitamin D3 1-alpha-hydroxylase and elongation of very long chain fatty acids protein 7-like) with RAS relative to FT treatment. Genes known to affect skin development, including keratin type I cytoskeletal 13-like and angiopoietin-related protein 7, were down-regulated in RAS compared to FT treated fish. Although no two RAS will share exactly the same environmental conditions, our comparison shows that early development of the skeleton and skin is hindered by the environment of the RAS used in this study and that a number of related genetic processes influencing skin and skeletal development are affected. These differences could affect the robustness of salmon at later life stages.

Salmon Calcitonin Attenuates Some Behavioural Responses to Nicotine in Male Mice.

The behavioural responses to nicotine involve appetite-regulatory hormones; however, the effects of the anorexigenic hormone amylin on reward-related behaviours induced by nicotine remain to be established. Previous studies have shown that the amylinergic pathway regulates behavioural responses to alcohol, amphetamine and cocaine. Here, we evaluated the effects of salmon calcitonin (sCT), an amylin and calcitonin receptor (CTR) agonist, on nicotine-induced locomotor stimulation and sensitisation as well as dopamine release in the nucleus accumbens (NAc) shell. Moreover, we investigated the effects of sCT on the acquisition and expression of nicotine-induced reward in the conditioned place preference (CPP) paradigm. Finally, we performed Western Blot experiments in an attempt to identify the levels of the amylin receptor components CTRa, CTRb, and RAMP1 in reward-related areas of mice responding differently to repeated injections of sCT and nicotine in the locomotor sensitisation test. We found that sCT blocked nicotine’s stimulatory and dopamine-releasing effects and prevented its ability to cause locomotor sensitisation. On the other hand, sCT did not alter nicotine-induced acquisition and expression of CPP. Lastly, sCT-nicotine treated mice from the locomotor sensitisation experiment displayed higher levels of total CTR, i.e. CTRa and CTRb together, in the reward-processing laterodorsal tegmental area (LDTg) of the brain compared to mice treated with vehicle-nicotine. Overall, the present data reveal that activation of CTR or/and amylin receptors attenuates certain nicotine-induced behaviours in male mice, further contributing to the understanding of appetite-regulatory peptides in reward regulation.

AM833, a Long Acting Amylin Analogue Induces Hypocalcemia in Young Rats via a Calcitonin Receptor Mediated Mechanism

Abstract Amylin is a peptide hormone that is co-secreted with insulin from pancreatic β-cells. It is member of the calcitonin family and activates amylin receptors (AMYRs) in the calcitonin receptor (CTR) family. During recent years, several AMYR agonists have been in clinical development for treatment of diabetes and obesity. In contrast to pramlintide, which is a short acting amylin analogue approved for use in combination with meal-time insulin in patients with diabetes, the more recent development candidates have been dual acting analogues activating both the CTR and the AMYRs. In rodents the CTR plays a significant role in calcium homeostasis, while in humans this has been associated with little long-term effect on the plasma calcium levels (Ganong, 1993; Longo et al., 2011); Rodents are usually chosen as first choice pre-clinical models for in vivo testing and introducing acute and profound hypocalcaemia can lead to general neurological and neuromuscular disturbances, which can be challenging in clinical development programs. AM833 (NNC0174-0833) is a long acting acylated dual acting amylin analogue activating both CTR and AMYRs. When combined with the long acting GLP-1 analogue semaglutide for weight management, both preclinical studies in rat models of obesity and clinical trials in humans with obesity have demonstrated significant weight loss potential. In young rats aged 7–9 weeks, single sc dose of 3–300 nmol/kg AM833 induced a reduction of total and ionized calcium of 40–50% from baseline with lowest level around 12 hours after dosing. A similar response was seen after administration of salmon calcitonin which also activates both CTR and AMYRs. Tachyphylaxis was introduced, with no effect on calcium found after sub chronic daily treatment for 2 weeks. Age played a key role in the sensitivity to calcium lowering with almost no effect found in 11 months old rats. When administering AM1213 (NNC0174-1213), a selective AMYR agonist with little activation of the CTR, no or little calcium lowering of around 10% was seen. These data indicated that the calcium lowering properties are CTR mediated. The induction of hypercalcemia was a rodent specific phenomenon as no calcium lowering outside of normal range was seen in young dogs and rabbits. Clinical data confirmed that CTR induced calcium lowering was not human relevant, but these data showed that understanding physiology and pharmacology in the animal models of investigation as well as human translational relevance is of outmost importance in the progress of an early development program.

GNAS Heterozygous Inactivation Differentially Affects Osteoclast-Specific Calcitonin Receptor Bioactivity in a Mouse Model of Albright Hereditary Osteodystrophy Based Upon Parental Inheritance

Abstract Albright hereditary osteodystrophy (AHO) is caused by the heterozygous inactivation of GNAS, encoding the α-stimulatory subunit (Gαs) of G protein-coupled receptors. Skeletal manifestations of AHO include adult short stature, brachydactyly and subcutaneous ossifications. AHO patients with maternally-derived GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) and are obese with resistance to hormones requiring Gαs (eg., PTH, TSH and GHRH) due to tissue-specific GNAS imprinting. Paternally-derived GNAS mutations cause pseudopseudohypoparathyroidism (PPHP) in which patients have AHO skeletal features but do not develop severe obesity or hormonal resistance. Mouse models have shown loss of Gα s signaling in osteoblasts or osteoclasts leads to osteopenia, and suggest AHO patients would display a reduced bone mineral density (BMD). Interestingly, PHP1A patients have been shown to have normal to increased BMD without any correlation to body mass index or serum PTH measurement. Based on the differences observed clinically and hormonally between PHP1A and PPHP, we hypothesize that there may also be distinctions in overall bone remodeling between these two disorders due to GNAS imprinting. This study addressed whether the heterozygous inactivation of Gnas differentially affects Gα s-receptor bioactivity within osteoclasts (OCs) based upon parental inheritance. Bone Marrow Macrophages (BMMs) were harvested from our laboratory’s AHO mouse model with either maternally-inherited (Gnas+/-m) mutations correlating to PHP1A or paternally-inherited (Gnas+/-p) mutations correlating to PPHP. BMMs were exposed to 10-7M salmon calcitonin (sCT), 10-5M forskolin or PBS for 6 hrs. OC receptor activity was measured by fluorescent microscopy to visualize actin ring morphology and RT-PCR analysis of Gα s-PKA signaling transcripts Crem and Ramp3. Forskolin treatment displayed no significant variations in OC ring morphology or Crem and Ramp3 mRNA expression between Gnas+/-m, Gnas+/-p and WT cultures. Both WT and Gnas+/-p OCs displayed appropriate responses to sCT, as indicated by a significant disruption in actin ring morphology and increased Crem and Ramp3 mRNA expression when compared to vehicle-treated controls. SCT-treated Gnas+/-m OCs, however, displayed only mild disruptions in actin ring morphology, and we observed significant reductions in Ramp3 expression compared to WT as well as reductions in Crem compared to WT and Gnas+/-p. These data suggest evidence of partial calcitonin resistance within Gnas+/-m OCs due to impaired Gα s- signaling. These data correlate with previous clinical observations of calcitonin resistance in PHP1A patients. Because these findings were observed only within Gnas+/-m cultures, future work is warranted to determine whether this impaired receptor activity may be attributed to partial Gnas imprinting within OCs or the myeloid lineage.

Analysis on application value of Fu's subcutaneous needling for intercostal pain after surgery of osteoporotic thoracic vertebral compression fracture

To observe the effect of Fu's subcutaneous needling therapy on pain degree, quality of life, serum 5-hydroxytryptamine (5-HT) and neuropeptide Y (NPY) in patients with intercostal pain after surgery for osteoporotic thoracic vertebral compression fracture (OVCF) and explore the application value of Fu's subcutaneous needling in treatment of post-operative intercostal pain. A total of 60 patients with intercostal pain after OVCF surgery were divided into a medication group and a Fu's subcutaneous needling therapy group, with 30 cases in each group. In the medication group, zoledronic acid injection, salmon calcitonin injection, calcitonin D tablets and mecobalamin tablets were prescribed. The duration of treatment was 8 weeks. In the Fu's subcutaneous needling therapy group, on the base of treatment as the medicine group, Fu's subcutaneous needling therapy was provided for 2 weeks，once every other day. The needle is inserted 6 cm away from the most painful point. Separately, in 2, 4 and 8 weeks of treatment, as well as in 16 weeks after treatment, the visual analogue scale (VAS) and the generic quality of life inventory-74 (GQOLI-74) were adopted to evaluate intercostal pain and quality of life in the patients of both groups and analyze the incidence of adverse reaction. Radioimmunoassay was used to measure the levels of serum 5-HT and NPY. Compared with the results before treatment, in 2, 4 and 8 weeks of treatment as well as in 16 weeks after treatment, VAS scores of both groups were all reduced (P<0.05), GQOLI-74 scores increased obviously (P<0.05) and the levels of serum 5-HT and NPY increased obviously (P<0.05) in the two groups. In 2, 4 and 8 weeks of treatment as well as in 16 weeks after treatment, VAS score in the Fu's subcutaneous needling therapy group was lower than that in the medication group (P<0.05), GQOLI-74 score and the levels of serum 5-HT and NPY were higher than those in the medication group (P<0.05). During the treatment and in 16 weeks after treatment, the difference was not significant in the incidence of adverse reaction between the two groups (P>0.05). Fu's subcutaneous needling therapy can relieve intercostal pain and improves the quality of life in the patients after surgery for OVCF.

Development of a highly sensitive chemiluminescence immunoassay for quantification of aggrecanase-generated ARGS aggrecan fragments in serum

ObjectiveCartilage degradation is a hallmark of osteoarthritis (OA). Aggrecan, a major proteoglycan of articular cartilage extracellular matrix (ECM), is degraded by ADAMTS-5 resulting in the release of ARGS-G2 fragments to synovial fluid and circulation. The aim was to quantify ARGS-G2 in the serum of OA patients using the huARGS immunoassay. MethodsThe immunoassay was produced under GMP conditions and the technical performance was assessed. The biological relevance of the immunoassay was assessed in the conditioned media from a bovine full-depth cartilage explant (BEX) model. The diurnal and inter-day variations of ARGS levels were evaluated in OA patients’ serum. Post-hoc analysis of huARGS was conducted in a sub-cohort of a phase III OA trial testing the safety and efficacy of oral salmon calcitonin. ResultsTechnical performance: huARGS demonstrated good technical performance. Biological relevance: ARGS release was induced by inflamatory facotrs stimulation compared to the vehicle group, reaching a peak at day 3 and gradually decreasing to base level at day 12. The ARGS release was suppressed by the addition of the ADAMTS-4/-5 activation inhibitor. Biological variation: No significant diurnal or inter-day effect was found. Phase III clinical trial: The participants in the lowest group (Q1) of baseline huARGS levels were more likely to progress radiographically than the highest group (Q4): OR 3.38[0.81-14.02]. ConclusionsThe huARGS shows good technical performance and low biological variation. It has the potential to aid drug development in various stages, both as a PD biomarker and identifying progressors who might be likely to respond to an OA drug.

Role of Electrostatic Interactions in Calcitonin Prefibrillar Oligomer-Induced Amyloid Neurotoxicity and Protective Effect of Neuraminidase.

Salmon calcitonin is a good model for studying amyloid behavior and neurotoxicity. Its slow aggregation rate allows the purification of low molecular weight prefibrillar oligomers, which are the most toxic species. It has been proposed that these species may cause amyloid pore formation in neuronal membranes through contact with negatively charged sialic acid residues of the ganglioside GM1. In particular, it has been proposed that an electrostatic interaction may be responsible for the initial contact between prefibrillar oligomers and GM1 contained in lipid rafts. Based on this evidence, the aim of our work was to investigate whether the neurotoxic action induced by calcitonin prefibrillar oligomers could be counteracted by treatment with neuraminidase, an enzyme that removes sialic acid residues from gangliosides. Therefore, we studied cell viability in HT22 cell lines and evaluated the effects on synaptic transmission and long-term potentiation by in vitro extracellular recordings in mouse hippocampal slices. Our results showed that treatment with neuraminidase alters the surface charges of lipid rafts, preventing interaction between the calcitonin prefibrillar oligomers and GM1, and suggesting that the enzyme, depending on the concentration used, may have a partial or total protective action in terms of cell survival and modulation of synaptic transmission.

A novel serological pharmacodynamic biomarker assessing type II collagen degradation in osteoarthritis patients

Purpose: A major hallmark of osteoarthritis (OA) is the altered cartilage metabolism and turnover. During cartilage erosion, type II collagen is cleaved by matrix metalloproteinases (MMPs) generating new protein fragments, called neo-epitopes. These fragments are released into the circulation and may reflect disease activity, and potentially be utilized for drug development as pharmacodynamic markers. Currently, there is a lack of type II collagen biomarkers reflecting pharmacologic responses to therapeutic interventions in OA. A novel immunoassay detecting a neo-epitope fragment of type II collagen (T2CM), cleaved by MMP-1 and MMP-13, has recently been developed. The assay is shown to be technically robust and highly specific for the T2CM neo-epitope. Studies have indicated that MMP-13 plays a critical role in type II collagen degradation in articular cartilage in OA and hence, T2CM may serve as a potential pharmacodynamic biomarker candidate in OA. The aim of this study was to evaluate the pharmacodynamic property of T2CM in OA patients treated with oral salmon calcitonin compared to placebo. Methods: T2CM levels were assessed in serum samples in a subset of the SMC study, a randomized, double-blind, multi-center, placebo-controlled study where knee OA patients (n=50) received 0.8 mg oral salmon calcitonin treatment twice daily for 24 months and compared to placebo (n=57). Serum samples were collected at baseline and after 1, 6, 12, and 24 months of treatment. Study samples were collected after informed consent and approved by local ethical committee in compliance with the Helsinki declaration of 1997. The statistical analysis included two-way ANOVA with Sidak’s post hoc test for difference between multiple comparisons of T2CM levels between the OA patients receiving salmon calcitonin treatment and placebo. Data were adjusted for differences in body mass index (BMI), sex, and age. Results: The mean age of OA patients treated with oral salmon calcitonin was 63.9 years (±SD 6.9) and 34% were female, and mean age of the placebo group was 63.1 years (±SD 6.1) and 45% female. The mean BMI of the treatment group and placebo was 30.2 (SD±5.1) and 27.9 (SD±3.3), respectively. T2CM levels were significantly decreased in OA patients receiving salmon calcitonin compared to the placebo group after 1, 6, and 24 months of treatment (p=0.0285; mean -61.49 ±SD 9.10 vs -33.19 ±SD 8.50, p=0.084; mean -66.35 ±SD 8.90 vs -41.46 ±SD 8.31, p=0.0035; mean -61.73 ±SD 9.49 vs -22.19 ±SD 8.76) (Fig.1). Conclusions: The novel T2CM assay measured significantly decreased levels of T2CM in OA patients treated with salmon calcitonin compared to placebo. This suggests that T2CM may have potential as a pharmacodynamic biomarker for type II collagen degradation. Currently, no disease-modifying drugs exist in OA. A validated pharmacodynamic biomarker for type II collagen degradation could promote the development of new disease-modifying OA drugs targeting cartilage repair or degradation.