Cellular Internalization of Human Calcitonin Derived Peptides in MDCK Monolayers: A Comparative Study with Tat(47-57) and Penetratin(43-58)

PURPOSE: The objective of this study was to evaluate key motif requirements of human calcitonin (hCT)-derived peptides for the permeation through the plasma membrane of MDCK monolayers, as epithelial model. METHODS: Truncated and sequence-modified fluorescent-labeled hCT-derived peptides were synthesized through Fmoc chemistry. Peptide uptake by confluent MDCK was observed by confocal laser scanning microscopy. The cytotoxic effect of the peptides on cellular integrity was followed by LDH release. For direct comparison we covered the cellular uptake of established cell penetrating peptides, Tat(47-57) and penetratin(43-58). RESULTS: Truncated sequences of hCT, from hCT(9-32) to hCT(18-32), penetrated the plasma membrane and demonstrated a sectoral, punctuated cytoplasmic distribution. The uptake process appeared to be temperature-, time- and concentration-dependent. Amino acid modifications of hCT(18-32) indicated that both the proline in position 23 and the positive charge of lysine in position 18 are crucial for peptide uptake. The reverse sequence hCT(32-18) did not penetrate the membrane, indicating the importance of sequence orientation. Tat(47-57) and penetratin(43-58) showed a similar punctuated cytoplasmic distribution in MDCK and HeLa cell lines. No relevant toxicity was observed. CONCLUSIONS: Selected hCT-derived peptides have cell penetrating properties. The uptake mechanism seems to involve an endocytic pathway.     

Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.     

Calcitonin-derived peptide carriers: mechanisms and application

Among the family of the so-called cell-penetrating peptides (CPP) sequences derived from the native peptide hormone human calcitonin (hCT) have recently proven to translocate different bioactive molecules across cellular membranes. Herein, we give an extensive summary of the development of hCT-derived carrier peptides, beginning with the therapeutic nasal administration of full-length hCT. Hence, N-terminally truncated hCT fragments were investigated and subsequently optimised to extend their field of application. The latest generation of hCT-derived carrier peptides are highly effective, branched peptides. The current state of the art is reviewed concerning the structural requirements, mechanistic assumptions and metabolic features of these peptides as well as experiments proving their excellent carrier potential.     

Side-chain lactam-bridge conformational constraints differentiate the activities of salmon and human calcitonins and reveal a new design concept for potent calcitonin analogues

We have recently reported the potent hypocalcemic effects of side-chain lactam-bridged analogues of human calcitonin (hCT) (Kapurniotu, A.; et al. Eur. J. Biochem. 1999, 265, 606-618). To extend these studies, we have now synthesized a new series of (Asp(17), Lys(21)) and (Asp(17), Orn(21)) side-chain bridged salmon calcitonin (sCT) and hCT analogues. The affinities of these analogues for the human calcitonin receptor, hCTR(I1)(-), and for rat-brain membrane receptors were assayed in competitive binding assays, and agonist potencies at the hCTR(I1)(-) receptors were assessed, using a cAMP-responsive gene-reporter assay. The bridged sCT analogues had activities similar to sCT itself. In contrast, an (Asp(17), Orn(21)) side-chain bridged hCT analogue, cyclo(17-21)-[Nle(8), Phe(12), Asp(17), Orn,(21) Tyr(22))-hCT, was 80 and 450 times more active than hCT in the hCTR(I1)(-) and rat-brain receptor binding assays, respectively, and was 90 times more potent than hCT and 16 times more potent than sCT in initiating receptor signaling. An uncyclized, isosteric analogue of this peptide was also more potent than hCT, demonstrating that the cyclization constraint and these single-residue substitutions enhance the activities of hCT in an additive fashion. This study demonstrates that the potency-enhancing effects of lactam-bridge constraints at hCT residues 17-21 are not transferable to sCT. We also show that, in comparison to the hCT analogues, sCT and its analogues are less potent agonists than expected from their hCTR(I1)(-) affinities. This suggests that it may be possible to preserve the efficient signal transduction of hCT while introducing additional receptor affinity-enhancing elements from sCT into our potent lactam-bridged hCT analogue, thereby creating new super-potent, hCT-based agonists.     

The use of cell-penetrating peptides for drug delivery

In the past decade, several peptides that can translocate cell membranes have been identified. Some of these peptides, which can be divided into different families, have short amino acid sequences (10-27 residues in length) and enter the cell by a receptor-independent mechanism. Furthermore, these peptides are capable of internalizing hydrophilic cargoes. Although the detailed mechanism by which these molecules enter cells is poorly understood, their ability to traverse the membrane into the cytoplasm has provided a new and powerful biological tool for transporting drugs across cell membranes.     

Degradation and Aggregation of Human Calcitonin In Vitro

Purpose. To investigate the degradation of human calcitonin (hCT) by enzymes or mucosa from different gastrointestinal (GI) compartments and evaluate the stabilization effect of a synthetic ionizable copolymer on the stability of hCT in an aqueous solution. These data are a prerequisite for the development of a hydrogel based colon-specific hCT delivery system. Methods. Luminal and brush border membrane (BBM) enzymes from the colon and small intestine (SI) of the rabbit were isolated and their enzymatic activity toward hCT in vitro was evaluated. Human fecalase was used to mimic the luminal enzymatic activity in the human colon and its degradation ability was assessed. Excised intact rabbit intestinal tissues from both the colon and the SI were used to study the degradation patterns of hCT by intact mucosa. Detection of intact human calcitonin was performed using gradient elution, reverse phase high-pressure liquid chromatography (RP-HPLC). The structure of the hCT fragments was determined by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) analysis. UV/VIS and fluorescence spectroscopy methods were used to evaluate the influence of a copolymer, possessing the same structure as the primary chains in hydrogels degradable in the colon, on the fibrillation process of hCT. Results. In vitro results showed that isolated luminal enzymes and BBM enzymes from the SI were more potent in degrading intact hCT, as expected. Moreover, BBM enzymes were far more abundant in the SI than in the colon. Compared with rabbit colonic luminal enzymes, the degradation potency of human fecalase was further abated. Intact mucosal studies revealed extensive degradation by the SI mucosa but not by the colonic mucosa. The primary structures of the peptide fragments were identified by MALDI-TOF MS analysis. Fibrillation studies of hCT indicated that acrylic acid-containing polymeric materials were able to decrease the aggregation of hCT in aqueous solutions. Conclusions. Reduced proteolytic activity suggests that the colon is an advantageous site for peptide delivery. The structures of hCT degradation products were identified and the participation of particular enzymes in the degradation process was suggested. In addition, it was determined that an acrylic acid-containing copolymer improved the physical stability of hCT in aqueous solution.     

In vitro metabolism of human and salmon calcitonins in rat liver and kidney evaluated by liquid chromatography-tandem mass spectrometry

1. Using LC-MS and LC-MS/MS, an in vitro study was conducted on the metabolism of human calcitonin (hCT) and salmon calcitonin (sCT) in rat liver and kidney to determine the rates of metabolism and the positions of hydrolytic cleavage in both peptides. 2. In lysosomal fractions of rat liver and kidney, hCT was degraded 9-12 times faster than sCT. Many metabolites of hCT were produced in the lysosomal fractions, whereas the metabolites of sCT were scarcely found. 3. In the case of the cytosolic fractions, three positions of initial endoproteolytic cleavage were found in hCT, leading to the production of many peptide fragments via subsequent exoproteolytic metabolism. The initial cleavage position of sCT could not be identified precisely, but it was postulated that the rate-determining step in the metabolism of sCT is the endoproteolytic hydrolysis. 4. The studies using pure proteases and protease inhibitors indicated that the metabolism of calcitonins proceeds by initial endoproteolytic cleavage and subsequent exoproteolytic digestion, catalysed by an aspartate-protease in lysosomes and by a metalloprotease and cysteine-protease in combination in the cytosol. 5. The result suggested that the higher in vivo pharmacological activity of sCT compared with that of hCT may be due to a slower metabolism of the former.     

Intracolonic bioavailability of human calcitonin in man

Summary Human calcitonin (hCT) injected into the lumen of the descending colon of normal human subjects was absorbed within minutes and could be recognized intact in plasma as shown by RIA in combination with reverse-phase HPLC.The absorption was low and variable, with bioavailabilities ranging from 0.01% to 2.7% relative to intravenously administered hCT (area under the concentrationtime curve). With intravenous hCT serum calcium was lowered and the fractional urinary excretion of calcium, phosphorus, sodium and chloride was significantly stimulated. With the intracolonic hCT, the fractional urinary excretions of calcium, sodium and chloride were also marginally stimulated relative to intracolonic vehicle (placebo).In conclusion, hCT is absorbed intact from the colon, but the bioavailability is low and highly variable.     

Metabolic Cleavage and Translocation Efficiency of Selected Cell Penetrating Peptides: A Comparative Study with Epithelial Cell Cultures

We investigated the metabolic stability of four cell penetrating peptides (CPPs), namely SAP, hCT(9-32)-br, [Palpha] and [Pbeta], when in contact with either subconfluent HeLa, confluent MDCK or Calu-3 epithelial cell cultures. Additionally, through analysis of their cellular translocation efficiency, we evaluated possible relations between metabolic stability and translocation efficiency. Metabolic degradation kinetics and resulting metabolites were assessed using RP-HPLC and MALDI-TOF mass spectrometry. Translocation efficiencies were determined using fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Between HeLa, MDCK and Calu-3 we found the levels of proteolytic activities to be highly variable. However, for each peptide, the individual degradation patterns were quite similar. The metabolic stability of the investigated CPPs was in the order of CF-SAP = CF-hCT(9-32)-br > [Pbeta]-IAF > [Palpha] and we identified specific cleavage sites for each of the four peptides. Throughout, we observed higher translocation efficiencies into HeLa cells as compared to MDCK and Calu-3, corresponding to the lower state of differentiation of HeLa cell cultures. No direct relation between metabolic stability and translocation efficiency was found, indicating that metabolic stability in general is not a main limiting factor for efficient cellular translocation. Nevertheless, translocation of individual CPPs may be improved by structural modifications aiming at increased metabolic stability.     

The role of hypoxia inducible factor-1α in the increased MMP-2 and MMP-9 production by human monocytes exposed to nickel nanoparticles

Nickel is an important economic commodity, but it can cause skin sensitization and may cause lung diseases such as lung fibrosis, pneumonitis, bronchial asthma and lung cancer. With development of nanotechnology, nano-sized nickel (Nano-Ni) and nano-sized titanium dioxide (Nano-TiO?) particles have been developed and produced for many years with new formulations and surface properties to meet novel demands. Our previous studies have shown that Nano-Ni instilled into rat lungs caused a greater inflammatory response as compared with standard-sized nickel (5 μm) at equivalent mass concentrations. Nano-Ni caused a persistent high level of inflammation in lungs even at low doses. Recently, several studies have shown that nanoparticles can translocate from the lungs to the circulatory system. To evaluate the potential systemic effects of metal nanoparticles, we compared the effects of Nano-Ni and Nano-TiO? on matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) gene expression and activity. Our results showed that exposure of human monocyte U937 to Nano-Ni caused dose- and time- dependent increase in MMP-2 and MMP-9 mRNA expression and pro-MMP-2 and pro-MMP-9 activity, but Nano-TiO? did not. Nano-Ni also caused dose- and time- related increase in tissue inhibitor of metalloproteinases 1 (TIMP-1), but Nano-TiO? did not. To determine the potential mechanisms involved, we measured the expression of hypoxia inducible factor 1α (HIF-1α) in U937 cells exposed to Nano-Ni and Nano-TiO?. Our results showed that exposure to Nano-Ni caused HIF-1α accumulation in the nucleus. Furthermore, pre-treatment of U937 cells with heat shock protein 90 (Hsp90) inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), prior to exposure to Nano-Ni significantly abolished Nano-Ni-induced MMP-2 and MMP-9 mRNA upregulation and increased pro-MMP-2 and pro-MMP-9 activity. Our results suggest that HIF-1α accumulation may be involved in the increased MMP-2 and MMP-9 production in U937 cells exposed to Nano-Ni.     

Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

Purpose Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a γ-zein related sequence. Methods Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. Results We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. Conclusion Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery. This work was supported by the Commission of the European Union (EU project on Quality of Life and Management of Living Resources, Project No. QLK2-CT-2001-01451.     

Relationships between hypocalcaemic and anorectic effect of calcitonin in the rat

Salmon calcitonin (sCT, 2 and 20 U/kg), porcine calcitonin (pCT, 20 and 40 U/kg) and human calcitonin (hCT, 20 and 40 U/kg) were injected subcutaneously to rats trained to eat their food during two hours each day. Food intake and serum Ca++ concentrations were determined at the end of 2h-feeding period. A long lasting anorectic effect was observed for 20 U/kg of sCT with a parallelism between hypocalcaemia and anorexia in the first 8 hours after treatment; on the contrary, rats continued to eat less than controls in the following hours when their serum Ca++ concentrations had risen to normal or even higher levels. As regards pCT and hCT, it was shown that these peptides reduced significantly meal size only for 1-2 hours when serum Ca++ levels were at their lowest levels for these peptides.     

Patent Evaluation: New Stable Forms of Human Calcitonin

Summary

Novelty: A new stable aqueous solution of human calcitonin (hCT) is disclosed which is said to remain free of hCT fibrils for at least twenty-four hours at 25°C. hCT is a potent hormone for the treatment of Paget's disease, hypercalcaemia and postmenopausal osteoporosis.

Biology: Extrapolated time of fibrillation (in years) is given for hCT concentrations of 3.3 and 6.6 mg/ml in various formulations. The stability of these formulations is said to be in the range twenty-six days to one hundred and eleven years; the latter for a mixture of 0.5% methyl cellulose, 0.01% benzethonium chloride and 0.001% acetic acid containing 3.3 mg/ml hCT.

Chemistry: A typical composition consists of dilute acetic acid at various concentrations, added to hCT powder and solubilized. The resulting solutions contain 5 mg/ml hCT at acetic acid concentrations of 0.0001%, 0.01%, 0.15 and 1.0%, and are said to be stable and perfectly clear after eight months. In vivo experiments appear to show that fresh hCT solutions and solutions stored for forty days have similar biological activity. Absorption spectra and HPLC experiments appear to show that there is no change in hCT properties as a result of storing hCT in aqueous solutions.     

Relationship between delta-9-tetrahydrocannabinol-induced arachidonic acid release and secretagogue-evoked phosphoinositide breakdown and Ca2+ mobilization of exocrine pancreas

We have previously shown that addition of exogenous arachidonic acid to pancreatic acinar cells inhibits the incorporation of myo-[3H]inositol into membrane phosphoinositides and causes a reduction in the steady state levels of [32P]phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). In the present study, delta-9-tetrahydrocannabinol (THC) was utilized to raise endogenous levels of arachidonic acid. In acinar cells simultaneously prelabeled with [3H]arachidonic acid and [32P]Pi, THC (1-20 microM) produced a concentration-dependent increase in free [3H]arachidonic acid release and a reduction in the steady state levels of [32P]Ptd-Ins4,5P2. THC (1-20 microM) also caused a concentration-dependent inhibition of myo-[3H]inositol trisphosphate accumulation, cytoplasmic Ca2+ level, and amylase secretion elicited by 0.1 microM caerulein. The findings that THC (20 microM) was unable to inhibit either the rise in [Ca2+]i elicited by ionomycin, or the secretory response to phorbol myristic acid or ionomycin, indicate that THC exerts a selective inhibitory effect on the phosphoinositide messenger system. These results support the postulate that endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover in exocrine pancreas.     

Cell death with atypical features induced by the novel antitumoral drug CHS 828, in human U-937 GTB cells

N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828), with promising antitumoral effects in vitro and in vivo, is currently in clinical Phase I and II studies. Its exact mechanism of action is unclear, but previous studies indicate that CHS 828 induces a controlled, delayed mode of cell death. The characteristics of the cell death process were investigated in vitro in the apoptosis-prone cell line U-937 GTB. Mitochondria showed hyperpolarization at 24 to 32 h and a subsequent late disruption of mitochondria membrane potential (Deltapsi(m)). Between 44 and 72 h of CHS 828 exposure, there was an increasing frequency of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) positive cells indicative of apoptosis, but caspase-3 was only modestly increased and caspases-8 and -9 showed no activation upon CHS 828 exposure. Furthermore, the morphology of exposed cells did not conform to classical apoptosis, and viability and morphology were unaffected by inhibition of caspases. Thus, CHS 828 induces several unexpected features in this system, suggesting a potentially novel mechanism of action.     

The role of hypoxia inducible factor-1α in the increased MMP-2 and MMP-9 production by human monocytes exposed to nickel nanoparticles

Abstract

Nickel is an important economic commodity, but it can cause skin sensitization and may cause lung diseases such as lung fibrosis, pneumonitis, bronchial asthma and lung cancer. With development of nanotechnology, nano-sized nickel (Nano-Ni) and nano-sized titanium dioxide (Nano-TiO₂) particles have been developed and produced for many years with new formulations and surface properties to meet novel demands. Our previous studies have shown that Nano-Ni instilled into rat lungs caused a greater inflammatory response as compared with standard-sized nickel (5 μm) at equivalent mass concentrations. Nano-Ni caused a persistent high level of inflammation in lungs even at low doses. Recently, several studies have shown that nanoparticles can translocate from the lungs to the circulatory system. To evaluate the potential systemic effects of metal nanoparticles, we compared the effects of Nano-Ni and Nano-TiO₂ on matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) gene expression and activity. Our results showed that exposure of human monocyte U937 to Nano-Ni caused dose- and time- dependent increase in MMP-2 and MMP-9 mRNA expression and pro-MMP-2 and pro-MMP-9 activity, but Nano-TiO₂ did not. Nano-Ni also caused dose- and time- related increase in tissue inhibitor of metalloproteinases 1 (TIMP-1), but Nano-TiO₂ did not. To determine the potential mechanisms involved, we measured the expression of hypoxia inducible factor 1α (HIF-1α) in U937 cells exposed to Nano-Ni and Nano-TiO₂. Our results showed that exposure to Nano-Ni caused HIF-1α accumulation in the nucleus. Furthermore, pre-treatment of U937 cells with heat shock protein 90 (Hsp90) inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), prior to exposure to Nano-Ni significantly abolished Nano-Ni-induced MMP-2 and MMP-9 mRNA upregulation and increased pro-MMP-2 and pro-MMP-9 activity. Our results suggest that HIF-1α accumulation may be involved in the increased MMP-2 and MMP-9 production in U937 cells exposed to Nano-Ni.     

新型降钙素的分子设计

以人和鲑鱼降钙素为先导物 ,根据多肽类药物设计原理 ,借助多肽蛋白质计算机分析软件辅助设计了一种新的人降钙素类似物 ,简称新型降钙素 .计算机分析结果表明 :该新型降钙素等电点提高 (pI 8.7) ,C 末端亲水性增加 ,抗原表位分析图谱与人降钙素的相同 .利用基因工程法制备新型降钙素 ,并经Westernblotting分析表明它与人降钙素分子的抗原性相似     

Superior local tolerability of human versus salmon calcitonin preparations in young healthy volunteers

Summary Possible local and systemic adverse effects following administration of salmon (sCT) and human (hCT) calcitonin (CT) have been evaluated in a double-blind, within-subject, comparative trial in 30 young, healthy volunteers. Each subject received 0.25 and 0.5 mg hCT and 100 IU sCT s.c.. Adverse effects and hypocalcaemia were recorded 1, 3 and 6 h after each injection.Significantly fewer local adverse reactions were observed after hCT (20 or 33%) than after sCT (80%), possibly due to the different vehicles employed (mannitol solution and acetic acid).The most frequent systemic adverse effects were gastrointestinal (nausea, vomiting), which occurred in 80% after 1 h, independently of the CT — preparation used. Hypocalcaemic changes were generally small and lasted longer after sCT.It is concluded that the hCT preparations were better tolerated locally than sCT in young, healthy volunteers, and that there were no differences in the systemic side effects or hypocalcaemic activity.     

Proteolysis of Human Calcitonin in Excised Bovine Nasal Mucosa: Elucidation of the Metabolic Pathway by Liquid Secondary lonization Mass Spectrometry (LSIMS) and Matrix Assisted Laser Desorption lonization Mass Spectrometry (MALDI)

Purpose. Two calcitonins, i.e. human calcitonin (hCT) and, for comparison, salmon calcitonin (sCT), were chosen as peptide models to investigate nasal mucosal metabolism. Methods. The susceptibility of hCT and sCT to nasal mucosal enzymes was assessed by in-and-out reflection kinetics experiments in an in vitro model based on the use of freshly excised bovine nasal mucosa, with the mucosal surface of the mucosa facing the peptide solution. The kinetics of CT degradation in the bulk solution was monitored by HPLC. Peptide sequences of the main nasal metabolites of hCT were analyzed by using both liquid secondary ionization mass spectrometry (LSIMS), following HPLC fractionation of the metabolites, and matrix-assisted laser desorption ionization mass (MALDI) spectrometry. For sCT, the molecular weights of two major metabolites were determined by LC-MS with electrospray ionization. Results. Both CTs were readily metabolized by nasal mucosal enzymes. In the concentration range studied metabolic rates were higher with hCT than with sCT. Presence of endopeptidase activities in the nasal mucosa was crucial, cleaving both calcitonins in the central domain of the molecules. Conclusions. Typically, initial metabolic cleavage of hCT in nasal mucosa is due to both chymotryptic- and tryptic-like endopeptidases. The subsequent metabolic break-down follows the sequential pattern of aminopeptidase activity. Tryptic endopeptidase activity is characteristic of nasal sCT cleavage.