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 carrier peptides

The recent discovery of carrier peptides offers new opportunities to translocate several bioactive molecules into the cytoplasm. Previous studies have shown that human calcitonin (hCT) and selected C-terminal sequences translocate in nasal epithelium. Moreover, the hCT(9-32) fragment was found to internalize efficiently a number of substances like fluorophores, nucleic acids or the enhanced green fluorescent protein (EGFP). In order to understand the uptake mechanism interactions of hCT(9-32) with membrane models of different lipid compositions have been investigated. From these studies it was possible to shed light on the conformational state of the peptide in the presence of membrane-like conditions. Further insight into the translocation mechanism was provided by fluorescence microscopy of truncated sequences of hCT that were shown to penetrate the plasma membrane and to distribute in a sectoral, punctuated pattern supporting an endocytotic internalization pathway as previously suggested.     

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.     

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.     

Enhanced physical stability of human calcitonin after methionine oxidation

Calcitonin is a blood-calcium-lowering peptide, present in different species, which inhibits the resorption of bone by osteoclasts. Human calcitonin (hCT) is one of the few calcitonin peptides, which contains a methionine residue; this residue is in position 8. Methionines are known to be readily oxidized to sulfoxides both in vivo and in vitro. The current work describes the effect of methionine oxidation on the physical stability of hCT. Aggregation kinetics of human calcitonin were studied at different pH values by intrinsic fluorescence spectroscopy, turbidity at 350 nm, microscopy analyses, Nile Red, and 1,8-ANS fluorescence emission. In all the experiments, methionine oxidation reduced the aggregation rate of human calcitonin. The effect of methionine oxidation was independent of pH. Fluorescence lifetime data also showed that the conformation of hCT in the aggregated state can be influenced by methionine oxidation. A hypothesis for the enhanced physical stability of oxidized hCT is presented and discussed.     

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.     

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.     

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.     

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     

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.     

The short term effect of peripherally administered brain-gut peptides on gastric acid secretion in rats.

Certain brain gut-peptides are known to either stimulate or inhibit gastric acid secretion in several species after direct injection into the central nervous system. However there is inconsistency of published results on the gastric acid secretory response to some of these peptides after peripheral administration in different experimental systems. Seven peptides, namely neurotensin (NT), substance P, cholecystokinin (CCK), thyrotropin releasing hormone (TRH), human calcitonin (hCT), rat calcitonin-gene-related peptide (rCGRP) and bombesin, all known to modulate gastric acid secretion after central administration, were initially screened for activity after peripheral (subcutaneous) injection of 10 micrograms/kg body weight in a single rat model. Peptides showing an effect were retested at lower doses. Despite the inherent variability of the gastric acid secretory response in the non-anaesthetized pylorus ligated rat, a standardized experimental design confirmed that reproducible and statistically valid results could be obtained. The technical feasibility of using a one hour collection period as might be appropriate for short acting peptides was demonstrated by the significant dose dependent inhibitory activity of salmon calcitonin. In this model, NT and substance P had no significant effect on either volume or concentration of acid secreted, CCK showed a slight stimulation of acid output, and TRH, hCT, rCGRP and bombesin all inhibited acid output; CGRP and bombesin were active at 10 and 100-fold lower doses. The potent and inhibitory activity of bombesin in this system is in disagreement with other publications reporting no effect or variable stimulatory effect in rats. Time and dose dependent responses in our rat system indicate that this apparent discrepancy may be explained by the short duration of action of bombesin.     

Autoradiographic localization of human calcitonin sensitive binding sites in rat brain.

Using 125I-salmon calcitonin (sCT) as a ligand, in vitro autoradiography of rat brain outlined specific anatomical localization of human calcitonin (hCT) sensitive binding sites. The results presented herein show that there are hCT sensitive binding sites in the ventral part of the lateral septum among the sCT specific binding sites distributed throughout the diencephalon.     

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.     

Conformational and topological requirements of cell-permeable peptide function

Cell-permeable peptide import recently was developed to deliver synthetic peptides into living cells for studying intracellular protein functions. This import process is mediated by an N-terminal carrier sequence which is the hydrophobic region of a signal peptide. In this study, the conformational consequence of the interaction of cell-permeable peptides with different mimetic membrane environments was investigated by circular dichroism analysis. We showed that cell-permeable peptides adopted a-helical structures in sodium dodecyl sulfate (SDS) micelles or aqueous trifluoroethanol (TFE). The potency of these peptides in forming helical structures is higher in an amphiphilic environment (SDS) than in a hydrophobic environment (TFE), suggesting that some hydrophilic molecules associated with the cell membrane may be involved in peptide import. We also studied topological requirements of cell-permeable peptide function. We demonstrated that peptides containing the carrier sequence in their C-termini can also be imported into cells efficiently. This important discovery can avoid repetitious synthesis of the membrane-translocating sequence for peptides with different functional cargoes and is potentially useful for developing a cell-permeable peptide library. Finally, we showed that, when a retro version of the carrier sequence was used, the peptide lost its translocating ability despite retaining a high content of a-helical structure in mimetic membrane environments. This suggests that the propensity of peptides to adopt a helical conformation is required but not sufficient for cellular import and that other structural factors such as the side-chain topology of the carrier sequence are also important. Our studies together contribute to the more rational design of useful cell-permeable peptides. ^© Munksgaard 1998.     

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.     

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.     

The role of chitosan on oral delivery of peptide-loaded nanoparticle formulation

Therapeutic peptides are conventionally administered via subcutaneous injection. Chitosan-based nanoparticles are gaining increased attention for their ability to serve as a carrier for oral delivery of peptides and vaccination. They offered superior biocompatibiltiy, controlled drug release profile and facilitated gastrointestinal (GI) absorption. The encapsulated peptides can withstand enzymatic degradation and various pH. Chitosan-based nanoparticles can also be modified by ligand conjugation to the surface of nanoparticle for transcellular absorption and specific-targeted delivery of macromolecules to the tissue of interest. Current research suggests that chitosan-based nanoparticles can deliver therapeutic peptide for the treatment of several medical conditions such as diabetes, bacterial infection and cancer. This review summarises the role of chitosan in oral nanoparticle delivery and identifies the clinical application of peptide-loaded chitosan-based nanoparticles.     

The absorption of human calcitonin from the transverse colon of man

Patients with a loop stoma were used to provide direct access to the transverse colon. The ease of delivery offers the chance to study absorption of human calcitonin (hCT) in a defined region of the large intestine difficult to access in healthy volunteers; i.v. infused hCT elicited a standard pharmacokinetic profile in eight loop stoma patients showing a biphasic elimination with half-lives of 11.5 ± 0.8 min and 33.7 ± 1.8 min. hCT administered via the loop stoma was absorbed across the transverse colonic mucosa in low amounts. The 10-mg dose achieved a mean maximum plasma concentration of 1242 ± 346 pg ml⁻¹, after 5–10 min with an absolute bioavailability of 0.22 ± 0.06%. We conclude that the transverse colon is a better site for the absorption of human calcitonin than the more distal regions of the colon. This could be a function of the transverse colonic epithelium. Alternatively, it could be due to the reduced levels of luminal debris and bacterial colonisation in the stoma patients compared with the previous studies carried out in the distal colon of healthy volunteers.     

Cell penetrating elastin-like polypeptides for therapeutic peptide delivery

Current treatment of solid tumors is limited by side effects that result from the non-specific delivery of drugs to the tumor site. Alternative targeted therapeutic approaches for localized tumors would significantly reduce systemic toxicity. Peptide therapeutics are a promising new strategy for targeted cancer therapy because of the ease of peptide design and the specificity of peptides for their intracellular molecular targets. However, the utility of peptides is limited by their poor pharmacokinetic parameters and poor tissue and cellular membrane permeability in vivo. This review article summarizes the development of elastin-like polypeptide (ELP) as a potential carrier for thermally targeted delivery of therapeutic peptides (TP), and the use of cell penetrating peptides (CPP) to enhance the intracellular delivery of the ELP-fused TPs. CPP-fused ELPs have been used to deliver a peptide inhibitor of c-Myc function and a peptide mimetic of p21 in several cancer models in vitro, and both polypeptides are currently yielding promising results in in vivo models of breast and brain cancer.