Effect of Methionine Sulfoxide on the Synthesis and Purification of Aggregation-Prone Peptides.

A two-step synthesis for methionine-containing hydrophobic and/or aggregation-prone peptides is presented that takes advantage of the reversibility of methionine oxidation. The use of polar methionine sulfoxide as a building block in solid-phase peptide synthesis improves the synthesis quality and yields the crude peptide, with significantly improved solubility compared to the reduced species. This facilitates the otherwise often laborious peptide purification by high-performance liquid chromatography. The subsequent reduction proceeds quantitatively. This approach has been optimised with the methionine-rich Tar-DNA-binding protein 43 (307-347), but is also more generally applicable, as demonstrated by the syntheses of human calcitonin and two aggregation-prone peptides from the human prion protein.

Synthesis, characterization and evaluation of bone targeting salmon calcitonin analogs in normal and osteoporotic rats.

In order to assess the therapeutic efficacy of an antiresorptive drug with imparted bone targeting potential using bisphosphonate (BP) conjugation and an improved pharmacokinetic profile using PEGylation, we synthesized, characterized and evaluated in vivo efficacy of bone-targeting PEGylated salmon calcitonin (sCT) analog (sCT-PEG-BP). sCT-PEG-BP was compared with non-PEGylated bone targeting sCT analog (sCT-BP) and unmodified, commercially available sCT. sCT-PEG-BP conjugates were characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis. The effect of PEG-BP or BP upon sCT secondary structure was examined by Circular Dichroism and sCT-PEG-BP was evaluated for in vitro bone mineral Hydroxyapatite (HA) binding ability and calcium salts specificity using a binding assay for bone HA and several calcium salts. Anti-calcitonin antibody binding ability of these analogs was determined using enzyme-linked immunosorbent assay (ELISA), by reacting bone targeting sCT analogs with calcium phosphate coated Osteologic® plates and detecting the bound sCT using anti-sCT antibody. Potential cytotoxicity of these compounds was evaluated in monocytic RAW 264.7 cells, and sCT bioactivity was evaluated using an in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells. Finally, in vivo efficacy of each compound was evaluated by determining the plasma levels of calcium after s.c. administration in normal rats, and in a rat model of Osteoporosis, secondary to ovariectomy (OVX). In vivo micro-computed tomography (micro-CT) was used to temporally map and quantify alterations in bone volume and bone mineral density (BMD) in the same animals at 1, 4, 8 and 12 weeks after OVX surgery. Sixteen 6 week old virgin female rats underwent OVX surgery followed by the daily s.c. injection of 2.5IU/kg/day sCT or equivalent analogs, and compared to four sham-operated, placebo treated control rats. Our results showed the chemical coupling of PEG-BP or BP to sCT altered its secondary structure without altering its antibody binding ability. sCT analogs retained strong sCT bioactivity, were non-toxic to RAW 264.7 cells in culture and elicited a comparable hypocalcemic effect to that of unmodified sCT in normal rats. Compared to marketed unmodified sCT, sCT-PEG-BP showed significantly improved efficacy in terms of preserving bone volume, BMD and trabecular micro-architecture in osteoporotic rats at the initial dose tested. Bisphosphonate-mediated targeting of PEGylated sCT to bone represents a new class of targeted antiresorptive compounds that has not previously been attempted.

Design, synthesis, characterization and in-vivo activity of a novel salmon calcitonin conjugate containing a novel PEG-lipid moiety.

OBJECTIVES: The aim of the study was to explore (1) the synthesis of a novel poly(ethylene glycol) modified lipid (PEG-lipid, PL) containing a chemically active tri-block linker, epsilon-maleimido lysine (Mal), and its conjugation with salmon calcitonin (sCT), and (2) the biophysical properties and activity of the resulting conjugate, Mal-PL-sCT, relative to the control, 2PEG-Mal-sCT, which comprises sCT conjugated with alpha-palmitoyl-N-epsilon-maleimido-L-lysine at cysteine 1 and cysteine 7, and PEG moieties at lysine 11 and lysine 18 via a conventional stepwise method. METHODS: The PEG-lipid was obtained by condensing palmitic acid derivative of epsilon-maleimido lysine with methoxy poly(ethylene glycol) amine. Under reductive conditions, the PEG-lipid readily reacted with sCT to yield the resultant compound, Mal-PL-sCT. KEY FINDINGS: Dynamic light scattering analyses suggested that Mal-PL-sCT and 2PEG-Mal-sCT exhibited robust helical structures with a high tendency to aggregate in water. Both compounds were more stable against intestinal degradation than sCT, although Mal-PL-sCT was less stable than 2PEG-Mal-sCT. However, 2PEG-Mal-sCT did not possess hypocalcaemic activity while Mal-PL-sCT retained the hypocalcaemic activity of sCT when it was subcutaneously injected in the rat model. Multiple functional groups may be conjugated to a peptide via a tri-block linker without the risk of obliterating the intrinsic bioactivity of the peptide. CONCLUSIONS: The resultant novel PEG-lipid has a potential role to optimize protein and peptide delivery.

Characterization of the adrenomedullin receptor acting as the target of a new radiopharmaceutical biomolecule for lung imaging.

Direct labeling of linear adrenomedullin (AM) with (99m)Tc ([(99m)Tc]AM) displayed excellent selectivity for imaging the pulmonary circulation system in dogs. Hence, we investigated this particular selectivity and characterized the binding sites found in dog lungs. AM and other peptides belonging to the calcitonin peptide family, including calcitonin-gene related peptide (CGRP), adrenomedullin-2 (AM2), amylin and pro-adrenomedullin N-terminal peptide (PAMP), were prepared by solid-phase peptide synthesis. Receptor binding assays were performed by using [(125)I]AM as a radioligand on dog lung homogenates. It was found that AM bound with potent affinity, displaying in fact a high and a low affinity binding site. Moreover, competition binding assays using peptide ligands showed the following ranking for displacement: AM>AM(13-52)>CGRP approximately AM2> or =AM(22-52)> or =AM2(16-47)>CGRP(8-37)>amylin approximately PAMP. Thus, these results strongly suggested that the AM binding site found in dog lungs and acting as a clearance receptor is mainly the adrenomedullin AM(1) receptor subtype. The pharmacophores underlying AM(1) binding affinity and specificity were studied by determining the key amino acids, the minimal peptide fragment, and some aspects of the secondary structures. So far, it appeared that the C-terminal segment of human AM is an essential feature for binding. Also, the alpha-helix secondary structure found in the AM molecule would facilitate the ligand recognition process with the AM receptor in dog lungs. Our results demonstrated that AM or some analogs or fragments could be suitable radiopharmaceutical agents for lung imaging.

Synthesis, characterization and in vivo activity of salmon calcitonin coconjugated with lipid and polyethylene glycol.

An irreversible lipidized salmon calcitonin (sCT) analog, Mal-sCT, was previously shown to have comparable hypocalcemic activity to sCT in vivo. This study reports on the synthesis, characterization and pharmacological activity of novel PEGylated Mal-sCT analogs. Mal-sCT, prepared by conjugating sCT via thio-ether bonds with aqueous-soluble palmitic acid derivative at Cys 1 and Cys 7, was reacted with mPEG-succinimide (mPEG-Suc, 5 kDa). The products were purified and then identified by MALDI-TOF MS and HPLC. Mal-sCT was conjugated with 1 (1PEG-Mal-sCT) or 2 (2PEG-Mal-sCT) PEG chains at Lys 11 and Lys 18, the former being the preferred site of conjugation at higher mPEG-Suc/Mal-sCT ratio. Circular dichroism analysis showed the PEGylated Mal-sCT analogs to possess a robust helical conformation, while size measurement by dynamic light scattering indicated a propensity of the peptides to self-aggregate in aqueous solutions. Both 1PEG-Mal-sCT and 2PEG-Mal-sCT were more stable in rodent intestinal fluids than sCT or Mal-sCT. However, 1PEG-Mal-sCT had comparable hypocalcemic activity to Mal-sCT when injected subcutaneously in the rat, while 2PEG-Mal-sCT was inactive. 1PEG-Mal-sCT was inactive when administered orally in the rat. This study suggested PEGylation of Mal-sCT increased the stability of the lipidized peptide to enzyme degradation, but did not enhance its hypocalcemic activity.

Quantum dot-carrier peptide conjugates suitable for imaging and delivery applications.

We developed multifunctional fluorescent nanoparticles suitable for the nonviral delivery of negatively charged molecules like RNA. Therefore, we incorporated the recently developed branched hCT-derived carrier peptide hCT(18-32)-k7 on the surface of luminescent quantum dots (QDs). Besides detailed characterization of our QD-peptide conjugates concerning stability, toxicity, and uptake mechanism. we used them for efficient RNA delivery into different cell lines. The results of our studies indicate the involvement of more than one endocytotic uptake pathway in the internalization process. Furthermore, we could show that the QD-peptide bioconjugates exhibit no effect on cell viability and possess high stability inside living cells. The efficacy of our newly designed constructs for oligonucleotide drug delivery is highlighted by the successful intracellular transport of Cy-3 labeled RNA. Moreover, by using the chemotherapeutic chloroquine the efficient release of the assemblies out of endosomes was demonstrated. These results prove that our multifunctional platforms are versatile tools for diagnostic and therapeutic imaging purposes applicable for biologically active siRNA or aptamer sequences.

Comparison of reversible and nonreversible aqueous-soluble lipidized conjugates of salmon calcitonin.

Reversible aqueous lipidization (REAL) at the interdisulfide bond has been shown to improve the deliverability of some peptide drugs. Recently, we developed a nonreversible aqueous lipidization method targeted at the interdisulfide bond of salmon calcitonin (sCT). The resultant derivative had comparable hypocalcemic activity to sCT after subcutaneous injection in rats, despite possessing significantly different biophysical properties. The purpose of this study was to conduct a comparative evaluation of the biophysical properties of the reversible aqueous-soluble lipidized sCT (REAL-sCT) and its corresponding nonreversible aqueous-soluble compound (Mal-sCT) with a view to correlate these properties to the bioactivities of the peptides. REAL-sCT and Mal-sCT were successfully synthesized, purified and identified. Both conjugates showed comparable retention times in a C-18 HPLC column, as well as robust helical structures and aggregation behavior in water, although REAL-sCT was shown by dynamic light scattering experiments to form larger aggregates than Mal-sCT in water. The larger particle size of REAL-sCT correlated with its stronger resistance to degradation by intestinal enzymes. Unlike Mal-sCT, REAL-sCT was rapidly converted to sCT in liver juice; however, the regenerated sCT appeared to degrade at a slower rate than unmodified sCT in the liver juice. Compared with sCT, REAL-sCT after subcutaneous injection as an aqueous solution at a dose of 0.15 mg/kg produced a prolonged hypocalcemic activity that lasted at least 24 h in the rat. Using a novel LC-MS/MS method that was developed for this study, we were able to show concomitant increases in REAL-sCT and sCT plasma concentrations with time, the latter prevailing at 10% the molar concentration of the former. In contrast, sCT was not present in the plasma following the subcutaneous injection of Mal-sCT, although a comparable hypocalcemic activity with shorter duration was observed. Oral administration of REAL-sCT and Mal-sCT as aqueous solutions at sCT equivalent dose of 5.0 mg/kg did not produce significant hypocalcemic activity. This study is the first thorough examination of the biophysical characteristics of the corresponding reversible and nonreversible aqueous-soluble lipidized peptide molecules. The results obtained should be useful for the development of the oral formulation of peptide and protein drugs.

Dendritic cell vaccination induces tumor epitope-specific Th1 immune response in medullary thyroid carcinoma.

The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC) and their resistance to classical therapies make it a prime candidate for adoptive immunotherapy. Highly potent antigen-presenting cells, namely dendritic cells (DCs), may serve as an interesting tool for anticancer vaccination. Here we report on the IN VITRO findings of a vaccination trial in five MTC patients, who were treated with a new DC generation protocol consisting of granulocyte-macrophage colony-stimulating factor and interferon-alpha (IFN-DCs). These cells were pulsed with tumor-specific calcitonin and administered twice. In two patients who responded to therapy we found a large increase (in mean 2.9+/-1.9%) of antigen-specific IFN-gamma-secreting CD4+ cells as well as an increase of granzyme B positive CD8+ cells (mean 2.2+/-0.2%) in the peripheral blood. In parallel, a decrease of CD4+/CD25+/FoxP3+ regulatory T cells was seen. Importantly, IN VITRO stimulation of PBMC with 10 different 15mer calcitonin peptides resulted in the identification of two HLA class II epitope regions within the central part of full-length calcitonin. These data were in accordance with the results drawn from the computer-based algorithm epitope prediction software SYFPEITHI. Measurement of different pro- and anti-angiogenic factors did not allow for a distinct outcome of prediction of the treated patients. In summary, we have demonstrated that immunization with IFN-DCs leads to a tumor epitope-specific immune response in MTC patients and may, therefore, represent a promising tool for future vaccination trials.

A synthetic peptide derived from mouse pituitary calcitonin cDNA sequence exhibits potent inhibition of prolactin secretion and prolactin mRNA abundance in primary mouse pituitary cells.

We have shown that gonadotrophs synthesize and secrete immunoreactive calcitonin (CT)-like peptide, and CT is a potent inhibitor of prolactin (PRL) secretion and gene transcription. CT cDNA cloned from LssT2 cells (pit-CT cDNA) exhibits 99% homology with mouse CT cDNA sequence, but exhibits four mismatches in the coding region of CT peptide (347-485 bp) with consequent changes in the amino acids at positions 5 and 17 of mouse CT. We have synthesized a putative 23 amino acid pit-CT peptide based on pit-CT cDNA sequence, and tested its effect on PRL secretion and mRNA abundance in primary mouse pituitary cells. The results suggest that synthetic pit-CT attenuates PRL mRNA abundance and inhibits PRL release from mouse anterior pituitary cells. Moreover, pit-CT is remarkably more potent than salmon (S)CT in attenuating PRL mRNA abundance. These results raise a possibility that this endogenous pituitary peptide may potentially serve as a therapeutic molecule for the treatment of prolactinomas.

Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures.

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.

Chemo-enzymatic synthesis and structure-activity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide.

Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation.

Stability of PEGylated salmon calcitonin in nasal mucosa.

The purpose of this study was to evaluate the stabilization of salmon calcitonin (sCT) by PEGylation in nasal mucosa. Degradation of native sCT in the homogenates of rat nasal mucosa was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The initial cleavage of sCT was due to tryptic-like endopeptidase activity, and the subsequent degradation followed the sequential pattern of aminopeptidase activity. To prepare PEGylated sCT resistant to the proteolytic degradation, the lysine residues susceptible to tryptic activity were selectively PEGylated by controlling reaction pH. The PEGylated sCT showed strong resistance against enzymatic degradation in rat nasal mucosa, with 56-fold prolonged half-life compared with that of native sCT. In the MALDI-TOF MS spectrum, the PEGylated sCT did not show any degradation peak for incubation of 120 min in the homogenates of rat nasal mucosa. The improved stability may be responsible for enhancing nasal absorption of PEGylated sCT.

Convenient synthesis of human calcitonin and its methionine sulfoxide derivative.

The human calcitonin peptide chain was assembled using Fmoc solid-phase peptide synthesis chemistry. The combinations of cleavage Reagent H with trans-[Pt(en)(2)Cl(2)](2+) and Reagents B, K, and R with trans-[Pt(CN)(4)Cl(2)](2-) provide convenient methods for the synthesis of human calcitonin and its methionine sulfoxide derivative; the formation of intramolecular disulfide bonds by the above Pt(IV) oxidants is essentially quantitative.

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.

Effect of carbohydrate structure on biological activity of artificially N-glycosylated eel calcitonin.

To reveal the function of the carbohydrate portion of glycopeptides and glycoproteins, we chemo-enzymatically synthesized artificially N-glycosylated derivatives of eel calcitonin and studied their three-dimensional structure and biological activity. The CD and NMR spectra in trifluoroethanol-H(2)O solution showed that the glycosylation did not change the three-dimensional structure. All the derivatives retained the strong in vivo hypocalcemic activity of calcitonin. However, the relative activity was dependent on the structure of the attached carbohydrate. The single GlcNAc attachment best enhanced the activity, while larger carbohydrates decreased the activity. This relative activity order of compounds could be partly explained by their calcitonin-receptor binding affinity, though the affinity of the GlcNAc derivative did not exceed that of calcitonin. The enhanced hypocalcemic activity of the GlcNAc derivative was explained by its altered biodistribution. The GlcNAc attachment caused calcitonin to escape from the trap at the liver during the early circulation. Thus, the glycosylation was shown to modulate the biological activity of calcitonin depending on the carbohydrate structure without a change in the peptide backbone conformation.

Synthesis of biologically active tritiated amylin and salmon calcitonin analogues.

Amylin is a hormone belonging to the calcitonin protein family of peptides. To facilitate receptor screening studies, alternatively radiolabeled and biologically active amylin and salmon calcitonin analogues were synthesized by reductive methylation. Free amino groups of amylin and salmon calcitonin were methylated by reaction of peptides with formaldehyde and sodium [(3)H]borohydride. Radioactively labeled peptides were purified by size exclusion chromatography followed by HPLC. Analysis by MALDI-TOF mass spectrometry of purified amylin and salmon calcitonin peptides revealed incorporation of both two and four tritiated methyl groups per peptide molecule. Specific activities of 22.6 and 23.2 GBq/mmol were measured for amylin and salmon calcitonin, respectively. Methylation of rat amylin and salmon calcitonin did not affect their biological activities as both retained their potency to inhibit insulin-stimulated glycogen synthesis in isolated rat soleus muscle. The synthesis of these tritiated analogues provides an alternative chemically stable radiolabeled ligand which may be useful in exploring receptor interactions within the calcitonin peptide family.

The peptides of alpha-aminosuberic acid I. New intermediates for synthetic studies.

The paper describes the synthesis of alpha-aminosuberic acid derivatives suitable for the synthesis of peptides. These include Z-, Boc- and Fmoc-protection on the alpha-amino group, benzyl ester, Boc-hydrazide and Z-hydrazide as well as the free carboxylic function in the side chain, and methyl ester, benzyl ester or free alpha-carboxylic group. Their use is demonstrated on the synthesis of the respective derivatives of Asu-Val-Leu. The enzyme catalyzed reaction was successfully used both as a route to L-Asu from the D,L-compound as well as for the direct synthesis of the optically active tripeptide derivative from the Z-D,L-Asu-OH.

Estudio farmacotécnico comparativo de siete especialidades farmacéuticas de calcitonina en aerosol para aplicación intranasal / Pharmacotechnical comparative study of seven finish products of intranasal spray calcitonin´s

Se efectúa un estudio comparativo de siete marcas de calcitonina aerosol intranasal desde el punto de vista farmacotécnico. Para ello se procede al análisis de los siguientes ensayos. pH de la disolución de calcitonina,estudio de posibles alteraciones organolépticas de la disolución en situaciones forzadas de temperatura (40 °C y 55 °C) y acción de la luz solar, volumen de dosificación, tipo de envase y características, alteración del envase sometido a situac iones forzadas de temperatura (40 °C y 55 °C) y acción de la luz solar, en envases de plástico observación del comportamiento a la permeabilidad en condiciones de baja y alta humedad ambiental, características de la válvula de dosificación, características del pulsador, exactitud de dosificación por pulsación (control de descarga), ángulo de pulverización, diámetro de salida del pulverizador, índice del caudal de salida (lectura "Solex'), dosis totales por unidad, uniformidad de masa por dosis, hermeticidad, texto impreso en el envase primario y en el envase secundario (AU)