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Mishandling of the Therapeutic Peptide Glucagon Generates Cytotoxic Amyloidogenic Fibrils

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Abstract

Purpose. Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application.

Methods. Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concen- trations ranging from 1 mg/ml to 80 mg/ml and then aged at 37°C. Amyloidogenic properties were assessed by circular dichroism (CD), electron microscopy (EM), staining with β-sheet-specific dyes, and size-exclusion chromatography (SEC). Cytotoxic characteristics were determined concomitantly.

Results. By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a β-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml.

Conclusions. These results indicated that glucagon was self-associated by its β-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as β-amyloid (Aβ)1-42 and prion protein fragment (PrP)106-126 including conformational change to a β-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.

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Author notes

  1. Satomi Onoue

    Present address: Pfizer Global Research and Development, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo, Aichi, 470-2393, Japan

Authors and Affiliations

  1. Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, 274-8510, Japan

    Satomi Onoue, Sumiko Iwasa & Takehiko Yajima

  2. Health Science Division, Itoham Foods Inc., Moriya, Ibaraki, 302-0104, Japan

    Satomi Onoue & Kazuhisa Kashimoto

  3. Applied Genome Informatics Division, Shizuoka Cancer Center Research Institute, Shizuoka, 411-8777, Japan

    Keiichi Ohshima

  4. Laboratory of Electron Microscopy, Showa University School of Medicine, Shinagawa, Tokyo, 142-8555, Japan

    Kazuhiro Debari & Keitatsu Koh

  5. Department of Anatomy I, Showa University School of Medicine, Shinagawa, Tokyo, 142-8555, Japan

    Seiji Shioda

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Onoue, S., Ohshima, K., Debari, K. et al. Mishandling of the Therapeutic Peptide Glucagon Generates Cytotoxic Amyloidogenic Fibrils. Pharm Res 21, 1274–1283 (2004). https://doi.org/10.1023/B:PHAM.0000033016.36825.2c

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