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Preparation and Characterization of PEGylated Amylin

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Abstract

Amylin is a pancreatic hormone that plays important roles in overall metabolism and in glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable for patients with diabetes who need to avoid glucose excursions. Protein conjugation with polyethylene glycol (PEG) has long been known to be a convenient approach for extending the biological effects of biopharmaceuticals. We have investigated the reactivity of amylin with methoxy polyethylene glycol succinimidyl carbonate and methoxy polyethylene glycol succinimidyl propionate, which have an average molecular weight of 5 kDa. The reaction, which was conducted in both aqueous and organic (dimethyl sulfoxide) solvents, occurred within a few minutes and resulted in at least four detectable products with distinct kinetic phases. These results suggest a kinetic selectivity for PEGylation by succinimidyl derivatives; these derivatives exhibit enhanced reactivity with primary amine groups, as indicated by an evaluation of the remaining amino groups using fluorescamine. The analysis of tryptic fragments from mono- and diPEGylated amylin revealed that conjugation occurred within the 1-11 amino acid region, most likely at the two amine groups of Lys1. The reaction products were efficiently separated by C-18 reversed phase chromatography. Binding assays confirmed the ability of mono- and diPEGylated amylin to interact with the amylin co-receptor receptor activity-modifying protein 2. Subcutaneous administration in mice revealed the effectiveness of monoPEG-amylin and diPEG-amylin in reducing glycemia; both compounds exhibited prolonged action compared to unmodified amylin. These features suggest the potential use of PEGylated amylin to restore basal amylin levels.

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Abbreviations

mPEG:

methoxy polyethylene glycol

mPEG-SC:

methoxy polyethylene glycol succinimidyl carbonate

mPEG-SPA:

methoxy polyethylene glycol succinimidyl propionate

MALDI-ToF-MS:

matrix-assisted laser desorption and ionization–time-of-flight mass spectrometry

RAMP:

receptor activity-modifying protein

References

  1. Hartter E, Svoboda T, Ludvik B, Schuller M, Lell B, Kuenburg E, et al. Basal and stimulated plasma levels of pancreatic amylin indicate its co-secretion with insulin in humans. Diabetologia. 1991;34(1):52–4.

    Article  PubMed  CAS  Google Scholar 

  2. Young A. Amylin: physiology and pharmacology. 1st ed. San Diego: Elsevier Academic; 2005.

    Google Scholar 

  3. Cooper GJS. Amylin and related proteins: physiology and pathophysiology. In: Jefferson LS, Cherring AD, editors. Handbook of physiology. New York: Oxford University Press; 2001.

    Google Scholar 

  4. Cooper GJ. Amylin and insulin co-replacement therapy for insulin-dependent (type I) diabetes mellitus. Med Hypotheses. 1991;36(3):284–8.

    Article  PubMed  CAS  Google Scholar 

  5. Cooper GJ, Willis AC, Clark A, Turner RC, Sim RB, Reid KB. Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc Natl Acad Sci USA. 1987;84(23):8628–32.

    Article  PubMed  CAS  Google Scholar 

  6. Cooper GJ, Willis AC, Reid KB, Clark A, Baker CA, Turner RC, et al. Diabetes-associated peptide. Lancet. 1987;2(8565):966.

    Article  PubMed  CAS  Google Scholar 

  7. Westermark P, Wernstedt C, O’Brien TD, Hayden DW, Johnson KH. Islet amyloid in type 2 human diabetes mellitus and adult diabetic cats contains a novel putative polypeptide hormone. Am J Pathol. 1987;127(3):414–7.

    PubMed  CAS  Google Scholar 

  8. Westermark P, Wernstedt C, Wilander E, Hayden DW, O’Brien TD, Johnson KH. Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells. Proc Natl Acad Sci USA. 1987;84(11):3881–5.

    Article  PubMed  CAS  Google Scholar 

  9. Cooper GJ, Leighton B, Dimitriadis GD, Parry-Billings M, Kowalchuk JM, Howland K, et al. Amylin found in amyloid deposits in human type 2 diabetes mellitus may be a hormone that regulates glycogen metabolism in skeletal muscle. Proc Natl Acad Sci USA. 1988;85(20):7763–6.

    Article  PubMed  CAS  Google Scholar 

  10. Young AA, Vine W, Gedulin BR, Pittner R, Janes S, Gaeta LS, et al. Preclinical pharmacology of pramlintide in the rat: comparisons with human and rat amylin. Drug Development Research. 1996;37:231–48.

    Article  CAS  Google Scholar 

  11. Westermark P, Engstrom U, Johnson KH, Westermark GT, Betsholtz C. Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation. Proc Natl Acad Sci USA. 1990;87(13):5036–40.

    Article  PubMed  CAS  Google Scholar 

  12. Guerreiro LH, Da SD, Ricci-Junior E, Girard-Dias W, Mascarenhas CM, Sola-Penna M, et al. Polymeric particles for the controlled release of human amylin. Colloids Surf B Biointerfaces. 2012;94:101–6.

    Article  PubMed  CAS  Google Scholar 

  13. Abuchowski A, Van ET, Palczuk NC, Davis FF. Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J Biol Chem. 1977;252(11):3578–81.

    PubMed  CAS  Google Scholar 

  14. Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov. 2003;2(3):214–21.

    Article  PubMed  CAS  Google Scholar 

  15. Larson N, Ghandehari H. Polymeric conjugates for drug delivery. Chem Mater. 2012;24(5):840–53.

    Article  PubMed  CAS  Google Scholar 

  16. Youn YS, Na DH, Lee KC. High-yield production of biologically active mono-PEGylated salmon calcitonin by site-specific PEGylation. J Control Release. 2007;117(3):371–9.

    Article  PubMed  CAS  Google Scholar 

  17. Veronese FM, Pasut G. PEGylation, successful approach to drug delivery. Drug Discov Today. 2005;10(21):1451–8.

    Article  PubMed  CAS  Google Scholar 

  18. Fee CJ. Protein conjugates purification and characterization. In: Veronese FM, editor. PEGylated protein drugs: basic science and clinical applications. 1st ed. Basel: Birkhäuser; 2009. p. 113–25.

    Chapter  Google Scholar 

  19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(259):680–5.

    Article  PubMed  CAS  Google Scholar 

  20. Freitas DS, Abrahao NJ. Biochemical and biophysical characterization of lysozyme modified by PEGylation. International Journal of Pharmaceutics. 2010;392:111–7.

    Article  CAS  Google Scholar 

  21. Scaramuzza S, Tonon G, Olianas A, Messana I, Schrepfer R, Orsini G, et al. A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: production and physicochemical characterization. J Control Release. 2012;164(3):355–63.

    Article  PubMed  CAS  Google Scholar 

  22. Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nature Methods. 2012;9:671–5.

    Article  PubMed  CAS  Google Scholar 

  23. Wojdyr M. Fityk: a general-purpose peak fitting program. Journal of Applied Crystallography. 2010;43:1126–8.

    Article  CAS  Google Scholar 

  24. Udenfriend S, Stein S, Bohlen P, Dairman W, Leimgruber W, Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972;178(63):871–2.

    Article  PubMed  CAS  Google Scholar 

  25. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biochem. 1976;72:248–54.

    Article  PubMed  CAS  Google Scholar 

  26. Strohalm M, Hassman M, Kosata B, Kodicek M. mMass data miner: an open source alternative for mass spectrometric data analysis. Rapid Communications in Mass Spectrometry. 2008;22(6):905–8.

    Article  PubMed  Google Scholar 

  27. Kusano S, Kukimoto-Niino M, Hino N, Ohsawa N, Okuda K, Sakamoto K, et al. Structural basis for extracellular interactions between calcitonin receptor-like receptor and receptor activity-modifying protein 2 for adrenomedullin-specific binding. Protein Sci. 2012;21(2):199–210.

    Article  PubMed  CAS  Google Scholar 

  28. Matozo HC, Santos MAM, Neto MD, Bleicher L, Lima LMTR, Iuliano R, et al. Low-resolution structure and fluorescence anisotropy analysis of protein tyrosine phosphatase eta catalytic domain. Biophys J. 2007;92(12):4424–32.

    Article  PubMed  CAS  Google Scholar 

  29. Panagiotidis G, Salehi AA, Westermark P, Lundquist I. Homologous islet amyloid polypeptide: effects on plasma levels of glucagon, insulin and glucose in the mouse. Diabetes Res Clin Pract. 1992;18(3):167–71.

    Article  PubMed  CAS  Google Scholar 

  30. Guerreiro L, Da Silva D, Mizurini DM, Sola-Penna M, Lima LMTR. Amylin induces hypoglycemia in mice. An Acad Bras Cienc. 2013;85(1):143–8.

    Article  Google Scholar 

  31. Ayala JE, Samuel VT, Morton GJ, Obici S, Croniger CM, Shulman GI, et al. Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice. Dis Model Mech. 2010;3(9–10):525–34.

    Article  PubMed  CAS  Google Scholar 

  32. Miller BT, Rogers ME, Smith JS, Kurosky A. Identification and characterization of O-biotinylated hydroxy amino acid residues in peptides. Analyt Biochem. 1994;219(2):240–8.

    Article  PubMed  CAS  Google Scholar 

  33. Miller BT, Kurosky A. Elevated intrinsic reactivity of seryl hydroxyl groups within the linear peptide triads His-Xaa-Ser or Ser-Xaa-His. Biochem Biophys Res Commun. 1993;196(1):461–7.

    Article  PubMed  CAS  Google Scholar 

  34. Banks PR, Paquette DM. Comparison of three common amine reactive fluorescent probes used for conjugation to biomolecules by capillary zone electrophoresis. Bioconjug Chem. 1995;6(4):447–58.

    Article  PubMed  CAS  Google Scholar 

  35. Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev. 2002;54(4):459–76.

    Article  PubMed  CAS  Google Scholar 

  36. Gilead S, Wolfenson H, Gazit E. Molecular mapping of the recognition interface between the islet amyloid polypeptide and insulin. Angew Chem Int Ed Engl. 2006;45(39):6476–80.

    Article  PubMed  CAS  Google Scholar 

  37. Shen Y, Joachimiak A, Rosner MR, Tang WJ. Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature. 2006;443(7113):870–4.

    Article  PubMed  CAS  Google Scholar 

  38. Leuthauser K, Gujer R, Aldecoa A, McKinney RA, Muff R, Fischer JA, et al. Receptor-activity-modifying protein 1 forms heterodimers with two G-protein-coupled receptors to define ligand recognition. Biochem J. 2000;351(Pt 2):347–51.

    Article  PubMed  CAS  Google Scholar 

  39. Tilakaratne N, Christopoulos G, Zumpe ET, Foord SM, Sexton PM. Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment. J Pharmacol Exp Ther. 2000;294(1):61–72.

    PubMed  CAS  Google Scholar 

  40. Hay DL, Christopoulos G, Christopoulos A, Poyner DR, Sexton PM. Pharmacological discrimination of calcitonin receptor: receptor activity-modifying protein complexes. Mol Pharmacol. 2005;67(5):1655–65.

    Article  PubMed  CAS  Google Scholar 

  41. Sexton PM, Morfis M, Tilakaratne N, Hay DL, Udawela M, Christopoulos G, et al. Complexing receptor pharmacology: modulation of family B G protein-coupled receptor function by RAMPs. Ann N Y Acad Sci. 2006;1070:90–104.

    Article  PubMed  CAS  Google Scholar 

  42. Christopoulos G, Perry KJ, Morfis M, Tilakaratne N, Gao Y, Fraser NJ, et al. Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product. Mol Pharmacol. 1999;56(1):235–42.

    PubMed  CAS  Google Scholar 

  43. Lakowicz JR. Principles of fluorescence spectroscopy. 2nd ed. New York: Plenum; 1999.

    Book  Google Scholar 

  44. Eisenstein M. More than insulin. Nat Biotechnol. 2011;29(9):782–5.

    Article  PubMed  CAS  Google Scholar 

  45. Eliaschewitz FG, Aita CA, Genzini T, Noronha IL, Lojudice FH, Labriola L, et al. First Brazilian pancreatic islet transplantation in a patient with type 1 diabetes mellitus. Transplant Proc. 2004;36(4):1117–8.

    Article  PubMed  CAS  Google Scholar 

  46. Thorel F, Nepote V, Avril I, Kohno K, Desgraz R, Chera S, et al. Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss. Nature. 2010;464(7292):1149–54.

    Article  PubMed  CAS  Google Scholar 

  47. Lutz TA. The role of amylin in the control of energy homeostasis. Am J Physiol Regul Integr Comp Physiol. 2010;298(6):R1475–84.

    Article  PubMed  CAS  Google Scholar 

  48. Young DA, Deems RO, Deacon RW, McIntosh RH, Foley JE. Effects of amylin on glucose metabolism and glycogenolysis in vivo and in vitro. Am J Physiol. 1990;259(3 Pt 1):E457–61.

    PubMed  CAS  Google Scholar 

  49. Wei L, Jiang P, Yau YH, Summer H, Shochat SG, Mu Y, et al. Residual structure in islet amyloid polypeptide mediates its interactions with soluble insulin. Biochemistry. 2009;48(11):2368–76.

    Article  PubMed  CAS  Google Scholar 

  50. Jiang P, Wei L, Pervushin K, Mu Y. pH-Dependent interactions of human islet amyloid polypeptide segments with insulin studied by replica exchange molecular dynamics simulations. J Phys Chem B. 2010;114(31):10176–83.

    Article  PubMed  CAS  Google Scholar 

  51. Pharmaceuticals A. NDA 21-332—Division of Pharmaceutical Evaluation-II. Office of Clinical Pharmacology and Biopharmaceutics. Symlin. Silver Spring: Food and Drug Administration; 2000.

    Google Scholar 

  52. Hashimoto M, Takada K, Kiso Y, Muranishi S. Synthesis of palmitoyl derivatives of insulin and their biological activities. Pharm Res. 1989;6(2):171–6.

    Article  PubMed  CAS  Google Scholar 

  53. Markussen J, inventor. Novo Industri A/S, assignee. Process for preparing esters of human insulin. Denmark patent US4343898; 1982.

  54. Markussen J, Havelund S, Kurtzhals P, Andersen AS, Halstrom J, Hasselager E, et al. Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs. Diabetologia. 1996;39(3):281–8.

    Article  PubMed  CAS  Google Scholar 

  55. Thibaudeau K, Leger R, Huang XC, Robitaille M, Quraishi O, Soucy C, et al. Synthesis and evaluation of insulin-human serum albumin conjugates. Bioconjugate Chemistry. 2005;16(4):1000–8.

    Article  PubMed  CAS  Google Scholar 

  56. Shechter Y, Mironchik M, Rubinraut S, Tsubery H, Sasson K, Marcus Y, et al. Reversible pegylation of insulin facilitates its prolonged action in vivo. European Journal of Pharmaceutics and Biopharmaceutics. 2008;70(1):19–28.

    Article  PubMed  CAS  Google Scholar 

  57. Drucker DJ, Dritselis A, Kirkpatrick P. Liraglutide. Nature Reviews Drug Discovery. 2010;9:267–8.

    Article  PubMed  CAS  Google Scholar 

  58. Hompesch M, Troupin B, Heise T, Elbroend B, Endahl L, Haahr H, et al. Time-action profile of insulin detemir and NPH insulin in patients with type 2 diabetes from different ethnic groups. Diabetes Obes Metab. 2006;8(5):568–73.

    Article  PubMed  CAS  Google Scholar 

  59. Wang YS, Youngster S, Bausch J, Zhang R, McNemar C, Wyss DF. Identification of the major positional isomer of pegylated interferon alpha-2b. Biochemistry. 2000;39(35):10634–40.

    Article  PubMed  CAS  Google Scholar 

  60. Abedini A, Singh G, Raleigh DP. Recovery and purification of highly aggregation-prone disulfide-containing peptides: application to islet amyloid polypeptide. Analyt Biochem. 2006;351(2):181–6.

    Article  PubMed  CAS  Google Scholar 

  61. Milton NG, Harris JR. Fibril formation and toxicity of the non-amyloidogenic rat amylin peptide. Micron. 2013;44:246–53.

    Article  PubMed  CAS  Google Scholar 

  62. Nilsson MR, Raleigh DP. Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin. J Mol Biol. 1999;294(5):1375–85.

    Article  PubMed  CAS  Google Scholar 

  63. Nilsson MR, Nguyen LL, Raleigh DP. Synthesis and purification of amyloidogenic peptides. Analyt Biochem. 2001;288(1):76–82.

    Article  PubMed  CAS  Google Scholar 

  64. Young A. Clinical studies. Adv Pharmacol. 2005;52:289–320.

    Article  PubMed  CAS  Google Scholar 

  65. Patil SM, Xu S, Sheftic SR, Alexandrescu AT. Dynamic alpha-helix structure of micelle-bound human amylin. J Biol Chem. 2009;284(18):11982–91.

    Article  PubMed  CAS  Google Scholar 

  66. Nanga RP, Brender JR, Xu J, Hartman K, Subramanian V, Ramamoorthy A. Three-dimensional structure and orientation of rat islet amyloid polypeptide protein in a membrane environment by solution NMR spectroscopy. J Am Chem Soc. 2009;131(23):8252–61.

    Article  PubMed  CAS  Google Scholar 

  67. Singh G, Brovchenko I, Oleinikova A, Winter R. Peptide aggregation in finite systems. Biophys J. 2008;95(7):3208–21.

    Article  PubMed  CAS  Google Scholar 

  68. Larson JL, Miranker AD. The mechanism of insulin action on islet amyloid polypeptide fiber formation. J Mol Biol. 2004;335(1):221–31.

    Article  PubMed  CAS  Google Scholar 

  69. Knight JD, Williamson JA, Miranker AD. Interaction of membrane-bound islet amyloid polypeptide with soluble and crystalline insulin. Protein Sci. 2008;17(10):1850–6.

    Article  PubMed  CAS  Google Scholar 

  70. Wiltzius JJ, Sievers SA, Sawaya MR, Eisenberg D. Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process. Protein Sci. 2009;18(7):1521–30.

    Article  PubMed  CAS  Google Scholar 

  71. Yonemoto IT, Kroon GJ, Dyson HJ, Balch WE, Kelly JW. Amylin proprotein processing generates progressively more amyloidogenic peptides that initially sample the helical state. Biochemistry. 2008;47(37):9900–10.

    Article  PubMed  CAS  Google Scholar 

  72. Cabaleiro-Lago C, Lynch I, Dawson KA, Linse S. Inhibition of IAPP and IAPP(20–29) fibrillation by polymeric nanoparticles. Langmuir. 2010;26(5):3453–61.

    Article  PubMed  CAS  Google Scholar 

  73. Rijkers DT, Hoppener JW, Posthuma G, Lips CJ, Liskamp RM. Inhibition of amyloid fibril formation of human amylin by N-alkylated amino acid and alpha-hydroxy acid residue containing peptides. Chemistry. 2002;8(18):4285–91.

    Article  PubMed  CAS  Google Scholar 

  74. Mazzaglia A, Micali N, Scolaro LM, Attanasio F, Magri A, Pappalardo G, et al. Aggregation properties of the peptide fragments derived from the 17–29 region of the human and rat IAPP: a comparative study with two PEG-conjugated variants of the human sequence. J Phys Chem B. 2010;114(2):705–13.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank Mr. Eduardo R. dos Santos (CEMBIO-IBCCF-UFRJ) for his technical assistance with the MALDI-ToF-MS measurements. This research was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Nanobiotec-CAPES 04/08, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), IMBEBB, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro Carlos Chagas Filho (FAPERJ), and PRONEX. The funding agencies had no role in the study design, data collection and analysis, or decision to publish or prepare the manuscript.

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Authors and Affiliations

  1. School of Pharmacy, Federal University of Rio de Janeiro (UFRJ, CCS), Bss34, Ilha do Fundão, 21941-590, Rio de Janeiro, Rio de Janeiro, Brazil

    Luiz Henrique Guerreiro, Mariana F. A. N. Guterres, Bruno Melo-Ferreira, Luiza C. S. Erthal, Priscilla Tinoco & Luís Maurício T. R. Lima

  2. Laboratory for Structural Biology (DIMAV), Brazilian National Institute of Metrology, Quality and Technology—INMETRO, Av. N. Sa. das Graças, 50, Xerém, Duque de Caxias, 25250-020, Rio de Janeiro, Brazil

    Marcela da Silva Rosa, Daniela Lourenço & Luís Maurício T. R. Lima

  3. National Institute of Science and Technology for Structural Biology and Bioimaging (INBEB-INCT), Federal University of Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil

    Luís Maurício T. R. Lima

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  1. Luiz Henrique Guerreiro
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  2. Mariana F. A. N. Guterres
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Correspondence to Luís Maurício T. R. Lima.

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Luiz Henrique Guerreiro and Mariana F. A. N. Guterres contributed equally to this work.

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Guerreiro, L.H., Guterres, M.F.A.N., Melo-Ferreira, B. et al. Preparation and Characterization of PEGylated Amylin. AAPS PharmSciTech 14, 1083–1097 (2013). https://doi.org/10.1208/s12249-013-9987-4

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