Radiolabeling of porcine, murine growth hormone and a potential antagonist G118R-mGH for biodistribution study

Amanda Palermo Nunes; Filipe Menezes; Larissa Andrade Almeida; Stephanie A.  Pomin; Patricia Lima Falcão; Carlos Soares

doi:10.15392/2319-0612.2024.2744

Authors

Nunes AP IPEN https://orcid.org/0009-0004-9684-9507 (unauthenticated)
Menezes F IPEN https://orcid.org/0009-0001-7830-0447 (unauthenticated)
Almeida LA IPEN https://orcid.org/0000-0002-0354-8384 (unauthenticated)
Stephanie A. Pomin IPEN https://orcid.org/0000-0001-5637-2303 (unauthenticated)
Falcão PL IPEN https://orcid.org/0000-0003-4640-9939 (unauthenticated)
Dr. Carlos Soares IPEN https://orcid.org/0000-0002-7982-1789 (unauthenticated)

DOI:

https://doi.org/10.15392/2319-0612.2024.2744

Keywords:

Growth Hormone , Radiolabeling, Iodine, SPECT-CT

Abstract

This study aimed to radiolabel porcine growth hormone (pGH), murine growth hormone (mGH), and its antagonist (G118R-mGH) with the iodine radioisotopes ¹³¹I and ¹²³I, to investigate the biodistribution and brain transport of these hormones. Radiolabeling was performed using the Chloramine T method, a protein iodination technique. The radiolabeled products were characterized by physicochemical techniques such as SDS-PAGE, size-exclusion high-performance liquid chromatography (HPLC-SE), and ascending paper chromatography, with measurements performed using single-photon emission computed tomography (SPECT). These measurements demonstrated high radiochemical purity, exceeding 95%, and preservation of molecular size. In vitro stability assays indicated that the radiotracers maintained their integrity for at least 24 hours. In vivo biodistribution studies in mice revealed distinct tissue distribution patterns for mGH and G118R-mGH, suggesting different uptake and metabolism mechanisms. The use of ¹²³I allowed for SPECT-CT imaging studies, which proved important for assessing the ability of mGH and G118R-mGH to cross the blood-brain barrier and distribute in specific regions of the brain. The results obtained in this work highlight the versatility of the radiolabeling protocol employed and its potential for investigating complex biological processes and application in research.

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Author Biography

Dr. Carlos Soares, IPEN

Graduado em Farmácia e Bioquímica pela Universidade de São Paulo (1989), realizou Mestrado (1995) e Doutorado (2000) em Tecnologia Nuclear - Aplicações pela Universidade de São Paulo. Atualmente é pesquisador do Instituto de Pesquisas Energéticas e Nucleares (IPEN-CNEN/SP) e professor de pós-graduação vinculado à Universidade de São Paulo. Com experiência em biotecnologia na expressão de proteínas recombinantes por bactéria e por células de mamífero. Atua principalmente no seguinte tema: síntese, purificação, caracterização e aplicações de hormônios recombinantes. Atualmente é Gerente do Centro de Biotecnologia do IPEN. Bolsista de Produtividade desen. Tec. e Extensão Inovadora do CNPq - Nível 2.

References

RANKE, M. B., & WIT, J. M. (2018). Growth hormone - past, present and future. Nat Rev Endocrinol, v. 14, n.5, p. 285-300, 2018.

FURIGO, I. C.; METZGER, M.; TEIXEIRA, P. D. S.; SOARES, C. R. J.; DONATO, J. Distribution of growth hormone-responsive cells in the mouse brain. Brain Structure and Function, v. 222, p. 222 – 341, 2017.

COCULESCO J. Blood-brain barrier for human growth hormone and insulin-like growth factor-I. J Pediatr Endocrinol Metab., v.12, n.2, p. 113-124, 1999.

NYBERG, F. Growth hormone in the brain: characteristics of specific brain targets for the hormone and their functions significance. Frontiers in Neuroendocrinology, v. 21, n. 4, p. 330-48, 2000.

VAIDYANATHAN, G.; ZALUTSKY, M. R. The radiopharmaceutical chemistry of the radioisotopes of iodine. Radiopharmaceutical Chemistry, v. 978, n. 3, p. 122, 2019.

YAMADA, A.; TRABOULSI, A.; DITTERT, L. W.; HUSSAIN, A. A. Chloramine-T in radiolabeling techniques. III. Radioiodination of biomolecules containing thioether groups. Analytical Biochemistry, v. 277, n. 2, p. 232-5, 2000.

ANDREWS, T. M.; ANDREWS, W. W.; BAKER, C. An investigation into the removal of enzymes from paper following conservation treatment. Journal of the American Institute for Conservation, v. 31, n. 3, p. 313-323, 1992.

KOPCHICK, J. J.; LIST, O. E.; KELDER, B.; GOSNEU, S. E.; BERRYMAN, E. D. Evaluation of growth hormone (GH) action in mice: discovery of GH receptor antagonists and clinical indications. Molecular and Cellular Endocrinology, v. 386, p. 34-45, 2014.

MODE, A.; TOLLET, P.; WELLS, T.; CARMIGNAC, D. F.; CLARK, R. G.; CHEN, W. Y.; KOPCHICK, J. J.; ROBINSON, I. C. The human growth hormone (hGH) antagonist G120RhGH does not antagonize GH in the rat, but has paradoxical agonist activity, probably via the prolactin receptor. Endocrinology, v. 137, n. 2, p. 447-54, 1996.

KOPCHICK, J. J.; PARKINSON, C.; STEVENS, E. C.; TRAINER, P. J. Growth hormone receptor antagonists: discovery, development, and use in patients with acromegaly. Endocrine Reviews, v. 23, n. 5, p. 623-46, 2002.

SOARES, C. R.; MORGANTI, L.; MILOUX, B.; LUPKER, J. H.; FERRARA, P.; BARTOLINI, P. High-level synthesis of human prolactin in Chinese-Hamster ovary cells. Biotechnology and Applied Biochemistry, v. 32 (Pt 2), p. 127-135, 2000.

MENEZES ACSC; SUZUKI MF; OLIVEIRA JE; FURIGO IC; DONATO J; RIBELA, MTCP; BARTOLINI, P; SOARES, C R J. Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space. Protein Expression and Purification. v. 131, p. 91-100, 2017.

BARTOLINI, P., & RIBELA, M. T. Influence of chloramine T iodination on the biological and immunological activity or the molecular radius of the human growth hormone molecule. Journal of immunoassay, v. 7, n. 3, p. 129–138, 1986.

GREENWOOD, F. C.; HUNTER, W. M.; GLOVER, J. S. The preparation of i-131-labelled human growth hormone of high specific radioactivity. The Biochemical journal, v. 89, n. 1, p. 114–123, 1963.

BISCAYART, P. L.; PALADINI, A. C.; VITA, N.; ROGUIN, L. P. Preparation of 125I-labeled human growth hormone of high quality binding properties endowed with long-term stability. Journal of immunoassay, v. 10, n. 1, p. 37–56, 1989.

FISKER S. Physiology and pathophysiology of growth hormone-binding protein: methodological and clinical aspects. Growth hormone & IGF, v. 16, n. 1, p. 1–28, 2006.

BERRY, R., MCGINNIS, G. R., BANERJEE, R. R., YOUNG, M. E., & FRANK, S. J. Differential tissue response to growth hormone in mice. FEBS open bio, v. 8, n. 7, p. 1146–1154, 2018.

MENEZES, F.; WASINSKI, F.; DE SOUZA, G. O.; NUNES, A. P.; BERNARDES, E. S.; DOS SANTOS, S. N.; DA SILVA, F. F. A.; PERONI, C. N.; OLIVEIRA, J. E.; KOPCHICK, J. J.; BROWN, R. S. E.; FERNANDEZ, G.; DE FRANCESCO, P. N.; PERELLÓ, M.; SOARES, C. R. J.; DONATO, J. The pattern of growth hormone action in the mouse brain. Endocrinology, v. 165, n. 7, 2024.