Journal of Nuclear Science, Engineering and Technology (JONSAT)
In cooperation with the Iranian Nuclear Society

Development of Radiolabeled Recombinant Erythropoietin for Receptor Studies

Document Type : Research Paper

Authors

A.R. Jalilian 1
H. H. Sadeghpour 2
M Akhlaghi 3
E Gholami 1

1 1. Nuclear Science and Technology Research Institute, AEOI, P.O.Box: 14155-1339, Tehran-Iran

2 Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, P.O.Box: 158371345, Shiraz - Iran

3 3. Research Institute for Nuclear Medicine, Tehran University of Medical Sciences, P.O.Box: 1411713137, Tehran - Iran

Abstract
The study of human erythropoietin receptor is of great importance in evolution, carcinogenesis as well as endocrine research. In this study the production and quality control of human recombinant erythropoietin (EPO) has been reported. EPO was successively labeled with [67Ga]-gallium chloride after conjugation with freshly prepared cyclic DTPA-dianhydride (ccDTPA). The best results of the conjugation were obtained by the addition of 0.5 ml of a EPO pharmaceutical solution (in phosphate buffer, pH=8) to a glass tube pre-coated with DTPA-dianhydride (0.01 mg) at 25°C with continuous mild stirring for 30 min followed by HPLC/RTLC control and biological biodistribution in normal rats. Under optimized conditions, radio-thin layer chromatography (RTLC), instant thin layer chromatography (ITLC) and high performance liquid chromatography (HPLC) showed overall radiochemical purity of higher than 96% (specific activity=300-500 MBq/mg, labeling efficiency 77%). Preliminary in vivo studies in normal rat specimen demonstrated a high liver, lung spleen, kidney as well as long bones uptake of the tracer; the fact which is consistent with the reported EPO receptor sites. This tracer can be used in nuclear medicine and biological studies for evaluation of EPO in vitro and/or in vivo.

Highlights

  1. M.J. Koury, S.T. Sawyer, S. Brandt, New insights into erythropoiesis, Curr Opinion Hematol (2002) 9: 93-100.

     

  2. D. Wen, J. Boissel, T. Tracy, R. Gruninger, L. Mulcahy, J. Czelusniak, M. Goodman, H. Bunn, Erythropoietin structure-function relationships: high degree of sequence homology among mammals, Blood (1993) 82: 1507-1516.

 

  1. H. Wu, X. Liu, R. Jaenisch, H.F. Lodish, Generation of committed erythroid BFU-E and CFU-E pro-genitors does not require erythropoietin or the erythropoietin receptor, Cell (1995) 83:59-67.

 

  1. C.S. Lin, S.K. Lim, V. D’Agati, F. Costantini, Differential effects of an erythropoietin receptor gene dis-ruption on primitive and definitive erythropoiesis, Genes Dev (1996) 10:154-164.

 

  1. H. Wu, S.H. Lee, J. Gao, X. Liu, M.L. Iruela-Arispe, Inactivation of erythropoietin leads to defects in cardiac morphogenesis, Development (1999) 126: 3597-3605.

 

  1. M.L. Brines, P. Ghezzi, S. Keenan, D. Agnello, N.C. De Lanerolle, C. Cerami, L.M. Itri, A. Cerami, Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury, Proc Natl Acad Sci U.S.A. (2000) 12; 97(19): 10526-31.

 

  1. A. Gorio, N. Gokmen, S. Erbayraktar, O. Yilmaz, L. Madaschi, C. Cichetti, A.M. Di Giulio, E. Vardar, A. Cerami, M. Brines, Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma, Proc Natl Acad Sci U.S.A. (2002) 9; 99(14): 9450-5.

 

  1. M. Celik, N. Gökmen, S. Erbayraktar, M. Akhisaroglu, S. Konakc, C. Ulukus, S. Genc, K. Genc, E. Sagiroglu, A. Cerami, M. Brines, Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury, Proc Natl Acad Sci U.S.A. (2002) 19; 99(4): 2258-63.

 

  1. D. Agnello, P. Bigini, P. Villa, T. Mennini, A. Cerami, M.L. Brines, P. Ghezzi, Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis, Brain Res (2002) 11; 952(1): 128-34.

 

 

  1. R. Bianchi, B. Buyukakilli, M. Brines, C. Savino, G. Cavaletti, N. Oggioni, G. Lauria, M. Borgna, R. Lombardi, B. Cimen, U. Comelekoglu, A. Kanik, C. Tataroglu, A. Cerami, P. Ghezzi, Erythropoietin both protects from and reverses experimental diabetic neuropathy, Proc Natl Acad Sci U.S.A. (2004) 20;101(3): 823-8.

 

  1. A.L. Sirén, M. Fratelli, M. Brines, C. Goemans, S. Casagrande, P. Lewczuk, S. Keenan, C. Gleiter, C. Pasquali, A. Capobianco, T. Mennini, R. Heumann, A. Cerami, H. Ehrenreich, P. Ghezzi, Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress, Proc Natl Acad Sci U.S.A. (2001) 27; 98(7):4044-9.

 

  1. S. Masuda, E. Kada, M. Nagao, R. Sasaki, In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells, Cytotechnology (1999) 29: 207-213.

 

  1. S.E. Juul, D.J. Ledbetter, A.E. Joyce, C. Dame, R.D. Christensen, Y. Zhao, V. DeMarco, Erythropoietin acts as a trophic factor in neonatal rat intestine, Gut (2001) 49: 182-189.

 

  1. A.L. Miller-Gilbert, S.H. Dubuque, B. Dvorak, C.S. Williams, J.G. Grille, S.S. Woodward, O. Koldovsky, P.J. Kling, Enteral absorption of erythropoietin in the suckling rat, Pediatr Res (2001) 50(2): 261-267.

     

  2. J.A. Widness, R.L. Schmidt, P. Veng-Pedersen, N.B. Modi, S.T. Sawyer, A sensitive and specific erythropoietin immunoprecipitation assay: application to pharmacokinetic studies, J Lab Clin Med (1992) 119(3): 285-94.

 

  1. A. Anagnostou, E.S. Lee, N. Kessimian, R. Levinson, M. Steiner, Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells, Proc Natl Acad Sci U S A (1990) 87(15): 5978-5982.

 

  1. A. Tojo, H. Fukamachi, T. Saito, M. Kasuga, A. Urabe, F. Takaku, Induction of the receptor for erythropoietin in murine erythroleukemia cells after dimethyl sulfoxide treatment, Cancer Res (1988) 48: 1818-22.

 

 

 

  1. Y. Yasuda, Y. Fujita, T. Matsuo, S. Koinuma, S. Hara, A. Tazaki, M. Onozaki, M. Hashimoto, T. Musha, K. Ogawa, H. Fujita, Y. Nakamura, H. Shiozaki, H. Utsumi, Erythropoietin regulates tumour growth of human malignancies, Carcinogenesis (2003) 24(6): 1021-19.

 

  1. S. Elliott, A.M. Sinclair, The effect of erythropoietin on normal and neoplastic cells, Biologics (2012) 6: 163-89, Epub 2012 Jun 27.

 

  1. P. Wu, N. Zhang, X. Wang, C. Zhang, T. Li, X. Ning, K. Gong, The erythropoietin/ erythropoietin receptor signaling pathway promotes growth and invasion abilities in human renal carcinoma cells, PLoS One (2012) 7(9): e45122.

 

  1. V. Stefanovic, P. Pakarinen, H. Alfthan, U.H. Stenman, A. Leminen, J. Pociuviene, A. Riska, M. Loukovaara, Erythropoietin is detectable in the ascitic fluid in patients with ovarian tumors, Int J Gynecol Cancer, (28 Sep, 2012) [Epub ahead of print].

 

  1. R.B. Firestone, V.S. Shirley, C.M. Baglin, J. Zipkin, In: Table of isotopes, 8th Edition, John Wiley and Sons, New York (1996) 1447.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. H. Sadeghpour, A.R. Jalilian, M. Akhlaghi, M. Kamali-Dehghan, M. Mirzaii, Preparation and biodistribution of [111In]-rHuEpo for erythropoietin receptor imaging, DOI: 10.1007/s10967-007-7212-1. Journal of Radioanalytical and Nuclear Chemistry, Vol. 278, 1 (2008) 117-112.

 

  1. D.J. Hnatowich, W.W. Layne, R.L. Child. Radioactive labeling of antibody: a simple and efficient method, Science (1983) 220: 613-619.

 

  1. W.C. Eckelman, S.M. Karesh, R.C. Reba, New compounds: fatty acid and long chain hydrocarbon derivatives containing a strong chelating agent, J Pharm Sci (1975) 64: 704-706.

 

  1. M.A. Green, M.J. Welch, Gallium radiopharmaceutical chemistry, Nucl Med Biol (1989) 16: 435-448.

 

  1. T. Ng, G. Marx, T. Littlewood, I. Macdougall, Recombinant erythropoietin in clinical practice, Postgrad Med J (2003) 79: 367-376 doi:10.1136/pmj.79.933.367 (Review).

Keywords

Radiogallium
EPO
Radiolabeling
Biodistribution
Radiotracer

  1. M.J. Koury, S.T. Sawyer, S. Brandt, New insights into erythropoiesis, Curr Opinion Hematol (2002) 9: 93-100.

     

  2. D. Wen, J. Boissel, T. Tracy, R. Gruninger, L. Mulcahy, J. Czelusniak, M. Goodman, H. Bunn, Erythropoietin structure-function relationships: high degree of sequence homology among mammals, Blood (1993) 82: 1507-1516.

 

  1. H. Wu, X. Liu, R. Jaenisch, H.F. Lodish, Generation of committed erythroid BFU-E and CFU-E pro-genitors does not require erythropoietin or the erythropoietin receptor, Cell (1995) 83:59-67.

 

  1. C.S. Lin, S.K. Lim, V. D’Agati, F. Costantini, Differential effects of an erythropoietin receptor gene dis-ruption on primitive and definitive erythropoiesis, Genes Dev (1996) 10:154-164.

 

  1. H. Wu, S.H. Lee, J. Gao, X. Liu, M.L. Iruela-Arispe, Inactivation of erythropoietin leads to defects in cardiac morphogenesis, Development (1999) 126: 3597-3605.

 

  1. M.L. Brines, P. Ghezzi, S. Keenan, D. Agnello, N.C. De Lanerolle, C. Cerami, L.M. Itri, A. Cerami, Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury, Proc Natl Acad Sci U.S.A. (2000) 12; 97(19): 10526-31.

 

  1. A. Gorio, N. Gokmen, S. Erbayraktar, O. Yilmaz, L. Madaschi, C. Cichetti, A.M. Di Giulio, E. Vardar, A. Cerami, M. Brines, Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma, Proc Natl Acad Sci U.S.A. (2002) 9; 99(14): 9450-5.

 

  1. M. Celik, N. Gökmen, S. Erbayraktar, M. Akhisaroglu, S. Konakc, C. Ulukus, S. Genc, K. Genc, E. Sagiroglu, A. Cerami, M. Brines, Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury, Proc Natl Acad Sci U.S.A. (2002) 19; 99(4): 2258-63.

 

  1. D. Agnello, P. Bigini, P. Villa, T. Mennini, A. Cerami, M.L. Brines, P. Ghezzi, Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis, Brain Res (2002) 11; 952(1): 128-34.

 

 

  1. R. Bianchi, B. Buyukakilli, M. Brines, C. Savino, G. Cavaletti, N. Oggioni, G. Lauria, M. Borgna, R. Lombardi, B. Cimen, U. Comelekoglu, A. Kanik, C. Tataroglu, A. Cerami, P. Ghezzi, Erythropoietin both protects from and reverses experimental diabetic neuropathy, Proc Natl Acad Sci U.S.A. (2004) 20;101(3): 823-8.

 

  1. A.L. Sirén, M. Fratelli, M. Brines, C. Goemans, S. Casagrande, P. Lewczuk, S. Keenan, C. Gleiter, C. Pasquali, A. Capobianco, T. Mennini, R. Heumann, A. Cerami, H. Ehrenreich, P. Ghezzi, Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress, Proc Natl Acad Sci U.S.A. (2001) 27; 98(7):4044-9.

 

  1. S. Masuda, E. Kada, M. Nagao, R. Sasaki, In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells, Cytotechnology (1999) 29: 207-213.

 

  1. S.E. Juul, D.J. Ledbetter, A.E. Joyce, C. Dame, R.D. Christensen, Y. Zhao, V. DeMarco, Erythropoietin acts as a trophic factor in neonatal rat intestine, Gut (2001) 49: 182-189.

 

  1. A.L. Miller-Gilbert, S.H. Dubuque, B. Dvorak, C.S. Williams, J.G. Grille, S.S. Woodward, O. Koldovsky, P.J. Kling, Enteral absorption of erythropoietin in the suckling rat, Pediatr Res (2001) 50(2): 261-267.

     

  2. J.A. Widness, R.L. Schmidt, P. Veng-Pedersen, N.B. Modi, S.T. Sawyer, A sensitive and specific erythropoietin immunoprecipitation assay: application to pharmacokinetic studies, J Lab Clin Med (1992) 119(3): 285-94.

 

  1. A. Anagnostou, E.S. Lee, N. Kessimian, R. Levinson, M. Steiner, Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells, Proc Natl Acad Sci U S A (1990) 87(15): 5978-5982.

 

  1. A. Tojo, H. Fukamachi, T. Saito, M. Kasuga, A. Urabe, F. Takaku, Induction of the receptor for erythropoietin in murine erythroleukemia cells after dimethyl sulfoxide treatment, Cancer Res (1988) 48: 1818-22.

 

 

 

  1. Y. Yasuda, Y. Fujita, T. Matsuo, S. Koinuma, S. Hara, A. Tazaki, M. Onozaki, M. Hashimoto, T. Musha, K. Ogawa, H. Fujita, Y. Nakamura, H. Shiozaki, H. Utsumi, Erythropoietin regulates tumour growth of human malignancies, Carcinogenesis (2003) 24(6): 1021-19.

 

  1. S. Elliott, A.M. Sinclair, The effect of erythropoietin on normal and neoplastic cells, Biologics (2012) 6: 163-89, Epub 2012 Jun 27.

 

  1. P. Wu, N. Zhang, X. Wang, C. Zhang, T. Li, X. Ning, K. Gong, The erythropoietin/ erythropoietin receptor signaling pathway promotes growth and invasion abilities in human renal carcinoma cells, PLoS One (2012) 7(9): e45122.

 

  1. V. Stefanovic, P. Pakarinen, H. Alfthan, U.H. Stenman, A. Leminen, J. Pociuviene, A. Riska, M. Loukovaara, Erythropoietin is detectable in the ascitic fluid in patients with ovarian tumors, Int J Gynecol Cancer, (28 Sep, 2012) [Epub ahead of print].

 

  1. R.B. Firestone, V.S. Shirley, C.M. Baglin, J. Zipkin, In: Table of isotopes, 8th Edition, John Wiley and Sons, New York (1996) 1447.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. H. Sadeghpour, A.R. Jalilian, M. Akhlaghi, M. Kamali-Dehghan, M. Mirzaii, Preparation and biodistribution of [111In]-rHuEpo for erythropoietin receptor imaging, DOI: 10.1007/s10967-007-7212-1. Journal of Radioanalytical and Nuclear Chemistry, Vol. 278, 1 (2008) 117-112.

 

  1. D.J. Hnatowich, W.W. Layne, R.L. Child. Radioactive labeling of antibody: a simple and efficient method, Science (1983) 220: 613-619.

 

  1. W.C. Eckelman, S.M. Karesh, R.C. Reba, New compounds: fatty acid and long chain hydrocarbon derivatives containing a strong chelating agent, J Pharm Sci (1975) 64: 704-706.

 

  1. M.A. Green, M.J. Welch, Gallium radiopharmaceutical chemistry, Nucl Med Biol (1989) 16: 435-448.

 

  1. T. Ng, G. Marx, T. Littlewood, I. Macdougall, Recombinant erythropoietin in clinical practice, Postgrad Med J (2003) 79: 367-376 doi:10.1136/pmj.79.933.367 (Review).

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