Türkan ERTAY, Perihan ÜNAK, Mine Şencan EREN, Fazilet Zümrüt MÜFTÜLER BİBER, Özhan ÖZDOĞAN, Özden ÜLKER, Reyhan YEŞİLAĞAÇ, Hatice Yıldız DURAK

A New 99mTc Labeled Peptide: 99mTc β-Casomorphin 6, Biodistribution and Imaging Studies on Rats

Peptide radiopharmaceuticals have an increasing significance in nuclear medicine practice. β-casomorphin is a digestive peptide with 6 amino acids (Tyr-Pro-Phe-Pro-Gly-Pro). N terminal amino acid chain mainly tyr-pro-phe-pro structured exogen opioid peptid type beta cosomorphin are μ-receptor agonistic activity with priority. Animal studies show that β-casomorphins can act as opioid receptor agonists. The aim of this study was to label β-casomorphin with 99m Tc and to examine its usefulness as an opioid receptor binding radiopharmaceutical in Albino Wistar rats and cancer cells. β-casomorphin was labeled with 99m Tc radionuclide using bifunctional chelating agent. Quality control studies were done by Instant Thin layer chromatography (ITLC) and High performance liquid chromatography (HPLC) methods. Binding efficiency of the compound was more than 99%. It was observed as stable for at least 3 h at room temperature. Lipophilicity was also performed for labeled molecule. Imaging studies for 99m Tc labeled molecule was done in rats by using gamma camera. For biodistribution studies; 99m Tc labeled molecule was injected to the rats from tail vein and radioactivity per gr weight of each organ was measured as count per second (cps). Receptor specificity was evaluated in imaging and biodistribution studies in experimental animals. Cell labeling studies were also performed on breast and ovarien cancer cells. In terms of evaluating the biodistribution of 99m Tc-β-casomorphin molecule in rats, uterus and ovary displayed high involvement. It was also confirmed by cell labeling studies. If the radiopharmaceutical is radiolabeled with therapeutic radionuclides it would be useful for therapy for uterus, ovary and breast tumors

99mTc Bağlı Yeni Bir Peptid: Ratlarda 99m Tc β-Kazomorfin 6 Molekülünün Biyodağılım ve Görüntüleme Çalışması

Peptit radyofarmasötikleri nükleer tıp pratiğinde giderek artan bir öneme sahiptir. β- kazomorfin 6 amino asit (Tyr-Pro-Phe-Pro-GIy-Pro) ile bir sindirim peptitidir. Tyr-Pro-Phe-Pro aminoasit zinciri ile başlayan ekzojen opioid peptid türevi β-kazomorfinler öncelikle μ-resptör agonistik aktiviteye sahiptir. Hayvan çalışmaları β-kazomorfin türevlerinin bir opioid reseptör agonisti olduğunu göstermiştir. Bu çalışmanın amacı, β-kazomorfin’i 99m Tc ile işaretleyerek opioid reseptörler radyofarmasötiği olarak kullanabilirliğini Albino Wistar ratlarda ve kanser hücrelerinde incelemektir. β-kazomorphin bifonksiyonel molekül kullanılarak 99m Tc radyonüklidi ile işaretlendi. Kalite kontrol çalışmaları, ince tabaka kromatografisi (ITLC) ve yüksek performanslı sıvı kromatografisi (HPLC) yöntemleri ile yapıldı. Bileşiğin işaretlenme verimi %99’dan fazla idi. Oda sıcaklığında, en az 3 saat boyunca stabil kaldığı tespit edildi. İşaretli molekülün lipofilite çalışması da gerçekleştirildi. 99mTc ile işaretli molekülün görüntüleme çalışmaları sıçanlarda gamma kamera kullanılarak gerçekleştirildi. Biyolojik dağılım çalışmaları için 99mTc işaretli molekül kuyruk veninden sıçanlara enjekte edildi ve her organın gr ağırlığı başına radyoaktivitesi sayım/saniye olarak ölçüldü. Molekülün reseptöre bağlanma özelliği deney hayvanlarında görüntüleme ve biyolojik dağılım çalışmaları ile değerlendirildi. Hücre işaretleme çalışmaları meme ve yumurtalık kanser hücreleri için yapıldı. 99m Tc β-kazomorfin molekülünün biyolojik dağılımı sıçanlarda değerlendirildiğinde, rahim ve yumurtalıkta yüksek tutulum gösterdiği görüldü. Molekülün reseptör etkinliği hücre işaretleme çalışmalarıyla da doğrulandı. Molekül tedavi radyonüklitleri ile işaretlendiğinde ise rahim ve yumurtalık tümörleri tedavisinde yararlı olabilecektir

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1. Fischman AJ, Babich JW, Strauss WH: A ticket to ride: Peptide radiopharmaceuticals. J Nucl Med, 34, 2253-2263, 1993.

2. Fani M, Maecke HR: Radiopharmaceutical development of radiolabelled peptides. Eur J Nucl Med Mol Imaging, 39, 11-30, 2012. DOI: 10.1007/ s00259-011-2001-z

3. Dong ZL, Wang F: Peptide-based radiopharmaceuticals for targeted tumor therapy. Curt Med Chem, 21, 139-152, 2014.

4. Giblin MF, Veerendra B, Smith CJ: Radiometallation of receptorspecific peptides for diagnosis and treatment of human cancer. In Vivo, 19, 9-29, 2005.

5. Ruzza P, Calderan A: Radiolabeled peptide-receptor ligands in tumor imaging. Expert Opin Med Diagn, 5, 411-424, 2011. DOI: 10.1517/ 17530059.2011.592829

6. Okarvi SM: Anti-tumour treatment, peptide-based radiopharmaceuticals and cytotoxic conjugates: Potential tools against cancer. Cancer Treat Rev, 34, 13-26, 2008. DOI: 10.1016/j.ctrv.2007.07.017

7. Behr T, Gotthardt M, Barth A, Behe M: Imaging tumors with peptide-based radioligands. Q J Nucl Med Mol Im, 45, 189-200, 2001.

8. Lee S, Xie J, Chen X: Peptide-based probes for targeted molecular imaging. Biochemistry 23, 1364-1376, 2010. DOI: 10.1021/bi901135x

9. Thakur ML, Pallela VR, Consigny PM, Rao PS, Vessileva-Belnikolovska D, Shi R: Imaging vascular thrombosis with Tc-99m labeled fibrin ?-chain peptide. J NucI Med, 41, 161-168, 2000.

10. Lister-James J, Moyer BR, Dean TV: Small peptides radiolabeled with 99mTc. Q J Nucl Med Mol Im, 40, 221-233, 1996.

11. Dijkraaf I, Wester HJ: Peptides, multimers and polymers, In, Semmler W, Schwaiger M (Eds): Moleculer Imaging II, Handb Exp Pharmacol. 61-92, Springer-Verlag Berlin Heidelberg, Germany, 2008.

12. Fichna J, Janecka A: Synthesis of target-specific radiolabeled peptides for diagnostic imaging. Bioconjugate Chem, 14, 3-17 2003. DOI: 10.1021/bc025542f

13. Trejtnar F, Laznicek M, Laznickova A, Mather SJ: Pharmacokinetics and renal handling of 99mTc-labeled peptides. J NucI Med, 41, 177-182, 2000.

14. Ertay T, Unak P, Tascı C, Biber FZ., Zihnioglu F, Durak H: Tc-99- exorphin C: A new peptide radiopharmaceutical for tumor imaging. JRNC, 265, 473- 479, 2005. DOI: 10.1007/s10967-005-0851-1

15. Kakali D, Susmita C, Santanu G, Bhart S, Mridula M: Synthesis and radiobiological evaluation of a new 99mTc-labeled small peptide: 99mTcYGGSLAK as imaging agent. J Label Compd Radiopharm, 54, 374-381, 2011. DOI: 10.1002/jlcr.1883

16. Ertay T, Unak P, Bekis R, Yurt F, Biber FZ, Durak H: New radiolabeled CCK-8 analogues [Tc-99m-GH-CCK-8 and Tc-99m-DTPA-CCK-8]: preparation and biodistribution studies in rats and rabbits. Nucl Med Biol 28, 667-678, 2001. DOI: 10.1016/S0969-8051(01)00196-2

17. Zhu Y, Pintar JE: Expression of opioid receptors and ligands in pregnant mouse uterus and placenta. Biol Reprod, 159, 925-932, 1998. doi: 10.1095/biolreprod59.4.925

18. Kornyei JL, Vertes Z, Oszter A, Kovacs KA, Rao CV, Vertes M: Opioid peptides inhibit the action of oestradiol on human myometrial cells in culture. Mol Hum Reprod, 5, 565-572, 1999. DOI: 10.1093/ MOLEHR/5.6.565

19. Volterra A, Restani P, Brunello N, Galli CL, Racagni G: Interaction of beta-casomorphins with multiple opioid receptors: İn vitro and in vivo studies in the newborn rat brain. Brain Res, 395, 25-30, 1986. DOI: 10.1016/0165-3806(86)90126-4

20. Brantl V, Teschemacher H, Henschen A, Lottspeich F: Novel opioid peptides derived from casein (?-casomorphins). I. Isolation from bovine casein peptone. Hoppe Seyler Z Physiol Chem, 360, 1211-1216, 1979.

21. Noni, ID, Cattaneo S: Occurrence of b-casomorphins 5 and 7 in commercial dairy products and in their digests following in vitro simulated gastro-intestinal digestion, Food Chemistry, 119, 560-566, 2010. DOI: 10.1016/j.foodchem.2009.06.058

22. Nguyen DD, Johnson SK, Busetti F, Solah VA : Formation and degradation of beta-casomorphins in dairy processing. Crit Rev Food Sci Nutr, 55, 1955-1967, 2015. DOI: 10.1080/10408398.2012.740102

23. Ertay, T, Unak P, Tascı C: Scintigraphic imaging with Tc-99m exorphin C in rabbits. Appl Radiat Isot, 62, 883-888, 2005. DOI: 10.1016/j. apradiso.2004.10.016

24. Meisel H: Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein. FEBS Lett, 196, 3363, 1986. DOI: 10.1016/0014-5793(86)80251-4

25. Suzuki S, Oldberg A, Hayman EG, Pierschbacher MD, Ruoslahti E: Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin. EMBO J, 4, 2519-24, 1985.

26. Buchwald P, Bodor N: Octanol-water partition of nonzwitterionic peptides: Predictive power of a molecular size-based model. Proteins, 30, 86-99, 1998. DOI: 10.1002/(SICI)1097-0134(19980101)30:1_____86::AIDPROT8>3.0.CO;2-I