대한핵의학회지 (The Korean Journal of Nuclear Medicine)

대한핵의학회 (The Korean Society of Nuclear Medicine)
권호 표지

농양 진단을 위한 $^{99m}Tc-HYNIC-PEG-liposomes$의 제조

Preparation of $^{99m}Tc-HYNIC-PEG-liposomes$ for Imaging of the Focal Sites of Infection

  • 홍준표 (연세대학교 보건과학대학 임상병리학과) ;
  • 오옥두 (연세대학교 보건과학대학 임상병리학과) ;
  • 김현석 (연세대학교 보건과학대학 임상병리학과) ;
  • 이은숙 (연세대학교 보건과학대학 임상병리학과) ;
  • 이태섭 (연세대학교 보건과학대학 임상병리학과) ;
  • 최태현 (연세대학교 보건과학대학 임상병리학과) ;
  • 최창운 (연세대학교 핵의학과) ;
  • 임상무 (연세대학교 핵의학과)
  • Hong, Jun-Pyo (Department of Medical Laboratory Science, College of Health Science, Yonsei University) ;
  • Awh, Ok-Doo (Department of Medical Laboratory Science, College of Health Science, Yonsei University) ;
  • Kim, Hyun-Suk (Department of Medical Laboratory Science, College of Health Science, Yonsei University) ;
  • Lee, Eun-Sook (Department of Medical Laboratory Science, College of Health Science, Yonsei University) ;
  • Lee, Tae-Sup (Department of Medical Laboratory Science, College of Health Science, Yonsei University) ;
  • Choi, Tae-Hyun (Department of Medical Laboratory Science, College of Health Science, Yonsei University) ;
  • Choi, Chang-Woon (Department of Nuclear Medicin, Yonsei University) ;
  • Lim, Sang-Moo (Department of Nuclear Medicin, Yonsei University)
  • 발행 : 2002.12.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

목적: BFCA 중 하나인 HYNIC을 제조하고, 염증부위에 선택적으로 국소화 되어 진단할 수 있는 PEG-liposome과 결합시켜 HYNIC-PEG-liposome을 제조하며, 제조된 HYNIC-PEG-liposome에 방사성 동위원소인 $^{99m}Tc$의 표지 방법을 확립하고자 하였다. 대상 및 방법: 6-Chloronicotinic acid를 출발물질로 하여 succinimidyl 6-BOC-hydrazinopyridine-3-carboxylic acid를 순차적으로 합성하였다. Triethylamine을 이용해 합성된 succinimidyl 6-BOC-hydrazinopyridine-3-carboxylic acid와 DSPE를 접합하였다. HYNIC-DSPE를 제조한 후에 EPC:PEG-DSPE: HYNIC-DSPE:cholesterol을 1.85:0.15 :0.07:1의 몰 비로 혼합하여 HYNIC-PEG-liposome을 제조하였다. 제조된 HYNIC-PEG-liposome에 $SnCl_2{\cdot}2H_2O$와 tricine을 이용하여 $^{99m}Tc$를 표지하였다. $^{99m}Tc$-HYNIC-PEG-liposome과 혈청을 섞어서 실온과 $37^{\circ}C$에서 24시간까지 안정성을 확인하였다. $^{99m}Tc$-HYNIC-PEG-liposome에 DTPA, cysteine, glutathione을 각각 과량의 몰 비로 섞고 $37^{\circ}C$에서 1시간 반응 후에 trans- chelation을 통해 안정성을 확인하였다. 결과: 6-Chloronicotinic acid를 출발물질로 하여 6-hydrazinopyridine-3-carboxylic acid, 6-BOC-hydrazinopyridine -3-carb-oxylic acid, succinimidyl-6-BOC-hydra-zino-pyri- dine-3-carboxylic acid를 합성하였으며 각각의 합성수율은 88.5%, 93.7%, 93.2%였고 최종수율은 77.3%였다. 제조된 HYNIC-PEG-liposome의 직경은 106 nm였고, PEG-liposome에 접합된 HYNIC의 농도는 1.08 nM이었다. HYNIC-PEG- liposome의 개수는 1 ml에 $5.2{\times}10^{14}$개가 존재하였고, PEG- liposome 한 개당 HYNIC은 약 1250개가 접합되었다. 제조된 HYNIC-PEG-liposome에 $SnCl_2{\cdot}2H_2O$와 tricine을 이용하여 $^{99m}Tc$를 표지하였으며 그 표지수율은 99%이상이었다. 혈청 내에서 24시간까지 93.5% 이상의 안정성을 나타내었다. DTPA, cysteine, glutathione을 각각 1000배의 몰 비를 첨가한 경우 $^{99m}Tc$-HYNIC- PEG-liposome의 방사화학적순도가 각각 98%, 96%, 99%으로서 안정하였다. 결론: BFCA중 하나인 HYNIC을 이용한 $^{99m}Tc$-HYNIC-PEG-liposome의 제조는 손쉬운 표지방법과 높은 표지수율 그리고 안정성을 나타낼 수 있는 방법으로서, 이를 염증부위에 선택적으로 국소화되어 염증의 진단에 유용하게 사용될 수 있을것으로 기대된다.

Purpose: A new linker, hydrazino nicotinamide (HYNIC), was recently introduced for labelling of liposome with $^{99m}Tc$. In this study we synthesized HYNIC derivatized PEG (polyethylene glycol)-liposomes radiolabeled with $^{99m}Tc$. Materials and Methods: In order to synthesize HYNIC-DSPE (distearoyl phosphatidyl ethanolamine) which is a crucial component for $^{99m}Tc$ chelation, first of all succinimidyl 6-BOC-hydrazinopyridine-3-carboxylic acid was synthesized from 6-chloronicotinic acid by three sequential reactions. A DSPE derivative of succinimidyl 6-BOC-hydrazinopyridine-3-carboxylic acid was transformed into HYNIC-DSPE by HCI/dioxane. HYNIC-PEG-liposomes were prepared by hydration of the dried lipid mixture of EPC (egg phosphatidyl choline): PEG-DSPE : HYNIC-DSPE:cholesterol (1.85:0.15:0.07:1, molar ratio). The HYNIC-PEG-liposomes were labeled with $^{99m}Tc$ in the presence of $SnCl_2{\cdot}2H_2O$ (a reducing agent) and tricine (a coligand). To investigate the level of in vivo transchelation of $^{99m}Tc$ in the liposomes, the $^{99m}Tc$-HYNiC-PES-liposomes were incubated with a molar excess of DTPA, cysteine or glutathione solutions at $37^{\circ}C$ for 1 hour. The radiolabeled liposomes were also incubated in the presence of human serum at $37^{\circ}C$ for 24 hours. Results: 6-BOC-hydrazinopyridine-3-carboxylic acid was synthesized with 77.3% overall yield. The HYNIC concentration in the PEG-coated liposome dispersion was 1.08 mM. In condition of considering the measured liposomal size of 106 nm, the phospholipid concentration of $77.5\;{\mu}mol/m{\ell}$ and the liposomal particle number of $5.2{\times}10^{14}$ liposomes/ml, it is corresponded to approximate 1,250 nicotinyl hydrazine group per liposome in HYNIC-PEG-liposome. The removal of free $^{99m}Tc$ was not necessary because the labeling efficiency were above 99%. The radiolabeled liposomes maintained 98%, 96% and 99%, respectively, of radioactivity after incubation with transchelators. The radiolabeled liposomes possessed above 90% of the radioactivity in serum. Conclusion: These results suggest that the HYNIC can be synthesized easily and applied in labelling of PEG-liposomes with $^{99m}Tc$.

키워드

참고문헌

  1. Kubota R, Yamada S, Ishiwata K, Tada M, IdoT, Kubota K. Cellular accumulation of 18F_labelled boronophenyl alanine depending onDNA synthesis and melanin incorporation: adouble-tracer microautoradiographic study ofB16 melanomas in vivo. Br J Cancer 1993;67(4):701-5.
  2. Babich JW, Tompkins RG, Graham W, BarrowSA, Fischman AJ. Localization of radiolabeledchemotactic peptide at focal sites of Escherichiacoli infection in rabbits: evidence for areceptor-specific mechanism. J Nucl Med 1997;38(8):1316-22.
  3. van der Lake CJ, Boerman OC, Oyen WJ, vande ven MT, Edwards DS. Technetium-99mlabeledchemotactic peptides in acute infectionand sterile inflammation. J Nucl Med. 1997;38(8):1310-5.
  4. Rennen HJ, Boerman OC, Oyen WJ. CorstensFR. Imaging infection/inflammation in the newmillennium. Eur J Nucl Med. 2001;28(2):241-52.
  5. Oyen WJ, Boerman OC, Storm G, van Bloois L,Koenders EB, Crommelin DJ, et al LabelledStealth liposomes in experimental infection: analternative to leukocyte scintigraphy? Nucl Med Cornrnun. 1996;17(9):742-8.
  6. Boerman OC, Oyen WJ, Storm G, Corvo ML,van Bloois L, van der Meer JW., et alTechnetium-99m-Iabeled liposomes to imageexperimental colitis in rabbits: comparison withtechnetium-99m-HMP AO-granulocytes and technetium-99m-HYNIC-IgG, J Nucl Med. 1997;39(12):2172-8.
  7. Woodle MC, Lasic DD. Sterically stabilizedliposomes. Biochi Biophy Acta. 1992; 1113(2):171-99.
  8. Woodle MC, Matthay KK, Newman MS,Hidayat JE, Collins LR, Redemann C, et alVersatility in lipid compositions showingprolonged circulation with sterically stabilizedliposomes. Biochi Biophy Acta. 1992;1105(2):193-200.
  9. Treat J, Greenspan A, Forst D, Sanchez JA,Ferrans VJ, Potkul LA, et al. Antitumor activityof liposomes-encapsulated doxorubicin in advancedbreast cancer: phase II study. J Nat Cancer [nst1990;82(21): 1706-10.
  10. Russell CD, Speiser AG. Complexes oftechnetium with hydroxy carboxylic acids:gluconic, glucoheptonic, tartaric, and citric. JNucl Med 1980;21(11):1086-90.
  11. Boerman OC, Oyen WJ, van Bloois L,Koenders EB, van der Meer JW, Corstens FH,et al. Optimization of technetium-99m-labeledPEG liposomes to image focal infection: effectsof particle size and circulation time, J Nucl Med1997 ;38(3) :489-93.
  12. Liu S, Edwards DS, Barrett JA. 99mTc labelingof highly potent small peptides. Bioconj Chern1997;8(5):621-36.
  13. Jeong JM, Kinuya S, Paik CH, Saga T, SoodVK, Carrasquillo JA, Neumann RD., et al.Application of high affinity binding concept toradiolabel avidin with Tc-99m labeled biotin andthe effect of pI on biodistribution. Nucl Med Bioi 1994;21(7):935-40.
  14. Jones GS, Elmaleh Jr DR, Strauss HW,Fischman AJ. 7,10-bis (2-mercapto-2-methyl)propyl-7,10-diazapalmitic acid: a novel, N2S2ligand for technetium-99m. Bioorg Med Chem1996 Lett. 6(20):2399-404.
  15. Milind Rajopadhye D, Scott Edwards Jeffrey P,Bourque, Timothy R. Carroll, Synthesis andtechnetum-99M labeling of cyclic GP IIB/IlIAreceptor antagonists conjugated to 4,5-bis (mercaptoacetamido)-pentanoic acid (MAPT). BioorgMed Chem Lett. 1996;6(15):1737-40.
  16. Edwards DS, Liu S, Barrett JA, Harris AR,Looby RJ, Ziegler MC., et al. New and versatileternary ligand system for technetium radiopharmaceuticals:water soluble phosphines andtricine as coligands in labeling a hydrazinonicotinamide-modified cyclic glycoprotein IIb/IIIareceptor antagonist with $^{99m}$Tc Bioconj Chem1997;8(2): 146-54.
  17. Goins BA, Phillips WT. The use of scintigraphicimaging as a tool in the development of liposomesformulations. Prog Lipid Res 2001;40:95-123.
  18. Oku N, Namba Y, Takeda A, Okada S. Tumorimaging with technetium-99m-DTP A encapsulatedin RES-avoiding liposomes. Nucl Med Bioi1993;20(4):407-12.
  19. Awasthi V, Goins B, Klipper R, Loredo R,Korvick D, Phillips WT. Imaging experimentalosteomyelitis using radiolabeled Iiposomes. JNucl Med 1998;39(6):1089-94.
  20. Babich JW, Graham W, Barrow SA, DragotakesSC, Tompkins RG, Rubin RH, et al. Technetium-99m-Iabeled chemotactic peptides: comparisonwith indium-Ill-labeled white bloodcells for localizing acute bacterial infection in the rabbit. J Nucl Med 1993;34(12):2176-81.
  21. Abrams MJ, Juweid M. tenKate CI., SchwartzDA, Hauser MM., Gaul FE, et al. Technbtium-99m-human polyclonal IgG radiolabeled via thehydrazino nicotinamide derivative for imagingfocal sites of infection in rats. J Nucl Med1990;31(12):2022-8.
  22. King TP, Zhao SW, Lam T. Preparation ofprotein conjugates via intermolecular hydrazonelinkage. Biochemistry 1986;25(19):5774-9.
  23. Enoch HG, Strittmatter P. Formation andproperties of 1000-A-diameter, single􀂽bilayerphospholipid vesicles. Proc Natl AcM Sci1979;76(1): 145-9.
  24. Allen TM, Hansen C, Rutledge J. liposomeswith prolonged circulation times: factorsaffecting uptake by reticuloendothelial and othertissues. Biochi Biophy Acta 1989;981(1):27-35.
  25. Gabizon AA. Selective tumor localization andimproved therapeutic index of anthra¢yclinesencapsulated in long-circulating liposomes.Cancer Res 1992;52(4):891-6.
  26. Bakker-Woudenberg lA, Lokerse AF, t􀂾n KateMT, Mouton JW, Woodle MC, St(jlrm G.liposomes with prolonged blood circulatlion andselective localization in Klebsiella pneumoniaeinfectedlung tissue. J Infect Dis 1993; 168(1):164-71.
  27. Goins B, Phillips WT, Klipper R. Bl0od-poolimaging using technetium-99m-labeled liposomesoJ Nucl Med 1996;37(8):1374-9.
  28. Laverman P, Dams ET, Oyen WJ, Storm G,Koenders EB, Prevost R, et al A novel methodto label liposomes with 991llTc by the hydrazinonicotinyl derivative. J Nucl Med 1999;40(1):192-7.