항체의 Cyclic DTPA를 이용한 99mTc 표지시 Polymer 형성과 체내 동태 변화

오옥두(Ok Doo Awh),임상무(Sang Moo Lim),우광선(Kwang Sun Woo),정위섭(Wee Sup Cung) 대한핵의학회 1993 핵의학 분자영상 Vol.27 No.2

N/A Technetium-99m labeling method using bifunctional chelat.ing agent cyclicDTPA has been evaluated with human polyclonal nonspecific IgG. IgG was conjugated with cyclic DTPA with various molar ratio. Reduction of Tc was done with Na,S,O, with various molar excesa Labeling efficiency and identification of polymer was confirmed with HPLC using TSK4000 SW column. Polymer was purified with 100 cm Sepharose 6LB column. Cultured 1x10' Staphylococcus aureus were injected into rat thigh 24 hours later labeled 1gG was injected, and in vivo distribution was observed 4 and 24 hours thereafter. Reduction of 99mTc was optimal with the 10000-50000 times molar excess of Na2S2O4, Polymer formation increased with increasing mloar excess of cyclic DTPA to IgG. Three step labeling-labeling DTPA conjugated IgG after reduction of 99mTc-rnade more polymer than two two step labeling-simultaneous mixing DTPA conjugated IgG, 99mTc and Na2S2O4,. Tc b]ood clearance and lower uptake in the abscess and other organa. IgG conjugated with 200 times molar excess of cyclic DTPA showed slower blood clearance with 200 times molar excess of cyclic DTPA showed slower blaod clearance than that of 200 times molar excess of cyclic DTPA showed slower blood clearance than that of 20 times molar excess. In the 99mTc labeling of IgG with cyclic DTPA for the immunoscintigraphy, obtimalllabeling condition should be chosen, and effect of the ' Tc labeled IgG polymer should be considered.

123I , 99mTc 사람 비특이 IgG 및 67Ga - Citrate의 실험동물에서 염증병소 섭취율의 비교

오옥두(Ok Doo Awh),임상무(Sang Moo Lim),이종두(Jong Doo Lee),우광선(Kwang Sun Woo),정위섭(Wee Sup Chung),서용섭(Yong Sup Seo) 대한핵의학회 1992 핵의학 분자영상 Vol.26 No.1

N/A 123I has ideal half life of 13 hours, suitable 159 keV gamma energy for imaging, and easy labeling methods. In Korea Cancer Center Hospital, 123I has been produced by MC-50 cyclotron. The purpose of this study is looking for good labeling condition of 123I and 99mTc to nonspecific human polyclonal IgG, and comparing these with 67Ga-citrate in the abscess bearing mice. Human polyclonal nonspecific IgG was labeled with 0.2 M phosphate buffer added 123I by chloramine T method. Human polyclonal nonspecific IgG was labeled with 99mTc-gluconate after activation with β-mercaptoethanol. In the abscess bearing mice, the radioactivity in the abscess was higher in 24 hours than 6 hours after injection. In the abscess, 123I nonspecific IgG had higher uptake than 99mTc-IgG or 67Ga-citrate. There was no significant difference in absecess uptake of 123I-IgG among 24, 72, 120 hours abscess age. Further clinical researches with 123I-nonspecific IgG, and other immunoscintigraphies using 123I are expected.

Taxol 유도체들의 생물학적 거동에 관한 연구

오옥두,유대웅,임상무 ( Ok Doo Awh,Dae Wung Yoo,Sang Moo Im ) 대한핵의학회 1997 핵의학 분자영상 Vol.31 No.4

This study was designed to prospect the 'In-labelled paclitaxel as tumor imaging agent. In order to provide a taxol molecule with a functional group which is able to chelate In-lll, taxol-DTPA conjugate and 2-hemisuccinyltaxol were synthesized by esterification of taxol at C-2 on C-13 carbon with DTPA anhydride and succinic anhydride, respectively. Synthesis yield of the taxol derivatives was 34% for taxol- DTPA and 80% for 2'-hemisuccinyltaxol. Cytotoxicity of the taxol derivatives were measured by MTT method toward cell lines HT29, B16, P388, and CT26. The cytotoxic activities of the taxol derivatives were maintained, although less active than taxol. Radiolabelling of the taxol derivatives were proceeded directly with InCh or indirectly with In-citrate(ligand-exchange method). The ligand-exchane methocl was not suitable because some precipitat:es appeared during the reaction. On the contrary, by direct radiolabelling methnd, we were able to obtain taxol DTPA--'In in 100% radiochemical yield. However, 2'-hemisuccinyltaxol was not labellecl by both methods. Yield and radiochemiral purity of the radiolabelled com- pound were determined by HPI.C, paper chromatography and instant thin layer chromatography. Taxol-DTPA-In was characterized to be hydrophilic by lipophi- licity test, and nearly non-adhesive to HT29, E316, P388, and CT26 by cell hinding affinity test. Binding affinity of the taxol-DTPA-'In complex to serum proteins was also examined by protein precipitation with 30% trichloroacetic acid. The results showed that 309o of the taxol-DTPA-'In complex binds with serum proteins.

지방육종형성 동물모델에서 123I-15-(p-iodophenyl)-3-R , S-methylpentadecanoic acid ( BMIPP ) 의 생체분포와 생체영상

최창운(Chang Woon Choi),임상무(Sang Moo Lim),이태섭(Tae Sup Lee),서용섭(Yong Sup Suh),우광선,정위섭(Wee Sup Chung),임수정(Soo Jung Lim),오옥두(Ok Doo Awh) 대한핵의학회 2001 핵의학 분자영상 Vol.35 No.5

N/A Purpose: 123I-labeled fatty acids have been used in the evaluation of regional myocardial energy metabolism. This study aimed to evaluate the usefulness of 123I-BMIPP as a liposarcorna-imaging agent. Materials and Methods: We compared in vitro uptakes between liposarcoma(SW872) and glioma(9L) cell lines, and examined biodistribution and in vivo images of 123I-BMIPP in liposarcoma-bearing nude mice. Cold-BMIPP was labeled with 123I using Cu2+ as catalyst. After purification by Sep-pak, radiochemical purity was determined by TLC. We compared cellular uptake between glioma and liposarcoma after incubation of 5, 10, 15, 30, 60, 120, and 180 mins with culture medium containing I-BMIPP. The difference in biodistribution was determined between non-feeding (water only) group for 18 hr and feeding group in normal mice (n=6/group) at 0.5, 2, and 24 hr. In liposarcoma-bearing nude mice model, liposarcoma, SW872, cell lines were injected subcutaneously into the left thigh of nude mice. The biodistribution of 123I-BMIPP was evaluated at 0.5, 2, and 24 hr (n=5 / group) and in vivo image of 123I-BMIPP was obtained with gamma camera at 2 and 24 hr in liposarcorna-bearing nude mice. Results: Radiolabeling yield and radiochemical purity were 95% and above 99%, respectively. SW872 cell line showed more increased uptake than 9L with 1.5 times at 180 mins. The clearance of 'I-BMIPP in various tissues was more delayed in the non-feeding group than in the feeding group, especially at delayed time (24 hr) in normal mice, and the major excreting organ was the gastrointestinal tract. In liposarcoma-bearing nude mice, tumor/blood ratio of 123I-BMIPP was 0.94, 0.75, and 1.38 and tumor/muscle ratio was 0.66, 1.53, and 1.11 at 0.5, 2, and 24hr, respectively. 123I-BMIPP was selectively localized in liposarcoma at 24 hr image. Conclusions: These results suggest that 123I-BMIPP can be used as a liposarcoma-imaging agent. (Korean J Nucl Med 200135:324-333)

Taxol 방사면역측정을 위한 125I-Iodotyraminehemisuccinyltaxol ( 125ITHT ) 의 제조

최창운(Chang Woon Choi),임상무(Sang Moo Lim),오옥두(Ok Doo Awh),이태섭(Tae Sup Lee),최태현(Tae Hyun Choi),김현석(Hyun Suk Kim),홍준표(Jun Pyo Hong),이은숙(Eun Sook Lee) 대한핵의학회 2002 핵의학 분자영상 Vol.36 No.2

목적: Taxol(Paclitaxel)은 난소암과 유방암의 치료제로 사용되고 있으며, 치료시 적절한 체내 혈중농도를 유지함으로서 치료효과를 극대화하기 위해서는 taxol의 혈중농도를 측정할 수 있는 방사면역측정시스템에 표지항원으로 사용할 수 있는 taxol 유도체의 방사성표지화합물을 합성하고, 이를 이용하여 방사면역측정법을 시행할수 있는지의 여부를 확인하고자 하였다. 대상 및 방법: Taxol과 succinic anhydride를 무수 pyridine을 용매로 하여 반응시켜 hemisuccinyltaxol을 합성하고, 합성된 hemisuccinyltaxol과 tyramine을 isobutyl-chloroformate를 coupling agent로 사용하여 tyraminehemisuccinyltaxol을 합성하고 HPLC로 분리정제 하였다. 산화제인 Chloramine-T(5.25 ㎎/ml, 10㎕)를 사용하여 tyraminehemisuccinyltaxol(4 ㎎/ml, 30㎕)에 ^125l(l mCi)를 방사성요오드화하고 HPLC를 이용하여 표지수율을 산정하였다. 정제된 tyraminehenisuccinyltaxol과 ^125l-iodotyraminehemisuccinyltaxol을 80% acetonitrile 수용액에 녹여 4℃와 37℃로 보관하면서, 각 시간대별로 화학적 순도와 방사화학적 순도를 결정하여 그 안정도를 HPLC를 이용하여 확인하였다. [^125l]Iodotyraminehemisuccinyltaxol를 방사성표지항원으로 사용하여 taxol에 대한 단클론항체(3G5A7)의 역가를 검정하였으며, 0∼100nM 농도범위에서 taxol 농도의 증가에 따른 표준투여응답곡선을 작성하였다. 결과: Hemisuccinyltaxol은 79.9%의 수율로 합성되었으며, tyraminehemisuccinyltaxol의 합성수율은 19.5%였다. ^125l-iodotyraminehemisuccinyltaxol의 표지수율은 93%이었다. tyraminehemisuccinyltaxol은 7일까지도 96.5% 이상의 순도를 보여 비교적 안정함을 확인하였으며, ^125l-iodotyraminehemisuccinyltaxol은 3일까지는 93.4% 이상으로 안정하였고 또한 7일 경과시에는 86.1% 이상의 순도를 보였다. taxol에 대한 단클론항체(3G5A7)의 역가를 검정하여 1:256의 역가를 나타냄을 확인하였으며, taxol 농도에 따른 표준투여응답곡선은 taxol과 방사표지 taxol 유도체간에 경쟁적으로 사용되어 직선성(R2=0.971)을 나타내었다. 결론: taxol의 경쟁적 방사면역측정법의 방사성 추적자로서 방사성표지 taxol 유도체인 ^125l-iodotyraminehemisuccinyltaxol을 이용한 방법이 유용함을 확인하였다. Purpose: Taxol(Paclitaxel), an antineoplastic agent, has been used in the treatment of ovarian and breast cancers. The determination of optimal Taxol concentrations in human serum was required for enhancing therapeutic effect and maintaining the appropriate Taxol level in blood. This study was aimed to synthesize radiolabeled Taxol derivatives as radiotracer in competitive radioimmunoassay for monitoring Taxol concentrations in blood and to determine the usefulness of its derivatives. Materials and Methods: Hemisuccinyltaxol(HT) was synthesized by esterification of Taxol with succinic anhydride. Tyraminehemisuccinyltaxol(THT) was synthesized by coupling of HT with tyramine using isobutylchloroformate as coupling agent and purified by HPLC. By using chloramine-T(5.25 ㎎/ml, 10㎕) as oxidant agent, THT(4 ㎎/ml, 30㎕) was labeled with ^125I(37 MBq, 1 mCi). To estimate the stability of purified THT, ^125I-iodotyraminehemisuccinyltaxol(^125ITHT) was dissolved in 80% acetonitrile aqueous solution, and the solution was incubated at 4℃ and 37℃ for 7 days. At various time intervals, the stability of THT and ^125ITHT was monitored. The tiler of Taxol monoclonal antibody, 3G5A7, was determined by competitive radioimmunoassay using ^125ITHT as a labeled antigen. A standard dose-response curve was demonstated by Taxol competitive radioimmunoassay. Results: HT and THT were synthesized with 79.9% and 19.5% yield, respectively. The labeling yield of ^125ITHT was 93%. After 7 days, the chemical purity of THT was 96.5% at 4℃, and 97.5% at 37℃. After 3 days, ^125ITHT was stable with 94.7% at 4℃ and 93.4% at 37℃. After 7 days, radiochermical purity was diminished to 88.1% at 4℃ and 86.1% at 37℃. The titer of Taxol monoclonal antibody, 3G5A7, was determined to 1:256. A standard dose-response curve demonstated good colinearity (R^2=0.971) as Taxol concentration-dependent manner. Conclusion: Competitive radioimmunoassay using ^125I-iodotyraminehemsuccinytaxol as radiotracer could be used to monitor for concentration of Taxol in the human serum.

림포신티그래피용 99mTc 표지황화안티몬 콜로이드 및 전분의 제조에 관한 연구

김재록(Jae Rok Kim),오옥두(Ok Doo Awh),박경배(Kyung Bae Park),홍성운(Seong Woon Hong),임상무(Sang Moo Lim) 대한핵의학회 1989 핵의학 분자영상 Vol.23 No.1

N/A For the development of Tc-99m-labelled antimony sulfide colloid and hydroxyethyl starch, various experiments such as preparation of colloid, control of the distribution of particle size, establishment of labelling conditions, determination of labelling yield and radiochemical purity, examination of stability, and organ imagings of rabbits etc. were carried out. 1) Antimony sulfide colloid was readily prepared by the reaction of aqueous solution of antimony potassium tartrate with hydrogen sulfide generated by treating ferrous sulfide with dilute sulfuric acid. The colloid could be stabilized by adding small amount of polyvinylpyrrolidone. 2) Electron microscopy analysis exhibited the distribution of colloid size in the range of 1∼15 nm with a major portion of 9 nm. The colloid solution was sterilized by membrane filtration (0.2 μm) and then stored at 4℃. This sterilized colloid was so stable that it was usable at least for one year. 3) The antimony sulfide colloid was labelled by adding sodium pertechnetate-Tc-99m solution to the reaction vial, followed by adding hydrochloric acid and then boiled for 30 min. The optimal pH of the reaction mixture was found to be in the range of 1.3∼1.4. Instant thin layer chromatography (ITLC) analysis showed high labelling yield of above 99.5%. This labelled colloid maintained high radiochemical purity of above 99% until 10 hours after labelling. 4) Animal studies showed high uptake of Tc-99m-Sb2S3 colloid at lymph vessels and nodes indicating a suitable agent for lymphoscintigraphy. Satisfactory results were also abtained in other clinical studies. 5) Hydroxyethyl starch (HES 0.6∼1.0%) was labelled with Na99mTO4, in the presence of SnCl2 with high labelling yield of above 99.5%. The optimal pH of the reaction mixture was in the range of 1.8∼2.0. Tc-99m-HES maintained high radiochemical purity of above 99% until 10 hours after labelling. 6) Animal studies showed that Tc-99m-HES migrated more rapidly from the injection sites into the lymph vessels than Tc-99m-Sb2S3 colloid while less amount of the former was uptaken at lymph nodes than that of the latter. Similar phenomenon was also observed in other clinical studies. As a result, Tc-99m-Sb2S3 colloid was found to be more effective lymphoscintigraphic agent than Tc-99m-HES.

유전자 영상용 HSV1-TK 기질의 합성

안순혁(Soon Hyuk Ahn),최창운(Chang Woon Choi),임상무(Sang Moo Lim),오옥두(Ok Doo Awh),최태현(Tae Hyun Choi) 대한핵의학회 2002 핵의학 분자영상 Vol.36 No.2

목적: 유전자 치료에서, 종양세포에 herpes simplex virus thymidine kinase 유전자를 발현시키고 이에 민감한 prodrug을 전신투여하여 발현된 종양세포에 특이적으로 집적되는 방법은 매우 효육적인 바업이다. 대표적인 prodrug인, IVDU, IVFRU는 herpes simplex virus type 1의 비침습적 영상화 방법에 응용되는 방사옥소 표지가능 화합물이다. 재료 및 방법: Trans-1-trimethylsilyl-2-tri-n-butylstannylethylene과 uridine 변형체상의 iodo-uracil을 Pd촉매하에서 반응시켜 표지전구체인 5-trimethylvinyl-deoxy-uridine과 5-trimethylsilylvinyl-deoxy-fluororibofuranosyl uracil을 합성하였다. 이것을 column chromatography의 방법으로 분리 정제하였다. 합성한 각 전구체를 ICl 담체를 이용하여 높은 수율로 표지하였고, HPLC를 사용하여 분리하였다. Radiohalogen exchange 방법은 비방사능을 낮게 할수록 표지에 효율적으로 알려져 있다. 이 방법으로 ICl 방법을 사용하여 담체를 포함하는 높은 방사능의 화합물을 얻을 수 있다. 결과: IVDU와 IVFRU 표지를 위한 전구체 합성수율은 각각 43, 72% 였다. ICl로 담체를 이용한 표지방법을 사용하여 IVDU와 IVFRU 표지수율은 98% 이상이었다. 결론: HSV1-tk를 이용한 유전자 영상용 기질의 전구체를 성공적으로 합성하였고, 95% 이상의 높은 표지수율의 유전자 영상용 방사성 추적자를 성공적으로 제조하였다. Purpose: In gene therapy, tumor cells expressing the herpes simplex virus thymidine kinase are sensitive to prodrugs. Potential prodrugs IVDU and IVFRU were synthesized and radiolabeled with radiodine for noninvasive imaging of herpes simplex virus type 1 gene expression. Material and Methods: 5-(2-trimethysilyl)vinyl-2`-deoxyuridine and 5-t(2-trimethylsilyl-2`-fluoro-2`-deoxyuridine, precursors of 5-(2-iodo)viny 1-2`-deoxy uridine(IVDU) and 5-(2-iodo)-2`-vinyl-2`-deoxy-2`-fluororibofuranosyl uracil(IVFRU), were synthesized from reaction of trans-1-trimethylsillyl-2-tri-n-butylstannylethylene with 5-iodo-2`-deoxyuridine and 5-iodo-2`-fluoro-2`-deoxyuridine, respectively, on the condition of Pd catalyst. These precursors were separated from reaction mixture by silica gel column chromatography method. Each precursor was radioiodinated with radioiodine by mixing with ICl oxidizing agent. These radioiodinated compounds were purified with HPKC. Radiohalogen exchange has been shown to be effective for the synthesis of products with lower specific activity. Similarly, carrier-added and high specific activity products have been isolated in respectable radiochemical yields using ICl method. Results: Synthetic yield of precursors, IVDU and IVFRU were 43% and 18%, respectively. Radiochemical purity of both compunds was over 98%. Conclusion: We synthesized precursors of IVDU and IVFRU for monitoring of HSV1-tk gene expression. Radiotracers were radioiodinated with high radiolabeling yield by ICl method. (Korean J Nucl Med 2002;36;102-109)

Alpha - hCG 측정을 위한 섬광 근접 측정법 ( Scintillation Proximity Assay ) 에 관한 연구

최태현(Tae Hyun Choi),최창운(Chang Woon Choi),임상무(Sang Moo Lim),정위섭(Wee Sup Chung),임수정(Soo Jeong Lim),이수진(Su Jin Lee),이태섭(Tae Sup Lee),오옥두(Ok Doo Awh),(Kwang Sun Woo) 대한핵의학회 2002 핵의학 분자영상 Vol.36 No.2

목적: 섬광 근접측정법은 항원 항체 반응 후 결합 분획과 유리분획을 분리하는 광정이 필요없다. 이러한 원리를 검체 내 hCG와 항 α hCG 항체간의 항원 항체 반응에 적용하고자 한다. 대상 및 방법: 항 α hCG 항체를 biotin과 결합시켜 SPA bead에 부착된 streptavidin과 부착 가능하게 만들었다. 이 측정법은 항 α hCG 항체가 부착된 SPA beads에 대해 혈청내 hCG와 표지항원인 [^125I]hCG간의 경쟁 반응을 기본 원리로 이용하였다. Biotin표지 항 α hCG항체를 [^125I]hCG 100㎕와 표준용액이나 환자 혈청 200㎕이 들어있는 실온에서 20분 방치하였다. 그리고 streptavidin이 붙은 SPA beads 20㎕를 바이알에 넣고 10분 더 방치한다. 환자 혈청의 수치를 표준 응답곡선을 통해 계산하였다. 결과: SPA측정법에 사용되는 방사성 핵종의 방사능 양에 따라 반응용액 속에서 SPA bead와 자유 방사성 핵종에 의한 배후 방사능이 측정값에 영향이 없음을 확인하였다. SPA 방법을 응용한 측정에서 적합한 표준 응답곡선을 얻었고, 실제 환자혈청에서의 hCG농도를 결정할 수 있었다. 결론: 이 실험을 통해 SPA 방법을 이용한 측정법이 임상진단에 유용하게 사용될 수 있을 것으로 사료된다. Purpose: Scintillation Proximity Assay (SPA) does not require the physical separation of receptor bound form from free form. SPA was applied to the study of interaction of human chorionic gonadotrpin (hCG) and anti-α hCG in serum. Materials and methods: Anti-α hCG was biotinylated for the binding to streptavidin. The assay was based on the simple competitive binding method between [^125l]hCG and the hCG in sample serum, with anti-α hCG-coated beads. Aliquots of biotinylated anti-α hCG were dispensed into scintillation vials containing 100㎕ [^125ㅣ]hCG and 200㎕ of either a standard concentration of hCG for preparation of standard curve or unknown sample, and oncubated for 20 min. at room temperature. Then 20㎕ streptavidin-coated beads were added to vials. and finaliy incubated for 10 min at room temperature. Values for unkncown samples were then calculated from the standard curve. Results: Optimal backfround counts were certificated using varied radioactivity of radionuclides. Appropriate standard curve was obtained from SPA method successively, and the concentration of hCG from unknown serum was determined by standard curve. The result from SPA assay was similar to that of RIA. Conclusion: This observation confims that SPA method could be useful for cilnical diafnosis.(Korean J Nucl Med 2002;36;133-139)

9-(4-[(18)F]Fluoro-3-hydroxymethylbutyl)guanine([(18)F]FHBG)의 합성과 헤르페스 단순 바이러스 티미딘 키나아제 이입 간암 세포주에서의 기초 연구

문병석 ( Byung Seok Moon ),이태섭 ( Tae Sup Lee ),이명근 ( Myoung Keun Lee ),이교철 ( Kyo Chul Lee ),안광일 ( Gwang Il An ),전권수 ( Kwon Soo Chun ),오옥두 ( Ok Doo Awh ),지대윤 ( Dae Yoon Chi ),최창운 ( Chang Woon Choi ),임상무 ( 대한핵의학회 2006 핵의학 분자영상 Vol.40 No.4

Expression of the Recombinant Single-Chain Anti-B Cell Lymphoma Antibody

임상무,--,--,-- THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 2003 Journal of biomedical laboratory sciences Vol.9 No.3

Recombinant single chain Fv (scFv) antibodies offer many advantages over mouse monoclonal antibodies such as faster clearance from blood, improved tumor localization, reduced human anti-mouse antibody (HAMA) response, and the availability to manipulate the scFv through genetic approaches. The recombinant phage display was constructed using lym-1 hybridoma cells as a source of genetic starting material. mRNA was isolated from the corresponding antibodies hybridoma cells. VH and VL cDNA were amplified with RT-PCR and linked with ScFy by linker DNA to form ScFv DNA, which then were inserted into phagemid pCANTAB5E. The phage of postive clones selected with tube containing raji lymphoma cell and infected by competent E. coIi HB2151 to express soluble scFv. The scFv lym-1 was secreted into the cytosol and culture supernatant and shown to be of expected size (approximately 32 kDa) by western blot. An active scFv lym-1 could be produced in E. coli with soluble form and high yield from hybridoma cell line, using phage display system. Immunoreactivity indicated that scFv lym1 showed a potential biding affinity against the raji lymphoma cell as its parental antibody (intact lym-1 Ab).