Synthesis of 99mTc-7-HYNIC-taxol and its biodistribution

임상병리학과/석사[한글] Taxol(Paclitaxel)은 주목과 나무인 Taxus brevifolia의 껍질에서 분리된 diterpenoid 구조의 항암물질로서, 세포 내 미세소관을 안정화시켜 세포분열을 G2/M 시기에서 정지시키는 효과적인 항암제로서, 난소암, 유방암 등 여러 종양의 치료에 효과적이다. 한편 Hydrazinonicotinamide(HYNIC)은 99mTc과 peptides을 표지하는데 유용하게 사용되는 bifunctional chelating agent(BFCA)이다.본 연구에서는 99mTc-7-HYNIC-taxol을 합성하여 이의 안정성, 단백질결합능, lipophilicity, transchelation, B16-F10 melanoma cell을 이식한 C57BL6 생쥐를 이용한 생체분포 및 γ-camera imaging 등을 실험하여 종양영상진단제로서의 이용가능성을 보고자 하였다. 6-Chloronicotinic acid를 출발 물질로 하여 6-hydrazinopyridine-3-carboxylic acid, 6-t-butyldicarbonylhydrazinopyridine -3-carboxylic acid(6-BOC-hydrazinopyridine-3-carboxylic acid)와 succinimidyl -6-BOC-hydrazinopyridine-3-carboxylic acid를 순차적으로 합성하였으며, 이때의 전 합성수율은 72.6%였다. 2'-O-(methoxyacetyl)taxol(2''-MAc-taxol)은 N-hydroxy succinimide와 methoxyacetyl chloride를 반응시켜 N-Hydroxysuccinimidyl methoxyacetate(MAc-NHS)를 합성한 후 taxol과 반응시켜 합성하였으며, 이때의 전 합성수율은 37.5%였다. 7-HYNIC-taxol은 succinimidyl-6-BOC- hydrazinopyridine-3-carboxylic acid와 2''-O-(MAc)taxol을 반응시켜 7-(t-BOC -HYNIC)-2''-O-(MAc)taxol을 합성한 후 imidazole과 2-propanol을 이용하여 MAc을, formic acid를 이용하여 t-BOC을 deprotecting하여 합성하였다. 7-HYNIC-taxol의 전 합성수율은 42.6%였다. 재결정 방법으로 정제된 7-HYNIC-taxol의 화학적 순도는 96.3%였으며 1H-NMR로 그 구조를 동정하였다. 생성물들의 정제는 silica gel 60을 이용한 column chromatography와 재결정 방법을 이용하였으며, HPLC를 사용하여 화학적 순도를 보았고 1H-NMR로 그 구조를 동정하였다. 합성된 7-HYNIC-taxol과 taxol의 세포독성능을 HT-29 cell을 이용하여 비교하였으며, IC50 값이 각각 90 nM, 210 nM로써 taxol 보다는 그 값이 낮음을 관찰하였다. 99mTc-7-HYNIC -taxol은 Na99mTcO4와 SnCl2·2H2O 환원제를 사용하여 합성하였으며, 이때의 표지수율은 99.9%였다. 99mTc-7-HYNIC-taxol은 37℃에서 24시간까지 94.4% 이상의 방사화학적 순도를 유지하는 안정한 방사성 표지화합물임을 확인하였다. 99mTc-7-HYNIC-taxol의 lipophilicity 실험에서 물층과 유기층에 대한 분포가 비슷함을 관찰하였으며, 혈청단백질결합능 실험에서 이 표지화합물의 39.9% 만이 혈청단백질과 결합함을 확인하였다. Transchelating 실험을 통해 99mTc-7-HYNIC- taxol이 다른 chelator에 영향을 거의 받지 않는 안정한 표지화합물임을 확인하였다. 99mTc-7-HYNIC-taxol의 B16-F10 melanoma를 이식한 C17BL6 생쥐에서의 생체분포실험에서 %ID/gram(percentage of injected dose per gram)으로 환산된 값이 주사후 1 시간에는 spleen, femur, lung, liver, kidneys 등의 순이었고, 6 시간에는 spleen, femur, tumor, liver, kidneys 등의 순이었고, 24 시간에는 femur, spleen, liver, kidneys, tumor 등의 순이었다. 종양대 혈액의 비율은 1 시간, 6 시간, 24 시간 후에 각각 1.75, 26.0, 2.87로 주사 후 6 시간이 가장 큰 값을 나타내었으며, 종양대 근육의 비율은 1 시간, 6 시간, 24 시간 후에 각각 12.2, 168, 15.0이었다. 또한 주사 후 1 시간, 6 시간, 24 시간에서 γ-camera 영상에서 tumor의 위치가 모두 관찰되었으나, 6 시간에서 가장 뚜렷한 tumor imaging을 볼수 있었다. 이상의 결과로부터 본 연구에서 합성한 99mTc-7-HYNIC- taxol은 앞으로 임상연구를 추가하면 종양영상 진단 방사성의약품으로써 이용가능성이 있을 것으로 본다. [영문]Taxol(Paclitaxel), a diterpenoid antineoplastic agent isolated from the bark of the Pacific yew Taxus brevifolia that stabilizes microtubules and arrests cells in the G2/M cell cycle phase, has shown anti-tumoral activity against many common cancers, including breast and ovarian cancer. HYNIC(hydrazinonicotinamide)-conjugated chemotatic peptides radiolabled with 99mTc have been demonstrated to be useful for tumor imaging. In this study, the potential value of 99mTc-labeled taxol derivatives has been evaluated as a tumor-imaging agent. 99mTc-7-HYNIC-taxol was synthesized and its biochemical behaviors, such as stability, protein bindability, lipophilicity and transchelation, were examined. Its biodistribution and resulting γ-camera images in C57BL6 mice bearing B16F10 melanoma were also examined.In order to synthesize 7-HYNIC-taxol, a precursor for 99mTc chelation, first of all, succinimidyl-6-BOC-hydrazinopyridine-3-carboxylic acid was synthesized from 6-chloronicotinic acid by three sequential reactions with 72.6% overall yield. N-Hydroxysuccinimidyl methoxyacetate(MAc-NHS) was then synthesized with 61.3% yield. The chemical purity of MAc-NHS was determined as 96.3% by HPLC. 2''-O-MAc-taxol was synthesized from MAc-NHS and taxol with 61.1% yield. 7-(t-BOC-HYNIC)taxol was synthesized by removal of MAc from 2''-O-(MAc)-7-(t-BOC-HYNIC)taxol. Then, 7-HYNIC-taxol was synthesized by removal of t-BOC from 7-(t-BOC-HYNIC)taxol with 42.6% yield. The chemical structure of 7-HYNIC-taxol was determined by a 1H-NMR spectrometer. The cytotoxicity of taxol, 2''-O-(MAc)taxol, and 7-HYNIC-taxol was examined by the MTT method using human colorectal adenocarcinoma cells (HT-29). IC50 values of taxol, 2''-O-(MAc)taxol, and 7-HYNIC-taxol were 90 nM, 910 nM, and 210 nM, respectively.The 7-HYNIC-taxol was labeled with 99mTc in the presence of SnCl2·2H2O as a reducing agent and tricine as a coligand with 99.9% yield. To estimate to stability of 99mTc-7-HYNIC-taxol, a solution of the 7-HYNIC-taxol radiotracer was added to the human serum and then allowed to stand at 37℃ for 24 hrs. After 24 hrs, radiochemical purity of 99mTc-7-HYNIC-taxol was 94.4%. According to the lipophilicity test, 99mTc-7-HYNIC-taxol was slightly more soluble in water than in organic solvent. The binidability of 99mTc-7-HYNIC-taxol to serum proteins was 39.9%. To investigate the vivo transchelation of 99mTc-7-HYNIC-taxol, a molar excess of DTPA or cysteine was added and then incubated at 37℃ for 1 hr. The 99mTc-7-HYNIC-taxol possessed over 86% of radiochemical purity after incubation with DTPA or cysteine. A trace amount of 99mTc-7-HYNIC-taxol was intravenously administered to C57BL6 mice bearing B16-F10 melanoma at footpad. The orders of 99mTc-7-HYNIC-taxol uptake were spleen>femur>lung>liver>kidneys at 1 hr post injection, spleen>femur>tumor>liver>kidneys at 6 hrs post injection, and femur>spleen>liver>kidneys>tumor at 24 hrs post injection. Tumor/blood ratios were 1.17, 26.0, and 2.87, and tumor/muscle ratios were 12.2, 168, and 15.0 in 1 hr, 6 hrs, and 24 hrs post injection, respectively. Images of the mice injected with the radiotrancer were taken by a gamma carmera after 1 hr, 6 hrs, and 24 hrs post injection. The images taken at 6 hrs post injection exhibited the best tumor image, resulting from efficient uptake of 99mTc-7-HYNIC-taxol by the melanoma cells, than those taken at the other time points. These results suggest that the 99mTc-7-HYNIC-taxol can be clinically utilized as a tumor-imaging agent.ope