以去唾液酸醣蛋白受體造影術測量鼠類肝臟殘存功能

Abstract

去唾液酸醣蛋白受體(Asialoglycoprotein receptor, ASGPR)存在肝細胞表面可以辨認醣蛋白的半乳醣基。本論文研究涵蓋兩部分，第一部分主要涵蓋第二-第四章節，係利用ASGPR可結合半乳醣基的特性，發展銦-111-乙二烯三胺五醋酸-六聚乳醣(111In diethylene triamine pentaacetic acid hexa-lactoside, 111In-DTPA-HL)造影標記與測量肝臟殘存功能的造影術；第二部分在第五章節，係比較銦-111-乙二烯三胺五醋酸-三聚半乳醣胺乙酸(111In diethylene triamine pentaacetic acid tri-galactosamine, 111In-DTPA-triGalNAc)和111In-DTPA-HL放射標誌與造影的必要條件。 第一部分實驗方法包括111In-DTPA-HL評估肝貯存量的專一性、靈敏度、準確度與閥值試驗，以及臨床前藥物動力學、組織分布、輻射劑量評估與延伸性急毒性試驗。本文以相對於正常鼠肝臟放射吸收讀值為肝貯存量。實驗結果顯示111In-DTPA-HL 73.64 +/- 7.11%聚積在正常鼠肝臟，而幾乎不存在肝癌區塊；以去唾液酸胎球蛋白(asialofetuin) 作為競爭試驗之抑制劑，當ASGPR受asialofetuin抑制，僅低於0.41 +/- 0.04% 111In-DTPA-HL存在肝臟。以20-80%部分切肝鼠進行造影，肝臟造影值和肝剩餘重量呈正相關(R2=0.8548)，說明其造影具一定準確度。以对羥基乙酰苯胺(acetaminophen)誘發急性肝炎，則相對正常肝貯存量只剩下19-45% ，且若是肝貯存量低於25%以下，一周內小鼠會死亡。藥動試驗顯示，111In-DTPA-HL 3-5分鐘快速且大量聚積在肝，半小時後代謝；因為有很好的肝標靶特性，由動物試驗推估成人劑量以單光子放射斷層掃描術可低到1 mCi，如此全身暴露劑量可減至1.1 mSv；由於單劑18F-FDG輻射吸收劑量(7-14 mSv)迄今並沒有任何毒性報導，如此推估111In-DTPA-HL也應該不致有輻射毒性或相關併發症。我們進一步發現單一肝臟細胞的放射活度吸收，在大小鼠是一樣的，但若以相同劑量(Ci)對肝臟吸收作圖，大鼠有高於小鼠4倍差異，也就是說大小鼠單一肝細胞在ASGPR的吞噬活性是相同的，但大鼠有高於小鼠4倍的受體數；這和衛福部食藥署指引所提出的大小鼠體表面積差異是相同的。臨床病理與組織病理試驗顯示以高於成人劑量10000倍的DTPA-HL沒有毒性反應，表示此藥劑具有高安全性。 第二部分的實驗方法包括111In-DTPA-triGalNAc和111In-DTPA-HL的合成、放射標誌與造影。實驗結果顯示以DTPA-HL/111In莫耳數比為10的條件，即可產製放射化學純度為100% 的111In-DTPA-HL，且比活度大於1000 uCi/ug。然而111In-DTPA-triGalNAc卻需要加一段六碳長鏈才能達到> 90%放射化學純度。於造影試驗，無論是111In-DTPA-HL或111In -DTPA-triGalNAc在大小鼠皆有肝標靶特性，但大小鼠需求之放射比活度不同。大鼠造影111In-DTPA-triGalNAc比活度即使低至4.6 uCi/ug仍可以看見肝臟有吸收，但做小鼠造影，111In-DTPA-triGalNAc比活度必須高於9.2 uCi/ug。 總結，111In-DTPA-HL似乎是一個良好、專一有準確靈敏的功能性肝貯存量的評估技術。依小鼠試驗，決定個體是否得以存活的肝貯存量閥值是25%。

The asialoglycoprotein receptor (ASGPR) on hepatocyte membranes recognizes the galactose residues of glycoproteins. There are two aims in this dissertation. One is to develop 111In-DTPA-hexa lactoside (111In-DTPA-HL) imaging biomarker and ASGPR scintigraphy for estimation of liver reserve (Chapters 2-4). The other is to compare 111In-DTPA-HL and 111In-DTPA- triGalNAc in the requirement of radiolabeling and imaging (Chapter 5). The result indicates a total of 73.64+/-7.11% of the injection dose accumulated in the normal liver tissue region, and radioactivity was barely detected in the hepatoma region. When asialoglycoprotein receptor was blocked using asialofetuin, a known ASGPR blockade, less than 0.41+/-0.04% of the injection dose was detected as background in the liver. Asialoglycoprotein receptor imaging data revealed a linear correlation between 111In-DTPA-HL binding and residual liver mass (R2=0.8548) in 20-80% of partially hepatectomized mice, demonstrating the accuracy of 111In-DTPA-HL imaging for measuring the functional liver mass. Asialoglycoprotein receptor imaging data in mice with liver failure induced using 600 mg/kg acetaminophen revealed 19-45% liver reserve relative to normal mice and a fatal threshold value of 25% liver reserve. The pharmacokinetics study showed 111In-DTPA-HL rapidly accumulated and largely concentrated in liver in 3-5 min, and metabolized gradually after 0.5 h. Based on the mice dose 4 uCi/20g in parallel-hole collimator type microSPECT, we predict 1 mCi of 111In-DTPA-HL should be suitable for first-in-human. The estimated total body dose was 2.98E-02 mSv/MBq which corresponds to a whole body dose of 1.1 mSv per 1 mCi administered dose. Since 18F-FDG ( 7-14 mSv each administration) is used routinely in clinical scans with no reports of toxicities, use of 111In-DTPA-HL should result in no toxicity or complications. There is no ASGPR endocytic activity discrimination in hepatocytes between rat and mouse. The observed difference in imaging comes from the difference of their total body surface area. There is no toxicity detected in rat at more than 10000-times the human dose. As to the comparative radiochemistry and microSPECT/CT imaging behavior of 111In-DTPA-HL and 111In-DTPA-triGalNAc, both of 111In-DTPA-HL and 111In-DTPA triGalNAc showed liver targeting characteristics either in mouse or in rat. However, they behaved quite different in radiochemistry. The 111In-DTPA-HL quite easily attained 100% radiochemical purity and more than 1000 uCi/ug specific radioactivity under room temperature with 10:1 molar ratio of ligand to 111In. However, 111In-triGalNAc needed heating to enhance the radiochemical yield unless extended with a C6 long arm. Interestingly, we found 111In-triGalNAc with 9.2 uCi/ug could be used for liver imaging either for rat and mouse; however, 4.6 uCi/ug specimen only could be used for liver imaging for rat, but not mouse. In conclusion, the 111In-DTPA-HL imaging method appears to be a good, specific visual sensitive and quantitative predictor of functional liver reserve. The diagnostic threshold for survival was at 25% liver reserve in mice.