高血脂兔心肌功能之研究及肝癌治療用核醫藥物之開發

Abstract

國人飲食及生活習慣的改變，使心血管疾病亦隨著歐美國家之腳步位居國人十大死因之前三名，其中以動脈粥狀硬化則為引起心血管疾病及死亡之主因，而高膽固醇血症則是血管粥狀硬化的重要元兇之一。有鑑於此，對高膽固醇的致病生理機轉做深入之瞭解實有其必要性。 由於高膽固醇與動脈粥狀硬化之關係已有許多詳細之研究，但有關高膽固醇對心肌交感神經之影響則甚少報告。本研究第一部份之研究重點在於: 探討高膽固醇食物對心臟交感神經功能之影響。我們選用三個月大之紐西蘭白兔做為實驗動物，將其分成二組，一組餵食普通之飼料(稱為控制組)，另一組則給予高膽固醇食物(稱為高膽固醇組)，在餵食前與餵食三個月後，分別注射123I-MIBG (123I-meta-iodobenzylguanidine)後，以核子醫學方法評估活體動物之交感神經功能。MIBG與臨床常用藥物Guanethidine之化學結構類似，已被證實與交感神經分泌物Norepinephrine(NE)利用相同神經再回收路徑及貯存之方法，當MIBG標幟上123I後，可用於診斷許多器官(包括心臟等)之交感神經完整性(integrity)及功能。經過三個月之餵食後，將所有的兔子犧牲，取其右心室乳突肌，分別以Isoproterenol(ISO)及NE評估其對心肌收縮力之增強作用。 實驗結果發現，高膽固醇組較同年齡之控制組有較高之心肌MIBG回收率，呈現較高之心肌對縱膈腔回收比值顯示其有較高之交感神經密度。123I-MIBG之心肌清除比值反應其交感神經之活性，高膽固醇組僅呈現較控制組稍微高但不具統計意義。心室肌肉等長收縮反應之試驗方面，發現ISO之濃度-反應曲線圖比起控制組明顯向右飄移。另外，我們將部份心室乳突肌先以cocaine前處理30分鐘使其阻斷交感神經未稍之回收功能後，與未經cocaine處理之乳突肌比較兩者對NE收縮反應之差異，發現經cocaine處理後之心肌，高膽固醇組之NE反應明顯較控制組大。針對本試驗之結果，我們的結論是兔子之高膽固醇血症會導致心肌之交感神經密度增加，且降低ISO的增強收縮力之作用，但在cocaine處理之心室乳突肌則會更增強其對NE之反應，本實驗結果顯示高膽固醇血症會引起神經重建(neuronal remodeling)，改變心臟對交感神經受體致活性之反應。 高膽固醇血症對心臟功能之影響除了導致血管粥狀動脈硬化外，並會導致心肌細胞膜之膽固醇含量增加而造成細胞膜結構或功能之改變。因此，本論文中除了探討交感神經系統功能之變化外，第二部份重點在於:探討不同胞外鈣離子濃度及不同刺激頻率時右心室乳突肌收縮力之表現，同時分析右心室細胞之鈣管道表現，並研究高膽固醇血症對細胞內之鈣離子調控基因及其相對蛋白的表現之影響。我們發現在胞外鈣離子濃度為2 mM並給予3 Hz之刺激頻率時，高膽固醇組之心室乳突肌收縮力較控制組弱，同時達到最大收縮力所需時間亦較控制組長。分析兩組之最大L-型鈣電流密度，高膽固醇組僅稍高於控制組但不具統計意義。以RT-PCR分析其mRNA的表現量，並以GAPDH的mRNA值將之標準化後，發現高膽固醇組sarcoplasmic reticulum Ca2+- ATPase (SERCA)的表現明顯低於控制組。同樣地，ryanodine receptor (RyR)亦有類似之結果。但相反地，NCX則在高膽固醇組表現量較高。Western blotting之實驗結果顯示SERCA的蛋白表現量降低而Na+-Ca2+ exchanger (NCX)則增加，另外dihydropyridine receptor的表現兩組間並無差異。 由上述心肌功能之研究發現，高膽固醇血症不只降低心肌等長收縮力之表現，同時也延長其收縮達到最大峰值之時間。在心室細胞之膜電位鉗定試驗中，我們也發現高膽固醇組之鈣電流密度僅稍大於控制組(但不具統計意義)且其鈣管道不活化曲線並不受高膽固醇之影響。由於高膽固醇對鈣電流之影響極微，因此，我們認為(1)SERCA及RyR之減少(2)NCX之增加，與高膽固醇血症會抑制最大心肌收縮力及延緩收縮力到達峰值之時間有密切關係。 另外，根據行政院衛生署的統計資料，除了心血管疾病外，癌症更是國人十大死因之首，開發肝癌之治療用藥更有其急迫性。對肝癌之治療雖然外科手術一般被認為是治療之首選方式，但一般而言，肝細胞癌之預後皆不樂觀，其它之治療方法例如化學療法、放射治療及經肝動脈栓塞結果也都不令人滿意。另一治療之選擇即為利用Lipiodol當作載體(carrier)可以攜帶化學治療用藥物或放射性同位素當作栓塞治療之用途則為目前國內外研究的焦點。因此，本研究之第三部份重點在於: 肝癌治療用藥物之開發研究。 Lipiodol是由罌粟子油提煉出來的一種碘化脂肪酸，Nakamura等人的實驗發現Lipiodol滯留於肝腫瘤相當長的時間，可用於肝腫瘤的診斷或栓塞治療。188Re為新近研發成功的治療用核種，它的半衰期(half-life)短，及放射能量適中，非常適合於放射治療與療程追蹤。99mTc-ECD(ethyl cyteinate dimer)為一臨床已使用的腦血流灌注造影劑，我們利用Tc化性與Re近似，且其配位子ECD具有親脂之特性，嘗試進行188Re標幟ECD之試驗以並發展Lipiodol之劑型，形成188Re- ECD/Lipiodol混合物，並植入肝癌細胞之大白鼠評估其是否具有肝癌治療之潛力。 依據我們目前之處方研究，比較好之188Re-ECD標幟方法為:(1)tartaric acid (TA)較EDTA更適於當螯合劑(chelating agent)；(2)每一反應瓶內含1 mg ECD, 15 mg SnCl2及5-10 mg TA。在上述條件下，可得約80%之標幟效率，而後經萃取及離心等步驟處理後，終產品之放射化學純度可超過94%。由生體分佈資料顯示肝動脈注射方式給予之188Re-ECD/Lipiodol主要分佈於肝腫瘤區域及正常之肝組織，兩者之Target/Nontarget比值由第一小時之13.21而後緩慢降低至第四十八小時約為6.84。在本研究中，我們發現利用體外系統測試188Re-ECD發現其可穩存於親脂性溶液之Lipiodol內。但是在體內時，仍有部份之188Re-ECD被游離出來，可能受到血液中特別之酯類分解酵素(esterase)代謝使其由血液及非腦部區域清除。由動物實驗生體分佈數據得知，肝動脈給藥之方式可以使188Re-ECD/Lipiodol在肝腫瘤區域有較高之放射性分佈，而在其它器官則相對僅有少量活性分佈。我們認為188Re-ECD/Lipiodol具有製備簡單之優點，同時具放射活性可較長時間停留腫瘤組織，未來具潛力發展成為肝腫瘤治療藥物之一。

Hypercholesterolemia is a risk factor commonly associated with atherosclerosis, coronary artery disease, and myocardial infarction. In order to assess the effect of hypercholesterolemia on cardiac sympathetic nerve function, New Zealand white rabbits were fed a normal diet (control group), or one enriched with 0.5% cholesterol (hypercholesterol group, HC group) for 3 months. Before and after the 3-month diet treatment, we performed serial imaging examinations and analyzed the uptake and washout ratio of 123I-meta-iodobenzylguanidine (123I-MIBG) from the myocardium by administration of 123I-MIBG through an ear vein. MIBG is an analog of guanethidine that shares the same neuronal uptake transport and storage mechanisms as norepinephrine (NE). When labeled with iodine-123, it can be used to image the integrity and function of adrenergic neurons in many organs, including the heart, with conventional planar or single photon emission computed tomography techniques. At the end of the experiments, the rabbits were sacrificed, and right ventricular strips were taken from their hearts. The inotropic response of the right ventricular strips to isoproterenol (ISO) and NE were evaluated. The cardiac MIBG uptake of the HC group, which is evaluated by using the heart/mediastinum (H/M) ratio, was higher than that of the age-matched control group. However, the washout ratios of 123I-MIBG did not statistically differ between the 2 groups. On pretreatment with cocaine, NE-enhanced contractility was greater in papillary muscles isolated from the HC group. The concentration-response curve to ISO was shifted to the right in the HC group, compared with that in the control group. In conclusion, hypercholesterolemia in rabbits resulted in an increase in sympathetic nerve density in the myocardium, a decrease in the inotropic response to ISO, and an increase in the inotropic response to NE in cocaine-treated myocardium. Both the in vivo and in vitro studies demonstrated the functional significance of neural remodeling induced by hypercholesterolemia. Although hypercholesterolemia resulted in a change of sympathetic function, little is known about the effect of hypercholesterolemia on cardiac contractile function. The objective of the further study was to examine the effect of hypercholesterolemia on myocardial contractility. Fifteen New Zealand white rabbits were fed with standard chow (control group) and the other 15 with cholesterol-enriched diet (HC group) for 12 weeks. The contractile response of ventricular muscle strips was measured in various extracellular calcium concentrations and at different pacing rate. The whole cell calcium current recording, mRNA and protein levels of cellular calcium handling proteins were also analyzed. In 2 mM Ca2+ and stimulated at 3 Hz, the contractile force of HC strips was less than that of the controls. The time to peak tension was longer in HC strips. Peak L-type calcium inward current density was slightly higher but not statistically significant in HC myocytes. The mRNA level of sarcoplasmic reticulum Ca+2-ATPase (SERCA) was significantly lower in the HC than that in controls, so was the ryanodine receptor (RyR). The mRNA of Na+/Ca+2 exchanger (NCX) was statistically higher in the HC group. Western blot experiments revealed that protein expression of SERCA in the HC strips decreased, but that of NCX increased. The protein expression of dihydropyridine receptor (DHPR) was similar between these two groups. From the myocardial functional experiment, it was shown that hypercholesterolemia not only decreased the isometric contractile force but also prolonged its time to peak tension. In the voltage-clamp experiments of ventricular myocytes, hyperchol- esterol emia slightly enhanced the L-type calcium current but had no effect on the inactivation curve. Because of little effect on calcium current, we conclude that the suppression of maximal contractile function and the prolongation of systolic contractile time course in hypercholesterolemia are probably mediated by a decreased SERCA and RyR and an increased NCX expression. In the third part of this dissertation, we tried to design a new hepatoma treatment agent by using a therapeutic isotope to label lipophilic ligand-ECD (ethyl cyteinate dimer). Primary hepato- cellular carcinoma (HCC) is the most common form of primary hepatic carcinoma, particularly in Asia and sub-Sahara Africa. Although surgery is usually considered the treatment of choice, the prognosis of HCC is very low. Other therapeutic modalities such as chemotherapy, radiotherapy and trans-hepatic arterial embolization (TAE) showed unsatisfactory results. Alternative methods of therapy using Lipiodol as a carrier for chemotherapeutic agents or radioisotopes are currently under investigation. Radiolabeled Lipiodol has been used in hepatoma therapy. In this report, we attempted to develop a new 188Re-ECD/Lipiodol radiopharmaceutical in which the chelating agent, ECD, is the constituent of the known brain perfusion agent, 99mTc-ECD, and to evaluate its stability and biodistribution in rats with hepatic tumors. Firstly, 188Re-ECD was prepared in a vial, followed by extraction with Lipiodol to get the final product, 188Re-ECD/Lipiodol. The optimal labeling conditions for 188Re-ECD were: (1) tartaric acid which is better than EDTA as a weak chelating agent; and (2) 15 mg of SnCl2, as the reducing agent, and 5-10 mg of tartaric acid in each vial had a better labeling yield. The radiochemical purity of 188Re-ECD/Lipiodol was more than 94 %. Twenty-four male Sprague-Dawley rats with liver tumors were sacrificed at 1, 24, and 48 hrs (eight rats each time) after an injection of approximately 7.4 MBq of 188Re-ECD/Lipiodol via the hepatic artery. The radioactivity in the liver tumor is significant higher following therapeutic arterial injection, and relatively low in other organs including the bone, spleen, brain, thyroid, stomach, muscle, blood and testis throughout this study. In conclusion, the new preparation of 188Re-ECD/Lipiodol is a candidate agent for the treatment of liver cancer.