請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18861
標題: | 含鐵檸檬酸接枝型幾丁聚醣應用於口服降血磷之研究 Study of Iron-Based Citric Acid Conjugated Chitosan as an Oral Phosphate Binder |
作者: | Shu-An Lee 李曙安 |
指導教授: | 林文貞(Wen-Jen Lin) |
關鍵字: | 慢性腎臟衰竭,高血磷,降血磷藥物,幾丁聚醣,檸檬酸,鐵, chronic kidney disease,hyperphosphatemia,phosphate binders,chitosan,citric acid,iron, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 慢性腎臟衰竭的病患通常需要搭配血液透析進行治療,血液透析後常常會伴隨著高血磷的副作用,高血磷會增加心血管併發症的致死率,故需要服用降血磷的藥物,而目前臨床上所使用的口服降血磷藥物仍然有許多缺點,最主要的缺點為藥物效價不佳,患者需要每天服用大劑量的藥物,使得病患的用藥順從性變差。新一代口服降血磷藥物以鐵作為吸附磷的活性成分,吸附胃腸道中的磷來達到降血磷的效果,雖然結果顯示新一代含鐵藥物可以達到和目前藥物相同的降血磷效果,但每天需要給予大劑量這個缺點仍然沒有克服。
本實驗選擇了幾丁聚醣當作主體,藉由幾丁聚醣的低毒性和可以結合金屬之特性,且結構上也與臨床的降血磷藥物Sevelamer相近,除此之外藉由接枝上自然界常見且毒性低的三元酸-檸檬酸,利用檸檬酸上的羧基,希望藉此增加化合物的鐵含量並加強降血磷的效果。本研究合成的部分主要分為兩個階段,第一階段為合成不同去乙醯化度的去聚合幾丁聚醣並與檸檬酸進行接枝反應,藉由去聚合和改變不同去乙醯化度來增加化合物的水溶性,並嘗試提高檸檬酸的接枝率,第二階段再利用上述合成的不同去乙醯化度之檸檬酸接枝型幾丁聚醣來與鐵進行結合。第一階段的去乙醯化度和檸檬酸接枝率利用紅外線分光光譜儀(ATR)和核磁共振光譜儀(NMR)進行確認,並使用膠體滲透層析(GPC)確認樣品的分子量和檸檬酸接枝的方式,並且利用紫外光/可見光分光光譜儀(UV/visible spectrophotometer)測量不同樣品在水中的穿透度比較各樣品的溶解度。第二階段進行鐵接合反應,樣品鐵含量測定是利用硫氰酸根(SCN-)和鐵結合後會形成紅色的複合物,此複合物在波長480 nm會有最大吸收,以鐵之檢量線進行樣品的鐵含量檢測。 製備好的樣品分別以體外和體內(in vitro and in vivo study)進行磷吸附試驗和鐵釋放試驗,體外試驗以pH 1.0、4.5和 7.5,在三種不同環境中2小時來測試樣品的磷吸附效果,並檢測樣品鐵釋放的情形,除了固定的pH環境外,也模擬了胃腸道中pH變化的環境,分別在pH 1.0,1小時、pH 4.5,1.5小時和pH 7.5,4.5小時,階段性pH變化之磷吸附試驗,樣品的磷含量測定是利用了鉬酸根離子(MoO42-)和磷結合後會形成藍綠色的複合物,此複合物在波長820 nm有最大吸收,以磷的檢量線進行樣品的磷含量檢測。體內試驗則是使用了10-15週齡的Wistar大鼠,將合成含鐵的樣品以固定比例0.25% w/w混入大鼠的飼料進行給藥,並且以臨床上常使用的口服降血磷藥物-碳酸鈣(CaCO3),給予相同的劑量當作對照組,每週抽血確認大鼠的血磷,並監測血鈣、血鐵、體重、外觀變化、食慾和活動力。 樣品合成的實驗結果顯示,不同去乙醯化度的接枝型檸檬酸去聚合幾丁聚醣,去乙醯化度分別為100%、75%、60%和40%,檸檬酸接枝率為20.5-30.5%,重量分子量(Mw)介於8,000-11,000 Da,第二階段的鐵接合反應,結果發現檸檬酸接枝型去聚合幾丁聚醣,每克樣品可接上270-315 mg鐵,比起不含有檸檬酸基的樣品,每克樣品只能接140-170 mg鐵,上升了67-110%,統計上具有顯著差異,證實了檸檬酸基可以增加樣品之含鐵量。 體外的磷吸附試驗結果顯示,在pH 1.0的環境下2小時,不含鐵之樣品幾乎沒有吸附磷的效果,證實了鐵為吸附磷之關鍵角色,而不含檸檬酸基的含鐵樣品,每克樣品能夠吸附磷約265-290 mg,但若是接枝檸檬酸的含鐵樣品,吸附磷的量可以提升至每克樣品吸附366-389 mg,統計上有顯著差異,但在pH 4.5和7.5環境時,發現含鐵樣品會沉澱出來而形成懸浮液,此時樣品的磷吸附效果較差,每克樣品磷吸附量約為88-120 mg和106-119 mg,而模擬胃腸道環境階段性改變pH值的磷吸附試驗,測試結果發現經過長時間且階段性pH值改變後,各樣品吸附磷的量並沒有明顯的改變,顯示了樣品吸附上磷之後,並不會因環境pH值變化而減少了原本磷的吸附量。 動物試驗結果,給藥三週後血磷從5.82 mg/dL下降至5.09 mg/dL(碳酸鈣組)和4.84 mg/dL (樣品組),證實本實驗樣品具有較好的降血磷效果,血鈣則是從2.58 mmol/L上升至2.72 mmol/L (碳酸鈣組)和2.69 mmol/L (樣品組),兩組血鈣無顯著差異,但樣品組別上升情況較輕微,血鐵則是從49.26 μmol/L下降至35.07 μmol/L (碳酸鈣組)和40.66 μmol/L (樣品組),給予實驗樣品的組別血鐵降低的程度較小,而兩組體重、食慾、外觀和活動力並無顯著差異,證實碳酸鈣和樣品無明顯副作用和毒性,從排泄物觀察到給予樣品組別糞便有些許變深的情況,此現象也能夠推測樣品(深褐色)吸附磷之後並不會被腸胃道吸收,而是會從糞便中排出,藉此抑制腸胃道吸收磷的量。 Patients with chronic kidney disease usually need to be treated with hemodialysis. Hemodialysis often accompanies with side effects of hyperphosphatemia. Hyperphosphatemia would increase the mortality of cardiovascular complications, so it is necessary to take phosphate binders. However, current clinical oral phosphate binders still have many disadvantages and the main one is poor potency. Patients need to take large doses of drugs daily, which would make patients’ compliance worse. A new generation of oral phosphate binders uses iron as the active ingredient to adsorb phosphate in the gastrointestinal tract to lower phosphorus level. Although the results show that the new generation of iron-based drugs can achieve the same effect as the current drugs, taking high daily doses is still not overcome. In this study, chitosan was chosen as the main structure based on its low toxicity and the ability of combining with metals. Besides, the structure of chitosan is similar to Sevelamer, a clinical phosphate binder. In addition, this research utilized citric acid- a tribasic acid, which is common in the nature and low toxicity, to conjugate with chitosan. Due to the carboxyl group on citric acid, it is expected to increase the iron content of the sample and enhance the effect of lowering blood phosphorus. The synthesis of this study was divided into two parts. The first part was the synthesis of depolymerized chitosan with different deacetylation degree and the conjugation reaction with citric acid. The purpose of depolymerization and varying deacetylation degrees is to increase the water-solubility of samples and conjugation ratio of citric acid. In the second part, citric acid conjugated depolymerized chitosan with different deacetylation degree were used to crosslink with the iron. The deacetylation degree and citric acid conjugation ratio of first part were confirmed by infrared spectroscopy (ATR) and nuclear magnetic resonance spectroscopy (NMR). Besides, the molecular weight and the conjugation way of citric acid were confirmed by gel permeation chromatography (GPC). And the solubility of each sample was compared by measuring the transmittance in water by UV/visible spectrophotometer. The iron content of second part samples was determined by thiocyanate (SCN-) which would combine with iron and form a red complex. The complex had the maximum absorption at wavelength of 480 nm and the iron content of samples was measured by the calibration curve. The prepared samples were carried out the phosphate adsorption test and the iron release test in vitro and in vivo. In vitro study, the samples were tested at pH 1.0, 4.5 and 7.5 three different environments for 2 hours for the phosphate adsorption ability and the iron release ratio. In addition to the fixed pH environment, the staged pH change of the phosphate adsorption test simulated the conditions in the gastrointestinal tract at pH 1.0, 1 hour, pH 4.5, 1.5 hours and pH 7.5, 4.5 hours. The phosphate adsorption of samples was determined by molybdate (MoO42-) which would combine with phosphate and form a blue-green complex. The complex had the maximum absorption at wavelength of 820 nm and the phosphate absorption of samples was measured by the calibration curve. In vivo study, 10-15 weeks old Wistar rats were used. The iron-based samples were mixed into rats’ diet at the ratio of 0.25% w/w. And gave the same dose of calcium carbonate (CaCO3) which was an oral phosphate binder commonly used in clinical practices as the control group. Blood sampled every week to confirm the phosphorus level and monitor calcium, iron, body weight, appearance changes, appetite and activity. The synthesis results of citric acid conjugated depolymerized chitosan with different deacetylation degree were shown that the deacetylation degree was 100%, 75%, 60% and 40%, respectively, the conjugation ratio of citric acid was 20.5% to 30.5%, and the molecular weight (Mw) was 8,000 to 11,000 Da. In the second part of the iron-crosslinking reaction, it was found that the citric acid conjugated samples could bind 270-315 mg iron per gram. Compared to the samples without citric acid groups which could only bind 140-170 mg iron per gram, the iron content increased 67% to 110%. And the difference of two groups was statistically significant, which confirmed that the citric acid groups could increase the iron content. In vitro phosphate adsorption experiments results showed that there was almost no phosphate adsorption on iron-free samples at pH 1.0 for 2 hours, which confirmed that iron played an important role for phosphate adsorption. The citric acid-free and iron-based samples adsorbed about 265-290 mg phosphate per gram. On the other hand, the iron-based samples with citric acid groups, the amount of adsorbed phosphate could be increased to 366-389 mg per gram. And the difference of two groups was statistically significant. However, at pH 4.5 and 7.5, the samples would precipitate and form a suspension. At this time, the phosphate adsorption capacity of all samples was poor and the amount of phosphate adsorption was about 88-120 mg and 106-119 mg per gram. And the results of phosphate adsorption test in the condition of staged pH change showed that after a long period of time and pH change, the amount of phosphate adsorption of each sample did not change significantly. It showed that the amount of phosphate adsorption on the sample would not be reduced by changing the pH value. In vivo study results showed that the serum phosphorus decreased from 5.82 mg/dL to 5.09 mg/dL (calcium carbonate group) and 4.84 mg/dL (sample group) after three weeks administration. The results showed that samples had better effect on lowering serum phosphorus. The serum calcium increased from 2.58 mmol/L to 2.72 mmol/L (calcium carbonate group) and 2.69 mmol/L (sample group). There was no significant difference between the two groups, but the increase of the sample group was minor. The serum iron decreased from 49.26 μmol/L to 35.07 μmol/L (calcium carbonate group) and 40.66 μmol/L (sample group), and the reduction of the sample group was minor. Besides, there was no significant difference in body weight, appetite, appearance and activity between two groups. It was observed that the feces of the sample group were slightly darker. This phenomenon was able to be estimated that the samples were not absorbed by the gastrointestinal tract, but were excreted from the feces, which inhibits the absorption of phosphate in gastrointestinal tract. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18861 |
DOI: | 10.6342/NTU201603779 |
全文授權: | 未授權 |
顯示於系所單位: | 藥學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-105-1.pdf 目前未授權公開取用 | 6.92 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。