基因重組人類表皮生長激素局部控釋輸藥系統之研究

Study of Recombinant Human Epidermal Growth Factor with Topical Controlled Drug Delivery System

楊智惠(Chih-Hui Yang)

指導教授 ： 蔡義弘

高雄醫學大學/藥學院/藥學研究所/博士(2004年)

摘要

表皮生長激素(Epidermal Growth Factor，簡稱EGF)為由53個氨基酸組成的polypeptide，其分子量為6216 daltons，因具有眾多的藥理活性作用， EGF被認為是臨床上極有開發為加速傷口癒合的治療藥物之價值。過去的研究曾報導有關EGF在不同溶液及體液的安定性，以及一些劑型的設計，但至目前為止，尚缺乏有系統的安定性評估及較理想的處方製劑出現，主要歸因於EGF的安定性不佳，及未考量使用基劑之合適性。因此，在本研究中，將進行一系列基因重組人類表皮生長激素(recombinant human Epidermal Growth Factor，簡稱rhEGF)局部使用之控釋輸藥系統設計及評估，包括rhEGF在物理、化學及酵素中安定性的探討；微脂粒製劑的製備及膠原蛋白傷口敷料的設計，以期得到一最佳化之rhEGF局部使用具控釋輸藥特性之處方，使rhEGF在投與時，不僅能具有持續釋放作用，且能發揮其生物活性。
經過一系列rhEGF之安定性評估顯示，在物理安定性方面，成功地建立以新穎之FTIR microspectroscopy來分析rhEGF之安定性，進而推估並篩選製備rhGF製劑的條件，經評估製備溫度必須控制在40oC以下；將pH值調整為中性及加入2 M的氯化鈉，皆可增加rhEGF之安定性；反之，若在含有結構干擾劑及碘離子的溶液中，則會降低rhEGF之安定性。在化學安定性方面，建立rhEGF在不同pH值及溫度下之反應速率，而篩選出較佳製備rhEGF之pH值及溫度。在酵素安定性方面，發現aminopeptidase inhibitor類之蛋白酶抑制劑中的Bestatin，可有效地保護EGF避免受到酵素的破壞。
除此之外，確定大白豬是比較接近人類皮膚，且可作為模擬人類燙傷模式之可替代性的實驗動物。且皮膚一經燙傷後，會誘發體內酸性之蛋白酶(capthesin B)及內生性rhEGF的增加，並會使rhEGF因體內誘發酵素含量的增加而加速分解。
在rhEGF局部使用控釋輸藥系統之設計方面，rhEGF的微脂粒製劑，在低膽固醇含量及帶負電的磷脂質的包埋下，包埋率約為25-75%，且顆粒大小約介於0.11~0.21 ?慆。而在經戊二醛交聯劑修飾後的含rhEGF膠原蛋白傷口敷料，可控制rhEGF的釋放速率，方便於選擇適合病患受傷傷口的大小，且具有網狀立體結構、良好的吸水性、機械強度及細胞相容性。此外，由體內動物傷口實驗結果，印證了當初設計的構想，成功地發展出rhEGF局部使用控釋輸藥系統，可有效地攜帶rhEGF，使其在臨床上使用時，能完全地發揮其生物活性，對於傷口的癒合，能有明顯的促進作用。

關鍵字

安定性 ； 人類表皮生長激素 ； 局部控釋輸藥系統 ； 傅利葉轉換紅外線顯微分光光譜法 ； 微脂粒 ； 膠原蛋白

並列摘要

Epidermal growth factor (EGF) is a polypeptide of 53 amino acid residues with a molecular weight of 6216. Based on it has many bioactivities, it has developed to accelerate wound healing as clinical pharmaceutical formulations. Although several approaches were reported the related studies about the stability of EGF in different solutions and body fluids and in progress to develop pharmaceutical formulations that allow administration of EGF effectively. Until present, there still had no appropriately optimized pharmaceutical formulations due to the instability of EGF and the used materials inappropriately. Herein we enclosed the stability of EGF completely including the physical, chemical and enzymatic stability of EGF, and choose the best prepared condition to design the optimize formulations of EGF, including liposome and collagen sponge, with topical controlled drug release system for bringing the EGF into full play.
In these studies, we successfully developed the FTIR micro- spectroscopy to analyze the stability of EGF in physical stability and choose the conditions for preparation. For examples, the temperature of preparation controlled below 40oC, the pHs controlled to natural and 2M sodium chloride added to EGF solution could increase the stability of EGF. It would decrease the stability of EGF in the solutions containing structure perturbants and iodide ion. In chemical stability of EGF, we discussed the reactive kinetics of EGF in different pHs and temperatures. In enzymatic stability of EGF, we found the aminopeptidase inhibitor, Bestatin, could avoid EGF to be broken from enzyme.
In addition, pig skins were the most closed to human skin and could be used to modify the burned model as the replaced animal models. It could induce the acid proteases, capthesin B, and increase the endocrine EGF of body after the skin burned. Then, due to the enzyme increased, EGF would be rapidly depredated.
In the design of EGF into topical controlled release system, the encapsulation efficacy of EGF-liposome formulation was about 25-75% under the low content of cholesterol and the negative charge of phospholipids encapsulated. And the particles size could be controlled between 0.11~0.21 ?慆. Besides, the EGF-collagen sponge modified with the cross-linkage reagent, glutaraldehyde, could be chosen by the patient’s requirement depended on the different size of wound and the released rate of EGF needed. And it contained 3D structure, nice water uptake, stronger mechanical strength, and cell stimulation ability. Therefore, we successfully developed the formulation of EGF with topical controlled release properties from the animal study. It could carry EGF sufficiently into full play and increase the wound healing.

並列關鍵字

topical controlled drug delivery system ； human epidermal growth factor ； FTIR ； liposome ； stability ； collagen

參考文獻

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