| 摘要: | Leuprolide acetate (LA) 胜肽藥物是 GnRHR agonist，應用於治療前列腺癌、子宮內膜異位、子宮肌瘤等疾病，需要長期給藥，故 leuprolide acetate 發展為緩釋劑型。由於 leuprolide acetate 為帶電親水性分子，加入油酸 (oleic acid, OA) 可以改變其溶解度。在文獻中，胜肽奈米管 (peptide nanotube, PNT) 可被應用於藥物載體。於先前研究中，本實驗室利用 Cyclo-(D-Trp-Tyr) 製備胜肽奈米管與 DNA 形成複合物，應用為 DNA 之載體。因此本實驗之研究目的是藉由油酸和環化雙胜肽 Cyclo-(D-Trp-Tyr) 胜肽奈米管發展 leuprolide acetate 的皮下或肌肉注射長效釋放劑型，由體外釋放實驗評估其可行性。
以 pyrene 為探針作為測量臨界微胞濃度 (critical micelle concentration, CMC) 之方法，於 leuprolide acetate 4.14mM 溶液中，油酸之 CMC 約為 5.38×10-3 mM。油酸/leuprolide acetate 複合物，OA : LA = 2 : 1，此莫爾比例下之 leuprolide acetate 在水中溶解度最低，為 6.07 ± 0.40%，因此選用此比例和胜肽奈米管形成複合物。胜肽奈米管/油酸/leuprolide acetate 複合物 (莫爾濃度比 PNT : OA : LA = 4 : 2 : 1) 之 leuprolide acetate 體外釋放實驗，於九十天釋放 15.36 ± 4.20% 之藥量，相較於油酸/leuprolide acetate 複合物藥物釋放較快。胜肽奈米管和油酸對於 leuprolide acetate 之包覆率與載藥率分別為 93.99 ± 0.33%、36.69 ± 0.08%。藉由雷射共軛焦顯微鏡、掃描式電子顯微鏡、原子力顯微鏡觀察胜肽奈米管/油酸/leuprolide acetate 複合物之型態。油酸/leuprolide acetate 複合物會形成球形物，胜肽奈米管為長條管狀構造，兩者形成複合物後，可以觀察到在胜肽奈米管的表面會有球形物附著其上，其胜肽奈米管長約 88.52 ± 29.82 μm、寬約 6.69 ± 2.92 μm、長寬比約 14.95 ± 7.74，其上之球型構造直徑約為 0.48 ± 0.16 μm 。之後藉由螢光分別標定胜肽奈米管與油酸/leuprolide acetate 複合物，Cy5-PNT、TM-Rhodamine-OA/LA，其螢光訊號有重疊現象，且在螢光共振能量轉移 (FRET) channel 也有螢光訊號，顯示形成胜肽奈米管/油酸/leuprolide acetate 複合物。
綜合以上結果，油酸/ leuprolide acatate 複合物分布於胜肽奈米管形成複合物，且胜肽奈米管/油酸/leuprolide acetate 複合物在體外釋放實驗中可以長效釋放leuprolide acetate。
Leuprolide acetate (LA), a GnRHR agonist, is a drug for treatment of prostate cancer, endometriosis, uterine fibroids. Because of the chronic administration of leuprolide acetate, formulations of leuprolide acetate were conceived to possibly assure a continuous release of drug. The solubility of leuprolide, a charged hydrophilic molecule, can be modulated by adding oleic acid (OA). The studies showed that peptide nanotubes (PNT) could be used as drug carriers. In our previous study, peptide nanotubes prepared from Cyclo-(D-Trp-Tyr) and DNA formed complex, which were used as carriers of DNA.
The aim of the study was to develop the sustained release formulation of leuprolide acetate by oleic acid and peptide nanotubes prepared from Cyclo-(D-Trp-Tyr) for subcutaneous injection or intramuscular injection, and to assess the feasibility of the formulation by in vitro release study.
Using pyrene as a probe to measure the critical micelle concentration (CMC), the CMC of OA was about 5.38×10-3 mM with leuprolide acetate 4.14 mM in the solution. The aqueous solubility of leuprolide acetate is the lowest, 6.07 ± 0.40 %, at the molar ratio of OA : LA = 2 : 1. After finding the optimal ratio of the OA/LA complex (OA : LA = 2 : 1), which were mixed with peptide nanotubes to form PNT/OA/LA complexes. Release profile of leuprolide acetate from PNT/OA/LA complexes were compared to the OA/LA complex, and a sustained release of LA from the PNT/OA/LA complex (molar ratio of PNT : OA : LA = 4 : 2 : 1) for 90 days by in vitro release study. There were about 15.36 ± 4.20% of LA release from the PNT/OA/LA complex in 90 days. The encapsulation efficiency and loading efficiency of leuprolide acetate were 93.99 ± 0.33% and 36.69 ± 0.08% in the PNT/OA/LA complex. Confocal Microscopy, Scanning Electron Microscopy (SEM), and Atomic Force Microscope (AFM) were used to investigate the morphology of complexes. The OA/LA complexes were spherical structures, and PNTs were cylindrical structures, after mixing both to form the PNT/OA/LA complex, there were spherical structures adsorbed on PNTs. The length, width and aspect ratio of the PNT/OA/LA complex were about 88.52 ± 29.82 μm, 6.69 ± 2.92 μm, 14.95 ± 7.74 and the diameter of spherical structures was about 0.48 ± 0.16 μm. Observe the relative position and fluorescence resonance energy transfer (FRET) between PNT and OA/LA in the complex under the laser confocal microscope by using Cy5 and TM-Rhodamine to individually label PNT and OA/LA.
The results showed that the OA/LA complexes were distributed on the PNTs, forming the PNT/OA/LA complex, and that a sustained release of LA from the PNT/OA/LA complex by in vitro release study. |
