Bowman B J, Ofner C M
Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia, PA 19104, USA.
Pharm Res. 2000 Oct;17(10):1309-15. doi: 10.1023/a:1026460023503.
Our laboratory has previously prepared gelatin/ methotrexate (MTX) conjugates containing mixed conjugation sites and by-product crosslinking, both of which may alter conjugate effectiveness. In this study, we prepared and evaluated gelatin/MTX conjugates having specific conjugate bond sites and minimal by-product crosslinking.
Opposite polarity conjugates were produced by coupling gelatin having blocked amino groups with MTX (G-MTX) and by coupling MTX having blocked amino groups with gelatin (M-GEL) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl. Amino groups were blocked using citraconic anhydride and deblocked under acidic conditions. Gelatin and MTX contents were determined spectrophotometrically. The stability of each conjugate was determined by evaluating their in vitro release of MTX in isotonic buffer at pH 7.4 and 37 degrees C for 7 days.
The G-MTX and M-GEL conjugates contained 21 and 1.2 mole MTX/mole gelatin and released 12 and 17% MTX by 7 days resulting in pseudo-first order release rate constants of 0.76x10(-3) and 1.0x10(-3) hr(-1), respectively. Alternate MTX species (< or =10%) were detected during the release study and were attributed to low molecular weight gelatin/MTX fragments and MTX polymers.
Gelatin/MTX conjugates having opposite conjugate bond polarities and minimal by-product crosslinking have been produced and slowly released MTX by hydrolytic cleavage indicating good stability for future cell culture studies.
我们实验室之前制备了含有混合共轭位点和副产物交联的明胶/甲氨蝶呤（MTX）共轭物，这两者都可能改变共轭物的有效性。在本研究中，我们制备并评估了具有特定共轭键位点且副产物交联最少的明胶/MTX共轭物。
使用1-乙基-3-（3-二甲基氨基丙基）碳二亚胺盐酸盐，通过将具有封闭氨基的明胶与MTX偶联（G-MTX）以及将具有封闭氨基的MTX与明胶偶联（M-GEL）来制备相反极性的共轭物。使用柠康酸酐封闭氨基，并在酸性条件下解封。通过分光光度法测定明胶和MTX的含量。通过评估它们在pH 7.4和37℃的等渗缓冲液中7天的MTX体外释放来确定每种共轭物的稳定性。
G-MTX和M-GEL共轭物分别含有21和1.2摩尔MTX/摩尔明胶，到7天时释放了12%和17%的MTX，导致假一级释放速率常数分别为0.76×10⁻³和1.0×10⁻³小时⁻¹。在释放研究期间检测到了替代的MTX种类（≤10%），这归因于低分子量的明胶/MTX片段和MTX聚合物。
已经制备了具有相反共轭键极性且副产物交联最少的明胶/MTX共轭物，并通过水解裂解缓慢释放MTX，表明其对于未来的细胞培养研究具有良好的稳定性。
