Yousefi Gholamhossein, Foroutan Seyed Mohsen, Zarghi Afshin, Shafaati Alireza
Department of Pharmaceutics, School of Pharmacy, Shaheed Beheshti University of Medical Sciences, 14155-6153, Iran.
Chem Pharm Bull (Tokyo). 2010 Feb;58(2):147-53. doi: 10.1248/cpb.58.147.
Methotrexate (MTX) is one of the most common anticancer drugs used for chemotherapy so far. However some problems such as high toxicity and short plasma half-life, have limited its use. To overcome these limitations, conjugation with polymers such as polyethylene glycol (PEG) is one the efficient approaches which has been attempted in recent years. The aim of the present study is to synthesize esters of MTX with PEGs of different molecular weights and to characterize their physicochemical properties. Polymeric esters (MTX-PEGs) of MTX with low, medium and high molecular weight PEGs (750 D, 5000 D and 35000 D, respectively) were synthesized using dicyclohexylcarbodiimide (DCC) as coupling agent and triethylamine (TEA) as catalyst. The purification was carried out using preparative TLC. Purified esters were characterized by UV, IR and (1)H-NMR spectroscopy methods and their thermal behavior was studied by differential scanning calorimetry (DSC). Also, an isocratic HPLC method with three mobile phase systems was set to determine the partition coefficient of the esters (log P). Gel permeation chromatography (GPC) was utilized for molecular weight determination of esters, which proved 1 : 1 conjugation of drug with each polymer. The stability and solubility of esters were determined in different pH values. The spectroscopy results indicated that esteric bond between MTX and PEGs were formed. The sharp endothermic peaks for MTX-PEGs were obtained in DSC which are similar to pure polymers, whilst a wide peak was observed for MTX. The values of log P for MTX-PEGs (+4.3, obtained by HPLC method) were remarkably different from log P of MTX (-1.4, obtained by shake-flask method). The stability results showed a pH range of 3-4 and an optimum polymer mw of 5000 for maximum stability of esters. A parabolic profile obtained from solubility studies that indicated the more solubility of MTX in alkaline condition (pH>5) and MTX-PEGs in acidic conditions (pH<5). Based on our results, it is concluded that MTX-PEGs were formed on an equimolar ratio of MTX and PEGs. The higher log P observed for the esters indicated dramatic physicochemical differences between MTX and its PEG conjugates and the higher stability and solubility in acidic medium showed a promising approach to improve the drug delivery of the conjugates, specially MTX-PEG5000 in the future.
甲氨蝶呤（MTX）是目前用于化疗的最常见抗癌药物之一。然而，诸如高毒性和血浆半衰期短等问题限制了其应用。为克服这些局限性，与聚乙二醇（PEG）等聚合物共轭是近年来尝试的有效方法之一。本研究的目的是合成MTX与不同分子量PEG的酯，并表征其理化性质。以二环己基碳二亚胺（DCC）为偶联剂、三乙胺（TEA）为催化剂，合成了MTX与低、中、高分子量PEG（分别为750 D、5000 D和35000 D）的聚合物酯（MTX-PEGs）。采用制备型薄层色谱进行纯化。通过紫外光谱、红外光谱和（1）H-NMR光谱法对纯化后的酯进行表征，并通过差示扫描量热法（DSC）研究其热行为。此外，建立了一种采用三种流动相系统的等度高效液相色谱法来测定酯的分配系数（log P）。利用凝胶渗透色谱法（GPC）测定酯的分子量，结果证明药物与每种聚合物以1:1的比例共轭。在不同pH值下测定酯的稳定性和溶解度。光谱结果表明MTX与PEGs之间形成了酯键。在DSC中，MTX-PEGs出现尖锐的吸热峰，与纯聚合物相似，而MTX出现宽峰。MTX-PEGs的log P值（通过高效液相色谱法测得为+4.3）与MTX的log P值（通过摇瓶法测得为-1.4）显著不同。稳定性结果表明，酯的最大稳定性对应的pH范围为3-4，最佳聚合物分子量为5000。溶解度研究得到的抛物线表明，MTX在碱性条件下（pH>5）溶解度更高，而MTX-PEGs在酸性条件下（pH<5）溶解度更高。根据我们的结果得出结论，MTX和PEGs以等摩尔比形成了MTX-PEGs。酯的log P值较高，表明MTX与其PEG共轭物之间存在显著的理化差异，且在酸性介质中具有更高的稳定性和溶解度，这显示了一种有望改善共轭物药物递送的方法，特别是未来的MTX-PEG5000。
