Malsch R, Harenberg J, Guerrini M, Torri G, Casu B, Heene D L
1st Department of Medicine, Medical University Clinic Mannheim, Germany.
Semin Thromb Hemost. 1994;20(2):182-92. doi: 10.1055/s-2007-1001902.
Unfractionated heparin and LMMH were substituted with different lipophilic organic compounds. Specifically endpoint attached (LMMH-tyramine and LMMH-tyramine-FITC) and nonspecifically substituted heparins (acylated heparins, and LMMH-biotin and LMMH-cholesterol hemisuccinate) were obtained. The lipophilically substituted heparins were analysed by HPSEC and showed different retention times, high peak purity, different UV/VIS absorbances, and areas under the absorbance time curve. The determination of the average molecular mass Mn, Mm, and Mz and the polydispersity P was performed by PAGE. The substituted heparins showed an increase in their molecular mass Mm, ranging from 2.9 to 129.7% unfractionated heparin and 3.9 to 224.0% (LMMH) compared with the parent compounds (unfractionated heparin and LMMH). The anticoagulant activity was measured by anti-Factor Xa. Lipophilically modified heparin had an aXa activity ranging from 52 to 168 U/mg (unfractionated) and 60 to 108 U/mg (LMMH) and antithrombin activity ranging from 31 to 270 U/mg (unfractionated) and 5 to 15 U/mg (LMMH). The thrombin generation inhibition assay demonstrated an effective anticoagulant potency of the modified compounds. They were neutralized by different amounts (1.1 to 4.1, w/w) of protamin. 1H NMR spectroscopy revealed the specific endpoint attachment of tyramine to LMMH and FITC to LMMH-tyramine. The lipophilically modified heparins showed intact anticoagulant properties and are now used for pharmacokinetic investigations.
普通肝素和低分子质量肝素（LMMH）被不同的亲脂性有机化合物所取代。具体而言，获得了末端连接的（LMMH-酪胺和LMMH-酪胺-异硫氰酸荧光素）以及非特异性取代的肝素（酰化肝素、LMMH-生物素和LMMH-胆固醇半琥珀酸酯）。通过高效体积排阻色谱（HPSEC）对亲脂性取代的肝素进行分析，结果显示其保留时间不同、峰纯度高、紫外/可见吸光度不同以及吸光度时间曲线下的面积不同。通过聚丙烯酰胺凝胶电泳（PAGE）测定平均分子量Mn、Mm和Mz以及多分散性P。与母体化合物（普通肝素和LMMH）相比，取代的肝素显示其分子量Mm增加，普通肝素增加幅度为2.9%至129.7%，LMMH增加幅度为3.9%至224.0%。通过抗Xa因子测定抗凝活性。亲脂性修饰的肝素的抗Xa活性范围为52至168 U/mg（普通肝素）和60至108 U/mg（LMMH），抗凝血酶活性范围为31至270 U/mg（普通肝素）和5至15 U/mg（LMMH）。凝血酶生成抑制试验证明了修饰化合物具有有效的抗凝效力。它们被不同量（1.1至4.1，w/w）的鱼精蛋白中和。1H核磁共振光谱揭示了酪胺与LMMH以及异硫氰酸荧光素与LMMH-酪胺的特异性末端连接。亲脂性修饰的肝素显示出完整的抗凝特性，目前用于药代动力学研究。
