Ramani Karthik, Miclea Razvan D, Purohit Vivek S, Mager Donald E, Straubinger Robert M, Balu-Iyer Sathy V
Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, New York 14260, USA.
J Pharm Sci. 2008 Apr;97(4):1386-98. doi: 10.1002/jps.21102.
Factor VIII (FVIII) is a multidomain protein that is deficient in hemophilia A, a clinically important bleeding disorder. Replacement therapy using recombinant human FVIII (rFVIII) is the main therapy. However, approximately 15-30% of patients develop inhibitory antibodies that neutralize rFVIII activity. Antibodies to epitopes in C2 domain, which is involved in FVIII binding to phospholipids, are highly prevalent. Here, we investigated the effect of phosphatidylserine (PS)-containing liposomes, which bind to C2 domain with high affinity and specificity, upon the immunogenicity of rFVIII. Circular dichroism studies showed that PS-containing liposomes interfered with aggregation of rFVIII. Immunogenicity of free- versus liposomal-rFVIII was evaluated in a murine model of hemophilia A. Animals treated with s.c. injections of liposomal-rFVIII had lower total- and inhibitory titers, compared to animals treated with rFVIII alone. Antigen processing by proteolytic enzymes was reduced in the presence of liposomes. Animals treated with s.c. injections of liposomal-rFVIII showed a significant increase in rFVIII plasma concentration compared to animals that received rFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between PS-containing bilayers and rFVIII may provide a basis for designing lipidic complexes that improve the stability, reduce the immunogenicity of rFVIII formulations, and permit administration by s.c. route.
凝血因子VIII（FVIII）是一种多结构域蛋白，在临床上重要的出血性疾病甲型血友病中缺乏。使用重组人FVIII（rFVIII）的替代疗法是主要治疗方法。然而，约15 - 30%的患者会产生中和rFVIII活性的抑制性抗体。针对参与FVIII与磷脂结合的C2结构域中表位的抗体非常普遍。在此，我们研究了以高亲和力和特异性与C2结构域结合的含磷脂酰丝氨酸（PS）脂质体对rFVIII免疫原性的影响。圆二色性研究表明，含PS脂质体干扰了rFVIII的聚集。在甲型血友病小鼠模型中评估了游离型与脂质体型rFVIII的免疫原性。与单独用rFVIII治疗的动物相比，皮下注射脂质体型rFVIII治疗的动物总滴度和抑制滴度较低。在脂质体存在下，蛋白水解酶的抗原加工减少。与单独接受rFVIII的动物相比，皮下注射脂质体型rFVIII治疗的动物rFVIII血浆浓度显著增加。基于这些研究，我们推测含PS双层与rFVIII之间的特定分子相互作用可能为设计脂质复合物提供基础，这些脂质复合物可提高稳定性、降低rFVIII制剂的免疫原性，并允许通过皮下途径给药。
