Development of a therapeutic anti-marinobufagenin antibody

虽然先兆子痫是产妇和胎儿死亡的主要原因，但其发病机制尚不完全清楚。内源性地黄样促心类固醇（CTS）与子痫前期的病理生理有关，临床观察表明，结合CTS的地高辛抗体Digibind可降低子痫前期患者的血压。最近，我们报道了严重PE患者血浆中肌蟾素（MBG）（一种蟾二烯内酯类血管收缩剂CTS）水平升高4倍。在本研究中，我们比较了正常胎盘和子痫前期胎盘中MBG的水平，并通过高效液相色谱（HPLC）检测了MBG和瓦巴因的抗体与子痫前期胎盘中纯化的物质的相互作用。MBG水平，而不是内源性瓦巴因水平，在子痫前期胎盘中显示出4倍的升高。内源性胎盘MBG样免疫反应物质反相高效液相色谱洗脱时间与正品MBG洗脱时间相同。以Digibind为基础的免疫分析没有检测到与含有乌阿拜样免疫反应物质的HPLC交叉反应性，但与保留时间与MBG和其他bufadienolides相似的HPLC组分交叉反应。2008年，我们还比较了Digibind和单克隆抗mbg抗体在两种PE大鼠模型中的体内疗效，即在妊娠14-20天补充NaCl致高血压的正常妊娠大鼠和糖尿病妊娠大鼠。在两种模型中，MBG水平均显著升高，抗MBG抗体降低血压和逆转Na/ k - atp酶（MBG的靶酶）抑制的能力超过Digibind。我们的研究结果表明，在子痫前期，全身和胎盘的MBG水平都显著升高，这表明MBG是该综合征患者免疫中和的潜在靶点。

Although preeclampsia is a major cause of maternal and fetal mortality, its pathogenesis is not fully understood. Endogenous digitalis-like cardiotonic steroids (CTS) are implicated in the pathophysiology of preeclampsia, as illustrated by clinical observations that Digibind, a digoxin antibody which binds CTS, lowers blood pressure in patients with preeclampsia. Recently we reported that plasma levels of marinobufagenin (MBG), a bufadienolide vasoconstrictor CTS, are increased four-fold in patients with severe PE. In the present study, we compared levels of MBG in normal and preeclamptic placentae, and tested antibodies against MBG and ouabain for their interaction with the material purified from preeclamptic placentae via high-performance liquid chromatography (HPLC). Levels of MBG, but not that of endogenous ouabain, exhibited a four-fold elevation in preeclamptic placentae. The elution time of endogenous placental MBG-like immunoreactive material from reverse-phase HPLC column was identical to that of authentic MBG. Immunoassay based on Digibind did not detect cross-reactivity with HPLC containing ouabain-like immunoreactive material, but cross-reacted with HPLC fractions having retention time similar to that of MBG and other bufadienolides. In 2008 we also compared the in vivo efficacy of Digibind and monoclonal anti-MBG antibodies in two rat models of PE, in normal and diabetic pregnant rats rendered hypertensive by NaCl supplementation during days 14-20 of gestation. In both models, levels of MBG were substantially elevated, and anti-MBG antibody exceeded Digibind in its capacity to lower blood pressure and to reverse inhibition of Na/K-ATPase, a target enzyme for MBG. Our results demonstrate that both systemic and placental levels of MBG are significantly elevated in preeclampsia, and suggest that MBG is a potential target for immunoneutralization in patients with this syndrome.