Marinobufagenin as a therapeutic target

首先，我们研究了Mg 2+离子是否可以增强DigiFab对PE与对照妊娠中离体红细胞NKA活性恢复的作用。我们已经检查了DigiFab、Mg 2+和两者的组合对PE患者的红细胞NKA活性的影响。在来自无并发症妊娠患者的红细胞中，我们比较了在不存在或存在Mg 2+或Mg 2+和DigiFab的组合的情况下MBG浓度增加的离体抑制活性。与对照组相比，PE患者血浆MBG升高3倍与红细胞NKA活性降低2.5倍相关。用Mg 2+或DigiFab预孵育红细胞增加NKA活性，并且Mg 2+和DigiFab两者的组合使NKA活性恢复至对照水平。Mg 2+使MBG在对照红细胞中的NKA抑制活性向右移动。Mg 2+和DigiFab的组合显著增强了DigiFab对MBG的免疫中和作用，使得NKA对MBG不太敏感。这些结果表明，镁能够增加DigiFab在PE中MBG诱导的NKA抑制的免疫中和中的功效，并且很有可能增强DigiFab的抗高血压作用，这可能在PE的抗高血压治疗中具有重要的临床应用。 在第二项研究中，我们首先测量了245例心力衰竭（HF）患者的血浆MBG水平，并进行了全面的临床、实验室和超声心动图评估。对全因死亡率、心脏移植和HF住院进行了5年的跟踪。 在我们的研究队列中，中位四分位数范围MBG为0.58（0.38-0.81）nM。较高的MBG与较高的髓过氧化物酶（MPO，r=0.42，p<0.0001）、BNP（r=0.25，p=0.001）和不对称二甲基精氨酸（ADMA，r=0.32，p<0.001）相关。MBG水平升高与右心室功能恶化相关（RV s：r=-0.39，p<0.0001），并预测不良临床结局的风险增加（MBG > 574 pM：HR 1.58（1.10-2.31），p=0.014），即使在调整了年龄、性别、糖尿病和缺血性病因后。在一项动物验证研究中，左前降支冠状动脉结扎心力衰竭模型导致MBG增加，而将MBG输注到小鼠体内4周导致MPO、ADMA和心脏纤维化显著增加。在心力衰竭的情况下，MBG血浆水平升高与右心室功能障碍相关，并在调整已建立的临床和生化风险因素的多变量模型中预测更差的长期临床结局。输注MBG似乎直接导致硝化应激和心脏纤维化增加。 在我们的第三项初级CRF患者（n=21; 9名女性和12名男性）的初步临床研究中，与健康对照组相比，MBG水平随着CRF分期的进展而逐渐增加，在血液透析阶段达到最高水平（2.15 +/- 0.26 nM vs. 0.18 +/- 0.01 nM）。在成人CRF患者（n=12; 56+/-4岁）中，MBG水平比健康对照组（n=9，年龄52 +/-5岁）增加3倍。成人CRF患者血液透析可降低血浆MBG浓度，但未降至对照水平。 总结：这些发现表明，PE和CRF患者中的MBG可能是目前正在人源化的3E 9抗MBG mAb的治疗应用的重要靶点。

First we studied, whether Mg2+ ions can potentiate the effect of DigiFab on the restoration of erythrocyte NKA activity ex vivo in PE vs. control pregnancy. We have examined the effect of DigiFab, Mg2+ and a combination of both on erythrocyte NKA activity in patients with PE. In erythrocytes from patients with uncomplicated pregnancies, we compared ex vivo inhibitory activity of incremental MBG concentration in the absence or presence of Mg2+, or combination of Mg2+ and DigiFab. Three-fold elevated plasma MBG in PE patients was associated with a 2.5-fold decrease of erythrocyte NKA activity vs. control. Preincubation of erythrocytes with Mg2+ or DigiFab increased NKA activity, and combination of both Mg2+ and DigiFab restored NKA activity to control level. NKA-inhibitory activity of MBG in control erythrocytes is shifted to the right by Mg2+. Combination of Mg2+ and DigiFab significantly potentiated immunoneutralizing effect of DigiFab on MBG, making NKA less sensitive to MBG. These findings indicate that magnesium is capable of increasing the efficacy of DigiFab in immunoneutralization of MBG-induced NKA inhibition in PE with a high probability to potentiate anti-hypertensive effect of DigiFab, which may have an important clinical application as antihypertensive therapy in PE. In a second study, we first measured plasma MBG levels and performed comprehensive clinical, laboratory, and echocardiographic assessment in 245 patients with heart failure (HF). All-cause mortality, cardiac transplantation, and HF hospitalization were tracked for 5 years. In our study cohort, median interquartile range MBG was 0.58 (0.38-0.81) nM. Higher MBG was associated with higher myeloperoxidase (MPO, r=0.42, p<0.0001), BNP (r=0.25, p=0.001), and asymmetric dimethylarginine (ADMA, r=0.32, p<0.001). Elevated levels of MBG were associated with measures of worse right ventricular function (RV s: r= -0.39, p<0.0001) and predicted increased risk of adverse clinical outcomes (MBG > 574 pM: HR 1.58 (1.10-2.31), p=0.014) even after adjustment for age, gender, diabetes mellitus, and ischemic etiology. In an animal validation study, a left anterior descending coronary artery ligation model of heart failure led to increases in MBG, while infusion of MBG into mice for 4 weeks led to significant increases in MPO, ADMA, and cardiac fibrosis. In the setting of heart failure, elevated plasma levels of MBG are associated with right ventricular dysfunction and predict worse long-term clinical outcomes in multivariable models adjusting for established clinical and biochemical risk factors. Infusion of MBG appears to directly contribute to increased nitrative stress and cardiac fibrosis. In our third preliminary clinical study in the junior CRF patients (n=21; 9 females and 12 males), levels of MBG progressively increased with an advancing CRF stage, reaching the highest levels at the hemodialysis stage (2.15 +/- 0.26 nM vs. 0.18 +/- 0.01 nM) compared to the healthy control. In adult CRF patients (n=12; 56+/-4 years), the level of MBG increased 3-fold vs. healthy control (n=9, age 52 +/- 5 years). Hemodialysis in adult CRF patients reduced plasma MBG concentration, but not to the control levels. Summary: These findings indicate that MBG in PE and CRF patients may be an important target for a therapeutic application of 3E9 anti-MBG mAb, which are currently in the process of humanization.