TP-0463518 is a highly potent HIF prolyl hydroxylase (PHD) inhibitor (IC50s: 13 nM and 18 nM for human and rat PHD2, respectively).

PHD2 (rat):18 nM (cell free), PHD1 (human):18 nM (cell free), PHD2 (human):13 nM (cell free), PHD3 (human):63 nM (cell free)

TP0463518 竞争性抑制人类 PHD2，Ki 值为 5.3nM。此外，TP0463518 也对人类 PHD1/3 表现出抑制作用，IC50 值分别为 18 nM 和 63 nM，以及对猴子 PHD2 的抑制作用，IC50 值为 22nM。

在正常的小鼠和大鼠中，TP0463518在5mg/kg和20mg/kg的剂量下显著提高了血清EPO水平。在5/6肾切除的慢性肾脏病模型大鼠中，TP0463518以10mg/kg的剂量也增加了血清EPO水平，血清EPO与血清TP0463518水平的相关系数为0.82。TP0463518的半衰期为5.2小时，且在5mg/kg的剂量下增加了EPO的曲线下面积(AUC)。

The PHDs inhibition studies were performed using fluorescence polarization.FITC-HIF and 2-oxoglutarate were mixed with enzyme solution in a reaction buffer (20 mM Tris-HCl [pH 7.5],5 mM KCl,1.5 mM MgCl2,10 μM FeSO4,2 mM ascorbic acid,1 mM DTT) with or without various concentrations of TP0463518.The concentrations of FITC-HIF and 2-oxoglutarate were twice the Km values of each enzyme.The reaction temperature was 30?C,and the reaction time was optimized to each PHD enzyme to obtain the initial velocity (9 to 20 min).At the end of the reaction,a stop solution containing 20 mM of EDTA and anti-hydroxylated HIF antibody was added to the reaction buffer.Then,the fluorescence (ex: 480 nm,em: 535 nm) was measured using EnVision to calculate the millipolarization (mP) value.The mP values and the corresponding hydroxylated HIF concentration were proportional,so we used the mP values as the activities.The IC50 values were calculated using SAS version 9.2 using a nonlinear least-squares method.To determine the mode of inhibition,the activity of PHD2 was measured with various concentrations of 2-oxoglutarate (0.025 to 8 μM) and TP0463518 (0 to 40 μM).Then the apparent Vmax and Km corresponding to each TP0463518 concentration were compared.The mode of inhibition was confirmed using a double reciprocal plot.

Nine-week-old Balb/c mice were randomly assigned to a vehicle or a 5 to 40 mg/kg dose of TP0463518 group. The mice were orally treated with 0.5% methylcellulose or a TP0463518 dosing suspension. Blood was collected at 6 h after administration from the orbital plexus under deep anesthesia, and euthanasia was performed without awakening. An aliquot of blood was mixed with EDTA, and the remaining blood sample was left to stand at room temperature for 15 min. The samples were then centrifuged (2130 x g for 10 min at 4°C) to prepare the plasma and serum.