Treprostinil Sodium (UT-15) is a potent DP1, IP and EP2 agonist (EC50: 0.6/1.9/6.2 nM).

RET:13 nM (Cell-free), VEGFR2:1 nM (Cell-free)

体外酶活性实验表明，Apatinib是一种比sunitinib更具选择性的VEGFR-2抑制剂，其IC50分别为0.001 μM和0.005 μM。Apatinib还能有效抑制Ret, c-kit和c-src的活性，其IC50分别为0.013 μM, 0.429 μM和0.53 μM。在高达10 μM的浓度下，Apatinib对EGFR, Her-2或FGFR1没有显著影响。Apatinib轻微抑制由20% FBS刺激的HUVEC增殖（IC50: 23.4 μM），而对由20 ng/mL VEGF刺激的HUVEC增殖有显著抑制作用（IC50: 0.17 μM）。在相同条件下，sunitinib的IC50值更低（分别为7.4 μM和0.034 μM）。1 μM Apatinib显著抑制了FBS诱导的HUVEC迁移。在1 μM浓度下，sunitinib也能抑制HUVEC迁移。Apatinib显著增强了在过表达ABCB1的KBv200、MCF-7/adr和HEK293/ABCB1细胞以及过表达ABCG2的S1-M1-80、MCF-7/FLV1000和HEK293/ABCG2-R2细胞中，ABCB1或ABCG2底物化合物的细胞毒性。与此相反，apatinib并未改变亲本细胞和过表达ABCC1细胞中特定化合物的细胞毒性。Apatinib显著增加了多药抗性（MDR）细胞内罗丹明123和多柔比星的积累。此外，apatinib还显著抑制了ABCB1和ABCG2与[(125)I]碘芳基偶氮吡唑相的光亲和标记，这种抑制作用是浓度依赖性的。Apatinib显着增加了ABCB1和ABCG2的ATP酶活性。然而，在产生MDR逆转的浓度下，apatinib并未显著改变ABCB1或ABCG2的蛋白或mRNA表达水平，也未改变AKT和细胞外信号调节激酶1/2（ERK1/2）的磷酸化。

The inhibitory activity of YN968D1 against tyrosine kinases was determined using ELISA methodology described previously. Her-2, c-kit, and c-src were activated intracellular protein tyrosine kinases expressed by Bab-to-Bac Baculovirus Expression Vector System and purified by Ni-NTA spin columns. The optical density was measured at 490 nm using VERSAmax. The inhibitory activity was expressed as IC50, which was calculated from three independent experiments by the Logit method [1].

Immunohistochemistry was used to determine vessel density by analyzing the expression of CD31, an endothelial marker. Briefly, nude mice xenografted with NCI-H460 tumor were treated with 200 mg/kg Apatinib by the oral garage for 14 days and tumor sections were prepared from formalin-fixed and paraffin-embedded tumor tissues. Slides were treated with 3% H2O2 for 10 min and then incubated in 2% goat serum for 20 min to block the nonspecific antibody binding. Slides were stained with the anti-CD31 antibody at room temperature for 2 h, followed by treatment with biotinylated goat anti-mouse IgG and SABC complex at 37C for 30 min. Finally, diaminobenzidine tetrachloride was used for color development and the slides were counterstained with hematoxylin. Positive cells in images were measured with Image-Pro Plus software [1].

The effects of YN968D1 on tumor growth were tested against various human tumors grown subcutaneously in BALB? cA nude mice. Tumor growth was initiated by subcutaneous inoculation of cells into mice. Tumors were allowed to establish and grow to 100–300 mm^3, at which time the mice were randomized into experimental groups. YN968D1 was administered once daily by oral gavage for the indicated periods. In combination treatment experiments, mice were administered YN968D1 alone by oral gavage; 5-FU, oxaliplatin, docetaxel and doxorubicin alone by intravenous injection; or YN968D1 in combination with each cytotoxic drug at the indicated dose and schedule. Tumor volume and body weight were monitored every other day or every 3 days, with the means indicated for groups of six (treated) or 12 (vehicle control) animals. Tumor volumes were determined by measuring the largest diameter (a) and its perpendicular (b) according to the formula (a ×b^2)? 2. The evaluation index for inhibition was the relative tumor growth ratio according to the equation: T?C (%) = mean increase of tumor volumes of treated groups?mean increase of tumor volumes of control groups ×100% [1].