VEGFR2 (IC50 = 1 nM); RET (IC50 = 13 nM)

Apatinib (YN968D1) exhibited a potent kinase suppression effect on VEGFR-2, c-kit, and c-src, as well as an inhibition of cellular phosphorylation of VEGFR-2, c-kit, and PDGFRβ. With an IC50 of 0.013 μM, 0.429 μM, and 0.53 μM, respectively, YN968D1 suppresses the activities of Ret, c-kit, and c-src. At concentrations up to 10 μM, YN968D1 did not significantly affect EGFR, Her-2, or FGFR1. In addition to blocking the budding of rat aortic ring, YN968D1 efficiently suppressed the proliferation, migration, and tube formation of human umbilical vein endothelial cells stimulated by FBS[1].

YN968D1 both by itself and in conjunction with chemotherapeutic agents efficiently and minimally harmed the growth of multiple well-established human tumor xenograft models in vivo[1].

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Apatinib was valid in tumor growth inhibition in vivo. The tumor volume decreased when compared with the control group (Figures 7a and b). In accordance with in vitro experiment, Figure 7c shows that Apatinib treatment increased the level of LC3-II and Bax, whereas the level of BCL-2 and VEGFR2 decreased in vivo. Immunohistochemistry showed that Apatinib decreased the expression of VEGFR2, p-STAT3 and BCL-2 in tumors formed by KHOS cells (Figure 7d). All the results revealed that Apatinib inhibited the growth of osteosarcoma in vivo [2].

Enzyme‐linked immunosorbent assay. [1]
The inhibitory activity of YN968D1 against tyrosine kinases was determined using ELISA methodology described previously. VEGFR‐2 and PDGFR were purchased commercially; 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]

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Apatinib, also known as YN968D1, is a newly developed selective inhibitor that can be taken orally and may have antiangiogenic and antineoplastic properties. Apatinib inhibits VEGFR2 by binding to it specifically. Apatinib also has a potent inhibitory effect on Ret, c-kit, and c-src activity, with IC50 values of 0.013 M, 0.429 M, and 0.53 M, respectively. Apatinib prevents PDGFRβ, c-kit, and VEGFR-2 from becoming phosphorylated in cells. Proliferation induced by 20 ng/mL VEGF is significantly inhibited by atainib (IC50 = 0.17μM).

In 96-well plates, the HUVEC were seeded. After incubating for 24 hours, the test agents (vehicle serving as the control) were added to the cells, along with 20 ng ⁄mL VEGF or 20% FBS, and left them for an additional 72 hours. The cells were first fixed with 10% trichloroacetic acid, then stained for 30 minutes at 37°C using 0.4% sulforhodamine B. Afterward, they were cleaned with 1% acetic acid wash. 520 nm optical density was measured after the complex was dissolved with the addition of Tris.[1]

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The CCK8 assay was used to evaluate the cell viability as described previously.40 The day before the experiment, the cells were seeded 5000 cells per well in 96-well plates. The cells were incubated with Apatinib at an indicated condition.[2]

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Apoptosis analysis and cell cycle: For cell-cycle assay, cells were fixed with 70% ethanol at −20 °C overnight, and stained with propidium iodide. For cell apoptosis analysis, cells were stained with the Annexin V/FITC Kit according to the manufacturer’s explanations and analyzed by flow cytometry after Apatinib treatment as described previously.[2]

tumor xenograft model (NCI-H460 human lung tumors, HCT 116 human colon tumors, or SGC-7901 human gastric tumors; BALB⁄cA nude mice)
50, 100 and 200 mg/kg
by oral gavage[1]

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Nude mouse human tumor xenograft model.  The effects of Apatinib (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 mm3, at which time the mice were randomized into experimental groups. YN968D1 was administered once daily by oral gavage for the indicated periods (Table 1). 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 (Table 2). Tumor volume and bodyweight 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 × b2)/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]

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A 4- to 6-week-old BALB/c nude mice were subcutaneously injected in the right flank with 2 × 106 KHOS cells. The mice were fed in specific pathogen-free conditions, and when a palpable mass developed, the mice were randomly divided into two sets and were administered DMSO or Apatinib 50 mg/kg orally daily for 30 days. The tumor was scaled every other day for 4 days. The tumor volume was counted by (length × width2/2). The mice were killed on the 13th day after the treatment. Tumor samples were prepared for western blot and IHC.[2]

Pharmacokinetic evaluation revealed GNE-3511 exhibited moderate (mouse, rat, and cynomolgus) to high (dog) in vivo plasma clearances, moderate volumes of distribution, short half-lives, and brain penetration sufficient to enable examination in animal models of neurodegeneration (Table 6). DLK inhibitor GNE-3511 was then tested in the mouse optic nerve crush model of axonal injury, which mimics the degeneration that occurs in glaucoma or optic neuropathy. Our previous studies demonstrated that loss of DLK expression resulted in protection of retinal ganglion cell neurons from degeneration as well as an attenuation of downstream signaling following injury. In this and other neuronal injury models, phosphorylation of c-Jun (p-c-Jun) is strongly induced by injury in a DLK/JNK dependent fashion and could thus be used as a pharmacodynamic readout of DLK inhibition in vivo. Animals were dosed orally with either inhibitor GNE-3511 at two dose levels or vehicle control 30 min prior to nerve crush injury. Six hours after insult, levels of p-c-Jun in retina were measured using a MSD assay. Treatment with inhibitor GNE-3511 resulted in a dose-dependent reduction of p-c-Jun present in retina (Figure 5).

[1]. YN968D1 is a novel and selective inhibitor of vascular endothelial growth factor receptor-2 tyrosine kinase with potent activity in vitro and in vivo. Cancer Sci. 2011 Jul;102(7):1374-80.

[2]. Apatinib promotes autophagy and apoptosis through VEGFR2/STAT3/BCL-2 signaling in osteosarcoma. Cell Death Dis. 2017 Aug; 8(8): e3015.

Rivoceranib is under investigation in clinical trial NCT02726854 (Apatinib as Second-line Treatment of Advanced Pancreatic Cancer).
Rivoceranib is an orally bioavailable, small-molecule receptor tyrosine kinase inhibitor with potential antiangiogenic and antineoplastic activities. Upon administration, rivoceranib selectively binds to and inhibits vascular endothelial growth factor receptor 2, which may inhibit VEGF-stimulated endothelial cell migration and proliferation and decrease tumor microvessel density. In addition, this agent mildly inhibits c-Kit and c-SRC tyrosine kinases.

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Angiogenesis is an important process in cell development, especially in cancer. Vascular endothelial growth factor (VEGF) signaling is an important regulator of angiogenesis. Several therapies that act against VEGF signal transduction have been developed, including YN968D1, which is a potent inhibitor of the VEGF signaling pathway. This study investigated the antitumor activity of YN968D1 (apatinib mesylate) in vitro and in vivo. YN968D1 potently suppressed the kinase activities of VEGFR-2, c-kit and c-src, and inhibited cellular phosphorylation of VEGFR-2, c-kit and PDGFRβ. YN968D1 effectively inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells induced by FBS, and blocked the budding of rat aortic ring. In vivo, YN968D1 alone and in combination with chemotherapeutic agents effectively inhibited the growth of several established human tumor xenograft models with little toxicity. A phase I study of YN968D1 has shown encouraging antitumor activity and a manageable toxicity profile. These findings suggest that YN968D1 has promise as an antitumor drug and might have clinical benefits.[1]

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The cure rate of osteosarcoma has not improved in the past 30 years. The search for new treatments and drugs is urgently needed. Apatinib is a high selectivity inhibitor of vascular endothelial growth factor receptor-2 (VEGFR2) tyrosine kinase, exerting promising antitumoral effect in various tumors. The antitumor effect of Apatinib in human osteosarcoma has never been reported. We investigated the effects of Apatinib in osteosarcoma in vitro and in vivo. Osteosarcoma patients with high levels of VEGFR2 have poor prognosis. Apatinib can inhibit cell growth of osteosarcoma cells. In addition to cycle arrest and apoptosis, Apatinib induces autophagy. Interestingly, inhibition of autophagy increased Apatinib-induced apoptosis in osteosarcoma cells. Immunoprecipitation confirmed direct binding between VEGFR2 and signal transducer and activator of transcription 3 (STAT3). Downregulation of VEGFR2 by siRNA resulted in STAT3 inhibition in KHOS cells. VEGFR2 and STAT3 are inhibited by Apatinib in KHOS cells, and STAT3 act downstream of VEGFR2. STAT3 and BCL-2 were downregulated by Apatinib. STAT3 knockdown by siRNA reinforced autophagy and apoptosis induced by Apatinib. BCL-2 inhibits autophagy and was apoptosis restrained by Apatinib too. Overexpression of BCL-2 decreased Apatinib-induced apoptosis and autophagy. Apatinib repressed the expression of STAT3 and BCL-2 and suppressed the growth of osteosarcoma in vivo. To sum up, deactivation of VEGFR2/STAT3/BCL-2 signal pathway leads to Apatinib-induced growth inhibition of osteosarcoma.[2]

C24H23N5O.CH4O3S

493.57798

397.19

C, 72.52; H, 5.83; N, 17.62; O, 4.03

811803-05-1

1218779-89-5 (HCl);1218779-75-9 (mesylate);811803-05-1;

11315474

Solid powder

4.1

2

5

6

30

608

0

WPEWQEMJFLWMLV-UHFFFAOYSA-N

InChI=1S/C24H23N5O/c25-17-24(11-1-2-12-24)19-5-7-20(8-6-19)29-23(30)21-4-3-13-27-22(21)28-16-18-9-14-26-15-10-18/h3-10,13-15H,1-2,11-12,16H2,(H,27,28)(H,29,30)

N-[4-(1-cyanocyclopentyl)phenyl]-2-(pyridin-4-ylmethylamino)pyridine-3-carboxamide

YN968D1; YN-968D1; YN 968D1; Rivoceranib; Apatinib; 811803-05-1; rivoceranib; Apatinib free base; Apatinib (free base); YN968D1; N-(4-(1-Cyanocyclopentyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide; N-[4-(1-cyanocyclopentyl)phenyl]-2-(pyridin-4-ylmethylamino)pyridine-3-carboxamide; Apatinib free base

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)