Anlotinib (hydrochloride)

Cell experiments:

Cell lines

EA.hy 926

Cell culture

The cells were routinely cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin, and subcultured in a humidified incubator at 37°C with 5% CO₂.

Reaction condition

1. Cell Viability Assay: The cells were seeded at a density of
1×10⁴ cells per well, treated with anlotinib at concentrations ranging from 0 to 1600 μM for 24 h. Subsequently, 0.5% MTT reagent was added for a further 4 h of incubation. After the formazan crystals were dissolved in DMSO, the absorbance at 570 nm was detected. The concentrations of 0.01 μM, 0.1 μM, and 1 μM anlotinib were identified as non-toxic doses with no significant impact on cell viability.
2. Migration Assay: In wound healing assays or Transwell assays, induction medium containing 1% FBS and supplemented with VEGF (20 ng/mL), PDGF-BB (100 ng/mL), or FGF-2 (20 ng/mL) was prepared. Anlotinib at concentrations of 0.01–1 μM was added to the induction medium, and the cells were incubated for 6 h.
3. Tube Formation Assay: The cells were pre-treated with anlotinib at 0.01–1 μM for 6 h first, then seeded into 96-well plates coated with Matrigel. The aforementioned induction factors were added, and the cells were incubated for 8 h to observe the formation of capillary-like tube structures.
4. Mechanism Verification (Western Blot): After being serum-starved in serum-free medium for 24 h, the cells were treated with anlotinib at 0.01–1 μM for 6 h, followed by stimulation with the corresponding induction factors for 10 min. The cells were harvested to extract total proteins, and the phosphorylation levels of target proteins were detected.

Results

The half-maximal inhibitory concentration (IC₅₀) of anlotinib against EA.hy 926 cells at 24 h was 30.26 μM. Anlotinib at concentrations of 0.01 μM, 0.1 μM, and 1 μM exerted no significant effect on cell viability, which were suitable for subsequent functional experiments.
1. Migration Inhibition: At a concentration of 1 μM, anlotinib achieved migration inhibition rates of 43.46% (wound healing assay) and 67.53% (Transwell assay) against VEGF-induced cell migration; 66.61% (wound healing assay) and 65.13% (Transwell assay) against PDGF-BB-induced migration; and 64.01% (wound healing assay) and 75.32% (Transwell assay) against FGF-2-induced migration. Notably, its inhibitory effect was superior to that of control drugs, including sunitinib and sorafenib.
2. Tube Formation Inhibition: Anlotinib inhibited the formation of capillary-like tube structures in endothelial cells in a concentration-dependent manner, and its inhibitory effect at 1 μM was significantly superior to that of the control drugs.
3. Signaling Pathway Inhibition: Anlotinib significantly reduced the phosphorylation levels of VEGFR2 (with a maximum inhibition rate of 56.37%), PDGFRβ (with an inhibition rate of 41.1%), and FGFR1 (with an inhibition rate of 45.0%), and blocked the activation of the downstream ERK signaling pathway.

Animal experiment:

Animal models

SD rats

Dosage form

Rat Aortic Ring Assay: No in vivo administration was performed. Rat aortic rings were cultured in vitro, and anlotinib at concentrations of 0.01 μM, 0.1 μM, and 1 μM was added, together with 50 ng/mL of VEGF/PDGF-BB/FGF-2 to induce microvessel sprouting.
Pharmacokinetic Experiment: The oral administration doses were 1.5 mg/kg, 3 mg/kg, and 6 mg/kg (via gavage); the intravenous administration dose was 1.5 mg/kg (via tail vein injection).
Tissue Distribution Experiment: A single oral dose of 3 mg/kg anlotinib was given (via gavage), and sample collection was conducted at 1 h, 4 h, 8 h, and 24 h after administration, respectively.
Safety Experiment: For the 14-day oral administration, the test dose for the median lethal dose (LD₅₀) reached 1735.9 mg/kg.

Results

Aortic Ring Angiogenesis: Anlotinib inhibited microvessel sprouting in a concentration-dependent manner. At a concentration of 1 μM, its inhibition rate was 15%–30% higher than that of control drugs such as sunitinib, and it exerted significant inhibitory effects on VEGF/PDGF-BB/FGF-2-induced sprouting.
Pharmacokinetics: The oral bioavailability was 28%–58%; the terminal half-life was 5.1±1.6 h; the total plasma clearance was 5.35±1.31 L·h⁻¹·kg⁻¹; the apparent volume of distribution was 27.6±3.1 L/kg; the plasma protein binding rate reached 97%. The proportion of unchanged drug excreted in urine, feces, and bile was low, and cytochrome P450-mediated oxidation was the main elimination pathway.
Tissue Distribution: The drug concentration in various tissue homogenates was significantly higher than that in plasma. Among them, the lung tissue had the highest exposure (197 times that of plasma), and the exposure levels in the liver, kidney, and heart were 49 times, 54 times, and 32 times that of plasma, respectively. Anlotinib can cross the blood-brain barrier, achieving brain concentrations comparable to those in plasma.
Safety: The median lethal dose (LD₅₀) of 14-day oral administration was 1735.9 mg/kg, which was much higher than the therapeutic dose. No obvious liver, kidney, or bone marrow damage was observed, and there was no reproductive or genetic toxicity.

References:

[1] Lin B, Song X, Yang D, Bai D, Yao Y, Lu N. Anlotinib inhibits angiogenesis via suppressing the activation of VEGFR2, PDGFRβ and FGFR1. Gene. 2018 May 15;654:77-86. doi: 10.1016/j.gene.2018.02.026. Epub 2018 Feb 14. Erratum in: Gene. 2020 Jan 10;723:144119. doi: 10.1016/j.gene.2019.144119. PMID: 29454091.
[2] Zhao Y, Liu Y, Shan J, Xu X, Zhang C, Liu Z, Li X, Zhong Z, Gao Y, Ren K, Jiao D, Ren J, Wu P, Jiang Y, Han X. Anti-inflammatory coupled anti-angiogenic airway stent effectively suppresses tracheal in-stents restenosis. J Nanobiotechnology. 2025 Jan 29;23(1):59. doi: 10.1186/s12951-024-03087-y. PMID: 39881307; PMCID: PMC11776288.
[3] Chen HM, Feng G. Use of anlotinib in intra-abdominal desmoplastic small round cell tumors: a case report and literature review. Onco Targets Ther. 2018 Dec 18;12:57-61. doi: 10.2147/OTT.S190333. PMID: 30588030; PMCID: PMC6302812.
[4] Zhong CC, Chen F, Yang JL, Jia WW, Li L, Cheng C, Du FF, Zhang SP, Xie CY, Zhang NT, Olaleye OE, Wang FQ, Xu F, Lou LG, Chen DY, Niu W, Li C. Pharmacokinetics and disposition of anlotinib, an oral tyrosine kinase inhibitor, in experimental animal species. Acta Pharmacol Sin. 2018 Jun;39(6):1048-1063. doi: 10.1038/aps.2017.199. Epub 2018 Apr 5. PMID: 29620050; PMCID: PMC6256268.

The IC50 of anlotinib on kinase activity

Targets

VEGFR2

PDGFRβ

FGFR1

IC₅₀

5.6±1.2 nM

8.7±3.4 nM

11.7±4.1 nM