Anlotinib Hydrochloride: Multi-Target Tyrosine Kinase Inhibitor for Advanced Anti-Angiogenic Research

Executive Summary: Anlotinib hydrochloride (CAS 1058157-76-8) is a next-generation, orally active small-molecule inhibitor that targets VEGFR2, PDGFRβ, and FGFR1 with nanomolar potency, making it a leading tool for anti-angiogenic and oncology research (Xie et al., 2018). It demonstrates superior inhibition of endothelial cell migration and capillary-like tube formation in vitro compared to sunitinib and sorafenib. Anlotinib’s pharmacokinetic profile supports rapid oral absorption, high tissue distribution, and strong plasma protein binding in preclinical models. Safety studies reveal a high median lethal dose (LD₅₀), low systemic toxicity, and negligible genotoxicity. APExBIO supplies Anlotinib (hydrochloride) as a rigorously characterized research reagent (APExBIO).

Biological Rationale

Angiogenesis is essential for tumor growth, invasion, and metastasis. Tumor cells rely on new blood vessel formation to obtain nutrients and oxygen once they exceed ~1 mm³ in size (Xie et al., 2018). Persistent and unregulated angiogenesis is a hallmark of cancer progression. Vascular endothelial growth factor (VEGF) signaling, particularly through its receptor VEGFR2, is a primary driver of pathological angiogenesis. Inhibiting this pathway abrogates tumor vascularization and growth. Unlike tumor cells, endothelial cells in tumor vasculature rarely acquire resistance, making anti-angiogenic interventions durable (DOI). Multi-target tyrosine kinase inhibitors (TKIs) such as Anlotinib can disrupt multiple pro-angiogenic pathways, including VEGF, PDGF, and FGF signaling, providing broader efficacy than single-target agents.

Mechanism of Action of Anlotinib (hydrochloride)

Anlotinib is a small-molecule TKI that selectively binds the ATP-binding pocket of VEGFR2, PDGFRβ, and FGFR1 tyrosine kinases. Its IC₅₀ values are 5.6 ± 1.2 nM (VEGFR2), 8.7 ± 3.4 nM (PDGFRβ), and 11.7 ± 4.1 nM (FGFR1), indicating high potency under standard biochemical assay conditions (25°C, pH 7.4) (Xie et al., 2018). Anlotinib inhibits VEGF/PDGF-BB/FGF-2-induced endothelial cell migration and capillary tube formation in a concentration-dependent manner. It suppresses the downstream ERK signaling pathway, impacting cell proliferation and survival. Compared to sunitinib, sorafenib, and nintedanib, Anlotinib demonstrates greater selectivity and potency against these angiogenesis-related kinases (see detailed mechanism).

Evidence & Benchmarks

• Anlotinib inhibits VEGFR2 kinase activity with an IC₅₀ of 5.6 ± 1.2 nM, outperforming sunitinib and sorafenib in matched assays (Xie et al., Table 1).
• In human endothelial cell (EA.hy 926) migration assays, Anlotinib displays concentration-dependent inhibition with superior efficacy over legacy TKIs (APExBIO insights).
• Capillary-like tube formation is significantly suppressed at low nanomolar concentrations (≤ 20 nM) in matrigel assays (DOI).
• In vivo, daily oral dosing with Anlotinib reduces tumor vascular density and can induce tumor regression in mouse xenograft models (Xie et al., 2018).
• Pharmacokinetic studies reveal rapid oral absorption, bioavailability of 28–58% in rats and 41–77% in dogs, and high plasma protein binding (93% in humans) (DOI).
• Safety evaluations show an LD₅₀ of 1735.9 mg/kg (oral, 14-day, rodents), with mild systemic effects and no significant organ/genetic toxicity (Xie et al., Supplemental).

This article extends the mechanistic focus of Redefining Tumor Angiogenesis Inhibition by adding quantitative IC₅₀ data and workflow parameters, and clarifies practical application points not covered in Mechanistic Innovation and Strategy, which emphasizes translational opportunities but not detailed pharmacokinetics.

Applications, Limits & Misconceptions

Anlotinib hydrochloride, as supplied by APExBIO, is optimized for research on anti-angiogenic mechanisms, endothelial biology, and signal transduction in cancer models. It is widely used in:

• Endothelial cell migration and matrigel tube formation assays.
• ERK pathway inhibition studies in cellular contexts.
• In vivo tumor angiogenesis and xenograft models.
• Pharmacokinetics and tissue distribution analyses.

For detailed assay guidance and integration tips, see the Anlotinib (hydrochloride) product page and GSKChem's workflow dossier, which this article updates with new evidence and stricter assay boundaries.

Common Pitfalls or Misconceptions

• Anlotinib is not suitable for therapeutic or diagnostic use; it is strictly for scientific research (APExBIO).
• Direct cytotoxicity to tumor cells is minimal at sub-micromolar concentrations; its primary effect is anti-angiogenic (Xie et al., 2018).
• Inhibition of kinases outside VEGFR2/PDGFRβ/FGFR1 occurs only at higher concentrations; off-target effects are low within recommended assay ranges.
• Results from animal models may not directly translate to clinical efficacy due to species-specific pharmacokinetics.
• Improper storage above -20°C or repeated freeze-thaw cycles may reduce compound stability and potency (APExBIO).

Workflow Integration & Parameters

Anlotinib hydrochloride (C8688) is provided as a high-purity powder for reconstitution. For in vitro studies, recommended stock concentration is 10 mM in DMSO. Working concentrations in cell-based assays typically range from 1–100 nM for endothelial cell migration and tube formation, and 1–10 μM for broader kinase profiling. In vivo dosing for mice is typically 1–3 mg/kg orally, once daily, referencing published xenograft protocols (DOI). Store at -20°C, desiccated, and protect from light. Avoid more than three freeze-thaw cycles.

For integration into multi-parameter screens or combination studies, consider parallel testing with sunitinib or nintedanib as benchmarks. Detailed mechanistic workflow recommendations are extended in Rewriting the Rules of Tumor Angiogenesis Inhibition, which this article updates with validated pharmacokinetic and safety data.

Conclusion & Outlook

Anlotinib hydrochloride is a rigorously validated, potent multi-target TKI for anti-angiogenic and oncology research. Its dual selectivity for VEGFR2, PDGFRβ, and FGFR1, favorable pharmacokinetic profile, and low toxicity position it as a flagship research tool. APExBIO’s C8688 kit enables reproducible, translationally relevant results in cellular, biochemical, and animal models. Continued mechanistic research and careful workflow design are essential for maximizing scientific value and avoiding common pitfalls.