Anlotinib Hydrochloride: Multi-Target VEGFR2/PDGFRβ/FGFR1 Inhibitor for Angiogenesis and Cancer Research

Executive Summary: Anlotinib hydrochloride (CAS 1058157-76-8), provided by APExBIO (SKU: C8688), is a next-generation small molecule inhibitor of VEGFR2, PDGFRβ, and FGFR1 with nanomolar potency (IC₅₀ values: 5.6–11.7 nM) (Xie et al., 2018). It inhibits endothelial cell migration and tube formation in vitro in a concentration-dependent manner and blocks the ERK signaling pathway downstream of its targets (Xie et al., 2018). Compared to sunitinib, sorafenib, and nintedanib, anlotinib shows superior inhibition of angiogenic processes (Xie et al., 2018). Pharmacokinetic data indicate rapid oral absorption, high plasma protein binding (93% in humans), and broad tissue distribution including tumor and brain (Xie et al., 2018). Safety studies demonstrate a high LD₅₀ (1735.9 mg/kg, oral, 14 days, rat) and minimal systemic or genetic toxicity (Xie et al., 2018).

Biological Rationale

Angiogenesis is essential for tumor growth, invasion, and metastasis. Tumor mass cannot expand beyond ~1 mm³ without neovascularization (Xie et al., 2018). Endothelial cells, which form the blood vessel lining, are genetically more stable than tumor cells and rarely develop resistance to anti-angiogenic therapy (Xie et al., 2018). VEGF/VEGFR2 signaling is the primary driver of pathological angiogenesis in solid tumors (Xie et al., 2018). Additional pro-angiogenic signals involve PDGF-BB/PDGFRβ and FGF-2/FGFR1 pathways. Inhibiting multiple receptor tyrosine kinases (RTKs) involved in angiogenesis is a validated research and therapeutic strategy. Monoclonal antibodies (e.g., bevacizumab, ramucirumab) and TKIs targeting these pathways are standard tools in cancer research. However, many TKIs lack selectivity, causing off-target toxicity and reduced efficacy (Xie et al., 2018).

Mechanism of Action of Anlotinib (hydrochloride)

Anlotinib hydrochloride is a small-molecule multi-target tyrosine kinase inhibitor designed to block the ATP-binding pocket of VEGFR2, PDGFRβ, and FGFR1. It exhibits potent inhibitory activity with IC₅₀ values of 5.6 ± 1.2 nM for VEGFR2, 8.7 ± 3.4 nM for PDGFRβ, and 11.7 ± 4.1 nM for FGFR1, as measured in kinase assays at 25°C, 10 mM Tris-HCl pH 7.5 (Xie et al., 2018). In endothelial cell assays (EA.hy 926, HUVEC), anlotinib inhibits VEGF/PDGF-BB/FGF-2-induced migration and capillary tube formation with nanomolar potency. It suppresses downstream ERK phosphorylation, blocking proliferative and migratory signaling (Xie et al., 2018). Anlotinib also impedes microvessel outgrowth in rat aortic ring assays and reduces vascular density in vivo in xenograft tumor models. Compared to sunitinib, sorafenib, and nintedanib, anlotinib demonstrates broader and stronger inhibition of angiogenic signaling at equivalent concentrations (Xie et al., 2018). These molecular and cellular effects are directly linked to its research applications in tumor angiogenesis inhibition and tyrosine kinase signaling pathway studies.

Evidence & Benchmarks

• Anlotinib inhibits VEGFR2 kinase activity with an IC₅₀ of 5.6 ± 1.2 nM (at 25°C, 10 mM Tris-HCl pH 7.5) (Xie et al., 2018).
• It suppresses VEGF-induced ERK phosphorylation in HUVEC at 1–10 nM concentrations (Xie et al., 2018, Fig. 3).
• In endothelial cell migration and tube formation assays, anlotinib shows >90% inhibition at 10–100 nM (EA.hy 926, 37°C, serum-free medium) (Xie et al., 2018, Table 2).
• Oral bioavailability in rats is 28–58% (5 mg/kg, fasted, plasma AUC), and 41–77% in dogs (10 mg/kg) (Xie et al., 2018, Table S4).
• Plasma protein binding in humans reaches 93% (37°C, pH 7.4) (Xie et al., 2018).
• Median lethal dose (LD₅₀) is 1735.9 mg/kg (oral, 14 days, rat), with mild toxicity and no genotoxicity (Xie et al., 2018, Table S6).
• Tissue distribution studies show high accumulation in lung, liver, kidney, heart, and tumor tissue; also crosses the blood-brain barrier (Xie et al., 2018, Table S5).
• Compared to sunitinib, anlotinib induces tumor regression in some xenograft models at equivalent dosing (10 mg/kg oral, daily, mouse) (Xie et al., 2018, Fig. 7).

For expanded mechanistic insights, see Anlotinib Hydrochloride: Mechanistic Insights and Translational Applications, which provides additional mechanistic context; this article updates with new benchmarks and direct product parameters.

This analysis clarifies and extends the strategic overview in Anlotinib Hydrochloride: Elevating the Multi-Target Tyrosine Kinase Inhibitor Standard by including new safety and tissue distribution data.

For a comparative perspective on anti-angiogenic potency, see Anlotinib Hydrochloride: Multi-Target Tyrosine Kinase Inhibitor, while the present article provides updated IC₅₀ values and pharmacokinetic parameters.

Applications, Limits & Misconceptions

Anlotinib (hydrochloride) is used in research settings to study:

• Anti-angiogenic mechanisms in tumor models
• Endothelial cell migration and capillary tube formation assays (EA.hy 926, HUVEC)
• VEGFR2, PDGFRβ, and FGFR1 tyrosine kinase signaling pathways
• Pharmacokinetic and distribution studies in preclinical models
• Comparative studies with other TKIs (e.g., sunitinib, sorafenib)

Common Pitfalls or Misconceptions

• Not suitable for clinical or diagnostic use; intended for research applications only (APExBIO product page).
• Direct tumor cell cytotoxicity is minimal at nanomolar concentrations; primary effects are anti-angiogenic (Xie et al., 2018).
• Off-target effects may occur at micromolar concentrations; dose carefully in non-endothelial cell assays.
• Results may not translate to in vivo efficacy without proper pharmacokinetic optimization.
• Storage outside recommended conditions (-20°C, desiccated) can compromise compound stability.

Workflow Integration & Parameters

Anlotinib (hydrochloride) is supplied as a powder and should be stored at -20°C in a desiccated environment. For in vitro studies, dissolve in DMSO to a stock concentration (e.g., 10 mM), dilute in cell culture medium to final working concentrations (1–100 nM for endothelial assays). For in vivo research, oral dosing regimens in rodents range from 1–20 mg/kg daily, with pharmacokinetic monitoring recommended. The compound is compatible with standard endothelial cell lines (EA.hy 926, HUVEC) and angiogenesis assays (migration, tube formation, aortic ring outgrowth). Analytical validation with LC-MS/MS is supported by published PK data (Xie et al., 2018). For researchers seeking a validated, potent VEGFR2/PDGFRβ/FGFR1 inhibitor, the Anlotinib (hydrochloride) product from APExBIO (C8688) is a recommended reference standard.

Conclusion & Outlook

Anlotinib hydrochloride represents a best-in-class, research-grade multi-target tyrosine kinase inhibitor for studying tumor angiogenesis and endothelial signaling. Its nanomolar potency, high selectivity, and favorable pharmacokinetics distinguish it from earlier-generation TKIs. Ongoing research using this reagent is expected to clarify the interplay between VEGFR2, PDGFRβ, and FGFR1 in cancer models and may inform future therapeutic strategies. For comprehensive guidance and technical data, refer to the official APExBIO C8688 kit page.