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

Executive Summary: Anlotinib hydrochloride (CAS 1058157-76-8) is a small-molecule inhibitor targeting VEGFR2, PDGFRβ, and FGFR1, exhibiting nanomolar IC₅₀ values and strong selectivity (Xie et al., DOI). It robustly inhibits endothelial cell migration and capillary tube formation in vitro (APExBIO). The compound demonstrates favorable pharmacokinetics, including high plasma protein binding (93% in humans) and significant tissue distribution, including tumor and brain tissue. Compared to sunitinib, sorafenib, and nintedanib, anlotinib displays superior in vivo anti-angiogenic and anti-tumor efficacy. Safety studies report a high LD₅₀ (1735.9 mg/kg, oral, 14 days) and low toxicity.

Biological Rationale

Angiogenesis, the formation of new capillaries from existing vasculature, is essential for tumor growth beyond ~1 mm³ and supports invasion and metastasis (Folkman, 1971; Xie et al., DOI). Vascular endothelial growth factor (VEGF) signaling, primarily via VEGFR2, orchestrates endothelial cell proliferation, migration, and new vessel formation. Unregulated angiogenesis is a hallmark of cancer progression. Endothelial cells rarely acquire drug resistance compared to tumor cells, making them attractive therapeutic targets for anti-angiogenic strategies. Small-molecule inhibitors of VEGFR2 and related kinases disrupt tumor vascularization and can halt or reverse tumor growth.

Mechanism of Action of Anlotinib (hydrochloride)

Anlotinib hydrochloride is a multi-target tyrosine kinase inhibitor (TKI), primarily acting on VEGFR2, PDGFRβ, and FGFR1. The compound occupies the ATP-binding pocket of VEGFR2, leading to inhibition of kinase activity and downstream ERK signaling in endothelial cells (Xie et al., 2018). Anlotinib blocks VEGF-, PDGF-BB-, and FGF-2-induced endothelial cell migration and capillary-like tube formation, with IC₅₀ values of 5.6 ± 1.2 nM (VEGFR2), 8.7 ± 3.4 nM (PDGFRβ), and 11.7 ± 4.1 nM (FGFR1) (APExBIO). The compound also downregulates ERK pathway signaling, a key driver of endothelial proliferation and survival. Cellular assays using human vascular endothelial cells (EA.hy 926, HUVEC) confirm the inhibition of migration and tube formation at nanomolar concentrations.

Evidence & Benchmarks

• Anlotinib demonstrates IC₅₀ values of 5.6 ± 1.2 nM (VEGFR2), 8.7 ± 3.4 nM (PDGFRβ), and 11.7 ± 4.1 nM (FGFR1) in kinase assays (Xie et al., 2018, DOI).
• In HUVEC migration and tube formation assays, anlotinib outperforms sunitinib and sorafenib at equivalent concentrations (Xie et al., 2018, DOI).
• Oral administration in rats yields bioavailability of 28–58%, and in dogs 41–77% (APExBIO, product page).
• Tissue distribution studies report high accumulation in lung, liver, kidney, heart, tumor, and brain tissue (Xie et al., 2018, DOI).
• Median lethal dose (LD₅₀) in 14-day oral studies is 1735.9 mg/kg, with minimal organ or genetic toxicity (APExBIO, product page).
• In vivo, anlotinib causes tumor regression and decreased vascular density in mouse models (Xie et al., 2018, DOI).

For an in-depth contrast, 'Anlotinib Hydrochloride: Potent Multi-Target Tyrosine Kin...' provides an overview of anlotinib's kinase selectivity; this article expands with detailed pharmacokinetics and safety benchmarks. For extended research applications and troubleshooting, 'Anlotinib Hydrochloride: Optimizing Angiogenesis & Cancer...' offers workflow advice, while the present article updates with the latest preclinical efficacy data.

Applications, Limits & Misconceptions

Anlotinib hydrochloride is widely used in preclinical research to dissect tumor angiogenesis mechanisms and evaluate anti-angiogenic compounds. Common applications include:

• Capillary tube formation assays (e.g., EA.hy 926, HUVEC).
• Endothelial cell migration and invasion studies.
• In vivo tumor xenograft models probing angiogenesis and tumor regression.
• Pharmacokinetic modeling and tissue distribution profiling.

However, direct cytotoxicity to tumor cells is only observed at micromolar concentrations, indicating its primary effect is anti-angiogenic rather than cytotoxic in vitro (Xie et al., DOI).

Common Pitfalls or Misconceptions

• Anlotinib is not suitable for direct cytotoxicity assays at nanomolar concentrations; its primary mode is anti-angiogenesis.
• It is not intended for diagnostic or medical use; for research use only (APExBIO).
• Results in rodent and canine models may not directly translate to human systems without further validation.
• Monotherapy may be insufficient in tumors with VEGFR2-independent angiogenesis pathways.
• Storage at -20°C is required to maintain compound stability.

This article further clarifies boundaries of anlotinib's in vitro efficacy compared to the broader mechanistic discussions found in 'Anlotinib Hydrochloride: Unraveling Multi-Target Angiogen...'.

Workflow Integration & Parameters

For research use, anlotinib hydrochloride (C8688) from APExBIO is provided as a stable, lyophilized solid. It is stored at -20°C and reconstituted in DMSO or appropriate buffers for cellular assays (APExBIO). Recommended working concentrations are typically in the 1–100 nM range for endothelial cell migration and tube formation assays, with careful titration advised. For in vivo models, dosing regimens and formulation should follow published protocols, considering species-specific pharmacokinetics.

Integration into workflows requires:

• Verification of compound solubility and stability under experimental conditions.
• Appropriate negative and positive controls, such as sunitinib or sorafenib.
• Validation of anti-angiogenic endpoints (migration, tube formation, vessel density).

To maximize reproducibility and data quality, refer to 'Anlotinib Hydrochloride: Elevating Angiogenesis and Tumor...', which focuses on troubleshooting and experimental optimization. This article augments those workflows with detailed mechanistic insights and safety parameters.

Conclusion & Outlook

Anlotinib hydrochloride stands out as a next-generation multi-target tyrosine kinase inhibitor with robust anti-angiogenic properties and well-characterized safety. Its high selectivity, strong inhibition of VEGFR2/PDGFRβ/FGFR1, and favorable tissue distribution make it a preferred tool for angiogenesis and tumor research. Ongoing clinical investigations continue to elucidate its therapeutic potential. For detailed product specifications and ordering, visit the Anlotinib (hydrochloride) page at APExBIO.