Lenvatinib metabolite
O-Demethyl Lenvatinib hydrochloride targets multiple receptor tyrosine kinases (RTKs) involved in angiogenesis and tumor growth, similar to its parent compound Lenvatinib. These targets include vascular endothelial growth factor receptors (VEGFR1, VEGFR2, VEGFR3), fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3, FGFR4), platelet-derived growth factor receptor (PDGFRalpha), KIT, and RET. By binding to the ATP-binding site of these kinases, the compound inhibits their kinase activity, blocking downstream signaling pathways (e.g., MAPK/ERK and PI3K/AKT pathways). This inhibits endothelial cell proliferation, migration, tube formation (angiogenesis), and tumor cell proliferation. The metabolite may have a similar or altered potency profile compared to the parent drug.

The in vitro activity of O-Demethyl Lenvatinib hydrochloride is expected to be similar to that of Lenvatinib, but specific data for the metabolite may not be fully characterized. Lenvatinib inhibits VEGFR2 kinase activity with an IC50 of approximately 4 nM, VEGFR3 with an IC50 of 5.2 nM, FGFR1 with an IC50 of 46 nM, and PDGFRalpha with an IC50 of 51 nM in cell-free assays. In cellular assays, Lenvatinib inhibits VEGF-stimulated HUVEC proliferation with an IC50 of ∼3 nM. O-Demethyl Lenvatinib is likely to retain inhibitory activity against these targets, though with possible differences in potency. The metabolite may also contribute to the overall pharmacological activity of Lenvatinib in vivo. The compound is an anticancer agent.

No specific in vivo data is provided for O-Demethyl Lenvatinib hydrochloride. The in vivo activity of the parent compound Lenvatinib is well-established. In mouse xenograft models of various human cancers (e.g., H146 small cell lung cancer, H460 NSCLC, HT-29 colon cancer, A375 melanoma), oral administration of Lenvatinib (10-100 mg/kg/day) significantly inhibits tumor growth and angiogenesis. Lenvatinib is approved for the treatment of radioactive iodine-refractory differentiated thyroid cancer (RR-DTC) and in combination with everolimus for advanced renal cell carcinoma (RCC), and as a first-line treatment for unresectable hepatocellular carcinoma (HCC). O-Demethyl Lenvatinib may contribute to the in vivo efficacy and safety profile of Lenvatinib therapy.

Cell-free kinase inhibition assays are used to determine the IC50 values of the compound against various RTKs. The assays are performed using recombinant kinase domains (e.g., VEGFR2, FGFR1, PDGFRalpha) and a peptide substrate (e.g., poly(Glu,Tyr) 4:1) in a buffer containing 50 mM HEPES (pH 7.4), 10 mM MgCl2, 2 mM MnCl2, 2 mM DTT, and 0.01% Tween-20. Varying concentrations of O-Demethyl Lenvatinib hydrochloride (0.01-1000 nM) are added and pre-incubated with the kinase for 10-15 minutes. The reaction is initiated by adding 10-50 microM ATP and incubated for 30-60 minutes at 30degC. The reaction is stopped by adding EDTA (final 25 mM) or a stop solution. Phosphorylated peptide is detected using an anti-phosphotyrosine antibody in an ELISA format or by time-resolved fluorescence resonance energy transfer (TR-FRET). Alternatively, the ADP-Glo kinase assay can be used. The IC50 is calculated from the dose-response curve. For comparative purposes, Lenvatinib is used as a positive control.

For cellular assays, HUVECs (human umbilical vein endothelial cells) are used to assess the anti-angiogenic activity of O-Demethyl Lenvatinib hydrochloride. HUVECs are seeded in 96-well plates (5×103 cells/well) and allowed to attach overnight. The medium is replaced with fresh medium containing O-Demethyl Lenvatinib hydrochloride (0.01-1000 nM) and incubated for 30 minutes, followed by stimulation with VEGF (10 ng/mL) or bFGF (20 ng/mL) for 10 minutes. Cell lysates are prepared, and the phosphorylation of VEGFR2 or downstream targets (ERK1/2, AKT) is measured by Western blotting or phospho-ELISA. For proliferation assays, HUVECs are seeded in 96-well plates (2×103 cells/well) and treated with O-Demethyl Lenvatinib (0.01-1000 nM) for 72 hours. Cell viability is measured using CellTiter-Glo. For anti-tumor cell activity, cancer cell lines (e.g., H146, H460, HT-29) are seeded in 96-well plates (5×103 cells/well) and treated with the compound (0.1-10,000 nM) for 72 hours. Cell viability is measured by MTT or CellTiter-Glo assay. The IC50 for cell proliferation inhibition is calculated.

The compound is a metabolite of Lenvatinib, so it is not typically administered directly in animal studies. For pharmacokinetic studies, Lenvatinib is administered to animals, and O-Demethyl Lenvatinib levels are quantified as part of the metabolite profiling. In a typical rat PK study, male Sprague-Dawley rats (n=6-8 per group) receive a single oral dose of Lenvatinib (10-50 mg/kg) in 0.5% methylcellulose. Blood is collected via tail vein at pre-dose, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, and 72 hours post-dose. Plasma is separated and frozen. O-Demethyl Lenvatinib levels are quantified by LC-MS/MS using a validated method. The metabolite-to-parent ratio (MPR) is calculated. For tissue distribution studies, animals are euthanized at selected time points, and tissues (liver, kidney, lung, tumor) are collected. The concentration of O-Demethyl Lenvatinib is measured in tissue homogenates. For in vivo efficacy, the parent drug Lenvatinib is administered, not the metabolite.

O-Demethyl Lenvatinib hydrochloride has a molecular formula of C20H18Cl2N4O4 and a molecular weight of 449.29 g/mol. The compound is supplied as a solid and should be stored at 4degC in a sealed container, protected from light and moisture, where it is stable for up to 3 years. For solution storage, it should be kept at -80degC for 6 months or -20degC for 1 month. The hydrochloride salt is used to enhance solubility. The compound is soluble in DMSO (10-20 mg/mL) and methanol. For in vivo studies, the parent drug is used; however, for analytical purposes, the metabolite can be dissolved in DMSO to prepare stock solutions for LC-MS/MS calibration standards. The product is for research use only.

This product is for research use only and is not for human therapeutic use. No specific toxicity data is available for this metabolite. However, O-Demethyl Lenvatinib is a major metabolite of Lenvatinib, which is an FDA-approved drug. The toxicity of Lenvatinib in humans includes hypertension, fatigue, diarrhea, nausea, proteinuria, and palmar-plantar erythrodysesthesia (hand-foot syndrome). The metabolite may contribute to these adverse effects. The hydrochloride salt is used in small quantities for research and is not expected to pose a significant hazard under normal handling conditions. Standard laboratory safety practices (gloves, lab coat, safety glasses) should be followed. Avoid inhalation of powder and contact with skin and eyes. The compound is not for human use.

[1]. Lenvatinib: A Promising Molecular Targeted Agent for Multiple Cancers. Cancer Control. 2018 Jan-Dec;25(1):1073274818789361.

[2]. Lenvatinib versus Bay 43-9006 in first-line treatment of patients with unresectable hepatocellularcarcinoma: a randomised phase 3 non-inferiority trial. Lancet. 2018 Mar 24;391(10126):1163-1173.

Lenvatinib is a small-molecule tyrosine kinase inhibitor that inhibits vascular endothelial growth factor receptors (VEGFR1-3), fibroblast growth factor receptors (FGFR1-4), platelet-derived growth factor receptor α (PDGFRα), stem cell factor receptor (KIT), and transfection rearrangement receptor (RET). These receptors are crucial for tumor angiogenesis, and lenvatinib inhibits tumor angiogenesis by suppressing the function of these receptors. Phase I clinical trials of lenvatinib were conducted simultaneously in Japan, Europe, and the United States, and tumor shrinkage effects were observed in thyroid cancer, endometrial cancer, melanoma, renal cell carcinoma, sarcoma, and colon cancer. Lenvatinib is a promising drug that has shown efficacy in treating a variety of solid tumors. Adverse reactions to lenvatinib treatment may include hypertension, proteinuria, diarrhea, and delayed wound healing. Managing these adverse events is also crucial for the use of lenvatinib. In this short review article, we summarize the current status, toxicities, and future prospects of lenvatinib in the treatment of thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, and lung cancer. [1]

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Background: Lenvatinib (an inhibitor of VEGF receptors 1–3, FGF receptors 1–4, PDGF receptor α, RET, and KIT) showed activity against hepatocellular carcinoma in a phase II clinical trial. We aimed to compare lenvatinib with sorafenib as first-line treatment for patients with unresectable hepatocellular carcinoma. Methods: This was an open-label, phase III, multicenter, non-inferiority trial that enrolled patients with unresectable hepatocellular carcinoma who had not received prior treatment for advanced disease at 154 research centers in 20 countries across Asia Pacific, Europe, and North America. Patients were randomized 1:1 using an interactive voice response system, with grouping factors including: geographic location; gross portal vein invasion, extrahepatic metastasis, or both; Eastern Cooperative Oncology Group (ECOG) performance status score; and weight. Patients received either oral lenvatinib (12 mg daily for those weighing ≥60 kg, 8 mg daily for those weighing <60 kg) or sorafenib (400 mg twice daily) for 28 days as one course of treatment. The primary endpoint was overall survival, defined as the time from randomization to death from any cause. Efficacy analysis followed the intention-to-treat principle, and safety analysis included only patients who received treatment. The non-inferiority margin was set at 1.08. This trial was registered at ClinicalTrials.gov under registration number NCT01761266. Results: Between March 1, 2013, and July 30, 2015, a total of 1492 patients were enrolled. 954 eligible patients were randomized to either the lenvatinib group (n=478) or the sorafenib group (n=476). The median survival in the lenvatinib group was 13.6 months (95% CI 12.1–14.9), which was non-inferior to the sorafenib group (12.3 months, 10.4–13.9; hazard ratio 0.92, 95% CI 0.79–1.06), meeting the non-inferiority criteria. The most common adverse events of any grade in the lenvatinib group were hypertension (201 cases [42%]), diarrhea (184 cases [39%]), decreased appetite (162 cases [34%]), and weight loss (147 cases [31%]); the most common adverse events of any grade in the sorafenib group were hand-foot syndrome (249 cases [52%]), diarrhea (220 cases [46%]), hypertension (144 cases [30%]), and decreased appetite (127 cases [27%]). Conclusion: In treatment-naïve patients with advanced hepatocellular carcinoma, lenvatinib was non-inferior to sorafenib in terms of overall survival. The safety and tolerability of lenvatinib were consistent with previous observations. [2]

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Lenvatinib (brand name Lenvima) is a multi-targeted TKI approved for the treatment of several cancers. O-Demethyl Lenvatinib is a major circulating metabolite in humans formed by oxidative N-demethylation, primarily by CYP3A4. The presence of this metabolite may contribute to the overall pharmacological effect and toxicity profile of Lenvatinib therapy. The compound can be used as a reference standard for metabolite quantification in pharmacokinetic and bioequivalence studies. The CAS number for O-Demethyl Lenvatinib (free base) is 417717-04-5. This product is not a clinically approved drug; it is a research chemical for use as a metabolite standard. Supplier information must not be included.

C20H18CL2N4O4

449.29

448.07051

O-Demethyl Lenvatinib;417717-04-5

168007119

Light yellow to yellow solid powder

KMZOGLZCTDVLLW-UHFFFAOYSA-N

InChI=1S/C20H17ClN4O4.ClH/c21-14-7-11(3-4-15(14)25-20(28)24-10-1-2-10)29-18-5-6-23-16-9-17(26)13(19(22)27)8-12(16)18;/h3-10,26H,1-2H2,(H2,22,27)(H2,24,25,28);1H

4-[3-chloro-4-(cyclopropylcarbamoylamino)phenoxy]-7-hydroxyquinoline-6-carboxamide;hydrochloride

O-Demethyl Lenvatinib (hydrochloride); O-Demethyl Lenvatinib hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO :~50 mg/mL (~111.29 mM)

Solubility in Formulation 1: ≥ 1.8 mg/mL (4.01 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 18.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)