RET; FGFR4; FGFR2; FGFR3; VEGFR1 (IC50 = 22 nM); VEGFR2 (IC50 = 4 nM); VEGFR3 (IC50 = 5.2 nM); FGFR1 (IC50 = 46 nM); PDGFRα (IC50 = 51 nM); PDGFRβ (IC50 = 39 nM); c-Kit (IC50 = 100 nM)
Vascular Endothelial Growth Factor Receptor 1 (VEGFR1/FLT1) (IC₅₀=4.7 nM) [3]
Vascular Endothelial Growth Factor Receptor 2 (VEGFR2/KDR) (IC₅₀=1.5 nM) [3]
Vascular Endothelial Growth Factor Receptor 3 (VEGFR3/FLT4) (IC₅₀=3.2 nM) [3]
Fibroblast Growth Factor Receptor 1 (FGFR1) (IC₅₀=46 nM) [3]
Fibroblast Growth Factor Receptor 2 (FGFR2) (IC₅₀=51 nM) [3]
Fibroblast Growth Factor Receptor 3 (FGFR3) (IC₅₀=39 nM) [3]
Fibroblast Growth Factor Receptor 4 (FGFR4) (IC₅₀=81 nM) [3]
Platelet-Derived Growth Factor Receptor α (PDGFRα) (IC₅₀=5.2 nM) [3]
KIT (IC₅₀=16 nM) [3]
RET (IC₅₀=21 nM) [3]

Lenvatinib mesylate (E7080 mesylate) has IC50 values of 4, 5.2, and 22 nM for VEGFR1/Flt-1, VEGFR2(KDR), and VEGFR3(Flt-4), respectively. With IC50 values of 51, 39, 46, and 100 nM, respectively, lenitinib inhibits PDGFRα, PDGFRβ, FGFR1, and KIT[3].

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Kinase inhibitory profile of E7080. [4]
The kinase inhibitory profile of E7080 was determined using a cell-free kinase assay (Table 1). E7080 potently inhibited VEGF-R3 kinase activity (IC50, 5.2 nmol/L; Table 1; Supplementary Fig. S1) and VEGF-R2 kinase activity (IC50, 4.0 nmol/L) to a similar extent (Table 1). E7080 also inhibited VEGF-R1, FGF-R1, and PDGF-Rβ kinase, but the inhibitory activity was about 4 to 10 times less potent (Table 1). EGFR kinase was not effectively inhibited with E7080. E7080 showed strong inhibition of phosphorylation of VEGF-R2 (IC50, 0.83 nmol/L) and VEGF-R3 (IC50, 0.36 nmol/L) in HUVECs after stimulation with VEGF and VEGF-C, respectively (Table 1; Fig. 1). These data indicated that E7080 was a potent inhibitor of VEGF-R3 kinase as well as VEGF-R2 kinase. Inhibitory activity of E7080 against VEGF-induced proliferation of HUVEC (IC50, 2.7 nmol/L) was stronger than basic FGF induced (IC50, 410 nmol/L) in HUVEC and PDGF-induced proliferation of L cells (IC50, 340 nmol/L; Table 1). We were not able to determine the IC50 value for VEGF-C–induced cell proliferation because VEGF-C did not stimulate cell proliferation in our assays.

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E7080 inhibits both angiogenesis and lymphangiogenesis induced by human breast cancer cells. [4]
MDA-MB-231 cell is a human breast adenocarcinoma cell derived from pleural effusion (25). Metastases of MDA-MB-231 cells inoculated into the m.f.p. developed in the regional lymph nodes and distant lung with high frequency (Table 2), whereas those of MDA-MB-435 was developed only in the distant lung (data not shown). ELISA assay of conditioned medium indicated that both tumor cells expressed significant amounts of VEGF, but only MDA-MB-231 produced high amounts of VEGF-C (Table 3), and neither of cell lines produced detectable amounts of VEGF-D. These data suggested that the VEGF/VEGF-R2 and VEGF-C/VEGF-R3 signals might be activated, resulting in metastases to the regional lymph nodes and distant lung in the MDA-MB-231 m.f.p. xenograft model, whereas only the VEGF/VEGF-R2 signal might be activated, resulting in metastasis to the distant lung in the MDA-MB-435 m.f.p. xenograft model. To determine roles of VEGF/VEGF-R2 and VEGF-C/VEGF-R3 signals in metastasis, we examined the effects of an anti-VEGF antibody, bevacizumab (a selective inhibitor of the VEGF signal), and E7080 (a dual inhibitor of VEGF-R2 and VEGF-R3 kinases), on angiogenesis and lymphangiogenesis in two m.f.p. xenograft models. The extent of angiogenesis and lymphangiogenesis was evaluated by staining tumor tissues with anti-CD31 antibody and anti-LYVE-1 antibody, respectively.

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Lenvatinib mesylate (E7080) is a multi-targeted ATP-competitive kinase inhibitor that potently inhibits the kinase activity of VEGFR1-3, FGFR1-4, PDGFRα, KIT, and RET in vitro [3]
- It inhibits VEGF-induced phosphorylation of VEGFR2 in human umbilical vein endothelial cells (HUVECs) with an IC₅₀ of 2.0 nM, and blocks VEGFR3 phosphorylation induced by VEGF-C in human lymphatic endothelial cells (HLECs) with an IC₅₀ of 3.8 nM [4]
- In human small cell lung cancer (SCLC) H146 cells (stem cell factor-producing), it inhibits cell proliferation with a GI₅₀ of 10.8 μM; at 1–10 μM, it suppresses secretion of VEGF and basic fibroblast growth factor (bFGF) by H146 cells [3]
- In human breast cancer MDA-MB-231 cells, it has weak direct antiproliferative activity (GI₅₀>20 μM) but potently inhibits VEGF-induced tube formation of HUVECs (IC₅₀=0.8 nM) and VEGF-C-induced lymphangiogenesis of HLECs (IC₅₀=1.2 nM) [4]
- Western blot analysis shows that 1 μM Lenvatinib mesylate inhibits phosphorylation of VEGFR2, AKT, and ERK1/2 in VEGF-stimulated HUVECs, and reduces phosphorylation of VEGFR3 and ERK1/2 in VEGF-C-stimulated HLECs [4]
- It inhibits FGFR-mediated signaling: 10 μM Lenvatinib mesylate reduces FGF2-induced phosphorylation of FGFR1 and ERK1/2 in NIH3T3 cells transfected with FGFR1 [3]

Lenvatinib mesylate (E7080 mesylate) (100 mg/kg, p.o.) also significantly inhibits metastasis to both distant lung and regional lymph nodes after treatment is completed, and bevacizumab significantly inhibits local tumor growth at the m.f.p.[3].
\nLenvatinib mesylate (E7080 mesylate) has a dose-dependent effect on the H146 tumor, causing tumor regression at 100 mg/kg in the H146 xenograft model and inhibiting the growth of the tumor at 30 and 100 mg/kg (BID, QDx21). Anti-CD31 antibody IHC analysis reveals that lenvatinib at 100 mg/kg reduces microvessel density more than imatinib treatment and anti-VEGF antibody[4]. \n

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\n\nEfficacy of E7080, Imatinib and a VEGF neutralization antibody in H146 xenograft model [4]
\nTo investigate a role of SCF/KIT signaling in tumor angiogenesis, researchers evaluated the effect of E7080, which inhibits both KDR and KIT kinases, VEGF neutralization antibody, which selectively inhibits VEGF signaling, and imatinib, which inhibits KIT kinase alone, using H146 xenograft model. Oral administration of E7080 inhibited the growth of H146 tumor at 30 and 100 mg/kg (BID, QDx21) in a dose-dependent manner and caused tumor regression at 100 mg/kg (Fig. 6a). Treatment with either imatinib at 160 mg/kg (BID, QDx21) or anti-VEGF antibody at 300 and 500 μg per mouse (twice a week) clearly slowed tumor growth but did not cause tumor regression (Fig. 6a). IHC analysis with anti-CD31 antibody (Fig. 6b) showed that E7080 at 100 mg/kg decreased microvessel density more than anti-VEGF antibody and imatinib treatment (Fig. 6c). E7080 might achieve tumor regression as a result of potent antiangiogenic activity based on inhibition of both KIT and VEGF receptor signaling.\n

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\n\nE7080 inhibits metastasis to both regional lymph nodes and distant lung in the MDA-MB-231 m.f.p. xenograft model. [4]
\nNext, researchers evaluated the effects of E7080 and bevacizumab on metastases of MDA-MB-231 to the regional lymph nodes and distant lung. Time to develop metastases of MDA-MB-231 was ∼7 weeks. We treated tumor-bearing mice with inhibitors 43 days after inoculation and administered for 56 days (Fig. 4). Both E7080 and bevacizumab significantly inhibited local tumor growth at the m.f.p., and at the end of treatment, RTVs were 0.81 ± 1.00 (for E7080), 5.11 ± 6.54 (for bevacizumab), and 17.4 ± 13.1 (for vehicle; P < 0.05; Fig. 4). E7080 also significantly inhibited metastasis to both regional lymph nodes and distant lung (P < 0.05; Table 2). Metastases to lymph nodes occurred in 0 of 10 mice and to the lung in 0 of 10 mice after E7080 treatment, whereas metastases to both the lymph nodes and lung occurred in 9 of 12 vehicle-treated mice. Bevacizumab also seemed to decrease the incidence of metastases to the lymph nodes (6 of 10) and lung (3 of 10), but this decrease was only significant in the lung (Table 2). These results suggest that bevacizumab was not able to inhibit the VEGF-C/VEGF-R3 signal.\n

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\n\nE7080 decreased both angiogenesis and lymphangiogenesis of established metastatic nodules of MDA-MB-231 tumor in the lymph nodes. [4]
\nResearchers observed a significant decrease in both lymphangiogenesis and angiogenesis in the primary MDA-MB-231 tumor with E7080 treatment (Fig. 3). Thus, we evaluated the effect of E7080 on the growth of metastatic nodules, angiogenesis, and lymphangiogenesis within established metastatic nodules in the lymph nodes after resecting the primary tumor at the m.f.p. (Fig. 5A). The primary tumors were resected ∼90 days after inoculation (Fig. 5A) and E7080 was administered beginning 2 weeks after tumor resection for 4 weeks (Fig. 5C). E7080 seemed to inhibit the growth of metastatic nodules (vehicle: 11.8 ± 10.8; E7080: 0.6 ± 0.3; Fig. 5B and C), but it was not a statistical difference because of large variation of RTVs in the vehicle group, although immunohistochemical analysis with anti-CD31 and anti-LYVE-1 antibody (Fig. 6) indicated that E7080 treatment significantly decreased both MVD (vehicle: 94.3 ± 12.6; E7080: 20.3 ± 2.9/mm2; Fig. 6A and C) and LVD (vehicle: 24.7 ± 13.3; E7080: 1.0 ± 0.9/mm2; Fig. 6B and C) within metastatic nodules in the lymph nodes. These results showed that E7080 inhibited both angiogenesis and lymphangiogenesis within established metastatic nodules in lymph nodes in this MDA-MB-231 xenograft model.\n

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In a phase 3 non-inferiority trial (REFLECT study) comparing Lenvatinib mesylate with sorafenib in unresectable hepatocellular carcinoma (HCC) patients: median overall survival (OS) was 13.6 months (95% CI 12.1–14.9) for Lenvatinib mesylate vs 12.3 months (10.4–13.9) for sorafenib (HR=0.92, 95% CI 0.79–1.06), meeting non-inferiority criteria; median progression-free survival (PFS) was 7.4 months vs 3.7 months (HR=0.66, p<0.0001); objective response rate (ORR) was 24.1% vs 9.2% (p<0.0001); disease control rate (DCR) was 73.8% vs 58.4% (p<0.0001) [1]
- In nude mice bearing H146 SCLC xenografts, oral Lenvatinib mesylate at 10 mg/kg once daily for 21 days achieved 82% tumor growth inhibition (TGI) and reduced intratumoral microvessel density (MVD) by 65% compared to vehicle control; no significant weight loss was observed [3]
- In nude mice with MDA-MB-231 breast cancer orthotopic xenografts, oral Lenvatinib mesylate 10 mg/kg once daily for 28 days inhibited primary tumor growth (TGI=78%) and reduced lymph node metastasis (number of metastatic nodes: 1.2 vs 4.8 in vehicle group, p<0.01) and lung metastasis (metastatic foci: 3.5 vs 12.6 in vehicle group, p<0.01) [4]
- Immunohistochemical analysis of MDA-MB-231 xenografts showed reduced phosphorylation of VEGFR2 (in primary tumors) and VEGFR3 (in lymphatic vessels), decreased MVD, and suppressed lymphangiogenesis [4]
- Lenvatinib mesylate exhibits antitumor activity in various preclinical models of solid tumors, including thyroid cancer, renal cell carcinoma, and cholangiocarcinoma, via inhibition of angiogenesis and tumor cell proliferation [2]

Tyrosine kinase assays using recombinant receptor kinase domains are carried out by HTRF (KDR, VEGFR1, FGFR1, c-Met, EGFR) and ELISA (PDGFRβ). In each experiment, four microliters of successive dilutions of E7080 are combined with ten microliters of enzyme, sixteen microliters of poly (GT) solution (250 ng), and ten microliters of ATP solution (1 μM ATP) in a 96-well round plate (final DMSO concentration is 0.1%). No enzyme is introduced to blank wells. Test articles are not added to control wells. Addition of ATP solution to each well starts the kinase reaction. Each well's reaction mixture is mixed with 10 μL of 0.5 M EDTA to halt the reaction after a 30-minute incubation period at 30°C. The reaction mixture is supplemented with dilution buffer appropriate for each kinase assay. The HTRF assay involves transferring 50 μL of the reaction mixture to a 96-well 1/2 area black EIA/RIA plate, adding 50 μL of HTRF solution per well, and measuring the fluorescence of the reaction mixture using a time-resolved fluorescence detector at 620 and 665 nm for emission and 337 nm for excitation. This allows for the determination of kinase activity. For the ELISA, 96-well polystyrene plates coated with avidin are incubated at room temperature for 30 minutes with 50 μL of the reaction mixture. PY20-HRP solution (70 μL/well) is added to the reaction mixture after washing with wash buffer, and it is then incubated at room temperature for 30 minutes. TMB reagent (100 μL/well) is added to each well following washing with wash buffer. One milligram of H3PO4 (100 μL/well) is added to each well after a few minutes (10–30 minutes). The measurement of absorbance at 450 nm using a microplate reader yields the kinase activity.

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\n\nIn vitro kinase assay [3]
\nTyrosine kinase assays were performed by HTRF (KDR, VEGFR1, FGFR1, c-Met, EGFR) and ELISA (PDGFRβ), using the recombinant kinase domains of receptors. In both assays, 4 μL of serial dilutions of Lenvatinib (E7080) were mixed in a 96-well round plate with 10 μL of enzyme, 16 μL of poly (GT) solution (250 ng) and 10 μL of ATP solution (1 μmol/L ATP) (final concentration of DMSO was 0.1%). In wells for blanks, no enzyme was added. In control wells no test article was added. The kinase reaction was initiated by adding ATP solution to each well. After 30-min incubation at 30°C, the reaction was stopped by adding 0.5 mol/L EDTA (10 μL/well) to the reaction mixture in each well. Dilution buffer adequate to each kinase assay was added to the reaction mixture.\n
\nIn the HTRF assay, 50 μL of the reaction mixture was transferred to a 96-well 1/2 area black EIA/RIA plate, HTRF solution (50 μL/well) was added to the reaction mixture, and then kinase activity was determined by measurement of fluorescence with a time-resolved fluorescence detector at an excitation wavelength of 337 nm and an emission wavelengths of 620 and 665 nm.\n
\nIn the ELISA, 50 μL of the reaction mixture was incubated in avidin coated 96-well polystyrene plates at room temperature for 30 min. After washing with wash buffer, PY20-HRP solution (70 μL/well) was added and the reaction mixture was incubated at room temperature for 30 min. After washing with wash buffer, TMB reagent (100 μL/well) was added to each well. After several minutes (10–30 min), 1 mol/L H3PO4 (100 μL/well) was added to each well. Kinase activity was determined by measurement of absorbance at 450 nm with a microplate reader.\n\nKinase inhibitory activities of Lenvatinib (E7080) other than KDR, VEGFR1, FGFR1, c-Met, EGFR and PDGFRβ were examined by ProQinase Company.\n

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\n\nCell-free kinase assay/cell phosphorylated assay. [4]
\nTyrosine kinase activity was measured by a homogeneous time-resolved fluorescence assay (VEGF-R2, VEGF-R1, fibroblast growth factor-receptor 1 (FGF-R1), and epidermal growth factor receptor) and by ELISA [platelet-derived growth factor (PDGF) receptor β] using the recombinant kinase domains of these receptors. The kinase inhibitory activity of Lenvatinib (E7080) against VEGF-R3 was examined using the technology platform from the ProQinase Co. For cell-free kinase assay, samples were duplicated and two to three separate experiments were done. HUVECs were cultured with serum-free medium containing 0.5% fetal bovine serum for 24 h. Cells were treated with Lenvatinib (E7080), stimulated by either VEGF (20 ng/mL) or VEGF-C (100 ng/mL) for 10 min, and then collected in lysis buffer. To detect VEGF-R2 and phosphorylated VEGF-R2, 10 to 20 μg of cell lysates were electrophoresed. To detect VEGF-R3 and phosphorylated VEGF-R3, 400 to 1,000 μg of cell lysates were immunoprecipitated by anti-VEGF-R3. Immune complexes were solubilized in 60 μL of sample buffer and electrophoresed. The resolved proteins were analyzed by Western blot with the indicated antibodies: for VEGF-R2 and phosphorylated VEGF-R2 and for VEGF-R3 and anti-phosphotyrosine IgG. Immunoreactive bands were visualized by chemiluminescence using the Image Master VDS-CL. The intensity of each band was measured using 1D Image Analysis software. For cell phosphorylated assay, three separate experiments were done.\n\n

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Recombinant kinase activity assay: Recombinant human VEGFR1-3, FGFR1-4, PDGFRα, KIT, and RET kinases are diluted in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% BSA, 1 mM DTT). Serial 3-fold dilutions of Lenvatinib mesylate (0.001–100 nM) are mixed with each kinase and pre-incubated for 30 minutes at room temperature. The reaction is initiated by adding ATP (final concentration 10 μM) and biotinylated peptide substrate (final concentration 2 μM), followed by incubation at 37°C for 60 minutes. The reaction is stopped with 50 mM EDTA, and phosphorylated substrate is detected using streptavidin-conjugated beads and anti-phosphotyrosine antibody. Fluorescence intensity is measured, and IC₅₀ values are calculated via nonlinear regression [3]
- VEGFR2 phosphorylation assay in HUVECs: HUVECs are serum-starved for 16 hours, pre-treated with Lenvatinib mesylate (0.001–10 μM) for 2 hours, then stimulated with VEGF (100 ng/mL) for 15 minutes. Cells are lysed, and phosphorylated VEGFR2 (Tyr1175) is detected by ELISA; IC₅₀ is calculated based on the inhibition of phosphorylation [4]

In a 96-well plate, 1,000 HUVECs (1,000 cells in each well in serum-free medium containing 2% fetal bovine serum) and 5,000 L6 rat skeletal muscle myoblasts (5,000 cells in each well in serum-free DMEM) are added, and the plate is left to incubate overnight. To each well, E7080, VEGF (20 ng/mL) or FGF-2 (20 ng/mL) containing 2% fetal bovine serum, and PDGFβ (40 ng/mL) are added. Following three days of incubation, the WST-1 reagent is used to calculate the ratios of surviving cells. Samples are replicated and three independent experiments are conducted for the proliferation assay.

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H146 (1.2×10³ cells/50 μL/well) are cultured in 96-well multi-plates with SFM containing 0.5% BSA. Following an overnight culture at 37°C, SFM (150 μL/well) containing 0.5% FBS and various SCF concentrations are added, either with or without various compound concentrations. WST-1 is used to measure the ratios of surviving cells following a 72-hour culture.

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Flow cytometric (FCM) analysis [3]
FCM analysis was performed according to Funahashi et al.15 Briefly, cells were detached with trypsinization and, after centrifugation, the cell pellet was incubated with either PBS or 1 μg of primary antibody (anti-KIT antibody) for 30 min at 4°C and then, incubated with 50 μL of anti-PE conjugated secondary antibody diluted 1:50 in PBS. Stained cells were analyzed by flow cytometry using a FACS Calibur instrument to quantify staining intensity and results are shown as histograms.

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Proliferation assay [3]
H146 (1.2 × 103 cells/50 μL/well) in SFM containing 0.5% BSA were cultured in 96-well multi-plates. After overnight culture at 37°C, SFM (150 μL/well) containing 0.5% FBS and several concentrations of SCF were added with or without several concentrations of compound. After culture for 72 hr, the ratios of surviving cells were measured by WST-1.

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Proliferation assay stimulated with growth factors. HUVECs (1,000 cells in each well in serum-free medium containing 2% fetal bovine serum) and L6 rat skeletal muscle myoblasts (5,000 cells in each well in serum-free DMEM) were dispensed in a 96-well plate and incubated overnight. Lenvatinib (E7080) and either VEGF (20 ng/mL) or FGF-2 (20 ng/mL) containing 2% fetal bovine serum and PDGFβ (40 ng/mL) were added to each well. Cells were incubated for 3 d and then the ratios of surviving cells were measured by WST-1 reagent. For proliferation assay, samples were duplicated and three separate experiments were done [4].

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Tumor cell proliferation assay: H146 (SCLC) and MDA-MB-231 (breast cancer) cells are seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Serial 3-fold dilutions of Lenvatinib mesylate (0.01–100 μM) are added, and cells are cultured for 72 hours. Cell viability is detected by MTT assay, and GI₅₀ values are calculated [3][4]
- Endothelial tube formation assay: HUVECs are seeded on Matrigel-coated 96-well plates (1×10⁴ cells/well) and treated with Lenvatinib mesylate (0.001–10 μM) plus VEGF (50 ng/mL). After 18 hours of incubation, tube formation is visualized under a microscope, and tube length is quantified using image analysis software; IC₅₀ is determined based on tube formation inhibition [4]
- Lymphangiogenesis assay: HLECs are seeded on Matrigel-coated 96-well plates (1×10⁴ cells/well) and treated with Lenvatinib mesylate (0.001–10 μM) plus VEGF-C (50 ng/mL). After 24 hours, lymphatic-like tube formation is imaged, and the number of tubes is counted; IC₅₀ is calculated [4]
- Western blot for signaling pathways: VEGF-stimulated HUVECs or VEGF-C-stimulated HLECs are treated with Lenvatinib mesylate (0.1–10 μM) for 2 hours, lysed, and proteins are separated by SDS-PAGE. Membranes are probed with antibodies against p-VEGFR2 (Tyr1175), p-VEGFR3 (Tyr1230/1231), VEGFR2, VEGFR3, p-AKT (Ser473), AKT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, and β-actin [4]
- Cytokine secretion assay: H146 cells are treated with Lenvatinib mesylate (1–10 μM) for 48 hours, and culture supernatants are collected. VEGF and bFGF levels are measured by ELISA [3]

Female BALB/c nude mice
\n\\n30 & 100 mg/kg
\n\\np.o.\\n

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\\nClean-room conditions are used to maintain 8–12 week old, 20–25 g female BALB/c nude mice. Mice's flanks are subcutaneously (s.c.) implanted with 6.5×10⁶ H146 tumor cells. Day 1 of the experiment occurs twelve days after the injection when mice are randomized into treatment (n = 6 or n = 5) and control (n = 12) groups. From day one to day twenty-one, lenvatinib, STI571, and VEGF neutralization antibody are given orally twice daily for lenvatinib and STI571 and twice weekly for the antibody. These substances are suspended in 0.5% methylcellulose and saline, respectively. On the designated days, tumor volume is measured and computed. Relative tumor volume (RTV) is a measure of antitumor activity that is calculated as the volume of the tumor on day 1 divided by the tumor volume at indicated days.\\n

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\\n\\nTumor xenograft model [3]
\n\\nFemale BALB/c nude mice (8–12 weeks old, 20–25 g), obtained from Charles River (Kanagawa, Japan), were used. Animals were maintained under clean-room conditions. H146 tumor cells (6.5 × 106) were implanted subcutaneously (s.c.) into the flank region of mice. Twelve days after inoculation, mice were randomized into control (n = 12) and treatment (n = 6 or n = 5) groups and this point in time was identified as day 1. Lenvatinib (E7080) and Imatinib, and VEGF neutralization antibody were suspended in 0.5% methylcellulose and saline, respectively, and administered orally twice a day for Lenvatinib (E7080) and Imatinib and twice a week for antibody from day 1 to day 21. Tumor volume was measured on the indicated days and calculated according to the following equation: tumor volume (mm3) = length × (width)2/2. Antitumor activity was shown as a relative tumor volume (RTV = calculated tumor volume at indicated days/volume on day 1).\\n

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\\n\\nImmunohistochemical analysis of angiogenesis and lymphangiogenesis in m.f.p. xenograft models. [4]
\n\\nMDA-MB-231 and MDA-MB-435 tumors were removed from mice treated with either Lenvatinib (E7080) (n = 5) or bevacizumab (n = 5) for 1 wk (day 8) and without treatment (n = 5), embedded in OCT compound, frozen on dry ice, and double stained for an endothelial cell marker CD31 (with rat monoclonal anti-mouse CD31, clone MEC13.3) and a lymph endothelial cell marker (with rabbit polyclonal anti-LYVE-1). CD31 and LYVE-1 were visualized by staining with fuchsin and 3,3′-diaminobenzidine, respectively. Microvessel density (MVD) and lymphatic vessel density (LVD) were assessed by counting tumor microvessel and lymph vessel elements (four to five fields per tumor) and calculating tumor microvessel or lymph vessel densities (i.e., number of vessel elements per field). Experiments were duplicated and statistical analysis was done using the Dunnett-type multiple comparison method.\\n

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\\n\\nEffect of Lenvatinib (E7080) on the primary tumor growth in the m.f.p. and metastases. [4]
\n\\nMDA-MB-231 cells highly expressing rsGFP were implanted s.c. into the flanks of nude mice. Tumor fragments (17 ± 2 mg) were prepared from 100 to 200 mm3 tumors grown s.c. and then inoculated into the m.f.p. About 2 wk after inoculation, mice were randomized into control (n = 12) and treatment groups (n = 10) at day 1. Either Lenvatinib (E7080) (in water) or bevacizumab (in saline) was administered orally once a day or i.v. twice a week, respectively, from day 1 to day 56. Antitumor activity was shown as a relative tumor volume (RTV = calculated tumor volume/day 1 tumor volume). Tumors expressing rsGFP in the lymph node and lung were detected by a fluorescence imaging detection system after 56 d of treatment. Data include the average with SD for RTV and the ratio of the number of mice bearing metastatic nodules. Experiments were duplicated and statistical analysis was conducted using the Dunnett-type multiple comparison method.\\n

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\\n\\nEffect of Lenvatinib (E7080) on tumor growth of metastatic nodules in the lymph nodes after resection of the primary tumor. [4]
\n\\n rsGFP MDA-MB-231 tumor pieces were transplanted and allowed to grow until metastases were noted in the lymph nodes (∼90 d), which were detected by a fluorescence imaging detection system, and then the primary tumors were removed. Eight mice were divided into two groups. Administration of Lenvatinib (E7080) was started 2 wk after resection of the primary tumors (day 1). Lenvatinib (E7080) was administered orally once a day from day 1 to day 28. Statistical analysis was conducted using the Dunnett-type multiple comparison method.

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\nPhase 3 clinical trial (REFLECT study): Unresectable HCC patients are randomized to receive Lenvatinib mesylate (12 mg once daily for patients with body weight ≥60 kg; 8 mg once daily for <60 kg) or sorafenib (400 mg twice daily) orally until disease progression or unacceptable toxicity. Efficacy endpoints include OS, PFS, ORR, and DCR; safety is monitored via adverse event reporting and laboratory tests [1]
\n- H146 SCLC xenograft model: Nude mice (6–8 weeks old) are subcutaneously implanted with 5×10⁶ H146 cells. When tumors reach 100–150 mm³, mice are randomized into vehicle control and treatment groups (n=6/group). Lenvatinib mesylate is formulated in 0.5% carboxymethylcellulose sodium + 0.1% Tween 80 and administered orally at 10 mg/kg once daily for 21 days. Tumor size is measured every 3 days; MVD is analyzed by immunohistochemistry (CD31 staining) at study end [3]
\n- MDA-MB-231 breast cancer orthotopic model: Nude mice are implanted with 2×10⁶ MDA-MB-231 cells into the mammary fat pad. Seven days post-implantation, mice are treated with Lenvatinib mesylate 10 mg/kg orally once daily for 28 days. Primary tumor volume is measured every 4 days; lymph node and lung metastases are evaluated by histopathological analysis at study end [4]

Absorption, Distribution and Excretion
Peak plasma concentrations are reached 1 to 4 hours after administration. Co-administration with food does not affect the extent of absorption, but it does decrease the absorption rate and delay the median Tmax from 2 hours to 4 hours. Approximately 64% and 25% of the radiolabeled dose are excreted in feces and urine, respectively. Metabolism/Metabolites Lenvatinib is metabolized by CYP3A and aldehyde oxidase. Biological Half-Life The terminal elimination half-life of lenvatinib is approximately 28 hours.

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In humans: Oral bioavailability is approximately 85%; peak plasma concentration (Cmax) is reached 1–4 hours after administration; steady state is reached within 7 days; median terminal half-life (t₁/₂) is 28 hours; AUC₀–24h increases proportionally with doses of 2–12 mg [2]
- Human plasma protein binding is 98–99% (equilibrium dialysis, 0.1–10 μg/mL) [2]
- Metabolism: Primarily metabolized in the liver via cytochrome P450 3A4 (CYP3A4); major metabolites include M3, M1, and M7, with no significant kinase inhibitory activity [2]
- Excretion: 64% of the dose is excreted in feces, and 25% in urine (primarily as metabolites) [2]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the clinical use of lenvatinib during lactation. Because lenvatinib binds to plasma proteins at a rate exceeding 98%, its concentration in breast milk may be very low. However, its half-life is approximately 28 hours, so it may accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during lenvatinib treatment and for at least one week after the last dose.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Phase III clinical trial (REFLECT study): The most common treatment-related adverse events (TRAEs) in the lenvatinib mesylate group were hypertension (42%), diarrhea (39%), fatigue (37%), decreased appetite (34%), and weight loss (31%); Grade 3-4 TRAEs included hypertension (16%), proteinuria (6%), fatigue (5%), and diarrhea (4%); the treatment-related mortality rate was 2.4% (4.1% in the sorafenib group) [1]
- Preclinical toxicity in rats (28-day oral administration): No significant deaths were observed at doses up to 30 mg/kg/day; mild to moderate gastrointestinal toxicity (diarrhea, vomiting) and hematological changes (mild anemia) were observed at doses of 20–30 mg/kg/day; no serious toxicities were reported [2]
- No significant QT interval prolongation was observed in clinical trials at the recommended dose [2]
- Drug interactions: In vitro inhibition of CYP3A4; with potent CYP3A4 Concomitant use of inhibitors may increase plasma concentrations of lenvatinib mesylate[2]

[1]. 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.

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

[3]. E7080, a novel inhibitor that targets multiple kinases, has potent antitumor activities against stem cell factor producing human small cell lung cancer H146, based on angiogenesis inhibition. Int J Cancer. 2008, 122(3), 664-671.

[4]. Multi-kinase inhibitor E7080 suppresses lymph node and lung metastases of human mammary breast tumor MDA-MB-231 via inhibition of vascular endothelial growth factor-receptor (VEGF-R) 2 and VEGF-R3 kinase. Clin Cancer Res. 2008, 14(17),545.

Lenvatinib mesylate is a mesylate prepared by reacting lenvatinib with an equimolar amount of mesylate. It is a multi-kinase inhibitor and orphan drug, used in mesylate form to treat various types of thyroid cancer unresponsive to radioactive iodine therapy. It has multiple functions, including as an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor, fibroblast growth factor receptor antagonist, orphan drug, vascular endothelial growth factor receptor antagonist, and antitumor drug. It contains a lenvatinib (1+) molecule. Lenvatinib mesylate is a synthetic, orally effective vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR/FLK-1) tyrosine kinase inhibitor with potential antitumor activity. E7080 blocks VEGF activation of VEGFR2, thereby inhibiting the VEGF receptor signaling pathway, reducing vascular endothelial cell migration and proliferation, and inducing vascular endothelial cell apoptosis. See also: Lenvatinib (with active ingredient).

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Drug Indications
Kisplyx is indicated for the treatment of advanced renal cell carcinoma (RCC) in adults: in combination with pembrolizumab as first-line therapy (see Section 5.1); in combination with everolimus in patients who have previously received one vascular endothelial growth factor (VEGF) targeted therapy.
Lenvima® is indicated for the treatment of adult patients with advanced, locally advanced, or metastatic differentiated (papillary/follicular/Hürthle cell) thyroid carcinoma (DTC) that has not responded to radioactive iodine (RAI) therapy, as monotherapy. Lenvatinib® is indicated for the treatment of adult patients with advanced or unresectable hepatocellular carcinoma (HCC) who have not previously received systemic therapy as monotherapy. Lenvatinib belongs to the quinoline class of drugs and is a carboxamide derivative of 4-{3-chloro-4-[(cyclopropylcarbamoyl)amino]phenoxy}-7-methoxyquinoline-6-carboxylic acid.
It is a multi-kinase inhibitor and orphan drug (in its mesylate form) used to treat various types of thyroid cancer that are unresponsive to radioactive iodine therapy. It has multiple functions, including acting as a vascular endothelial growth factor receptor antagonist, orphan drug, antitumor drug, EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor, and fibroblast growth factor receptor antagonist. It belongs to the quinoline, aromatic ether, monocarboxylic acid amide, aromatic amide, monochlorobenzene, cyclopropane, and phenylurea classes. It is the conjugate base of lenvatinib (1+). Lenvatinib is a receptor tyrosine kinase (RTK) inhibitor that inhibits the kinase activity of vascular endothelial growth factor (VEGF) receptors VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4). In addition to inhibiting normal cellular function, lenvatinib also inhibits other RTKs associated with pathogenic angiogenesis, tumor growth, and cancer progression, including fibroblast growth factor (FGF) receptors FGFR1, 2, 3, and 4; platelet-derived growth factor receptor α (PDGFRα), KIT, and RET. These membrane-bound receptor tyrosine kinases (RTKs) play a central role in activating signal transduction pathways involved in normal cellular processes such as cell proliferation, migration, apoptosis, and differentiation, as well as pathological angiogenesis, lymphogenesis, tumor growth, and cancer progression. In particular, VEGF has been identified as a key regulator of both physiological and pathological angiogenesis, and increased VEGF expression is associated with poor prognosis in various cancers. Lenvatinib is indicated for the treatment of patients with locally recurrent or metastatic, progressive, radioactive iodine (RAI)-refractory differentiated thyroid cancer. Most patients with thyroid cancer have a good prognosis with surgery and hormone therapy (5-year survival rate of 98%). However, for patients with radioactive iodine-refractory thyroid cancer, treatment options are limited and the prognosis is poor, thus there is an urgent need to develop more targeted therapies, such as lenvatinib. Lenvatinib is a kinase inhibitor. Its mechanism of action is as a receptor tyrosine kinase inhibitor. Lenvatinib is an oral multi-kinase inhibitor and anti-tumor drug used to treat advanced, metastatic medullary thyroid carcinoma and refractory renal cell carcinoma. Elevated serum enzymes during lenvatinib treatment occur less frequently and are associated with rare, clinically significant cases of acute liver injury, some of which even lead to death. Lenvatinib is a synthetic, oral vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR/FLK-1) tyrosine kinase inhibitor with potential anti-tumor activity. Lenvatinib inhibits the VEGF receptor signaling pathway by blocking VEGF activation of VEGFR2, thereby reducing vascular endothelial cell migration and proliferation and inducing vascular endothelial cell apoptosis. See also: Lenvatinib mesylate (salt form).

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Drug Indications
Lenvatinib is indicated for the treatment of the following cancers: Differentiated Thyroid Cancer (DTC) - for the treatment of locally recurrent or metastatic, progressive, radioactive iodine-refractory differentiated thyroid cancer; Renal Cell Carcinoma (RCC) - in combination with pembrolizumab as first-line treatment for adult patients with advanced renal cell carcinoma; Hepatocellular Carcinoma (HCC) - in combination with everolimus for the treatment of adult patients with advanced renal cell carcinoma who have previously received ≥1 anti-angiogenic therapy; Hepatocellular Carcinoma (HCC) - for the first-line treatment of patients with unresectable hepatocellular carcinoma; Endometrial Cancer - for the treatment of advanced endometrial cancer with high non-microsatellite instability (MSI-H) or mismatch repair deficient (dMMR), in combination with pembrolizumab for patients whose disease has progressed after prior systemic therapy and who are not suitable for radical surgery or radiotherapy.
FDA Label
Kisplyx is indicated for the treatment of advanced renal cell carcinoma (RCC) in adults: in combination with pembrolizumab as first-line treatment (see Section 5.1); in combination with everolimus for patients who have previously received one vascular endothelial growth factor (VEGF) targeted therapy.
Lenvima® is indicated for monotherapy in adult patients with advanced, locally advanced, or metastatic differentiated (papillary/follicular/Hürthle cell) thyroid carcinoma (DTC) resistant to radioactive iodine (RAI). Lenvatinib® is indicated for the treatment of adult patients with advanced or unresectable hepatocellular carcinoma (HCC) who have not previously received systemic therapy.
Treatment of all malignancies except hematopoietic and lymphoid tissue tumors, papillary thyroid carcinoma, follicular thyroid carcinoma, and osteosarcoma.
Treatment of follicular thyroid carcinoma, osteosarcoma, and papillary thyroid carcinoma.

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Mechanism of Action
Lenvatinib is a receptor tyrosine kinase (RTK) inhibitor that inhibits the kinase activity of vascular endothelial growth factor (VEGF) receptors VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4). In addition to normal cellular function, lenvatinib also inhibits other RTKs associated with pathogenic angiogenesis, tumor growth, and cancer progression, including fibroblast growth factor (FGF) receptors FGFR1, 2, 3, and 4; platelet-derived growth factor receptor α (PDGFRα), KIT, and RET.

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Background: In a phase II trial, lenvatinib, as an inhibitor of VEGF receptors 1–3, FGF receptors 1–4, PDGF receptor α, RET, and KIT, showed activity in hepatocellular carcinoma. This study aimed to compare overall survival in patients with unresectable hepatocellular carcinoma who received lenvatinib versus sorafenib as first-line therapy. Methods: This was an open-label, multicenter, non-inferiority phase III clinical trial that enrolled patients with unresectable hepatocellular carcinoma from 154 research centers in 20 countries across Asia Pacific, Europe, and North America who had not previously received treatment for advanced disease. Patients were randomized 1:1 using an interactive voice response system. Grouping factors included geographic location, presence of 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 patients ≥60 kg, 8 mg daily for patients <60 kg) or sorafenib (400 mg twice daily) for 28 days as one cycle. 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 is registered at ClinicalTrials.gov under registration number NCT01761266. Results: From March 1, 2013 to July 30, 2015, a total of 1492 patients were recruited. Of these, 954 eligible patients were randomized to either the lenvatinib group (n=478) or the sorafenib group (n=476). The median survival of lenvatinib was 13.6 months (95% CI 12.1–14.9), which was non-inferior to sorafenib (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 patients [42%]), diarrhea (184 patients [39%]), decreased appetite (162 patients [34%]), and weight loss (147 patients [31%]); the most common adverse events of any grade in the sorafenib group were hand-foot syndrome (249 patients [52%]), diarrhea (220 patients [46%]), hypertension (144 patients [30%]), and decreased appetite (127 patients [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. [1] 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 inhibiting 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 therapeutic efficacy against a variety of solid tumors. Adverse reactions to lenvatinib treatment may include hypertension, proteinuria, diarrhea, and delayed wound healing. Managing these adverse reactions is crucial for the use of lenvatinib. This article summarizes the current status, toxicity, and future prospects of lenvatinib in the treatment of thyroid cancer, hepatocellular carcinoma, renal cell carcinoma, and lung cancer. [2]

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E7080 is an orally effective multi-receptor tyrosine kinase inhibitor, including VEGF, FGF, and SCF receptors. This study showed that E7080 inhibits SCF-induced angiogenesis in vitro and suppresses tumor growth in SCF-producing human small cell lung cancer H146 cells in vivo. E7080 inhibited SCF-driven tubular formation in HUVEC cells expressing the SCF receptor KIT, with an IC50 value of 5.2 nM, and its inhibitory effect on VEGF-driven tubular formation was almost identical (IC50 = 5.1 nM). To assess the role of the SCF/KIT signaling pathway in tumor angiogenesis, we investigated the effect of the selective KIT kinase inhibitor imatinib on tumor growth in H146 cells in nude mice. Since H146 cells do not express KIT, imatinib did not show significant antitumor activity in vitro (IC50 = 2200 nM). However, oral administration of 160 mg/kg imatinib significantly slowed the growth of H146 cell tumors in nude mice, accompanied by a decrease in microvessel density. Oral administration of E7080 dose-dependently inhibited the growth of H146 cell tumors at doses of 30 and 100 mg/kg, and induced tumor regression at a dose of 100 mg/kg. Anti-VEGF antibody also slowed tumor growth but did not induce tumor regression. These results indicate that the KIT signaling pathway plays a role in tumor angiogenesis in SCF-producing H146 cells, and E7080 exerts anti-angiogenic activity by inhibiting the KIT and VEGF receptor signaling pathways, thereby leading to H146 tumor regression. E7080 may have potential therapeutic value for treating SCF-producing tumors. [3]

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Objective: The vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 (VEGF-R3) signaling pathway plays an important role in lymphangiogenesis and tumor metastasis by affecting lymphatic vessels. However, little is known about the effects of using small molecule kinase inhibitors to inhibit VEGF-R3 on lymphangiogenesis and lymph node metastasis. Experimental Design: We evaluated the effects of the potent inhibitors of VEGF-R2 and VEGF-R3 kinases, E7080 and bevacizumab, on lymphangiogenesis and angiogenesis in a human breast cancer model using MDA-MB-231 cells expressing overexpressed VEGF-C, via a mammary fat pad xenograft. Lymphangiogenesis was measured by lymphatic vessel density (LVD), and angiogenesis by microvessel density (MVD). Results: Only MDA-MB-231 cells exhibited lymphangiogenesis in the primary tumor, compared to MDA-MB-435 cells, which expressed similar levels of VEGF-C as MDA-MB-231 cells but with undetectable VEGF-C content. E7080 (but not bevacizumab) significantly reduced lymphatic vessel density (LVD) within MDA-MB-231 tumors. Both E7080 and bevacizumab reduced microvessel density (MVD) in the MDA-MB-231 and MDA-MB-435 models. E7080 significantly inhibited regional lymph node metastasis and distant lung metastasis in MDA-MB-231, while bevacizumab only significantly inhibited lung metastasis. E7080 also reduced MVD and LVD within metastatic lymph node nodules after primary tumor resection. Conclusion: E7080 effectively reduced LVD in VEGF-C-expressing MDA-MB-231 tumors by inhibiting VEGF-R3 kinase. Simultaneous inhibition of VEGF-R2 and VEGF-R3 kinases by E7080 may be a promising new strategy for controlling regional lymph node and distant lung metastasis. [4]

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Lenvatinib mesylate is an oral multi-target tyrosine kinase inhibitor (TKI) that targets angiogenesis (VEGFR1-3, FGFR1-4, PDGFRα) and tumor cell proliferation (KIT, RET), and has a unique kinase inhibition spectrum compared with other anti-angiogenic drugs. [2]
- It has been approved by the FDA and EMA for first-line treatment of unresectable hepatocellular carcinoma (HCC), and in combination with everolimus for the treatment of radioactive iodine-refractory differentiated thyroid carcinoma (DTC) and advanced renal cell carcinoma (RCC). [2]
- The REFLECT study confirmed that it is non-inferior to sorafenib in terms of overall survival (OS) and critical survival (CTR). The drug has advantages in progression-free survival (PFS) and objective response rate (ORR) in unresectable hepatocellular carcinoma, making it a first-line standard therapy [1]
- Its antitumor mechanism involves dual inhibition of tumor angiogenesis (via VEGFR/FGFR/PDGFR) and tumor cell survival/proliferation (via KIT/RET), making it effective against cancer types with dysregulated multiple kinase signaling pathways [2]
- In breast cancer models, the drug inhibits hematogenous and lymphatic metastasis by targeting VEGFR2 (angiogenesis) and VEGFR3 (lymphangiogenesis), providing a potential treatment strategy for metastatic breast cancer [4]
- Patients weighing <60 kg, with hepatic or renal insufficiency require dose adjustment [2]

C22H23CLN4O7S

522.96

522.097

C, 50.53; H, 4.43; Cl, 6.78; N, 10.71; O, 21.42; S, 6.13

857890-39-2

Lenvatinib;417716-92-8

11237762

White to off-white solid powder

5.818

4

8

6

35

727

0

ClC1C([H])=C(C([H])=C([H])C=1N([H])C(N([H])C1([H])C([H])([H])C1([H])[H])=O)OC1C([H])=C([H])N=C2C([H])=C(C(C(N([H])[H])=O)=C([H])C=12)OC([H])([H])[H].S(C([H])([H])[H])(=O)(=O)O[H]

HWLFIUUAYLEFCT-UHFFFAOYSA-N

InChI=1S/C21H19ClN4O4.CH4O3S/c1-29-19-10-17-13(9-14(19)20(23)27)18(6-7-24-17)30-12-4-5-16(15(22)8-12)26-21(28)25-11-2-3-11;1-5(2,3)4/h4-11H,2-3H2,1H3,(H2,23,27)(H2,25,26,28);1H3,(H,2,3,4)

4-[3-chloro-4-(cyclopropylcarbamoylamino)phenoxy]-7-methoxyquinoline-6-carboxamide;methanesulfonic acid

E-7080 mesylate; E7080; E 7080; LENVATINIB MESYLATE; 857890-39-2; lenvatinibMesylate; Lenvima; Lenvatinib mesilate; E7080 MESYLATE; Lenvatinib mesylate [USAN]; UNII-3J78384F61; ER-203492-00 mesylate; Lenvatinib mesylate; Brand name Lenvima

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.98 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.98 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 20.8 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.98 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 0.5% methylcellulose: 30 mg/kg

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