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); FGFR2; FGFR3; FGFR4; RET

Lenvatinib (E7080) has IC50s of 4, 5.2, and 22 nM for VEGFR1 (Flt-1), VEGFR3 (Flt-4), and VEGFR2 (KDR), in that order. FGFR1, PDGFRβ, KIT, and TCGF are all inhibited by lentinib, with IC50 values of 51, 39, 46, and 100 nM, respectively[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.

Lenvatinib (E7080) (100 mg/kg, p.o.) also significantly inhibits metastasis to both distant lung and regional lymph nodes after treatment concludes, in addition to significantly inhibiting local tumor growth at the m.f.p.[3]. Lenvatinib (E7080) causes tumor regression in the H146 xenograft model at 100 mg/kg and dose-dependently suppresses the growth of the H146 tumor at 30 and 100 mg/kg (BID, QDx21). Lenvatinib at 100 mg/kg reduces microvessel density more than anti-VEGF antibody and STI571 treatment, according to IHC analysis using anti-CD31 antibody[4].

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Efficacy of E7080, Imatinib and a VEGF neutralization antibody in H146 xenograft model [4]
To 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.

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E7080 inhibits metastasis to both regional lymph nodes and distant lung in the MDA-MB-231 m.f.p. xenograft model. [4]
Next, 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.

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E7080 decreased both angiogenesis and lymphangiogenesis of established metastatic nodules of MDA-MB-231 tumor in the lymph nodes. [4]
Researchers 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.

Recombinant kinase domains of receptors are used in HTRF (KDR, VEGFR1, FGFR1, c-Met, EGFR) and ELISA (PDGFRβ) tyrosine kinase assays. In both assays, 10 μL of enzyme, 16 μL of poly (GT) solution (250 ng), and 10 μL of ATP solution (1 μM ATP) are combined with 4 μL of serial dilutions of E7080 in a 96-well round plate (final concentration of DMSO is 0.1%). Enzyme is not added to blank wells. There is no test article added to control wells. Each well receives an addition of ATP solution to start the kinase reaction. The reaction is terminated by adding 0.5 M EDTA (10 μL/well) to the reaction mixture in each well following 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. Following washing with wash buffer, the reaction mixture is incubated at room temperature for 30 minutes before PY20-HRP solution (70 μL/well) is added. In each well, 100 μL of TMB reagent is added following washing with wash buffer. Each well receives 100 μL of 1 M H₃PO₄ after a few minutes (10–30 minutes). By measuring absorbance at 450 nm with a microplate reader, kinase activity can be identified.

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In vitro kinase assay [3]
Tyrosine 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.
In 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.
In 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. Kinase inhibitory activities of Lenvatinib (E7080) other than KDR, VEGFR1, FGFR1, c-Met, EGFR and PDGFRβ were examined by ProQinase Company.

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Cell-free kinase assay/cell phosphorylated assay. [4]
Tyrosine 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.

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].

Clean-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.

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Tumor xenograft model [3]
Female 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).

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Immunohistochemical analysis of angiogenesis and lymphangiogenesis in m.f.p. xenograft models. [4]
MDA-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.

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Effect of Lenvatinib (E7080) on the primary tumor growth in the m.f.p. and metastases. [4]
MDA-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.

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Effect of Lenvatinib (E7080) on tumor growth of metastatic nodules in the lymph nodes after resection of the primary tumor. [4]
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.

Absorption, Distribution and Excretion
Time to peak plasma concentration occurred from 1 to 4 hours post­dose. Administration with food did not affect the extent of absorption, but decreased the rate of absorption and delayed the median Tmax from 2 hours to 4 hours.
Following administration of a radiolabeled dose, approximately 64% and 25% of the radiolabel were eliminated in the feces and urine, respectively.

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Metabolism / Metabolites
Lenvatinib is metabolized by CYP3A and aldehyde oxidase.

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Biological Half-Life
The terminal elimination half­life of lenvatinib is approximately 28 hours.

Hepatotoxicity
In large clinical trials of lenvatinib, elevations in serum aminotransferase levels were common, occurring in 52% of patients. Values greater than 5 times the upper limit of normal (ULN), however, occurred in only 3% to 5% of recipients. Serum alkaline phosphatase elevations were also common occurring in 28% of patients and were above 3 times ULN in 2%. In addition, fatal hepatic failure was reported in 3 of 1160 patients treated in preregistration clinical trials and another patient developed symptomatic but self-limited acute hepatitis with jaundice. The degree of relatedness of these events to lenvatinib therapy, however, was not defined. In the product label for lenvatinib, serum ALT, AST and alkaline phosphatase elevations are listed as adverse reactions, and acute hepatitis is mentioned as a rare occurrence. Monitoring of serum enzymes before, every 2 weeks for 2 months and monthly thereafter during treatment is recommended with dose reduction or discontinuation depending upon the degree and persistence of the abnormalities.
Likelihood score: D (possible cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of lenvatinib during breastfeeding. Because lenvatinib is more than 98% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 28 hours and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during lenvatinib therapy and for at least 1 week after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
In vitro binding of lenvatinib to human plasma proteins ranged from 98% to 99%.

[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.

Pharmacodynamics
Based on x-ray crystallography and kinetic interaction studies, lenvatinib binds to the adenosine 5'-triphosphate binding site of VEGFR2 and to a neighbouring region via a cyclopropane ring and thereby inhibits tyrosine kinase activity and associated signalling pathways.

C21H19CLN4O4

426.85

426.109

C, 59.09; H, 4.49; Cl, 8.30; N, 13.13; O, 14.99

417716-92-8

Lenvatinib mesylate;857890-39-2;Lenvatinib-d4;Lenvatinib-d5

9823820

Off-white to light yellow solid powder

1.5±0.1 g/cm3

627.2±55.0 °C at 760 mmHg

333.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.697

3.39

3

5

6

30

634

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]

WOSKHXYHFSIKNG-UHFFFAOYSA-N

InChI=1S/C21H19ClN4O4/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/h4-11H,2-3H2,1H3,(H2,23,27)(H2,25,26,28)

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

E-7080; E7080; E 7080; ER-203492-00; Lenvatinib; 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

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: ≥ 0.64 mg/mL (1.50 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 2: 0.5% methylcellulose: 30 mg/kg

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Solubility in Formulation 3: 6.67 mg/mL (15.63 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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