VEGFR2 (IC50 = 6.5 nM); VEGFR3 (IC50 = 15 nM); EphB2 (IC50 = 24 nM); VEGFR1 (IC50 = 30 nM); PDGFRα (IC50 = 40 nM)

AV-951 is a novel derivative of urea and quinoline. AV-951 prevents endothelial cell proliferation and the VEGF-dependent activation of mitogen-activated protein kinases.[1]
KRN951 is a novel tyrosine kinase inhibitor for VEGFRs with antitumor angiogenesis and antigrowth activities. KRN951 potently inhibited VEGF-induced VEGFR-2 phosphorylation in endothelial cells at in vitro subnanomolar IC50 values (IC50 = 0.16 nmol/L). It also inhibited ligand-induced phosphorylation of platelet-derived growth factor receptor-beta (PDGFR-beta) and c-Kit (IC50 = 1.72 and 1.63 nmol/L, respectively). KRN951 blocked VEGF-dependent, but not VEGF-independent, activation of mitogen-activated protein kinases and proliferation of endothelial cells. In addition, it inhibited VEGF-mediated migration of human umbilical vein endothelial cells.[1]

Studies conducted in vivo demonstrate that AV-951, particularly when administered orally at a dose of 1 mg/kg, also reduces micro vessel density and suppresses VEGFR2 phosphorylation levels in tumor xenografts. In athymic rats, AV-951 almost completely inhibits the growth of tumor xenografts (TGI>85%).[1] Another study using a peritoneal disseminated tumor model in rats demonstrates that AV-951 can extend the survival of the tumor-bearing rats up to 53.5 days after the MST. When applied to various human tumor xenografts, such as lung, breast, colon, ovarian, pancreatic, and prostate cancer, AV-951 exhibits antitumor activity.[2]

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Following p.o. administration to athymic rats, KRN951 decreased the microvessel density within tumor xenografts and attenuated VEGFR-2 phosphorylation levels in tumor endothelium. It also displayed antitumor activity against a wide variety of human tumor xenografts, including lung, breast, colon, ovarian, pancreas, and prostate cancer. Furthermore, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) analysis revealed that a significant reduction in tumor vascular hyperpermeability was closely associated with the antitumor activity of KRN951. These findings suggest that KRN951 is a highly potent, p.o. active antiangiogenesis and antitumor agent and that DCE-MRI would be useful in detecting early responses to KRN951 in a clinical setting. KRN951 is currently in phase I clinical development for the treatment of patients with advanced cancer.[1]

The IC50 values of AV-951 against various recombinant receptor and nonreceptor tyrosine kinases, such as VEGFR1, VEGFR2, VEGFR3, c-Kit, PDGFRβ, Flt-3, and FGFR1, are ascertained by conducting cell-free kinase assays in quadruplicate using 1 μM ATP.

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Kinase selectivity. [1]
Cell-free kinase assays were done in quadruplicate with 1 μmol/L ATP to determine the IC50 values of Tivozanib (AV951; KRN-951) against a variety of recombinant receptor and nonreceptor tyrosine kinases. Recombinant enzymes were obtained from ProQinase GmbH. [1]
Cell-based assays were done to determine the ability of Tivozanib (AV951; KRN-951) to inhibit ligand-dependent phosphorylation of receptor tyrosine kinases as described previously. Briefly, the cells were starved overnight in appropriate basic medium containing 0.5% fetal bovine serum (FBS). Following the addition of KRN951 or 0.1% DMSO, the cells were incubated for 1 hour and then stimulated with the cognate ligand at 37°C. Receptor phosphorylation was induced for 5 minutes except for VEGFR-3 (10 minutes), c-Met (10 minutes), and c-Kit (15 minutes). All the ligands used in the assays were human recombinant proteins, except for VEGF-C, a rat recombinant protein. Following cell lysis, receptors were immunoprecipitated with appropriate antibodies and subjected to immunoblotting with phosphotyrosine. Quantification of the blots and calculation of IC50 values were carried out as described previously. [1]

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Mitogen-activated protein kinase activation. [1]
This was evaluated as described previously. Briefly, HUVECs were starved for 16 hours in a basic medium (EBM-2) containing 0.5% FBS. Following incubation with Tivozanib (AV951; KRN-951) for 1 hour, HUVECs were stimulated with 50 ng/mL VEGF, 25 ng/mL basic fibroblast growth factor or 20 ng/mL EGF. Cell lysates were subjected to SDS-PAGE followed by immunoblotting of phosphorylated MAPKs with phosphorylated p44/42 mitogen-activated protein kinase (MAPK) antibody.[1]

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Permeability assay and detection of VEGFR‐2 phosphorylation.[2]
The effects of malignant ascites on endothelial cell permeability and VEGFR‐2 phosphorylation, and the inhibitory activity of Tivozanib (AV951; KRN-951) on these effects, were evaluated. Ascites samples taken from a vehicle‐treated peritoneal disseminated tumor model on day 25 were pooled and the resulting supernatant was used in these assays. We examined propidium iodide uptake as a measure of permeability in an in vitro assay. VEGFR‐2 phosphorylation was determined by western blotting. For the permeability assay, HUVEC at 90% confluence in culture were serum starved overnight in a basic medium (EBM‐2) containing 0.5% fetal bovine serum. These cells were then washed with phosphate‐buffered saline, and malignant ascites and a 10‐nM concentration of Tivozanib (AV951; KRN-951) were added to the culture plates. Medium alone or 50 ng/mL VEGF without ascites served as the internal controls. After 7 h incubation, the cells were harvested and treated with propidium iodide (1 µg/mL), and subjected to FACS analysis. Permeability was assessed by measuring the uptake of propidium iodide by the HUVEC. For western blotting, HUVEC were treated in the same way except that the stimulation time with ascites of 10 min. Following cell lysis, VEGFR proteins were immunoprecipitated with an anti‐VEGFR‐2 antibody and then subjected to immunoblotting with an antiphosphotyrosine antibody, as described previously.[2]

The ability of AV-951 to inhibit ligand-dependent phosphorylation of tyrosine kinase receptors is assessed using assays based on human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts. In the proper basic medium with 0.5% fetal bovine serum (FBS), the cells are starved for the duration of the next day. The cells are stimulated with the cognate ligand at 37 °C after being incubated for an hour with either AV-951 or 0.1% DMSO. With the exception of VEGFR3, c-Met, and c-Kit, which are induced for 10 and 15 minutes, respectively, receptor phosphorylation lasts for five minutes. VEGF-C, a rat recombinant protein, is the only ligand utilized in the assays that is not a human recombinant protein. After cell lysis, receptors are phosphotyrosine-immunoblotted after being immunoprecipitated with the proper antibodies. Both the blot quantification and IC50 value computation are completed.

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Endothelial cell proliferation.[1]
HUVECs were seeded in M-199 containing 5% FBS in collagen-coated 96-well plates at a density of 4,000 cells/200 μL/well. After 24 hours, Tivozanib (AV951/KRN-951) was added followed by 20 ng/mL VEGF or 10 ng/mL bFGF, and the cells were cultured for 72 hours. [3H]thymidine (1 μCi/mL) was added and the cells were cultured for a further 12 hours. Cells were then harvested and their radioactivity was measured with a Liquid Scintillation Counter. [1]

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Chemotaxis assay. [1]
HUVEC migration was assessed using 96-well microchamber plates. Cells were starved for 5 hours in EBM-2 containing 0.1% bovine serum albumin (BSA). Then, cells were harvested, resuspended in EBM-2 containing 0.1% BSA, and placed in the upper chamber. Cell migration was initiated by placing medium containing 10 ng/mL VEGF, 0.1% FBS, and 0.1% BSA to the bottom chamber. When indicated, Tivozanib (AV951/KRN-951) was added to both the upper and lower chambers. After 22 hours of incubation, cells were stained with 4 μg/mL calcein AM in HBSS. Fluorescence in the cells that had migrated through the pores of the fluorescence blocking membrane was directly measured through the bottom of the chambers in a fluorescence plate reader at excitation/emission wavelengths of 485/530 nm. [1]

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Cytotoxicity assays. [1]
These assays were done as described previously. Briefly, cells were seeded in 96-well plates and cultured in medium containing 10% FBS. Tivozanib (AV951/KRN-951) was added ∼24 hours after the start of culture and the cells were then incubated for 72 hours. WST-1 reagent was used for the detection of cell viability.[1]

Mice: The athymic rats receive a subcutaneous injection of cancer cells in their right flank. Tumors up to 1,500 mm3 are surgically removed, and smaller pieces (20–30 mg) are s.c. implanted into the right flank of rats exposed to radiation. Beginning on day zero of randomization, oral administration of KRN951 (0.2 or 1 mg/kg) or the vehicle is administered. Using Vernier calipers, tumor volume is measured and computed twice a week.

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Tumor xenograft models. Athymic rats (RH-rnu/rnu) were used. Twenty-four hours after whole body irradiation with a γ-source (7 Gy, Co60), cancer cells were s.c. inoculated into the right flank of the rats. Once established, tumors of ∼1,500 mm3 were surgically excised and smaller tumor fragments (20-30 mg) were s.c. implanted in the right flank of irradiated rats. Oral administration of Tivozanib (AV951/KRN-951) (0.2 or 1 mg/kg) or vehicle was initiated at the day of randomization (day 0). Tumor volume was measured twice weekly with Vernier calipers, and calculated as (length × width2) × 0.5. Relative tumor volume (RTV) was calculated by the formula: RTV at day x = tumor volume at day x / tumor volume at day 0. Percentage tumor growth inhibition (TGI%) was calculated as described previously. Statistical analysis of RTV was done using the unpaired t test. DCE-MRI. Athymic rats (RH-rnu/rnu) were s.c. implanted with fresh Calu-6 tumor fragments. Rats were randomized when the tumors reached a volume of 274 to 287 mm3 (day −1). Once-daily p.o. administration of Tivozanib (AV951/KRN-951) or vehicle was initiated the day after randomization (day 0) and continued for 2 weeks (days 0-13). MRI experiments were carried out at 1.5 T on a whole body magnet equipped with a flexible receiver coil (circularly polarized). DCE-MRI acquisitions were done on day −1 (before the start of treatment), day 2, day 13, and day 21. On days 2 and 13, the rats were imaged 4 hours after p.o. administration of Tivozanib (AV951/KRN-951). The rat tail vein was cannulated for contrast agent injection before placing the animals in the magnet. During the experiment, rats were anesthetized by an i.m. injection of a mixture of ketamine and xylazine (2/1, v/v, 70 and 15 mg/kg, respectively). The anesthetized rats were placed in a cradle supine position inside the resonator. The exact position of the rats was assessed by a scout imaging sequence. [1]

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Measurement of tumor vessel diameter. During the MRI study, an additional three groups of Calu-6 tumor-bearing rats (RH-rnu/rnu, three rats per group) were used for measurement of tumor vessel diameter using the fluorescent dye H33342 (24). Rats were treated with Tivozanib (AV951/KRN-951) (0.2 or 1 mg/kg) or vehicle for 14 days (from day 0 to day 13) and were sacrificed 1 minute after the i.v. injection of H33342 (20 mg/kg) at day 13. The tumors were removed and 10 μm cryosections prepared from five levels of each tumor separated by at least 200 μm. Tumor sections were studied under UV illumination using a Nikon epifluorescence microscope to identify blood vessels with a surrounding halo of fluorescent H33342-labeled cells. The lumen enclosed by the halos was measured as the vessel diameter using Win ROOF software. Statistical analysis was done using the Mann-Whitney test.[1]

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Histologic analysis of smooth muscle actin–positive pericyte coverage of tumor vessels. Calu-6 tumor xenografts were established in athymic rats by s.c. implantation of cells. Rats were randomized when tumor volumes reached an average of 273 to 275 mm3 and then treated p.o. with Tivozanib (AV951/KRN-951) or vehicle for 2 weeks. Immunofluorescence staining of pericytes in tumors was done with Cy3-conjugated monoclonal anti-α-smooth muscle actin antibody following the staining of endothelial cells with anti-CD31 antibody. Tissue images were captured digitally at ×100 magnification with LSM 510 systems. Six fields per section (0.8489 mm2 each) were randomly analyzed, excluding peripheral surrounding connective tissues and central necrotic tissues. The number of CD31-positive objects and those surrounded by the region positive for α-smooth muscle actin were quantified using Win ROOF software after blind-coding the histology slides to avoid operator bias.[1]

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Pharmacokinetic analysis of Tivozanib (AV951/KRN-951). Athymic rats (F344/N JcL-rnu, four females per group) received Tivozanib (AV951/KRN-951) p.o. and blood samples were collected from their tail vein at predetermined intervals up to 72 hours postdose. An appropriate amount of internal standard material, KRN633, was added to each serum sample. Serum samples were deproteinated with acetonitrile and supernatants were analyzed by high-performance liquid chromatography–tandem mass spectrometry. Pharmacokinetic variables were calculated by noncompartmental analysis. The serum concentration of Tivozanib (AV951/KRN-951) at steady state after repeated p.o. administrations of a 0.2 mg/kg dose was simulated as described previously [1]

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Tivozanib (AV951/KRN-951) was suspended in vehicle (0.5% methyl cellulose in distilled water) and stored at 4°C. Fresh solutions were prepared weekly.[2]
Experimental design.  Rats inoculated with RCN‐9 cells were assigned randomly to three groups and given daily oral doses of Tivozanib (AV951/KRN-951) (1 or 3 mg/kg) or a 0.5% methylcellulose vehicle control. These treatments commenced at 4 or 14 days after tumor transplantation, and continued for 10 or 11 days, respectively. At the end of the treatment periods, the rats were killed and tumor progression was evaluated. Ascites were also collected and their volumes were measured. Each transparent window in the mesentery surrounded by fatty tissue was observed microscopically. The percentages of the mesenteric windows with a vasculature and the number of tumor nodules with or without a vasculature on the mesenteric windows were then counted. [2]
In a subsequent survival study, rats inoculated with RCN‐9 cells were assigned randomly to vehicle‐treated or 1 mg/kg Tivozanib (AV951/KRN-951)‐treated groups (n = 10 per group). Separate treatments then commenced from the day of tumor inoculation or at 14 days after this transplantation. The results were plotted using Kaplan–Meier methods and the differences in survival were analyzed by log‐rank test. A P‐value of <0.05 was considered statistically significant. [2]

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Tumor vessel imaging.  RCN‐9 cell‐inoculated rats with and without Tivozanib (AV951/KRN-951) treatment were anesthetized and injected intravenously with fluorescein isothiocyanate‐labeled dextran (molecular weight 200 000). After the animals had been killed, the mesenteries were fixed with 4% paraformaldehyde and placed on glass slides. The vasculature associated with each mesenteric window was then photographed microscopically. The number of vessel joints and paths (as vessel bifurcation characteristics), the areas and lengths of vessels (as the angiogenesis density), and the tortuosity of the vasculatures were recorded objectively and evaluated quantitatively using an angiogenesis image analyzer (Kurabo, Osaka, Japan). Four rats were used in these experiments from each group and 12–15 different fields from each animal were analyzed. [2]

Absorption, Distribution and Excretion
The median Tmax of tivozanib is 10 hours, however, can range from 3 to 24 hours. A pharmacokinetic study in 8 healthy subjects revealed a Cmax and AUC for radiolabeled tivozanib of 12.1 ± 5.67 ng/mL and 1084 ± 417.0 ng·h/mL, respectively. Steady-state tivozanib concentrations are achieved at concentrations 6-7 times higher the normal dose.
Tivozanib is primarily excreted in the feces. After oral ingestion of a radiolabeled 1.34 mg dose of tivozanib in healthy volunteers, 79% of the administered dose was found in the feces (with 26% unchanged) and 12% was found in the urine solely as metabolites.
Tivozanib has an apparent volume of distribution (V/F) of 123 L.
The apparent clearance (CL/F) of tivozanib is approximately 0.75 L/h.

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Metabolism / Metabolites
Tivozanib is primarily metabolized by CYP3A4. After oral ingestion of a radiolabeled 1.34 mg dose of tivozanib in healthy volunteers, unchanged tivozanib accounted for 90% of the radioactive drug detected in serum.

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Biological Half-Life
The half-life of tivozanib is about 111 hours according to prescribing information. Information from clinical studies reveals a half-life of 4-5 days.

Hepatotoxicity
In the published preregistration clinical trials of tivozanib, rates of serum ALT or AST elevations ranged from 10% to 29%, with 1% to 4% of treated patients having elevations above 5 times the upper limit of normal (ULN). Instances of clinically apparent liver injury including deaths with hepatic failure were reported in some clinical trials, but in all cases were attributed to hepatic metastases or to other underlying liver diseases. Since its approval and more widespread clinical use, there have been no reports of clinically apparent liver injury or hepatic failure attributed to tivozanib, but it has had limited clinical use.
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

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Protein Binding
In vitro, the protein binding of tivozanib is mainly bound to albumin at a rate of ≥ 99%.

[1]. Cancer Res. 2006 Sep 15;66(18):9134-42.

[2]. Cancer Sci. 2008 Mar;99(3):623-30.

1-[2-chloro-4-[(6,7-dimethoxy-4-quinolinyl)oxy]phenyl]-3-(5-methyl-3-isoxazolyl)urea is an aromatic ether.
Renal cell carcinoma (RCC) is responsible for 3% of cancer cases and is one of the 10 most common cancers in adults. The average age of diagnosis is between age 65 to 74. Tivozanib, also known as FOTIVDA, is a kinase inhibitor developed to treat adult patients with relapsed or refractory advanced renal cell carcinoma (RCC) after prior failed systemic therapies. It was approved on March 10, 2021 by the FDA. Marketed by Aveo Oncology, tivozanib is a promising therapy for individuals with RCC who have not been treated successfully with other therapies.
Tivozanib is a Kinase Inhibitor. The mechanism of action of tivozanib is as a Tyrosine Kinase Inhibitor.
Tivozanib is a small molecule multi-kinase inhibitor that is used in the treatment of relapsed or refractory renal cell carcinoma. Tivozanib is associated with transient and usually mild elevations in serum aminotransferase during therapy and but has not been linked instances of clinically apparent acute liver injury.
Tivozanib is an orally bioavailable inhibitor of vascular endothelial growth factor receptors (VEGFRs) 1, 2 and 3 with potential antiangiogenic and antineoplastic activities. Tivozanib binds to and inhibits VEGFRs 1, 2 and 3, which may result in the inhibition of endothelial cell migration and proliferation, inhibition of tumor angiogenesis and tumor cell death. VEGFR tyrosine kinases, frequently overexpressed by a variety of tumor cell types, play a key role in angiogenesis.
See also: Tivozanib Hydrochloride (has salt form); tivozanib hydrochloride anhydrous (is active moiety of).

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Drug Indication
Tivozanib is approved in the USA for the treatment of relapsed or refractory renal cell carcinoma in adult patients who have undergone two or more systemic therapies. In the UK and other countries, is indicated as first line therapy of adults with advanced renal cell carcinoma (RCC) and VEGFR and mTOR pathway inhibitor-naïve patients after disease progression following one previous treatment with cytokine therapy for advanced disease.
Fotivda is indicated for the first line treatment of adult patients with advanced renal cell carcinoma (RCC) and for adult patients who are VEGFR and mTOR pathway inhibitor-naïve following disease progression after one prior treatment with cytokine therapy for advanced RCC. , , Treatment of advanced renal cell carcinoma. ,

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Mechanism of Action
The VHL mutation-HIF upregulation-VEGF transcription is the main pathway implicated in the growth of renal cell carcinoma. Vascular endothelial growth factor receptors (VEGFR receptors) are important targets for tyrosine kinase inhibitors, which halt the growth of tumours. Tivozanib is a tyrosine kinase inhibitor that exerts its actions by inhibiting the phosphorylation of vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2 and VEGFR-3 and inhibits other kinases such as c-kit and platelet derived growth factor beta (PDGFR β). The above actions inhibit tumour growth and progression, treating renal cell carcinoma.

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Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis by stimulating the proangiogenic signaling of endothelial cells via activation of VEGF receptor (VEGFR) tyrosine kinases. Therefore, VEGFRs are an attractive therapeutic target for cancer treatment. In the present study, we show that a quinoline-urea derivative, KRN951, is a novel tyrosine kinase inhibitor for VEGFRs with antitumor angiogenesis and antigrowth activities. KRN951 potently inhibited VEGF-induced VEGFR-2 phosphorylation in endothelial cells at in vitro subnanomolar IC50 values (IC50 = 0.16 nmol/L). It also inhibited ligand-induced phosphorylation of platelet-derived growth factor receptor-beta (PDGFR-beta) and c-Kit (IC50 = 1.72 and 1.63 nmol/L, respectively). KRN951 blocked VEGF-dependent, but not VEGF-independent, activation of mitogen-activated protein kinases and proliferation of endothelial cells. In addition, it inhibited VEGF-mediated migration of human umbilical vein endothelial cells. Following p.o. administration to athymic rats, KRN951 decreased the microvessel density within tumor xenografts and attenuated VEGFR-2 phosphorylation levels in tumor endothelium. It also displayed antitumor activity against a wide variety of human tumor xenografts, including lung, breast, colon, ovarian, pancreas, and prostate cancer. Furthermore, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) analysis revealed that a significant reduction in tumor vascular hyperpermeability was closely associated with the antitumor activity of KRN951. These findings suggest that KRN951 is a highly potent, p.o. active antiangiogenesis and antitumor agent and that DCE-MRI would be useful in detecting early responses to KRN951 in a clinical setting. KRN951 is currently in phase I clinical development for the treatment of patients with advanced cancer.[1]

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We assessed the antitumor efficacy of KRN951, a novel tyrosine kinase inhibitor of vascular endothelial growth factor receptors, using a rat colon cancer RCN-9 syngeneic model in which the tumor cells are transplanted into the peritoneal cavity of F344 rats. KRN951 treatments that commenced 4 days after tumor transplantation (day 4) significantly inhibited tumor-induced angiogenesis, the formation of tumor nodules in the mesenteric windows, and the accumulation of malignant ascites. Moreover, KRN951 treatments initiated on day 14, by which time angiogenesis and malignant ascites have already been well established, resulted in the regression of newly formed tumor vasculatures with aberrant structures and also in the apparent loss of malignant ascites by the end of the study period. Quantitative analysis of the vessel architecture on mesenteric windows revealed that KRN951 not only regressed, but also normalized the tumor-induced neovasculature. Continuous daily treatments with KRN951 significantly prolonged the survival of rats bearing both early stage and more advanced-stage tumors, compared with the vehicle-treated animals. The results of our current study thus show that KRN951 inhibits colon carcinoma progression in the peritoneal cavity by blocking tumor angiogenesis, ascites formation, and tumor spread, thereby prolonging survival. Moreover, these studies clearly demonstrate the therapeutic effects of KRN951 against established tumors in the peritoneal cavity, including the regression and normalization of the tumor neovasculature. Our findings therefore suggest that KRN951 has significant potential as a future therapeutic agent in the treatment of peritoneal cancers with ascites.[2]

C22H21CLN4O6

472.878344297409

472.114

682745-40-0

Tivozanib;475108-18-0; 682745-40-0 (hydrate); 682745-41-1 (HCl hydrate)

71433801

White to off-white solid powder

3

8

6

33

631

0

O(C1C=CC(NC(=O)NC2=NOC(C)=C2)=C(Cl)C=1)C1=CC=NC2C=C(OC)C(OC)=CC1=2.O

VTWZGSZTIGEYIC-UHFFFAOYSA-N

InChI=1S/C22H19ClN4O5.H2O/c1-12-8-21(27-32-12)26-22(28)25-16-5-4-13(9-15(16)23)31-18-6-7-24-17-11-20(30-3)19(29-2)10-14(17)18;/h4-11H,1-3H3,(H2,25,26,27,28);1H2

1-[2-chloro-4-(6,7-dimethoxyquinolin-4-yl)oxyphenyl]-3-(5-methyl-1,2-oxazol-3-yl)urea;hydrate

Tivozanib (hydrate); 682745-40-0; 1-[2-chloro-4-(6,7-dimethoxyquinolin-4-yl)oxyphenyl]-3-(5-methyl-1,2-oxazol-3-yl)urea;hydrate; N-[2-chloro-4-[(6,7-dimethoxy-4-quinolinyl)oxy]phenyl]-N'-(5-methyl-3-isoxazolyl)-urea,monohydrate; Tivozanib (hydrate)?; CHEMBL4784300;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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