VEGFR1/FLT1 (IC50 = 0.1 nM); VEGFR2/Flk1 (IC50 = 0.18 nM); VEGFR2/KDR (IC50 = 0.2 nM); VEGFR3 (IC50 = 0.1 nM-0.3 nM nM); PDGFRβ (IC50 = 1.6 nM)

Axitinib may inhibit VEGF-mediated endothelial cell viability, tube formation, and downstream signaling in addition to cellular autophosphorylation of VEGFR. Variable cell lines with IC50 values of >10,000 nM (IGR-N91), 849 nM (IGR-NB8), 274 nM (SH-SY5Y), and 573 nM (non-VEGF stimulated HUVEC) are all inhibited by axitinib.[2]

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Axitinib potently inhibits cellular VEGF RTK activities in vitro. [2]
In transfected or endogenous RTK-expressing cells, axitinib potently blocked growth factor-stimulated phosphorylation of VEGFR-2 and VEGFR-3 with average IC50 values of 0.2 and 0.1 to 0.3 nmol/L, respectively (Fig. 2A; Table 1). Cellular activity against VEGFR-1 was 1.2 nmol/L (measured in the presence of 2.3% bovine serum albumin), equivalent to an absolute IC50 of ∼0.1 nmol/L, based on protein binding of axitinib. The potency against murine VEGFR-2 (Flk-1) in Flk-1-transfected NIH-3T3 cells was 0.18 nmol/L, similar to that of its human homologue. Axitinib showed ∼8- to 25-fold higher IC50 against the closely related type III and V family RTKs, including PDGFR-β (1.6 nmol/L), KIT (1.7 nmol/L), and PDGFR-α (5 nmol/L; Table 1); nanomolar concentrations of axitinib blocked PDGF BB-mediated human glioma U87MG cell (PDGFR-β-positive) migration but not proliferation (data not shown). In contrast, axitinib had much weaker target and functional activity against FGFR-1 (Fig. 1A and B; Table 1). With up to 1 μmol/L concentrations, axitinib showed minimal activity against Flt-3 in RS;411 cells and RET in TT cells (data not shown). A similar trend was observed for enzymatic inhibitory activity (Ki) against recombinant tyrosine kinases of the aforementioned receptors (data not shown). Importantly, axitinib had little inhibition against “off-target” protein kinases; at a concentration of 1 μmol/L (∼1,000-fold of the Ki for VEGFR-2) across three kinase panels of ∼100 protein kinases (Pfizer in-house; Upstate and Dundee panels), axitinib inhibited only five protein kinases: Abl, Aurora-2, Arg, AMPK, Axl, and MST2 (≥60% inhibition). Finally, axitinib exhibited no significant activity in a broad protein kinase screen (Cerep; data not shown).

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Axitinib inhibits VEGF-mediated endothelial cell survival, migration, and tube formation. [2]
Axitinib showed potent inhibition of VEGF-stimulated but not basic FGF-stimulated HUVEC survival with ∼1,000-fold selectivity for VEGFR-2 versus FGFR-1 receptors (Fig. 2B). The average IC50 value for VEGFR-2 derived from the functional assays (0.24 ± 0.09 nmol/L) was similar to that obtained in the cellular receptor phosphorylation assays (Table 1), confirming that receptor antagonism led to a functional inhibition by the compound. In addition, axitinib dose-dependently inhibited spheroidal endothelial tube formation in a three-dimensional fibrin matrix system (Fig. 2C; Supplementary Fig. S1). Higher compound concentrations than other types of assays were required to inhibit tube formation because of the presence of the higher serum level (4-8% FBS) in the system.

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Axitinib inhibits intracellular signal transduction in endothelial cells. [2]
Axitinib rapidly and dose-dependently reduced the phosphorylation of Akt, endothelial nitric oxide synthase (eNOS), and extracellular signal-regulated kinase 1/2 (ERK1/2), key downstream signaling molecules of VEGF (Fig. 2D), with IC50 values similar to that of the inhibition of VEGFR-2. This suggests that the reduction in Akt, eNOS, and ERK1/2 phosphorylation may be due to antagonism of upstream VEGFRs by axitinib.

Axitinib shows primary inhibition against orthotopically transplanted models of colon cancer (HCT-116), melanoma (M24met), and renal cell carcinoma (SN12C).[1] In IGR-N91 fenografts, axitinib reduces the Mean Vessels Density (MVD) to 21 from 49 in controls and delays the tumor growth by 11.4 days when compared to the controls (p.o. 30 mg/kg).[2] In the BT474 breast cancer model, axitinib at doses of 10-100 mg/kg dramatically suppresses growth and alters the tumor microvasculature.[3] In a variety of tumor types, such as melanoma, thyroid cancer, non-small cell lung cancer, and renal cell carcinoma, axitinib has demonstrated single-agent activity.

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The present studies were designed to determine the pathophysiologic consequences of both single and combined treatments using fractionated radiotherapy plus Axitinib/AG-013736, a receptor tyrosine kinase inhibitor that preferentially inhibits vascular endothelial growth factor receptors. DU145 human prostate xenograft tumors were treated with (a) vehicle alone, (b) Axitinib/AG-013736, (c) 5x2 Gy/wk radiotherapy fractions, or (d) the combination. Automated image processing of immunohistochemical images was used to determine total and perfused blood vessel spacing, overall hypoxia, pericyte/collagen coverage, proliferation, and apoptosis. Combination therapy produced an increased tumor response compared with either monotherapy alone. Vascular density progressively declined in concert with slightly increased alpha-smooth muscle actin-positive pericyte coverage and increased overall tumor hypoxia (compared with controls). Although functional vessel endothelial apoptosis was selectively increased, reductions in total and perfused vessels were generally proportionate, suggesting that functional vasculature was not specifically targeted by combination therapy. These results argue against either an AG-013736- or a combination treatment-induced functional normalization of the tumor vasculature. Vascular ablation was mirrored by the increased appearance of dissociated pericytes and empty type IV collagen sleeves. Despite the progressive decrease in tumor oxygenation over 3 weeks of treatment, combination therapy remained effective and tumor progression was minimal. [1]

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Target modulation in vivo by Axitinib. [2]
Acute axitinib treatment rapidly and significantly reduced retinal vascular VEGFR-2 phosphorylation. One hour after the second dose, retinal VEGFR-2 phosphorylation was reduced by 80% to 90% compared with that of the control tissues (Fig. 3A, left). Six and 24 to 32 h post-dose, the phospho-VEGFR-2 levels returned to ∼50% and 100% of the control, respectively. Levels of phospho-VEGFR-2 inversely correlated with axitinib plasma concentrations over the study time course. The EC50 value for the inhibition of VEGFR-2 phosphorylation was 0.49 nmol/L (or 0.19 ng/mL, the unbound value corrected for plasma protein binding; Fig. 3A, right). [2]

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Axitinib also inhibited murine VEGFR-2 phosphorylation in angiogenic vessels of xenograft tumors of the M24met; M24met tumors secrete high VEGF-A, are highly vascularized, and do not express functional human VEGFR-2. A single oral dose of axitinib (100 mg/kg) markedly suppressed murine VEGFR-2 phosphorylation for up to 7 h compared with control tumors (Fig. 3B). Phosphorylation of downstream ERK1/2 was also measured from the same samples. Compared with the control, partial inhibition of ERK1/2 signal was observed in treated tissues as early as 30 min post-dose and remained inhibited for at least 7 h (Fig. 3C). [2]

Axitinib rapidly inhibited VEGF-induced vascular permeability in the skin of mice; the inhibition was dose-dependent and directly correlated with drug concentration in mice (Fig. 3D). Pharmacokinetic/pharmacodynamic analysis indicated an unbound EC50 of 0.46 nmol/L (Supplementary Fig. S2). Similar inhibitory effects were also shown in the skin of MV522 tumor-bearing mice without exogenous VEGF-A stimulation (data not shown). [2]

Taken together, the required in vivo pharmacologic concentration (Ctarget) based on the inhibition of vascular VEGFR-2 phosphorylation and VEGF-mediated permeability is ∼0.5 nmol/L (unbound), which translates to a Ctarget of ∼100 nmol/L (or 40 ng/mL, total concentration) in humans.

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Axitinib inhibits tumor growth and angiogenesis in mice. [2]
Axitinib inhibited the growth of human xenograft tumors in mice (Table 2). Axitinib produced dose-dependent growth delay regardless of initial tumor size, model type, or implant site. Importantly, axitinib exhibited primary tumor inhibition and distant metastasis control in orthotopically implanted tumors such as M24met (melanoma), HCT-116 (colorectal cancer), and SN12C (renal cell carcinoma). A dose-dependent growth inhibition in the MV522 tumor model is shown (Fig. 4A). Tumor growth inhibition (TGI) was associated with central necrosis, reduction in microvessel density (CD31 staining) and Ki-67, and increased caspase-3 in the tumor (Fig. 4B; Supplementary Fig. S3). Similar effects were observed in all tumor types examined regardless of tumor type and RTK expression. In summary, axitinib treatment produced consistent antitumor efficacy across various tumor types and this activity is associated with reduction in vascular angiogenesis and tumor proliferation and increase in tumor apoptosis.

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Determination of ED50 and Ceff. [2]
The efficacious dose resulting in 50% antitumor efficacy (ED50) was determined using the MV522 model. MV522 tumor cells do not express functional VEGF or PDGF RTKs. In addition, the tumors have a moderate growth rate, making it an ideal model to evaluate the antiangiogenesis-associated ED50 of axitinib. Based on the relationship between dose and the corresponding TGI (Fig. 4A), the ED50 was determined to be 8.8 mg/kg twice daily (Supplementary Fig. S4) and a 30 mg/kg twice daily dose level corresponded to an ED70 in this model. The range of in vivo efficacious concentration (Ceff) corresponding to a 50% TGI was determined by evaluating the relationship between TGI (Fig. 4A) and plasma concentrations. Based on Cmin (trough concentration), the estimated unbound Ceff was determined to be 0.28 nmol/L (or 0.11 ng/mL; Fig. 4C, left); based on Cave (average concentration across 24 h), the calculated unbound Ceff was determined to be 0.85 nmol/L (0.33 ng/mL; Fig. 4C, right). Thus, the Ceff value range (0.28-0.85 nmol/L) is in agreement with the Ctarget value (0.5 nmol/L) obtained from in vivo target modulation studies. [2]

The relationship between dose and target inhibition was further analyzed based on pharmacokinetic profiles, IC50 values, Ctarget, and Ceff. Plasma concentrations at 10 mg/kg (ED50 dose) and 30 mg/kg (ED70 dose) were both above and near Ctarget (for VEGFRs) and Ceff (TGI-based) during the majority of the day (Fig. 4D). However, the plasma concentrations at these doses only allowed a total of ∼5 and 12 h coverage over the cellular IC50 of PDGFR-β, respectively (anti-PDGFR-based Ctarget or Ceff from in vivo studies is not available). Based on this analysis, the antitumor efficacy at 10 mg/kg in the MV522 model (VEGFR-null, PDGFR-null) appeared to be mainly driven from vascular VEGFR inhibition by Axitinib. In the same model, infusions of axitinib achieved a near maximal antitumor efficacy (80%) that was associated with a steady-state plasma concentration greater than the cellular IC50 for VEGFRs but below the cellular IC50 for PDGFR-β (data not shown).

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Axitinib enhances antitumor efficacy of chemotherapeutic agents in multiple tumor models. [2]
The antitumor efficacy of axitinib was assessed in combination with docetaxel (in LLC and human breast cancer models), carboplatin (in a human ovarian cancer model), or gemcitabine (in a human pancreatic cancer model). These models were chosen because they have only low or moderate sensitivity to chemotherapies in mice. [2]

In the LLC model, Axitinib (10 or 30 mg/kg orally twice daily) in combination with a maximally tolerated dose of docetaxel (40 mg/kg once a week) enhanced tumor growth delay, defined as the increase in the median time to the end point (TTE) in a treatment group compared with the control group. A TTE (a measure of disease progression) is defined as tumor size = 1,500 mm3 or animal moribund due to tumor burden or metastasis. A 54% or 100% tumor growth delay was obtained for docetaxel plus 10 or 30 mg/kg axitinib versus a 9%, 30%, and 60% for docetaxel alone and 30 and 60 mg/kg axitinib alone, respectively (data not shown). Docetaxel plus axitinib significantly delayed disease progression compared with docetaxel alone (Fig. 5A). In the MDA-MB-435/HAL-luc model, axitinib (30 mg/kg) and docetaxel (5 mg/kg; 25% of murine maximally tolerated dose) produced a robust tumor growth delay as shown by the reduction of tumor bioluminescent signal (Supplementary Fig. S5) and increase in the number of complete responders compared with either single agent alone (data not shown). [2]

The antitumor efficacy of Axitinib in combination with gemcitabine was investigated against various dosing schedules in the gemcitabine-resistant BxPC-3 human pancreatic cancer model (Fig. 5B). In one study, single-agent gemcitabine (140 mg/kg i.p., days 1, 4, 7, and 10, either one-cycle or three-cycle treatments) or axitinib (30 mg/kg orally twice daily) delayed tumor growth. In the groups receiving gemcitabine plus axitinib, the “early dosing” of axitinib (day 1, group 5) produced a greater tumor growth delay than “late dosing” [starting axitinib on day 11 (group 6) or 16 (group 7) after initiation of gemcitabine] regardless of the number of gemcitabine cycles; with the same axitinib regimen, three gemcitabine treatment cycles (group 8, 9, 10) produced a greater efficacy than one gemcitabine treatment cycle (group 5, 6, 7). Alternating dose of the two agents (group 12) or early termination of axitinib (group 11) resulted in a significant compromise in tumor growth delay compared with coadministration and continuous twice daily dosing of axitinib.

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Combination of Axitinib and bevacizumab produced significant antimetastasis activity in M24met model. [2]
The ability of axitinib to enhance bevacizumab efficacy in the orthotopically implanted and spontaneous metastasis human melanoma M24met tumor model was investigated; M24met tumors do not express functional RTKs (data not shown). Most importantly, circulating human VEGF-A, the ligand for bevacizumab, was found to be >95% of total circulating VEGF-A in vivo in this model. [2]

In this study, both Axitinib (60 mg/kg orally twice daily) and bevacizumab (5 mg/kg i.v., 2xqw) exhibited moderate single-agent activity against lymph node tumor metastasis. The combination of the two agents significantly improved antimetastasis efficacy assessed based on reduction of lymph node tumor mass (Fig. 5C), antiangiogenesis (Supplementary Fig. S6), and proliferation index of metastatic lymph node tumors (Supplementary Fig. S7). In addition, the combination therapy significantly prolonged animal survival measured by reduction of time to progression (TTE), with a 13-day TTE for both single agents versus the control and a 20-day TTE for combination therapy versus the control (Fig. 5D). As expected, dosing with bevacizumab or bevacizumab plus axitinib, but not axitinib single-agent treatment, significantly reduced free plasma human VEGF-A (data not shown).

Generated are porcine aorta endothelial (PAE) cells that overexpress full-length VEGFR2, PDGFRβ, Kit, and NIH-3T3 cells that overexpress murine VEGFR2 (Flk-1) or PDGFRα. To prepare ELISA capture plates, 100 μL/well of 2.5 μg/mL anti-VEGFR2 antibody, 0.75 μg/mL anti-PDGFRβ antibody, 0.25 μg/mL anti-PDGFRα antibody, 0.5 μg/mL anti-KIT antibody, or 1.20 μg/mL anti-Flk-1 antibody are coated on the 96-well plates. Next, an ELISA is used to measure RTK phosphorylation [2].

A 96-well plate is seeded with 5 × 10⁴ cells, and the cells are cultured for a full day. Concentrations of axitinib ranging from 1 nM to 10 μM are added to the cells. MTS tetrazolium substrate is used to measure cell viability after 72 hours, and IC50 values are computed.

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Three-dimensional spheroidal tube formation assay [2]
Five hundred human microvascular endothelial cells were added to EGM-2 medium containing 0.24% methylcellulose and transferred to U-bottomed 96-well plates to form a spheroid overnight. Approximately 50 spheroids were collected and mixed with 2 mg/mL fibrinogen solution containing 4% to 8% fetal bovine serum (FBS) with or without compound in the 48-well plates coated with thrombin (5,000 units/mL). The resulting three-dimensional fibrin gel was covered with EGM-2 containing 4% to 8% FBS and incubated at 37°C. Endothelial tube formation was observed daily under an inverted microscope.

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Immunoprecipitation and immunoblotting [2]
Endothelial or tumor cells were starved for 18 h in the presence of either 1% FBS (HUVEC) or 0.1% FBS (tumor cells). Axitinib was added and cells were incubated for 45 min at 37°C in the presence of 1 mmol/L Na3VO4. The appropriate growth factor was added to the cells, and after 5 min, cells were rinsed with cold PBS and lysed in the lysis buffer and a protease inhibitor cocktail. The lysates were incubated with immunoprecipitation antibodies for the intended proteins overnight at 4°C. Antibody complexes were conjugated to protein A beads and supernatants were separated by SDS-PAGE. The Super Signal West Dura kit was used to detect the chemiluminescent signal.

Mice and Rats: Mice bearing 400–600 mm³ M24met xenograft tumors receive either a single Axitinib dose or 0.5% carboxymethylcellulose/H₂O as a control. Samples of blood and tumor tissue are obtained for VEGFR-2 and pharmacokinetic analyses. The Bradford colorimetric assay is used to measure the total protein concentrations in tumor tissues.
Axitinib (30 mg/kg) is injected intraperitoneally twice into six-day-old Sprague-Dawley rats. Retinal tissue is extracted and lysed, animals are sacrificed, and immunoprecipitation and immunoblotting experiments are carried out. The Alpha Imager 8800 is used for densitometry analysis, and ECL-Plus is used for detection.

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In vivo target modulation [2]
VEGFR-2 phosphorylation inhibition in the rat development model. Six-day-old Sprague-Dawley rats were given two i.p. injections of Axitinib. Animals were sacrificed, retinas were collected and lysed, and immunoprecipitation/immunoblotting experiments were done as described above. ECL-Plus was used for detection and densitometry analysis was done using the Alpha Imager 8800.
VEGFR-2 phosphorylation inhibition in xenograft tumors. Mice with M24met xenograft tumors (400-600 mm3) were administered with a single dose of Axitinib or the control (0.5% carboxymethylcellulose/H2O). Blood and tumor tissue samples were collected for pharmacokinetic and VEGFR-2 measurements. Total protein concentrations in tumor tissues were determined using the Bradford colorimetric assay. Procedures for immunoprecipitation/immunoblotting and ELISA were as described above.

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Axitinib pharmacokinetics [2]
Plasma concentrations of Axitinib were quantitatively determined by a triple-quadruple mass spectrometer equipped with a high-performance liquid chromatography system (Agilent 1100) using a Phenyl column (Zorbax Eclipse XDB, 5 μm particle size, 50 × 2.1 mm) under isocratic conditions of 60:40 water/acetonitrile containing 0.1% formic acid. Data were collected under multiple reaction monitoring mode of m/z 387.3→356.2 for axitinib and m/z 394.2→360.2 for the internal standard, Axitinib/AG-013736-d7. The method quantified for axitinib over the range of 1 to 1,000 ng/mL in mouse plasma. The area under the plasma concentration-time curve of axitinib was calculated using the linear trapezoidal rule.

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Skin vascular permeability assay in naive or tumor-bearing mice [2]
The assay was done according to Miles and Miles with some modifications. nu/nu mice (n = 5-8) received a single oral dose of Axitinib followed by an injection of 30 μL Evan's blue dye through the tail vein. Thirty minutes later, murine VEGF-A (400 ng in 10 μL PBS) or PBS was injected into the trunk area posterior to the shoulder of the animal. Four hours later, the skin region immediately surrounding the blue color area was dissected and immersed in 1 mL formamide. Evan's blue was extracted by incubating the tissues in formamide at 56°C for 24 h. Vascular permeability was quantified by measuring light absorbance at 620 nm.

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Mouse xenograft models [2]
In general, tumor cells in FBS-depleted medium were implanted s.c. into the right flank region of athymic mice, except for the following: the M24met cells were implanted intradermally in a 50 to 100 μL volume in BALB/C severe combined immunodeficient mice; the procedures for orthotopic implantation of HCT-116-GFP and SN12C-GFP tumors have been described elsewhere; the A375 cells were implanted in the presence of 10% Matrigel; the LLC tumors were inoculated either using the suspension cells or 2 × 2 mm viable tumor fragments via the Trocar needles. Unless otherwise specified, mice were randomized when the average tumor was ∼100 mm3 (9-12 per group). Tumor volumes were measured three times per week by electronic calipers and calculated according to the following equation: 0.5 × [length × (width)2]. Treatment usually lasted for 2 to 4 weeks or until tumors reached 1,500 mm3. The procedure for tumor bioluminescent imaging and quantification using the IVIS Imaging System has been reported elsewhere.

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Treatments. Axitinib/AG-013736, a receptor kinase inhibitor of VEGFRs and, at higher doses, PDGFRs (IC50 = 0.1 nmol/L for VEGFR-1, 0.2 nmol/L for VEGFR-2, 0.1–0.3 nmol/L for VEGFR-3, and 1.6 nmol/L for PDGFRβ; ref. 18), was provided by Pfizer Global Research and given once daily by gavage in a volume of 0.13 mL. Control animals received 0.5% carboxymethylcellulose drug carrier. Irradiations were done on nonanesthetized mice using a 137Cs source operating at 2.4 Gy/min. Mice were confined to plastic jigs with tumor-bearing legs extended through an opening in the side, allowing local irradiations. Fractionated doses were given in five daily 2 Gy fractions per week (omitting weekends). For combination treatments, radiotherapy was delivered first, and Axitinib/AG-013736 was given within ∼4 h. Mice were sacrificed, and tumors were excised and then quick frozen (using liquid nitrogen) following 1, 2, or 3 weeks of treatment. [1]

Absorption, Distribution and Excretion
Following administration of 5 mg axitinib, maximum plasma concentrations are reached in approximately 2.5 to 4.1 hours. Axitinib is primarily excreted unchanged in feces (41%), with 12% being unchanged axitinib. An additional 23% is excreted in urine, mostly as metabolites. The volume of distribution is 160 liters. The mean clearance of axitinib is 38 liters per hour. Metabolism/Metabolites Axitinib is primarily metabolized in the liver. CYP3A4 and CYP3A5 are the major hepatic enzymes, while CYP1A2, CYP2C19, and UGT1A1 are minor enzymes. Biological Half-Life The half-life of axitinib is 2.5 to 6.1 hours.

Hepatotoxicity
Elevated serum transaminase levels were common in large clinical trials of axitinib, with an incidence rate as high as 25%. However, values exceeding 5 times the upper limit of normal (ULN) were uncommon, with an incidence rate of only 1% to 2%. Furthermore, no clinically visible liver injury caused by axitinib has been reported in premarket studies or larger-scale use after approval. Nevertheless, regular monitoring of liver function is recommended during axitinib treatment. Probability score: E (Not yet confirmed, but suspected as a cause of clinically visible liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information on the use of axitinib during lactation. Because axitinib binds to plasma proteins at a rate exceeding 99%, its concentration in breast milk is likely to be low. The manufacturer recommends discontinuing breastfeeding during axitinib treatment and for 2 weeks after the last dose. When axitinib is used in combination with avelumab or pembrolizumab, please refer to the relevant records in LactMed.
◉ 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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Protein binding
Axitinib has a high plasma protein binding rate, exceeding 99%, primarily binding to albumin, followed by α1-acid glycoprotein.

[1]. The addition of AG-013736 to rractionated radiation improves tumor response without functionally normalizing the tumor vasculature. Cancer Res. 2007 Oct 15;67(20):9921-8.

[2]. Nonclinical antiangiogenesis and antitumor activities of axitinib (AG-013736), an oral, potent, and selective inhibitor of vascular endothelial growth factor receptor tyrosine kinases 1, 2, 3. Clin Cancer Res. 2008 Nov 15;14(22):7272-83.

[3]. Metabolic Symbiosis Enables Adaptive Resistance to Anti-angiogenic Therapy that Is Dependent on mTOR Signaling. Cell Rep. 2016 May 10;15(6):1144-60.

Axitinib is an indazole compound with a 2-(pyridin-2-yl)vinyl substituted at the 3-position and a 2-(N-methylaminocarboxyl)phenylthioyl substituted at the 6-position. It is used to treat advanced renal cell carcinoma after failure of first-line systemic therapy. It possesses antitumor activity and is a tyrosine kinase inhibitor and vascular endothelial growth factor receptor antagonist. It belongs to the indazole, pyridine, aryl thioether, and benzamide classes of compounds. Axitinib is a second-generation tyrosine kinase inhibitor whose mechanism of action is through selective inhibition of vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, VEGFR-3). Through this mechanism, axitinib can block angiogenesis, tumor growth, and metastasis. It has been reported that axitinib is 50-450 times more potent than first-generation VEGFR inhibitors. Axitinib is an indazole derivative, most commonly marketed under the name Inlyta®, and is available in oral formulations. Axitinib is a kinase inhibitor. Its mechanism of action is as a receptor tyrosine kinase inhibitor. Axitinib is an oral tyrosine kinase inhibitor that selectively targets vascular endothelial growth factor (VEGF) receptors -1, -2, and -3 for the treatment of advanced renal cell carcinoma. Axitinib treatment typically causes a transient increase in serum transaminases, but this is usually mild and asymptomatic. No clinically significant cases of acute liver injury have been found with axitinib. Axitinib is an orally bioavailable tyrosine kinase inhibitor. It exerts its anti-angiogenic effect by inhibiting the pro-angiogenic cytokines vascular endothelial growth factor (VEGF) and platelet-derived growth factor receptor (PDGF). It is a benzamide and indazole derivative that acts as a tyrosine kinase inhibitor of VEGF receptors. It is used to treat advanced renal cell carcinoma. Drug Indications: For the treatment of renal cell carcinoma, and its use/treatment in pancreatic and thyroid cancer is under investigation. FDA Label: Inlyta is indicated for the treatment of adult patients with advanced renal cell carcinoma (RCC) who have failed prior therapy with sunitinib or cytokines. Mechanism of Action Axitinib selectively blocks tyrosine kinase receptors VEGFR-1 (vascular endothelial growth factor receptor), VEGFR-2, and VEGFR-3. Pharmacodynamics Axitinib inhibits cancer progression by suppressing angiogenesis and blocking tumor growth. In summary, current findings indicate that combination therapy does not induce normalization of tumor angiogenesis. In this tumor model, AG-013736 and its combination therapy did not tighten pericytes but rather loosened the connections between pericytes and blood vessels, as well as between pericytes and the basement membrane. Treatment significantly reduced total vascular density and functional vascular density, but contrary to the normalization hypothesis, overall tumor hypoxia gradually worsened. Despite reduced oxygenation, tumor progression was minimal during the 3-week combination therapy, likely due to persistent vascular destruction and inhibition of new blood vessel growth. Further research is needed to generalize these measurements to other tumor models and determine whether other dosing regimens can enhance the treatment response. [1]

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Objective: Axitinib (AG-013736) is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases 1 through 3, currently in clinical development for the treatment of solid tumors. This article comprehensively describes the in vitro properties and activities, in vivo anti-angiogenic effects, antitumor efficacy, and translational pharmacology data of axitinib. Experimental Design: The potency, kinase selectivity, pharmacological activity, and antitumor efficacy of axitinib were evaluated in a variety of non-clinical models. Results: Axitinib inhibited cellular autophosphorylation of the VEGF receptor (VEGFR) with a picomolar IC50 value. Screening of multiple kinases and proteomes showed that axitinib was selective for VEGFR. Axitinib blocked VEGF-mediated endothelial cell survival, tubular formation, and downstream signal transduction of kinases mediated by endothelial nitric oxide synthase, Akt, and extracellular signal-regulated kinases. Following twice-daily oral administration, axitinib produces sustained and dose-dependent antitumor efficacy, which is associated with blocking VEGFR-2 phosphorylation, vascular permeability, angiogenesis, and simultaneous induction of tumor cell apoptosis. Compared with monotherapy, axitinib combined with chemotherapy drugs or targeted therapies enhances antitumor efficacy in various tumor models. Dosage regimen studies in a human pancreatic tumor xenograft model showed that co-administration of axitinib with gemcitabine, without interruption of axitinib administration or shortening of the axitinib administration time, yielded the best antitumor efficacy. The effective drug concentrations predicted in non-clinical studies were consistent with the actual clinically achieved concentration range. Although axitinib inhibits platelet-derived growth factor receptor and KIT with nanomolar potency in vitro, based on pharmacokinetic/pharmacodynamic analysis, at current clinical exposure levels, axitinib primarily functions as a VEGFR tyrosine kinase inhibitor. Conclusion: Axitinib's selectivity and potency against VEGFR, as well as its potent non-clinical activity, make it a promising candidate for improving cancer treatment. [2]

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Targeting tumor angiogenesis with VEGF inhibitors has shown significant efficacy in some human tumor and mouse cancer models, but the efficacy is short-lived and limited by unconventional adaptive/escape resistance mechanisms. In one mouse model, potent angiogenesis inhibitors induced compartmentalization of cancer cells around residual blood vessels. Glucose and lactate transporters GLUT1 and MCT4 were induced in a HIF1α-dependent manner in distal hypoxic cells, indicating the initiation of the glycolytic pathway. Tumor cells near blood vessels expressed lactate transporters MCT1 and p-S6, the latter reflecting the mTOR signaling pathway. Normally oxygenated cancer cells take up and metabolize lactate, enhancing glutamine metabolism through lactate catabolism, thereby upregulating the mTOR signaling pathway. Thus, a metabolic symbiosis is established when angiogenesis is inhibited: hypoxic cancer cells take up glucose and export lactate, while normally oxygenated cancer cells take up and break down lactate. Inhibition of mTOR signaling disrupts this metabolic symbiosis and leads to upregulation of glucose transporter GLUT2. [3]

C22H18N4OS

386.47

386.12

C, 68.37; H, 4.69; N, 14.50; O, 4.14; S, 8.30

319460-85-0

Axitinib-13C,d3;1261432-00-1;Axitinib-d3;1126623-89-9

6450551

white to off-white solid powder

1.4±0.1 g/cm3

668.9±55.0 °C at 760 mmHg

213-215ºC

358.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.728

4.15

2

4

5

28

557

0

O=C(C1=C(SC2=CC3=C(C(/C=C/C4=CC=CC=N4)=NN3)C=C2)C=CC=C1)NC

RITAVMQDGBJQJZ-FMIVXFBMSA-N

InChI=1S/C22H18N4OS/c1-23-22(27)18-7-2-3-8-21(18)28-16-10-11-17-19(25-26-20(17)14-16)12-9-15-6-4-5-13-24-15/h2-14H,1H3,(H,23,27)(H,25,26)/b12-9+

N-methyl-2-[[3-[(E)-2-pyridin-2-ylethenyl]-1H-indazol-6-yl]sulfanyl]benzamide

AG 013736; Axitinib; 319460-85-0; AG-13,736; axitinibum; C9LVQ0YUXG; UNII-C9LVQ0YUXG; NSC-757441; N-methyl-2-((3-((1E)-2-(pyridin-2-yl)ethenyl)-1H-indazol-6-yl)sulfanyl)benzamide; AG013736; Axitinib; AG-013736; Brand name: Inlyta

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: ≥ 2.08 mg/mL (5.38 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 (5.38 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (5.38 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% CMC: 30mg/mL

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Solubility in Formulation 5: 8.33 mg/mL (21.55 mM) in 20% HP-β-CD/10 mM citrate pH 2.0 (add these co-solvents sequentially from left to right, and one by one), clear solution; Need ultrasonic and adjust pH to 3 with H2O.

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