Src (IC50 = 2.7 nM); v-Abl (IC50 = 30 nM); EGFR (IC50 = 66 nM); c-Kit (IC50 = 200 nM)

An oral Src inhibitor called Saracatinib (AZD0530) has been shown to have strong antimigratory and anti-invasive properties in vitro. It also prevents bladder cancer metastases in a mouse model. Saracatinib's antiproliferative efficacy varies between cell lines (IC50 0.2-10 μM). Saracatinib exhibits varying antiproliferative efficacy in a variety of human cancer cell lines expressing endogenous Src and potently inhibits the proliferation of Src3T3 murine fibroblasts. IC50 values of 0.2-0.7 μM are obtained for five human cancer cell lines examined with Saracatinib, representing the tumor types colon, prostate, lung, and leukemia, indicating submicromolar growth suppression. Saracatinib suppresses the Bcr-Abl-driven human leukemia cell line K562's proliferation in 3-day MTS cell proliferation tests, with an IC50 of 0.22 μM. Saracatinib decreases human lung cancer A549 cells' movement in the microdroplet migration assay in a concentration-dependent manner (IC50 0.14 μM)[1].

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Inhibition of isolated protein kinase activity [1]
Saracatinib (AZD0530) potently inhibited Src and the other Src tyrosine kinase family members investigated (c‐Yes, Fyn, Lyn, Blk, Fgr, and Lck), with high selectivity observed against a panel of other protein kinases involved in signal transduction (Table 1), including Csk, the intracellular negative regulator of Src activation. The only other notable activities observed were versus Abl (in common with other ATP‐competitive Src inhibitors (Golas et al., 2003; Lombardo et al., 2004)) and versus activating mutant forms of the EGFR (L858R and L861Q).

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Inhibition of cell proliferation [1]
Saracatinib (AZD0530) potently inhibited the in vitro proliferation of Src3T3 mouse fibroblasts and demonstrated variable antiproliferative activity in a range of human cancer cell lines containing endogenous Src (Table 2). Sub micromolar growth inhibition of five of the human cancer cell lines tested with AZD0530 (tumor types: colon, prostate, lung, and leukemia) was observed with IC50 values of 0.2–0.7μM. In 3‐day MTS cell proliferation assays (Promega G3580), AZD0530 inhibited in vitro proliferation of the Bcr–Abl‐driven human leukemia cell line K562 with an IC50 of 0.22μM.

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Inhibition of cell migration [1]
In the microdroplet migration assay, Saracatinib (AZD0530) reduced the migration of human lung cancer A549 cells in a concentration‐dependent manner (IC50 0.14μM; Figure 1A). AZD0530 at concentrations of 0.01–0.5μM demonstrated a clear dose‐dependent antimigratory effect compared with untreated controls in monolayer scratch assays of human breast cancer MDA‐MB‐231 cells (Figure 1B). AZD0530 also dose dependently inhibited the EGF‐ and collagen‐stimulated migration of NBT‐II bladder cancer cells (Figure 1C). Moreover, at a single concentration of 0.25μM, AZD0530 consistently inhibited migration in monolayer scratch assays of bladder cancer cell lines (31%, 37%, and 78% inhibition versus untreated controls in T24, SCaBER, and 1A6 cell lines, respectively).

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Inhibition of cell invasion [1]
Saracatinib (AZD0530) treatment significantly impaired the invasion of HT1080 cells through a 3‐dimensional (3D) collagen matrix (Figure 1D). In contrast to other 3D invasion assays, our assay (see Methods) monitors the proportion of total cells within a 3D matrix invading beyond a specified depth (60μm), thereby distinguishing repressed cellular invasion from cell loss that may be due to impaired adhesion, proliferation, or excessive cell death. This method confirms that AZD0530 exposure produces a significant and dose‐dependent inhibition of the invasive properties of HT1080 cells.

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Inhibition of cell scattering [1]
Saracatinib (AZD0530) 1μM completely inhibited EGF‐induced cell scattering in NBT‐II bladder cancer cells, restoring cell–cell adhesion and the localization of desmoplakin to desmosomal junctions (Figure 2A).

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Inhibition of paxillin phosphorylation [1]
Saracatinib (AZD0530) dose dependently inhibited phosphorylation of the Src substrate paxillin in NBT‐II bladder cancer cells (Figure 2B). Moreover, AZD0530 treatment (1μM) induced a relocalization of paxillin from the cell membrane into the cell cytoplasm (Figure 2C) further supporting a role for Src in the early development of cell migratory behavior.

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Effects on the EGFR [1]
Given the moderate inhibitory effect observed for Saracatinib (AZD0530) against the EGFR in the isolated kinase assay (IC50 66nM; Table 1), further studies were performed to investigate AZD0530 effects on the EGFR. In a head‐to‐head assay measuring EGFR phosphorylation in KB cells, AZD0530 exhibited an IC50 of 1.25μM compared with 11nM for gefitinib, an EGFR tyrosine kinase inhibitor. In isolated kinase assays, AZD0530 exhibited IC50s of 5 and 4nM respectively, for EGFR point‐mutant isoforms L858R (Millipore 14‐626) and L861Q (Millipore 14‐627). Effects on the EGFR L858R isoform were further investigated in PC‐9 cells, a non‐small‐cell lung cancer (NSCLC) cell line harboring this EGFR mutation. AZD0530 inhibited growth of PC‐9 cells with an IC50 of 0.23μM in 3‐day MTS cell proliferation assays (Promega G3580).

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Enzyme kinetics [1]
Pre‐incubation of Src kinase with 100nM Saracatinib (AZD0530) (in the presence of 160μM ATP) resulted in >95% inhibition of Src activity. A subsequent 10‐fold dilution to 10nM AZD0530 in the presence of a high concentration of ATP (1.60mM) led to a regain of Src activity, with only 30% inhibition, suggesting that inhibition was reversible. A similar degree of inhibition was observed for the same final concentrations of AZD0530 and ATP without the dilution step. The reversibility studies implied that Saracatinib (AZD0530) was competitive with ATP, as has been reported for other anilinoquinazolines (Bridges, 2001). ATP‐competitive inhibition was confirmed in experiments where both the concentration of ATP and that of AZD0530 were varied at fixed concentrations of the substrate, Src II peptide. The estimated inhibition constant, K is was 1.5 and the 95% confidence interval (CI) was 1.2, 1.9nM. This is lower than the IC50 of 2.7nM reported in Table 1 because it relates to a situation in the absence of competing ATP. AZD0530 was shown to follow pure noncompetitive kinetics when the concentration of Src II peptide was varied at 250μM ATP, with an apparent K i′ of 10nM (95% CI 8.4, 12.0; Figure 3A and B), which corresponds to a K i of 1.1nM (95% CI 0.9, 1.3) in the absence of ATP. Many kinases have structural differences between their active and inactive forms (Huse and Kuriyan, 2002). Inactivated Src adopts a closed conformation and AZD0530 binds approximately 10‐fold more weakly to inactive Src (K d 11nM; 95% CI 9.3, 14).

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Dual c-Met and Src inhibition is more effective than either inhibitor alone [2]
MD-MSCs treated with saracatinib (0.5 μmol/L) together with increasing cabozantinib concentrations had a three-fold decrease in the IG50 of cabozantinib (0.6 μmol/L, compared with 2.2 μmol/L for cabozantinib alone), and more than five-fold selectivity over WT-MSCs (IG50 = 3.4 μmol/L; Fig. 4A). Treatment of MD-MSCs with Saracatinib (AZD0530) (0.5 μmol/L) and increasing cabozantinib concentration resulted in decreased phosphorylation levels of c-Met(Y1234/1235), Erk1/2, and Akt(T308) after 6 hours (Fig. 4B). After 24 hours of the combination treatment, MD-MSCs had decreased cyclin D1 levels and increased p27 levels at lower cabozantinib concentrations than those treated with cabozantinib alone (Fig. 4C).

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Dual c-Met and Src inhibition reduces viability of primary VS cells [2]
We investigated the efficacy of this drug combination in decreasing viability of primary VS cells obtained from two patients. VS01 was a sporadic VS with a heterozygous duplication of NF2 exon 5 and a homozygous duplication of exon 7. VS02 was from an NF2 patient with an exon 14 deletion. VS cells were cultured with cabozantinib (2 μmol/L) and Saracatinib (AZD0530) (2 μmol/L), alone or in combination, for 48 hours. For both VS01 and VS02, the combination treatment reduced VS cell viability by approximately 35%–40% compared with vehicle (0.3% DMSO) and was significantly more effective than saracatinib alone. In addition, for VS01 cells, the combination treatment was more effective than cabozantinib alone (Fig. 4D).

Treatment with Saracatinib (AZD0530) potently and dose-dependently suppresses the proliferation of transplanted Src3T3 fibroblasts under the skin in mice and rats. Significant tumor growth inhibition is observed in both models at doses ≥6 mg/kg/day (60% in mice and 98% in rats versus animals treated with vehicle), and complete tumor growth inhibition is observed at the highest doses investigated (100% in rats and mice at 25 mg/kg/day and 10 mg/kg/day, respectively) [1].

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Src3T3 allografts and xenografts [1]
Saracatinib (AZD0530) treatment potently inhibited the proliferation of subcutaneously transplanted Src3T3 fibroblasts in mice (Figure 4B) and rats (Hennequin et al., 2006) in a dose‐dependent manner. In both models, significant inhibition of tumor growth was seen at doses ≥6mg/kg/day (60% inhibition in mice [P<0.01] and 98% inhibition in rats [P<0.001] versus animals treated with vehicle) and, at the maximum doses investigated, complete tumor growth inhibition was observed (100% inhibition at 25mg/kg/day in mice and 10mg/kg/day in rats) (Hennequin et al., 2006).

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Human tumor xenografts [1]
Once‐daily oral dosing of Saracatinib (AZD0530) resulted in a moderate growth delay in 4/10 xenograft models tested (Table 3). In the remainder of the xenograft panel, AZD0530 treatment failed to inhibit primary tumor growth. Representative xenograft growth delay curves for an AZD0530 growth‐inhibition‐sensitive (Calu‐6) and an AZD0530 growth‐inhibition‐insensitive model (LoVo) are shown in Figure 4C and D, respectively. AZD0530 did not affect any aspect of tumor vascularity in xenograft models as assessed by blood vessel number or CD31 positive vessels (data not shown).

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NBT‐II bladder cancer metastasis model [1]
In nude mice bearing subcutaneous xenografts of NBT‐II bladder cancer cells, once‐daily Saracatinib (AZD0530) treatment at doses of 10, 25, and 50mg/kg/day resulted in a small, non significant delay in xenograft growth at the highest dose only (50mg/kg/day; Figure 4E). In contrast, all three doses of AZD0530 resulted in a reduction in the number of mice from which tumor colonies could be grown from mesenteric lymph node extracts (Figure 5A). In an additional experiment, this antimetastatic effect of AZD0530 did not appear to be affected by a 7‐day delay between cell inoculation and drug administration. One out of seven mice developed metastases in a group that started treatment with AZD0530 50mg/kg/day 7 days after tumor cell inoculation, and 1/7 developed metastases in a group that started treatment with the same dose immediately after cell inoculation.

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Phosphorylation of Src kinase substrates FAK and paxillin [1]
Levels of phosphorylated FAK (pY861) and paxillin (pY31), assessed by immunohistochemistry using phosphospecific antibodies, were reduced markedly in tumors from animals bearing both growth‐inhibition‐sensitive and growth‐inhibition‐insensitive xenografts after 14–28 days' treatment with Saracatinib (AZD0530) 50mg/kg/day (Figure 5B–D). Antibodies detecting non‐phospho epitopes of FAK or paxillin showed no effect on the expression of either protein (data not shown).

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Dual c-Met and Src inhibition slows MD-MSC growth in vivo [2]
To evaluate the efficacy of cabozantinib and Saracatinib (AZD0530) in vivo, luciferase-expressing MD-MSCs were grafted into the sciatic nerves of NSG mice, and the graft size was monitored by bioluminescence imaging (BLI). Upon confirmation of successful grafting (Supplementary Fig. S2A), mice were assigned into vehicle, cabozantinib (12.5 mg/kg/day), saracatinib (25 mg/kg/day), and combination (cabozantinib and saracatinib at 12.5 mg/kg/day and 25 mg/kg/day, respectively) treatment cohorts. BLI showed that grafts in the combination-treated group had a significantly slower growth rate compared with those in the single-agent groups (Fig. 5A and B). Although the vehicle-treated allografts had a 160-fold increase in BL signal over 14 days, the grafts treated with saracatinib or cabozantinib had a 50- and 60-fold increase in BL signal, respectively. Significantly, the allografts from the combination group had only a 25-fold increase in BL signal after 14 days of treatment (Fig. 5C). The reduction in BL signals correlated with lower tumor weights in the combination-treated group compared with the vehicle group (Fig. 5D; Supplementary Fig. S2B).

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Dual c-Met and Src inhibition modulates FAK and ERK signaling pathways in allografts [2]
To assess the signaling pathways modulated by drug treatments, allograft sections were analyzed by immunohistochemistry. Similar to the in vitro findings, Saracatinib (AZD0530) decreased the FAK phosphorylation levels, and cabozantinib decreased the ERK1/2 phosphorylation levels compared with vehicle controls (Fig. 5E). These changes were also observed in the combination-treated allografts. Moreover, CD31 staining of endothelial cells was decreased in grafts treated with cabozantinib compared with vehicle controls, indicating that cabozantinib targets the vasculature in vivo as well (Fig. 5E). Grafts from mice treated with cabozantinib or saracatinib, alone or in combination, had fewer cyclin D1 and Ki67-positive cells compared with those from vehicle-treated mice. In addition, grafts in the combination-treated group had elevated cleaved caspase 3 staining, compared with those in the vehicle and single treatment groups (Fig. 5E), supporting the conclusion that the drug combination induces apoptosis of MD-MSCs in vivo.

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Dual c-Met and Src inhibition induces MD-MSC apoptosis [2]
To confirm the in vivo findings, we studied caspase-dependent apoptosis in cultured MD-MSCs. Caspase 3/7 activity was triggered by cabozantinib or Saracatinib (AZD0530) alone only when administered at high doses (3 μmol/L saracatinib or 10 μmol/L cabozantinib) for 19 to 24 hours (Fig. 6A). However, cotreatment of MD-MSCs with saracatinib (0.5 μmol/L) and increasing cabozantinib concentrations induced caspase 3/7 activity at 100-fold lower concentrations of cabozantinib compared with cabozantinib administered alone (0.1 μmol/L vs. 10 μmol/L; Fig. 6A). Similarly, MD-MSCs treated with cabozantinib (0.5 μmol/L) and increasing saracatinib concentrations induced caspase 3/7 activity at approximately 30-fold lower saracatinib concentrations compared with saracatinib administered alone (0.1 μmol/L vs. 3 μmol/L; Fig. 6A). A membrane asymmetry assay confirmed that the drug combination induced a larger apoptotic cell population than either drug alone (Fig. 6B). Treatment with 2 μmol/L saracatinib resulted in 6.2% apoptotic cells, 4.8% dead cells, and 88.4% live cells, whereas treatment with 2 μmol/L cabozantinib had 7.7% apoptotic cells, 9% dead cells, and 82.8% live cells. The combination treatment increased the apoptotic population to 20% after 19 hours (Fig. 6C). When administered alone, saracatinib did not induce cleavage of caspase 3. Cabozantinib induced caspase cleavage at 1μmol/L whereas in the presence of 0.5 μmol/L saracatinib, caspase cleavage was detected at 0.1 μmol/L cabozantinib (Fig. 6D). Collectively, our results indicate that although the individual drugs promote a G1 cell-cycle arrest of MD-MSCs, saracatinib and cabozantinib combination is cytotoxic and promotes caspase 3/7-dependent apoptosis.

Isolated protein kinase assay [1]
Inhibition of tyrosine kinase activity was examined using an enzyme‐linked immunosorbent assay (ELISA) with recombinant catalytic domains of a panel of receptor and non‐receptor tyrosine kinases (in some cases only part of the catalytic domain was used). This method has been described previously (Plé et al., 2004).  Saracatinib (AZD0530) dose ranges varied depending on the activity versus the particular kinase tested, but were typically 0.001–10μM.
Specificity assays against a panel of serine/threonine kinases were performed using a filter capture assay with 32P. Briefly, multidrop 384 plates containing 0.5μL  Saracatinib (AZD0530) or controls (dimethyl sulfoxide [DMSO] alone or pH 3.0 buffer controls) were incubated with 15μL of enzyme plus peptide/protein substrate for 5min before the reaction was initiated by the addition of 10μL of 20mM Mg.ATP. For all enzymes the final concentration was approximated to the Michaelis constant (K m). Assays were carried out for 30min at room temperature before termination by the addition of 5μL orthophosphoric acid. After mixing, the well contents were harvested onto a P81 Unifilter plate, using orthophosphoric acid as the wash buffer. Microcal Origin software was used to interpolate IC50 values by nonlinear regression.

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Enzyme kinetics [1]
Investigation of the reversibility and the mechanism of  Saracatinib (AZD0530) inhibition was conducted using a full‐length activated human Src (phosphorylated at tyrosine 416, Upstate Biotech, Dundee, UK) in a continuous, coupled assay adapted from Jenkins (1991). ATP and peptide substrate (Src II peptide) concentrations were varied in turn (ATP 40–1280μM; Src II peptide 100–800μM), in conjunction with AZD0530 (0–30nM), at saturating concentrations of the non‐varied substrate (ATP 1.6mM; Src II peptide 1.0mM). The binding affinity of AZD0530 for inactivated Src (phosphorylated at tyrosine 527, not tyrosine 416) was measured using a BIAcore inhibition‐in‐solution assay (Karlsson et al., 2000). The assay followed competition binding between AZD0530 and an immobilized ureidoquinazoline (Sullivan et al., 2005) for binding to Src. Data analysis was performed by unweighted nonlinear regression using GraFit, version 5 and an F‐test (Mannervik, 1982) was used to identify the most suitable equation.

Cell lines [1]
The cell lines used were mouse NIH 3T3 fibroblasts engineered to overexpress a constitutively active form of human Src (Src3T3) with a point mutation at the negative regulatory tyrosine site in the c‐terminal domain (Y530F) (Plé et al., 2004) and human cancer cell lines containing endogenous Src (Table 2). Src3T3 fibroblasts form colonies in soft agar and grow subcutaneously in immunocompromised athymic rats and mice in vivo, while the wild‐type parental 3T3 cells do not. Src3T3 cells will grow in medium containing as little as 0.5% fetal calf serum (FCS; used in the assay conditions), whereas wild‐type non‐Src‐transfected 3T3 cells will not grow in these low serum conditions. AZD0530 functional activity against Abl kinase was assessed in an in vitro proliferation assay using K562 human leukemia cells known to be growth driven through Bcr–Abl activity (Lozzio and Lozzio, 1975). NBT‐II rat bladder cancer cells were sourced and cultured as described previously (Boyer et al., 2002).

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Cell proliferation assay [1]
Cell proliferation was assessed using a colorimetric 5‐bromo‐2′‐deoxyuridine (BrdU) Cell Proliferation ELISA kit (Roche Diagnostics GmbH), as described previously (Plé et al., 2004). Briefly, cells were plated onto 96‐well plates (1.5×104cells/well), the following day 0.039–20μM  Saracatinib (AZD0530) in DMSO (at a final concentration of 0.5%) was added and the cells were incubated for 24h. The cells were pulse labeled with BrdU for 2h and fixed. Cellular DNA was then denatured with the provided solution and incubated with antiBrdU peroxidase for 90min. Following three washes with phosphate‐buffered saline, tetramethylbenzidine substrate solution was added and the plates were incubated on a plate shaker for 10–30min until the positive control absorbance at 690nm was approximately 1.5 absorbance units.

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EGFR phosphorylation assay [1]
KB (nasopharyngeal carcinoma) cells were seeded at 5000cells/well in 96‐well plates and cultured for 72h in Rosewell Park Memorial Institute (RPMI) 1640 media with 10% FCS, followed by 24h' incubation with serum‐free RPMI 1640. Cells were treated with compound for 90min at concentrations ranging from 0 to 10μM. Cells were incubated with 15ng/mL EGF ligand (concentration required to increase receptor phosphorylation to 90% of maximum) for 5 min prior to lysis. Levels of phosphorylated EGFR were measured using the human phospho‐EGFR Duoset ELISA kit.

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Microdroplet migration (chemokinesis) assay [1]
The microdroplet migration assay assesses the ability of test compounds to inhibit the random motility (chemokinesis) of human epithelial A549 lung cancer cells, which were routinely cultured in Dulbecco's modified Eagle's medium (DMEM)+10% FCS. The assay method has been described in full previously (Plé et al., 2004). Briefly, A549 cells (2×107/mL) were suspended in warm DMEM (37°C) containing 0.3% agarose. The suspension was pipetted into 96‐well plates (2μL/well) and chilled briefly on ice to allow the agarose microdroplet to gel. When set, 90μL of chilled RPMI 1640 medium was added to each well, followed by 10μL  Saracatinib (AZD0530) (0.02–5μM). The plates were incubated for 72h at 37°C to allow migration to occur. Cell migration was measured by taking several equidistant measurements from around the agar droplet to the migrating front of the motile cells. An illustration of the microdroplet migration assay is shown in Figure 1A, indicating how cell migration was measured.

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Monolayer scratch assay [1]
Human MDA‐MB‐231 breast cancer cells and T24, SCaBER, and 1A6 bladder cancer cells were grown as monolayers in adapted six‐well tissue culture plates. Confluent monolayers were gently scraped with a sterile pipette tip to form a scratch and  Saracatinib (AZD0530) (0.01–0.5μM) was added to the wells in medium (phenol red‐free DMEM supplemented with 10% charcoal/dextran‐treated FCS and 1% l‐glutamine). Cell movement back into the area of the scratch was recorded by time‐lapse photography over 18h.

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NBT‐II cell adhesion and migration assays [1]
The effects of  Saracatinib (AZD0530) on EGF‐ and collagen‐induced motility of NBT‐II cells were assessed by videomicroscopy as described previously (Valles et al., 2004). The effect of AZD0530 on EGF‐induced cell dispersion was evaluated by immunofluorescence with anti‐desmoplakin antibodies to monitor the EGF‐induced loss of desmosomes from the cell periphery, as reported before (Boyer et al., 1997).

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Assessment of paxillin phosphorylation [1]
The effects of  Saracatinib (AZD0530) on paxillin phosphorylation was assessed in NBT‐II cells that had been stimulated with 100ng/mL EGF for 15min in the presence of increasing amounts of  Saracatinib (AZD0530). Paxillin tyrosine phosphorylation was revealed by immunoblotting with polyclonal antibodies against phospho Y118‐paxillin and total paxillin.

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3D invasion assay [1]
Fibrillar collagen type I gels (1mg/mL final concentration) were prepared by neutralizing a solution of collagen type I with 1/10 volume 10× DMEM concentrate, diluted to a final concentration of 1× with distilled H2O, to which 1/11 volume 0.1N NaOH was added. Fibrillar collagen gels (80μL) were set within the upper chamber of a 24‐well transwell insert, above an 8μm pore‐size polycarbonate filter (Costar, Corning Inc.) at 37°C for 18h prior to cell seeding. Human HT1080 fibrosarcoma cells (ATCC CCL 121; DMEM, supplemented with 0.2% FCS and 2mM l‐glutamine) were seeded on top of a collagen gel in an upper transwell chamber (1×104cells per transwell). DMEM supplemented with 10% FCS and 2mM l‐glutamine (750μL total volume) was placed in the lower chamber to provide a chemotactic gradient.  Saracatinib (AZD0530) (0.1, 1, and 10μM) or vehicle (0.1% DMSO) was added to the cells prior to seeding and also to media within the lower chamber. Transwells were incubated at 37°C for 72h and then incubated with 10μM Hoechst 33342 in serum‐free DMEM for 30min at 37°C. Invading cells were visualized by confocal microscopic analysis using a Bio‐Rad Radiance 2000 multiphoton confocal illumination unit attached to a Nikon Eclipse inverted microscope. Quantification of cell invasion was performed with modification to the protocol as described previously (Carragher et al., 2006). Briefly, using a 20× objective, optical sections were scanned at 20μm intervals from the collagen gel surface. Image‐Pro analysis software was applied to identify the number of positive pixels (Hoechst stain above a threshold) and segment into individual nuclei per optical section. The accumulated sum of the positive nuclei present in the optical sections between 60 and 200μm below the top of collagen gels was expressed as a percentage of the total number of positive nuclei on top and within the collagen gel. This value, therefore, represents the proportion of total cells within each sample invading beyond a depth of 60μm and up to 200μm.

\n\nXenograft studies [1]
\nFemale athymic mice (nu/nu: Alpk; Alderley Park) and rats (RH‐rnu/rnu) were housed and maintained as previously described (Wilkinson et al., 2007). Src3T3 and human tumor lines (as indicated in Table 3) were inoculated subcutaneously in the left flank of animals. Tumor growth was monitored by bi‐dimensional caliper measurements twice weekly. The tumor volume was calculated by the following formula: (length×width)×√(length×width)×(π/6) and supported by excision and weighing of tumors at the end of the studies. Dosing started when the average tumor volume reached 0.2–0.5cm3 (except MDA‐MB‐231 and HT29; see Table 3). Animals were treated once daily by oral gavage with either vehicle alone or  Saracatinib (AZD0530) 6.25–50mg/kg for 10–91 days (Table 3). Tumor growth inhibition was calculated as described previously (Wedge et al., 2005). For pharmacokinetic and pharmacodynamic analysis animals were humanely sacrificed and samples (plasma and tumor) were collected. Tumor samples were homogenized with 5 volumes of water and extracted with chloroform. Plasma and tumor samples were analyzed for  Saracatinib (AZD0530) concentration using high‐performance liquid chromatography with tandem mass spectrometric detection after solid‐phase extraction.\n

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\n\nMetastasis studies [1]
\nFemale nude mice (nu/nu Swiss strain) aged 7 weeks old were injected subcutaneously with 5×106 NBT‐II cells. Treatment with  Saracatinib (AZD0530) at 10 (n=14), 25 (n=14) or 50mg/kg/day po (n=7) was initiated on the same day as cell inoculation. In an additional cohort of seven mice, treatment with  Saracatinib (AZD0530) 50mg/kg/day po was initiated one week after inoculation. Treatment with  Saracatinib (AZD0530) continued for 2 months. Control mice received vehicle alone. Tumor growth was assessed weekly by measuring two perpendicular diameters with a caliper. Tumor volume (V) was calculated as follows: V=a 2×b/2 where a=tumor width (mm) and b=tumor length (mm). When the tumors reached a volume of 1500–2000mm3, the mice were sacrificed and lymph nodes harvested. The presence of macroscopic metastases in the lymph nodes, liver, and lungs was assessed at autopsy. To assess the presence of micrometastases, fragments of lymph nodes or other organs (liver, lungs) were placed into culture and examined microscopically at various times after seeding for the presence of epithelial foci derived from metastatic cells.

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\n\nPharmacokinetics [2]
\nNSG (NOD.Cg-Prkdcscid Il2rgtmlWjl/SzJ) mice were used. Three mice were dosed by oral gavage (20 mg/kg dasatinib, 25 mg/kg  Saracatinib (AZD0530), or 40 mg/kg cabozantinib in a vehicle containing 1.25% polyethylene glycol, 2.5% Tween-80, and 5% DMSO) and one mouse received vehicle only. At the indicated times, mice were sacrificed.\n

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\n\nOrthotopic sciatic nerve allograft model [2]
\nMale and female NSG mice were bred in house and used at 6 to 8 weeks of age. Following anesthesia with isoflurane (1–3% in oxygen), the right sciatic nerve was injected with 5,000 luciferase-expressing MD-MSCs in 3 μL of L-15 prf media. Wounds were sealed with Vetbond. Mice were imaged with an In Vivo Imaging System 15 minutes after intraperitoneal injection of luciferin (150 mg/kg) to confirm engraftment, were assigned to treatment groups (Day 0) and began daily treatment with the vehicle (as above),  Saracatinib (AZD0530) (25 mg/kg), cabozantinib (12.5 mg/kg), or a combination of saracatinib and cabozantinib (25 and 12.5 mg/kg, respectively). Mice were imaged with IVIS every 7 days and were sacrificed at day 14. Tumor grafts and contralateral sciatic nerves were removed, weighed and photographed, followed by fixation overnight in 4% paraformaldehyde and storage in 30% sucrose/0.02% azide.

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\n

Dissolved in 0.5% hydroxypropyl methylcellulose, 0.1% Tween 80; 25 mg/kg; Oral gavage

CB17 mice are implanted with DU145 cells.

Saracatinib reduces the activity of MD-MSCs and has good nerve penetration [2]. A pharmacokinetic study of aracatinib (25 mg/kg oral dose) in NSG mice showed that plasma and nerve concentrations of aracatinib peaked at 0.5 hours and had a half-life of 4.4 hours, but the concentration in nerves remained stable at approximately 300-400 ng/g for at least 8 hours (Figure 3D). Saracatinib selectively reduced the activity of MD-MSCs (IG50 = 0.3 μmol/L), but its maximum effect was low, at approximately 40%-50%, while the maximum effect of dasatinib was 80% (Figure 3E). Compared with total Src levels, aracatinib reduced the phosphorylation level of Src (Y416) and reduced the phosphorylation level of Src-dependent FAK (Y576) and Src/FAK-dependent paxillin (Y118) at 6 hours after treatment (Figure 3F). Twenty-four hours after treatment with Saracatinib, the p27 level in MD-MSCs was higher than that in the control group (Figure 3G).

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Pharmacokinetics[1]
Six hours after oral administration, the plasma concentration of AZD0530 increased proportionally to the dose (Figure 4A).

Saracatinib exhibits a toxicity profile that involves multiple organ systems, with its underlying toxicokinetics largely driven by metabolic bioactivation.
From a toxicokinetic perspective, Saracatinib is metabolized in the body primarily by the cytochrome P450 3A4 enzyme. This metabolic process leads to bioactivation, forming reactive intermediates such as ortho-quinone and iminium species. These reactive metabolites have the potential to covalently modify cellular proteins, a mechanism that is strongly implicated in the pathogenesis of drug-induced organ toxicities, particularly hepatotoxicity.
In terms of clinical toxicity, Saracatinib has been associated with a range of adverse events observed in trials.
Hepatotoxicity is a significant concern, with documented elevations in liver enzymes including aspartate aminotransferase (AST), alanine transaminase (ALT), and gamma-glutamyltransferase (GGT). These elevations have, in some cases, reached Grade 3 severity, indicating dose-limiting liver injury.
Pulmonary toxicity has also been reported, manifesting as severe conditions such as hypoxia (dangerously low blood oxygen levels), pneumonia, and acute respiratory distress syndrome (ARDS), including instances of Grade 4 hypoxia.
Gastrointestinal effects are very common, with diarrhea and nausea being frequently reported.
Hematological toxicities are prevalent as well, including lymphopenia, neutropenia, leukopenia, and febrile neutropenia.
Other common adverse effects include fatigue, decreased appetite, and electrolyte imbalances like hyponatremia.
Regarding tolerability, the maximum tolerated dose in cancer trials was established at 175 mg daily. However, this dose is associated with significant toxicities. For repurposed applications, such as in Alzheimer's disease, a lower dose of 100-125 mg daily is being investigated for a more manageable safety profile. Interestingly, early preclinical in silico models had predicted Saracatinib to be non-hepatotoxic, highlighting the importance of clinical findings and later bioactivation studies in fully understanding its toxic potential.

[1]. Preclinical anticancer activity of the potent, oral Src inhibitor AZD0530. Mol Oncol, 2009, 3(3), 248-261.

[2]. Combination Therapy With c-Met and Src Inhibitors Induces Caspase-Dependent Apoptosis of Merlin-Deficient Schwann Cells and Suppresses Growth of Schwannoma Cells. Mol Cancer Ther. Mol Cancer Ther. 2017 Nov;16(11):2387-2398.

Saracatinib difumarate is the difumarate of aracatinib, an orally effective 5,7-substituted aniline quinazoline compound with anti-invasive and antitumor activities. Saracatinib is a dual-specific inhibitor of Src and Abl protein tyrosine kinases, which are overexpressed in chronic myeloid leukemia cells. The drug binds to these tyrosine kinases and inhibits their activity, thereby affecting cell motility, migration, adhesion, invasion, proliferation, differentiation, and survival. Specifically, Saracatinib inhibits Src kinase-mediated osteoclast-mediated bone resorption. Saracatinib belongs to the quinazoline class of compounds, with its quinazoline ring substituted at positions 4, 5, and 7 by (5-chloro-2H-1,3-benzodioxane-4-yl)amino, (oxacyclopenten-4-yl)oxy, and 2-(4-methylpiperazin-1-yl)ethoxy, respectively. It is a dual inhibitor of tyrosine kinases c-Src and Abl (IC50 values of 2.7 nM and 30 nM, respectively). Saracatinib was initially developed by AstraZeneca for the treatment of cancer, but in 2019 it was granted orphan drug designation by the U.S. Food and Drug Administration (FDA) for the treatment of idiopathic pulmonary fibrosis (IPF). IPF is a lung disease that leads to lung scarring (fibrosis). It has antitumor activity and is an EC 2.7.10.2 (nonspecific protein tyrosine kinase) inhibitor, radiosensitizer, autophagy inducer, apoptosis inducer, and anticoronavirus drug. It belongs to the quinazoline class, secondary amino compounds, N-methylpiperazine class, aromatic ether class, oxacyclohexane class, benzodioxane class, organochlorine class, and diether class.
Saracatinib has been investigated for the treatment of cancer, osteosarcoma, ovarian cancer, fallopian tube cancer, and primary peritoneal cancer. Saracatinib is an orally potent 5,7-substituted aniline quinazoline compound with anti-invasive and antitumor activities. It is a dual-specific inhibitor of Src and Abl, protein tyrosine kinases overexpressed in chronic myeloid leukemia cells. The drug binds to these tyrosine kinases and inhibits their activity, thereby affecting cell motility, migration, adhesion, invasion, proliferation, differentiation, and survival. Specifically, Saracatinib inhibits Src kinase-mediated osteoclast-mediated bone resorption. AZD0530 is an orally administered Src inhibitor that exhibits potent anti-migration and anti-invasive activity in vitro and inhibits metastasis in a mouse bladder cancer model. The in vitro antiproliferative activity of AZD0530 varies by cell line (IC50 from 0.2 to 10 μM). AZD0530 inhibited tumor growth in four of ten xenograft tumor models and dynamically inhibited in vivo phosphorylation of Src substrates paxillin and FAK in both growth-inhibiting and growth-inhibiting xenograft tumors. AZD0530’s in vitro activity against NBT-II bladder cancer cells was consistent with inhibition of cell migration and stabilization of cell-cell adhesion. These data suggest that AZD0530 has major anti-invasive pharmacological effects and may limit tumor progression in a variety of cancers. AZD0530 is currently in a Phase II clinical trial. [1]

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The data presented in this paper support the mechanism by which endogenous Src plays a role in promoting an aggressive cell phenotype by modulating cell adhesion, migration, and invasion. AZD0530 inhibited the proliferation of cancer cells in constitutively active Src fibroblast tumor models and certain human tumor cell lines both in vitro and in vivo. The lack of consistent anti-proliferative activity and the potential mechanisms of this diverse response require further investigation in other tumor models. The effects on invasion models are even more pronounced: AZD0530 inhibits cancer cell invasion and migration in vitro and suppresses the phosphorylation of proteins controlling migration both in vitro and in vivo. In an in vivo model of bladder cancer metastasis, AZD0530 suppressed the number of lymph node metastases detected by colony formation assays, thus reproducing the effects previously observed in this model using dominant-negative Src and Csk constructs (Boyer et al., 2002). We and other researchers have recently demonstrated the anti-metastatic activity of AZD0530 in pancreatic cancer models (Green et al., 2005) and human colorectal cancer in situ models (Phillips et al., 2007). Combined with the results presented in this paper, these data suggest that AZD0530 may provide clinical benefit by inhibiting tumor cell migration and invasion, thereby preventing or delaying tumor progression. AZD0530 is currently in Phase II clinical trials. [1] Neurofibromatosis type 2 (NF2) is a neurological tumor caused by the inactivation of the merlin tumor suppressor encoded by the NF2 gene. Bilateral vestibular schwannomas are the diagnostic marker for NF2. Currently, the mainstream treatment for NF2-related tumors is limited to surgery and radiotherapy; however, off-label use of targeted molecular therapies is becoming increasingly common. This article investigates drugs targeting two kinases activated in NF2-related schwannomas, c-Met and Src. We demonstrated that treatment of mouse Schwann cells (MD-MSCs) lacking merlin protein with the c-Met inhibitor cabozantinib or the Src kinase inhibitors dasatinib and salatinib resulted in G1 phase cell cycle arrest. However, when MD-MSCs were treated with a combination of cabozantinib and salatinib, they exhibited caspase-dependent apoptosis. In an orthotopic allogeneic mouse model, the combination therapy also significantly inhibited the growth of MD-MSCs, with an inhibition rate more than 80% lower than that of the control group. In addition, compared with the control group, the cell viability of human vestibular schwannoma cells carrying NF2 mutations decreased by 40% after treatment with cabozantinib and salatinib. This study shows that simultaneous inhibition of the c-Met and Src signaling pathways in MD-MSCs can induce apoptosis and reveals a fragile pathway that can be used to develop NF2 therapy. [2] This study shows that simultaneous inhibition of c-Met and Src can induce apoptosis in merlin-deficient mouse Schwann cells cultured in vitro and allogeneic transplanted, and reduce the growth of primary VS cells carrying NF2 mutations. The drug combination also reduced the in vitro viability of two merlin-deficient human Schwann cell lines and one human benign meningoma cell line (Supplementary Figure S4), confirming that the effect is independent of cell line or species. ERK and Src-FAK kinases both converge in the PI3K/Akt pathway; inhibitors of this pathway can induce NF2-related apoptosis. Given the severe toxicity of directly targeting PI3K with edrallixib, and the possibility that simultaneous inhibition of the c-Met-ERK and Src-FAK pathways with cabozantinib and dasatinib/salatinib may promote apoptosis in schwannomas with fewer adverse reactions, the preclinical data provided in this study support the possibility of further investigation into the dual inhibition of c-Met and Src as a potential therapy for NF2-related schwannomas. [2]

C35H40CLN5O13

774.17

773.231

C, 54.30; H, 5.21; Cl, 4.58; N, 9.05; O, 26.87

893428-72-3

Saracatinib;379231-04-6

44195703

Off-white to light yellow solid powder

3.311

5

18

12

54

862

0

CN1CCN(CC1)CCOC2=CC3=C(C(=NC=N3)NC4=C(C=CC5=C4OCO5)Cl)C(=C2)OC6CCOCC6.C(=C/C(=O)O)\C(=O)O.C(=C/C(=O)O)\C(=O)O

OQXMMAQEMJUIQM-LVEZLNDCSA-N

InChI=1S/C27H32ClN5O5.2C4H4O4/c1-32-6-8-33(9-7-32)10-13-35-19-14-21-24(23(15-19)38-18-4-11-34-12-5-18)27(30-16-29-21)31-25-20(28)2-3-22-26(25)37-17-36-22;2*5-3(6)1-2-4(7)8/h2-3,14-16,18H,4-13,17H2,1H3,(H,29,30,31);2*1-2H,(H,5,6)(H,7,8)/b;2*2-1+

(E)-but-2-enedioic acid;N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(oxan-4-yloxy)quinazolin-4-amine

SARACATINIB DIFUMARATE; 893428-72-3; AZD0530 DIFUMARATE; Saracatinib fumarate; Saracatinib difumarate [USAN];

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)

DMSO: ~83 mg/mL (~107 mM)

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.)