EGFR (IC50 = 4 nM); ErbB2 (IC50 = 4 nM); HER3 (IC50 = 4 nM)

AZD8931 exhibits varying levels of potency in comparison to NSCLC and SCCHN cell lines. With a GI50 of 0.1 nM, AZD8931 exhibits high sensitivity to PC-9 cells (which have an EGFR activating mutation), while its activity against NCI-1437 cells is low, with a GI50 exceeding 10 μM. In PE/CA-PJ41, PE/CA-PJ49, DOK, and FaDu cells, AZD8931 shows greater efficacy against phospho-EGFR, phospho-erbB2, and phospho-erbB3 than either lapatinib or gefitinib does.[1]

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AZD8931 shows potent in vitro inhibition of EGFR and erbB2 tyrosine kinases. AZD8931 shows greater inhibition of ligand-stimulated EGFR phosphorylation in cells than gefitinib or lapatinib. AZD8931 also potently inhibits erbB2- and erbB3-mediated signaling in vitro. AZD8931 shows a distinct pattern of tumor cell growth inhibition in NSCLC and head and neck squamous cell carcinoma cell panels.[1]

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AZD8931 inhibits EGFR pathway activity. AZD8931 inhibits proliferation and induces apoptosis in human IBC cells.[2]

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As an alternative, the pan-HER inhibitor AZD8931 was used to block HER3 in CRC cells. Because we determined that other HER receptors were not involved in EC CM activation of HER3 in CRC cells, the effects of the pan-HER inhibitor on EC CM-induced cell survival were mainly due to HER3 inhibition. AZD8931 nearly complete inhibition of HER3 phosphorylation in CRC cells (Fig. 4C). Moreover, we used MTT assay to determine the effects of AZD8931 on CRC cell viability when cultured with LPEC CM and 5-FU (Fig 5A, C, E). The relative numbers of viable CRC cells were significantly increased by LPEC-1 CM compared to those with CRC CM, as expected. 5-FU or AZD8931 single agent treatments decreased CRC cell viability in both CRC CM and LPEC-1 CM. When CRC cells were treated in combination of 5-FU and AZD8931, the relative numbers of viable cells were as low as half of that from single agent treatment. We then used AZD8931 to determine the effects of blocking HER3 on chemoresistance in CRC cells (Fig. 5B, D, F). AZD8931 alone led to an insignificant change in apoptosis in CRC cells incubated in either CRC or LPEC-1 CM. 5-FU alone induced CRC cell apoptosis but to a less extent in LPEC-1 CM than in CRC CM. In contrast, levels of apoptosis were significantly higher in cells treated with 5-FU and AZD8931 than in those with 5-FU alone, even in cells incubated with LPEC-1 CM. This data suggest that inhibiting HER3 by AZD8931 blocked LPEC-1 CM-induced chemoresistance in CRC cells [4].

AZD8931 exhibits antitumor activity in xenografts of PC-9, BT474c, Calu-3, LoVo, and FaDu. After acute treatment, AZD8931 may lower p-Akt, Ki67 expression, and p-ERK in BT474c xenografts. Additionally, AZD8931 induces the M30 apoptosis marker. Additionally, in LoVo xenografts, AZD8931 exhibits a higher proapoptotic effect than both lapatinib and gefitinib.[1]

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Sapitinib (AZD-8931) inhibits the growth of EGFR-sensitive and erbB2-sensitive human tumor xenograft models [1]
The ability of AZD8931 to inhibit human tumor xenograft growth in mice was tested in a variety of models with different sensitivities to agents targeting either EGFR or erbB2. Oral dosing of AZD8931 significantly inhibited BT474c (breast), Calu-3 (NSCLC), LoVo (colorectal), FaDu (SCCHN), and PC-9 (NSCLC) tumor xenograft growth (Fig. 4A, B, C, D, and E), showing that AZD8931 is active in xenograft tumor models responsive to EGFR inhibition alone (LoVo and PC-9) or EGFR or erbB2 inhibition (BT474c, Calu-3, and FaDu). No xenograft model selectively dependent on erbB2 alone was available. Furthermore, we found that lapatinib was not significantly active in LoVo (Supplementary Table S1) and was only modestly active in the EGFR mutant model PC-9 (Fig. 4E), even at maximum well-tolerated dose. In contrast, AZD8931 inhibited PC-9 tumor volume by 145% at a dose (6.25 mg/kg bid), well below its maximum well-tolerated dose (50 mg/kg bid; Supplementary Table S1; Fig. 4E). Lapatinib was significantly active in the EGFR and erbB2 inhibitor–sensitive xenograft models BT474c, Calu-3, and FaDu, which is consistent with an erbB2-selective mode of action. A summary of all antitumor data generated across these five models can be seen in Supplementary Table S1.

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Sapitinib (AZD-8931) causes pharmacodynamic changes in proliferation and apoptosis markers in human tumor xenograft models [1]
Having determined the antitumor activity profile of these compounds, we evaluated their in vivo mechanism of action using specific EGFR, or EGFR- and erbB2-driven models. Using the BT474c xenograft model as a high erbB2-expressing example, pharmacodynamic analysis by IHC showed significant inhibition of EGFR (P = 0.04), erbB2 (P = 0.024), and erbB3 (P < 0.001) phosphorylation with AZD8931 compared with control (Fig. 5A). Furthermore, a significant reduction in the downstream signaling biomarker p-AKT (P = 0.002) and a significant decrease in Ki67 expression (P < 0.001) by IHC ex vivo analysis were also observed with AZD8931 (Fig. 5A). A reduction in the expression of p-ERK was also detected with AZD8931 compared with vehicle control xenografts, but this did not reach statistical significance.

Apoptosis was assessed by M30 evaluation. The M30 antibody recognizes a specific caspase cleavage site within cytokeratin (CK) 18 that is not detectable in the native CK18 of normal cells, and is a measure of an early apoptotic event. AZD8931, versus vehicle control xenografts, caused a significant induction of the M30 apoptosis marker (P = 0.002) at 1 h after four doses, which recovered to near control levels at 4 h after the fourth dose (Fig. 5B).

Pharmacodynamic activities for AZD8931, gefitinib, and lapatinib were also compared using the FaDu (Fig. 5C) and LoVo (Fig. 5D) xenograft models, which do not overexpress erbB2. AZD8931, gefitinib, and lapatinib all showed significant inhibition of EGFR and erbB2 phosphorylation in the FaDu xenograft model (P < 0.001; Fig. 5C). AZD8931 (P < 0.0003) and gefitinib (P = 0.03) were also able to inhibit erbB3 phosphorylation significantly compared with control. In contrast, lapatinib did not significantly inhibit the phosphorylation of erbB3 in this model. Apoptosis was then assessed in the LoVo model, and AZD8931 showed significant induction of apoptosis compared with control as measured by the levels of cleaved caspase-3 (P < 0.001; Fig. 5D). Furthermore, AZD8931 also showed a significantly greater proapoptotic effect compared with gefitinib (P = 0.003) and lapatinib (P < 0.001). The pharmacokinetic and pharmacodynamic relationship for AZD8931 was assessed using the LoVo xenograft model (Fig. 5E). There was a direct relationship between the total plasma levels of AZD8931 and the inhibition of EGFR phosphorylation observed.

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Sapitinib (AZD-8931) inhibits the tumor growth of human IBC models [2]
Previous study has shown that AZD8931 inhibits human tumor xenograft growth with different sensitivities to agents targeting either EGFR or HER2 in a variety of models including one human breast cancer cell line BT-474, which expresses ER/PgR, high levels of HER2, and moderate levels of EGFR. Here, we determine the effects of AZD8931 alone or combined with paclitaxel on the growth of human IBC cells in vivo in SCID mice. Toward this goal, the tumors were orthotopically grown in the mammary fat pads of SCID mice and monitored by caliper measurement twice weekly. The changes in tumor volume following different treatments for both SUM149 and FC-IBC-02 cell lines are shown in Figure 4A and C. The tumor growth curves represent the group mean values over the course of 33 days for SUM149 xenograft and 26 days for FC-IBC-02 xenograft. AZD8931 alone significantly suppressed the xenografted tumor growth of SUM149 (P = 0.002; Figure 4A) and FC-IBC-02 (P < 0.001; Figure 4C) cells compared with the control group. The dose of AZD8931 at 25 mg/kg was chosen based on previous study. Paclitaxel alone also delayed tumor growth over treatment compared with the control group in both xenografted human IBC models, but the effect of inhibition was much less than that seen in the AZD8931 alone group. The combination of paclitaxel + AZD8931 was more effective at delaying tumor growth than the control and other treatment groups in both xenografted IBC models. The difference was significant for paclitaxel + AZD8931 versus paclitaxel alone in SUM149 (P = 0.01; Figure 4A) and FC-IBC-02 (P < 0.01; Figure 4C). However, the difference was not statistically significant compared with AZD8931 alone.

In addition, we also examined the weight of xenografted tumors at the end of study. The inhibitory pattern of tumor size following different treatments was very similar to that seen in tumor growth curves in both IBC models. The combination of paclitaxel + AZD8931 was more effective at reducing tumor sizes than all of the other treatment groups. The difference was also significant for paclitaxel + AZD8931 versus paclitaxel alone in SUM149 (P = 0.008; Figure 4B) and FC-IBC-02 (P = 0.001; Figure 4D) models. Compared with AZD8931 alone, the difference was marginally significant for SUM149 tumors (P = 0.056) and FC-IBC-02 tumors (P = 0.07).Finally, we examined the expression of total EGFR, HER2, HER3, phosphorylated EGFR, phosphorylated HER2, and phosphorylated HER3 in SUM149 xenografted tumors by immunohistochemistry. As expected, high level expression of EGFR and low levels of HER2 and HER3 expression were observed in both AZD8931-treated and control tumors. The expression of phosphorylated EGFR, HER2, and HER3 was inhibited in AZD8931-treated tumors compared with control tumors (Figure 5A). The average of pathologist’s H-score for both membrane and cytoplasmic staining was shown in Figure 5B. Together, we conclude that AZD8931 significantly inhibits tumor growth in HER2 non-amplified IBC xenograft models by inhibiting EGFR, HER2 and HER3 phosphorylation. The combination of paclitaxel + AZD8931 was more effective than single agent paclitaxel or AZD8931 alone at delaying tumor growth.

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Sapitinib (AZD-8931) showed potent tumor growth inhibition in various xenograft models, driven by EGFR alone (LoVo, PC9) or EGFR and HER2 (BT474C, Calu3, and FaDu) with concomitant pharmacodynamic changes (e.g., phosphorylated EGFR, HER2, and/or HER3).8 Compound 2/Sapitinib (AZD-8931) was evaluated head to head against 1 and 7 in the LoVo mouse xenograft model: based on high potency against EGFR, better exposure in mouse, and better plasma fraction unbound, it showed better tumor growth inhibition (76% inhibition at 100 mg/kg oral dose once daily vs 36% for 1 and 28% for 7 at the same dose, Figure 4).

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HER3 inhibition blocked EC CM-induced CRC tumor in vivo [4]
In order to validate the effects of EC CM on CRC cell growth in vivo, we used a proof-of-principle subQ xenograft tumor model with Luciferase-labeled HCP-1 cells. HCP-1 cells were pretreated with either CRC or LPEC-1 CM and then injected subQ in an inoculation mixture of concentrated CM and Matrigel. As a result, HCP-1 tumors injected with LPEC-1 CM had significantly greater tumor burden and volume over time (Suppl. Fig. 6A—C) compared to the control group injected with CRC CM. After tumors were harvested, HCP-1 tumors treated with LPEC-1 CM were significantly larger and weighed more than those treated with HCP-1 control CM (Suppl. Fig. 6D—F). The effects of blocking HER3 on CRC tumor growth were further determined by the subQ xenograft tumor model with treatment of the HER3 inhibitor Sapitinib (AZD-8931). After HCP-1 cells were injected subQ in the mixture of CM and Matrigel as described above, mice were then treated with either vehicle or AZD8931 by gavage and the tumor growth was monitored over time (Fig. 6). Our results showed that LPEC-1 CM treated tumors led to significantly greater tumor growth, as expected. More importantly, AZD8931 significantly inhibited the tumor growth in both CRC CM and LPEC-1 CM-treated CRC tumors compared with tumors without AZD8931 treatment (Fig. 6B). In addition, CRC tumors that were treated by AZD8931 had significantly lower tumor weight compared with tumors not treated with AZD8931, leading to ~2-fold decrease compared to CRC CM treated tumors, and > 4-fold decrease compared to LPEC-1 CM treated tumors (Fig. 6C).

Isolated kinase assays [1]
Human EGFR and erbB2 intracellular kinase domains have been cloned and expressed in the baculovirus/Sf21 system. Using the ELISA method, the inhibitory activity of Sapitinib (AZD-8931) is assessed with ATP at Km concentrations (0.4 mM for erbB2 and 2 mM for EGFR).[1]

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In vitro EGFR phosphorylation studies [1]
KB cells were grown in RPMI 1640 containing 10% FCS for 72 h, serum starved (0% FCS) for 24 h, and then incubated with Sapitinib (AZD-8931), gefitinib, or lapatinib for 90 min before stimulating for 5 min with 15 ng/mL recombinant EGF to increase EGFR phosphorylation to 90% of max (ED90) to allow interassay comparison. The cells were lysed in RIRA buffer [50 mmol/L tris (pH 7.4), 1% NP40, 0.25% deoxycholate, 150 mmol/L NaCl, 1 mmol/L sodium orthovanidate, 1 mmol/L EDTA, and Roche protease inhibitor cocktail] and EGFR phosphorylation was measured by a sandwich ELISA.

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In vitro drug sensitivity testing—cell viability assay [1]
To determine their antiproliferative activity against cell lines grown in vitro, Sapitinib (AZD-8931), gefitinib, and lapatinib were tested in a panel of NSCLC and SCCHN cell lines. Cells were incubated for 96 h with a suitable range of concentrations of drug to ensure accurate estimation of the inhibitor concentration required to give 50% growth inhibition (GI50; typically between 0.001-10 μmol/L). Viable cell number was determined by 4 h of incubation with MTS Colorimetric Assay reagent and absorbance measured at 490 nm on a spectrophotometer. Each experiment was carried out in triplicate for each drug concentration and data are presented as geometric means. Sensitivity groupings of GI50 data were <1 μmol/L (classed as sensitive), 1 to 7 μmol/L (classed as intermediate), and >7 μmol/L (classed as resistant).

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Receptor tyrosine kinase (RTK) array [4]
RTK array kit was used and the assay was performed according to the manufacturer’s instructions. In brief, 0.5 ×106 HCP-1 cells were incubated in 1% FBA medium overnight, and then treated with control or LPEC-1 CM for 30 minutes. Cell lysates were prepared with lysis buffer from the kit and 300 μg total proteins from each group were loaded to the membranes. Intensities of spots for P-EGFR, P-MET, and P-HER3 on the same film were measured and compared with the reference spots by ImageJ version 1.47.

AZD8931 is tested in a panel of NSCLC and SCCHN cell lines to ascertain its antiproliferative activity against cell lines grown in vitro. AZD8931 (0.001-10 μM) is added to cells and incubated for 96 hours. After incubating the MTS Colorimetric Assay reagent for four hours, the absorbance at 490 nm is measured using a spectrophotometer to determine the viable cell count.

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Cell proliferation and apoptotic assay [2]
SUM149 and FC-IBC-02 cells (2 × 103) were seeded in triplicate in a 96-well plate and cultured overnight. Cells were treated with Sapitinib (AZD-8931) at indicated concentration for 72 hrs. Cell proliferation was monitored at the indicated times, absorbance at 490 nm was measured using a microplate reader using the MTS assay according to the manufacturer’s instruction. Apoptotic cells were measured by Annexin V staining. Cells (1 × 105) were treated with 1 μM Sapitinib (AZD-8931) for 48 and 72 hrs. Cells were harvested and labeled with Annexin V-PE and 7-amino-actinomycin D (7-AAD) according to the manufacturer’s instructions. The samples were then analyzed by Guava system on a GuavaPC personal flow cytometer.

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MTT assay [4]
CRC cells were seeded at 3,000 cells/well in 96-well plates, cultured in 1% FBS overnight and then treated with CM for the indicated times. When the HER3 inhibitor Sapitinib (AZD-8931) (2 μM), HER3 antibody MM-121 (125 μg/ml), or 5-FU (2 μg/ml) were used, cells were pretreated with AZD8931 or MM-121in 1% FBS medium overnight, and then cultured with or without 5-FU and AZD8931 or MM-121 in CM for 72 hours. Cell viability was assessed by adding MTT substrate (0.25% in PBS, Sigma) in growth medium (1:5 dilution) to cells for 1 hour at 37 °C. Cells were washed with PBS and 50 μl DMSO was added. Optical density was measured at 570 nm and relative MTT was presented as % of control.

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Cell apoptosis [4]
CRC cells were cultured in 1% FBS overnight and then cultured with or without 5-FU with CM for 48 hours (HCP-1 cells) or 72 hours (HT29 and SW480). When the HER3 inhibitor Sapitinib (AZD-8931) was used, cells were pretreated with AZD8931 in 1% FBS medium overnight, and then cultured with or without 5-FU and AZD8931 in CM. Cell apoptosis was determined using the FITC Annexin V Apoptosis Detection Kit I as described before. In brief, single suspended cells were double-stained with FITC Annexin V and propidium iodide and analyzed by fluorescence-activated cell sorting. Double-positive cells were counted as apoptotic cells and presented as a % of the total population.

The mice used are severe combined immunodeficient and Swiss nude (nu/nu genotype). A 1% (v/v) solution of polyoxyethylenesorbitan monooleate (Tween 80) in deionized water is used to suspend Sapitinib (AZD-8931), GW572016, and ZD1839. Once (qd) or twice daily (bid), animals are given Sapitinib (AZD-8931) (6.25-50 mg/kg), GW572016 (100 mg/kg), ZD1839 (100-150 mg/kg), or vehicle control by oral gavage. Tumor growth characteristics dictate the duration of each study, with studies coming to an end when tumors reach less than 1 cm³. Tumor volume and percentage tumor growth inhibition are determined, and a standard t test is used to statistically analyze any changes in tumor volume (a P value of less than 0.05 is deemed statistically significant).

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All human tumor xenografts were established by s.c. injecting 0.1 mL tumor cell suspension (between 4 × 106 and 9 × 107 cells) mixed 1:1 with Matrigel, with the exception of LoVo, which did not include Matrigel, and Calu-3, which used 30% Matrigel. The BT474c cell line was subcloned from the human breast cell line BT474. For the BT474c model, nude mice were implanted with estradiol 0.36 mg 60-d release pellets (Innovative Research) 24 h before being implanted with tumor cells. In all models, the animal weight and tumor volumes (as determined using bilateral caliper measurements) were monitored twice weekly, as previously described. Group sizes (n = 8-17/group) for each xenograft model were determined by power analysis, and randomization occurred when tumors reached the determined size (>0.2 cm3). Sapitinib (AZD-8931), lapatinib, and gefitinib were suspended in a 1% (v/v) solution of polyoxyethylenesorbitan monooleate (Tween 80) in deionized water. Animals were given Sapitinib (AZD-8931) (6.25-50 mg/kg), lapatinib (100 mg/kg), gefitinib (100-150 mg/kg), or vehicle control once (qd) or twice daily (bid) by oral gavage. The duration of each study was determined by tumor growth characteristics, with studies ending once tumors reached ∼1 cm3. Tumor volume and percentage tumor growth inhibition were calculated as previously described and statistical analysis of any change in tumor volume was carried out using a standard t test (P value of lower then 0.05 was considered to be statistically significant).

To determine the pharmacodynamic effects of Sapitinib (AZD-8931), lapatinib, or gefitinib treatment, groups of tumor-bearing mice (typically n = 5/group) were humanely culled following dosing and blood and tumors removed. Frozen tumor samples were lysed in 6-fold (volume/weight) RIRA Buffer (containing 1% phosphatase inhibitors 1 and 2 and protease inhibitor) using a Polytron homogenizer. The protein content of all tumor lysates was evaluated using detergent-compatible protein assay reagents. Phospho-EGFR, phospho-erbB2, and phospho-erbB3 levels were analyzed in tumor samples using the multiplex MesoScale Discovery kit or by ELISA as described above. Pharmacodynamic effects were also examined by immunohistochemistry (IHC). Antigen retrieval was done on formalin-fixed, paraffin-embedded tumor sections.

For pharmacokinetic studies, plasma samples were processed and Sapitinib (AZD-8931) concentrations were determined by high performance liquid chromatography tandem mass spectrometry. For the complete summary of xenograft models, treatment and IHC protocols, pharmacodynamic sampling, image analysis details, and statistical methods see Supplementary Materials and Methods. [1]

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UM149 and FC-IBC-02 (3 × 106) cells were suspended in 200 μL of 1:1 ratio of phosphate-buffered saline/matrigel and orthotopically injected into the mammary fat pads of six week old female C.B-17 severe combined immunodeficient (SCID) mice. Tumor volume was calculated from the formula TV = L*W*H*0.5236 where L, W, and H are the tumor dimensions in three perpendicular dimensions by caliper measurement. When tumor volumes were approximately 50 mm3 for SUM149 cells or 80 mm3 for FC-IBC-02 cells, the mice were randomly allocated into four groups (5 mice per group) and treatments were initiated. Sapitinib (AZD-8931) was suspended in a 1% (v/v) solution of polyoxyethylenesorbitan monooleate (Tween 80) in deionized water and given once daily by oral gavage at 25 mg/kg for 4 weeks. Paclitaxel solution was diluted in saline and given twice weekly by subcutaneously injection at 10 mg/kg. The control-group received 1% Tween 80 vehicle treatment. Mice were sacrificed at 33 days (SUM149) or 26 days (FC-IBC-02) post treatments. Tumors were surgically removed and weighed. [2]

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Tumor-bearing mice were treated with Sapitinib (AZD-8931) at 50 mg/kg/day for 4 days. Tumors were removed and fixed at 4 hrs after fourth dose. Formalin-fixed paraffin-embedded tumors were cut onto glass slides and processed for immunohistochemical (IHC) staining as previously described. In brief, antigen retrieval was performed on formalin-fixed, paraffin-embedded tumor sections. A polymer detection system (DAKO Envision + K4007) was used for secondary detection and sections were counterstained with Carazzi’s hematoxylin. Semiquantitative scoring was carried out by light microscopy by a pathologist (CW) for immunohistochemical brown staining on a four point scale (0+, none; 1+, weak; 2+, moderate; 3+, strong) and for percentage (%) distribution, to calculate an H-Score (sum of 1 x% 1+, 2 x% 2+, and 3 x% 3+). Cytoplasmic and membrane staining was recorded. [2]

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HCP-1 cells labeled with a CMV-driven luciferase reporter were pretreated with CM for 24 hours and then suspended in an inoculation matrix (1:1 mix of growth-factor-reduced Matrigel and concentrated HCP-1 or LPEC-1 CM) and injected subcutaneously into the right flanks of athymic/nude mice (1×106 cells in 100 μl/injection, n=10 mice/group). After injection, tumor burden was assessed by bioluminescence with the In Vivo Imaging System (IVIS) and D-Luciferin substrate according to the manufacturer’s instructions. Tumor volumes were measured with a caliper. Owning to Hurricane Harvey, we could measure bioluminescence with IVIS only on Day 4 and Day 15, and we measured tumor volumes with a caliper on Day 4, Day 11, and Day 15 after injection. Sapitinib (AZD-8931) was suspended in 1% (v/v) solution of polyoxyethylenesorbitan monooleate (Tween 80) in deionized water and gavaged once daily from Day 1 at 25mg/kg in 100μl per mouse, with 1% Tween 80 only for control groups. All mice were euthanized when 3 mice from any group had tumor size reached 1,000mm3, and tumors were harvested for weighing. [4]

Sapitinib (AZD-8931) exhibited no CYP P450 inhibition (IC50 > 10 μM against 1A2, 2C9, 2C19, 2D6, and 3A4). It displayed high fraction unbound in rat and human plasma (higher than 1 or 7). The higher fraction unbound seen with the 2-fluoro 3-chloro aniline 2/Sapitinib (AZD-8931) compared to the 4-fluoro 3-chloro aniline 7 is likely linked to the reduced lipophilicity associated with this aniline (Table 4) and is a pattern observed with a wider set of matched pairs.
Furthermore, 2/Sapitinib (AZD-8931) exhibited a pH dependent aqueous solubility due to the two ionizable sites (piperidine pKa 6.7, quinazoline pKa 5.0; aqueous solubility at pH 6.8 (phosphate buffer): 17 μM) and good permeability as measured in CaCo-2 cells (Papp,A to B 15.9 × 10–6 cm·s–1 at 10 μM). Compound 2 displayed favorable oral pharmacokinetics in rat and dog (low clearance and good bioavailability, see Table 5) and low human hepatocyte turnover (Clint < 4.5 μL/min/106 cells). In nude mouse after oral administration at 50 mg/kg, 2 showed improved exposure (AUC: 66 μM·h) compared to 7 and 1 (AUC: 19 μM·h for both). [3]

[1]. AZD8931, an equipotent, reversible inhibitor of signaling by epidermal growth factor receptor, ERBB2 (HER2), and ERBB3: a unique agent for simultaneous ERBB receptor blockade in cancer. Clin Cancer Res. 2010 Feb 15;16(4):1159-69.

[2]. AZD8931, an equipotent, reversible inhibitor of signaling by epidermal growth factor receptor (EGFR), HER2, and HER3: preclinical activity in HER2 non-amplified inflammatory breast cancer models. J Exp Clin Cancer Res. 2014 May 30;33:47.

[3]. Discovery of AZD8931, an Equipotent, Reversible Inhibitor of Signaling by EGFR, HER2, and HER3 Receptors. ACS Med Chem Lett. 2013 May 31;4(8):742-6.

[4]. Endothelial Cells Promote Colorectal Cancer Cell Survival by Activating the HER3-AKT Pathway in a Paracrine Fashion. Mol Cancer Res. 2019 Jan;17(1):20-29.

Sapitinib is a member of the class of quinazolines that is 4-amino-7-methoxyquinazoline in which the amino group has been substituted by a 3-chloro-2-fluorophenyl group and in which position 6 of the quinoline ring has been substituted by a {1-[2-(methylamino)-2-oxoethyl]piperidin-4-yl}oxy group. Sapitinib is a dual tyrosine kinase inhibitor (TKI) of epithelial growth factor receptors (EGFR) HER2 and HER3. It has a role as an epidermal growth factor receptor antagonist and an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor. It is a member of quinazolines, a member of piperidines, a member of monofluorobenzenes, a member of monochlorobenzenes, an aromatic ether, a secondary amino compound and a tertiary amino compound.
Sapitinib has been used in trials studying the treatment and basic science of Neoplasms, Breast Cancer, Breast Neoplasms, Metastatic Cancer, and Metastatic Breast Cancer, among others.
Sapitinib is an erbB receptor tyrosine kinase inhibitor with potential antineoplastic activity. erbB kinase inhibitor AZD8931 binds to and inhibits erbB tyrosine receptor kinases, which may result in the inhibition of cellular proliferation and angiogenesis in tumors expressing erbB. The erbB protein family, also called the epidermal growth factor receptor (EGFR) family, plays major roles in tumor cell proliferation and tumor vascularization.

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Purpose: To test the hypothesis that simultaneous, equipotent inhibition of epidermal growth factor receptor (EGFR; erbB1), erbB2 (human epidermal growth factor receptor 2), and erbB3 receptor signaling, using the novel small-molecule inhibitor AZD8931, will deliver broad antitumor activity in vitro and in vivo. Experimental design: A range of assays was used to model erbB family receptor signaling in homodimers and heterodimers, including in vitro evaluation of erbB kinase activity, erbB receptor phosphorylation, proliferation in cells, and in vivo testing in a human tumor xenograft panel, with ex vivo evaluation of erbB phosphorylation and downstream biomarkers. Gefitinib and lapatinib were used to compare the pharmacological profile of AZD8931 with other erbB family inhibitors. Results: In vitro, AZD8931 showed equipotent, reversible inhibition of EGFR (IC(50), 4 nmol/L), erbB2 (IC(50), 3 nmol/L), and erbB3 (IC(50), 4 nmol/L) phosphorylation in cells. In proliferation assays, AZD8931 was significantly more potent than gefitinib or lapatinib in specific squamous cell carcinoma of the head and neck and non-small cell lung carcinoma cell lines. In vivo, AZD8931 inhibited xenograft growth in a range of models while significantly affecting EGFR, erbB2, and erbB3 phosphorylation and downstream signaling pathways, apoptosis, and proliferation. Conclusions: AZD8931 has a unique pharmacologic profile providing equipotent inhibition of EGFR, erbB2, and erbB3 signaling and showing greater antitumor activity than agents with a narrower spectrum of erbB receptor inhibition in specific preclinical models. AZD8931 provides the opportunity to investigate whether simultaneous inhibition of erbB receptor signaling could be of utility in the clinic, particularly in the majority of solid tumors that do not overexpress erbB2.[1]

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Introduction: Epidermal growth factor receptor (EGFR) overexpression has been associated with prognostic and predictive value in inflammatory breast cancer (IBC). Epidermal growth factor receptor 2 (HER2) overexpression is observed at a higher rate in IBC compared with noninflammatory breast cancer. Current clinically available anti-HER2 therapies are effective only in patients with HER2 amplified breast cancer, including IBC. AZD8931 is a novel small-molecule equipotent inhibitor of EGFR, HER2, and HER3 signaling. In this study, we investigated the antitumor activity of AZD8931 alone or in combination with paclitaxel using preclinical models of EGFR-overexpressed and HER2 non-amplified IBC cells. Methods: Two IBC cell lines SUM149 and FC-IBC-02 derived from pleural effusion of an IBC patient were used in this study. Cell growth and apoptotic cell death were examined in vitro. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. AZD8931 was given by daily oral gavage at doses of 25 mg/kg, 5 days/week for 4 weeks. Paclitaxel was subcutaneously injected twice weekly. Results: AZD8931 significantly suppressed cell growth of IBC cells and induced apoptosis of human IBC cells in vitro. Significantly, we showed that AZD8931 monotherapy inhibited xenograft growth and the combination of paclitaxel + AZD8931 was demonstrably more effective than paclitaxel or AZD8931 alone treatment at delaying tumor growth in vivo in orthotopic IBC models. Conclusion: AZD8931 single agent and in combination with paclitaxel demonstrated signal inhibition and antitumor activity in EGFR-overexpressed and HER2 non-amplified IBC models. These results suggest that AZD8931 may provide a novel therapeutic strategy for the treatment of IBC patients with HER2 non-amplified tumors.[2]

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Deregulation of HER family signaling promotes proliferation and tumor cell survival and has been described in many human cancers. Simultaneous, equipotent inhibition of EGFR-, HER2-, and HER3-mediated signaling may be of clinical utility in cancer settings where the selective EGFR or HER2 therapeutic agents are ineffective or only modestly active. We describe the discovery of AZD8931 (2), an equipotent, reversible inhibitor of EGFR-, HER2-, and HER3-mediated signaling and the structure-activity relationships within this series. Docking studies based on a model of the HER2 kinase domain helped rationalize the increased HER2 activity seen with the methyl acetamide side chain present in AZD8931. AZD8931 exhibited good pharmacokinetics in preclinical species and showed superior activity in the LoVo tumor growth efficacy model compared to close analogues. AZD8931 is currently being evaluated in human clinical trials for the treatment of cancer.[3]

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The regulation of colorectal cancer cell survival pathways remains to be elucidated. Previously, it was demonstrated that endothelial cells (EC) from the liver (liver parenchymal ECs or LPEC), the most common site of colorectal cancer metastases, secrete soluble factors in the conditioned medium (CM) that, in turn, increase the cancer stem cell phenotype in colorectal cancer cells. However, the paracrine effects of LPECs on other colorectal cancer cellular functions have not been investigated. Here, results showed that CM from LPECs increased cell growth and chemoresistance by activating AKT in colorectal cancer cells in vitro. Using an unbiased receptor tyrosine kinase array, it was determined that human epidermal growth factor receptor 3 (ERBB3/HER3) was activated by CM from LPECs, and it mediated AKT activation, cell growth, and chemoresistance in colorectal cancer cells. Inhibition of HER3, either by an inhibitor AZD8931 or an antibody MM-121, blocked LPEC-induced HER3-AKT activation and cell survival in colorectal cancer cells. In addition, CM from LPECs increased in vivo tumor growth in a xenograft mouse model. Furthermore, inhibiting HER3 with AZD8931 significantly blocked tumor growth induced by EC CM. These results demonstrated a paracrine role of liver ECs in promoting cell growth and chemoresistance via activating HER3-AKT in colorectal cancer cells. IMPLICATIONS: This study suggested a potential of treating patients with metastatic colorectal cancer with HER3 antibodies/inhibitors that are currently being assessed in clinical trials for various cancer types.[4]

C31H33CLFN5O11

706.07

705.184912

C, 52.73; H, 4.71; Cl, 5.02; F, 2.69; N, 9.92; O, 24.92

1196531-39-1

848942-61-0; 1196531-39-1 (fumurate)

44470103

Typically exists as solids at room temperature

ClC1=CC=CC(=C1F)NC1=C2C(C=C(C(=C2)OC2CCN(CC(NC)=O)CC2)OC)=NC=N1.OC(/C=C/C(=O)O)=O.OC(/C=C/C(=O)O)=O

AZD-8931 fumarate; Sapitinib difumurate; 1196531-39-1; AZD8931 diFuMaric acid; AZD8931 difumaricacid; diFuMaric acid; AZD-8931 difumaricacid; RD1QAE9R4N; UNII-RD1QAE9R4N;

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.)