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Purity: ≥98%
Defactinib HCl (formerly known as VS-6063 HCl, PF-04554878 HCl) is a novel, potent, selective, and orally bioactive small molecule inhibitor of the FAK (focal adhesion kinase), it inhibits the phosphorylation of FAK at the Tyr397 site in a time- and dose-dependent manner. FAK is an essential component of numerous oncogenic pathways and is a nonreceptor tyrosine kinase. It has been documented that several tumor types, such as ovarian, colon, and breast cancers, have elevated FAK expression. Defactinib may therefore have antitumor effects because it inhibits FAK. Tumor cell migration, proliferation, and survival may be inhibited by defactinib's inhibition of FAK, which also inhibits integrin-mediated activation of multiple downstream signal transduction pathways, such as ERK, JNK/MAPK, and PI3K/Akt.
| Targets |
FAK
|
|---|---|
| ln Vitro |
Defactinib (VS-6063) inhibits FAK phosphorylation at the Tyr397 site in a time- and dose-dependent manner. AKT and YB-1 levels in taxane-resistant cell lines are decreased by defactinib, according to RPPA data. The dose-dependent inhibition of pFAK (Tyr397) expression in all cell lines is statistically significant when Defactinib is used. pFAK (Tyr397) expression is inhibited by defactinib for three hours, and by 48 hours, expression gradually returns[1].
Effect of FAK Inhibition on Sensitivity to PTX [1] We first tested the in vitro effects of Defactinib/VS-6063 on FAK phosphorylation. The expression of pFAK (Tyr397) was statistically significantly inhibited by VS-6063 in a dose-dependent manner in all cell lines (Figure 1A; Supplementary Figure 1A, available online). VS-6063 inhibited pFAK (Tyr397) expression within 3 hours, with a gradual return of expression by 48 hours (Figure 1B; Supplementary Figures 1B and 2, available online). Supplementary Figure 3 (available online) represents a typical experiment in which statistical analysis of triplicate experiments showed no statistically significant changes in FAK phosphorylation at the other residues tested (Tyr 576/577, Tyr 925, or Tyr 861). Because Pyk2 and FAK are approximately 60% identical in the central catalytic domain, we also tested Tyr402, Tyr 579/580, and Tyr 881 within PYK2. Phosphorylation inhibition of Tyr402 was observed only in the HeyA8 cells, with no phosphorylation residues changed in the HeyA8-MDR cell line after VS-6063 (1 µM) treatment for 1 hour. VS-6063/Defactinib as a single agent (0–1 µM) did not affect the growth of any of the cells or their taxane-resistant counterparts (Supplementary Figure 4, available online). However, the combination of PTX and VS-6063 (1 µM) was more effective than either agent alone. PTX cytotoxicity was 2.1- to 4.9-fold greater in combination with VS-6063 compared with PTX alone (Figure 1C; Supplementary Figure 1C, available online). The combination effect of PTX and VS-6063 was evaluated with combination index by the Chou–Talalay method (20). Interactions between PTX and VS-6063 were synergistic in both HeyA8 and HeyA8-MDR cells (Figure 1D). Simultaneous exposure to doses of PTX and VS-6063 at ratios of 1:1000 for HeyA8 and 1:10 for HeyA8-MDR had a synergistic inhibitory effect on cell growth, with combination index values of 0.953 and 0.705, respectively. No synergy was noted between PTX and VS-6063 in SKOV3ip1 or SKOV3-TR cells, but an additive inhibitory effect on proliferation was noted (data not shown). Next, we tested the effects of VS-6063/Defactinib based therapy on proliferation and apoptosis. Compared with PTX alone, the combination of VS-6063 (1 µM) with PTX (at the median inhibitory concentration [IC50] levels of PTX for the SKOV3 and HeyA8 cells of 8.5 and 6.3 nmol/L, respectively) decreased the proliferation rate in both taxane-sensitive and taxane-resistant cells (Figure 1E; Supplementary Figure 1D, available online). For apoptosis, the greatest effects were observed with combined PTX and VS-6063 (Figure 1F; Supplementary Figure 1E, available online). These findings suggest that VS-6063 in combination with PTX had at least an additive effect on both cell proliferation and apoptosis. PTX increased the proportion of the cell population in the G2 cell cycle fraction to 51.0% ± 0.4% in the HeyA8 cells compared with control (34.3% ± 0.3%; P < .001), and when combined with VS-6063 treatment, the percentage of cells in the G2 phase statistically significantly increased to 60.8% ± 0.9% (P < .001 compared with PTX). HeyA8-MDR cells were not arrested in G2 phase in either the PTX group or the combination group (P = .05) (Figure 1G; Supplementary Figure 1F, available online). Impact of FAK Inhibition on Downstream Signaling [1] Because FAK is known to signal through beta 1 integrin, we first tested whether beta 1 integrin levels were affected by Defactinib/VS-6063, but no statistically significant changes were noted (Supplementary Figure 8, available online). To identify potential signaling pathways downstream of FAK in taxane-resistant cell lines, RPPAs were used and analyzed (DAVID Bioinformatics Resources; http://david.abcc.ncifcrf.gov/). In the VS-6063-treated group, 53 of the 161 proteins analyzed demonstrated a statistically significant change compared with the untreated group in both resistant cell lines (P < .05) (Figure 3A; Supplementary Table 1, available online). We found that the AKT pathway was the most involved pathway, including pFAK (Tyr 397), pAKT (Thr308), GSK-3 alpha/beta (Ser21/9), p27(T198), p70S6K (Thr389), PRAS40 (T246), and pYB-1(Ser102). Among these, pYB-1 (Ser102) was the most statistically significantly decreased protein (36.6% decreased by Defactinib/VS-6063 treatment; P < .001). Given the potential role of YB-1 in oncogenic and drug-resistance pathways, we focused on the potential relationship between FAK and YB-1 in subsequent studies.Figure 3B shows that PTX increased nuclear YB-1 expression, whereas PTX combined with VS-6063 decreased its expression in the HeyA8-MDR cells. Immunofluorescence studies also revealed similar findings (Figure 3C). FAK inhibition suppresses the malignant progression of ESCC cells [2] Because FAK is hyperactivated in several ESCC cell lines, including KYSE150, KYSE180, KYSE30, KYSE410, KYSE450, KYSE510, Colo680, and KYSE70,9 we measured the growth inhibitory effect of Defactinib on these ESCC cell lines using MTS assay. As shown in Figure 1A, Defactinib treatment for 72 h dose-dependently decreased the viability of these indicated ESCC cell lines (Figure 1A). We further observed the anti-invasive or migratory ability of defactinib in KYSE410 and KYSE510 using Transwell assay. As shown in Figure 1B,C, defactinib dose-dependently inhibited the migration and invasion of indicated ESCC cells. Taken together, these results indicate that defactinib exerts excellent antitumor effects in ESCC cells. Next, FAK enhances the activity of PI3K/AKT pathway via interacting with PI3K catalytic subunit-p85. As shown in Figure 2E,F, defactinib dose-dependently disrupted the interaction between FAK and PI3K p85 subunit after 4 or 24 h treatment. In conclusion, the kinetics and magnitude of PI3K/AKT pathway inhibition suggest that this effect has an important role in the defactinib response. Effective inhibition of the downstream gene network by Defactinib [2] To further understand molecular effects elicited by Defactinib treatment, we analyzed the transcriptomic profile of KYSE410 cells incubated with 10 μM defactinib from 4 to 24 h (Figure 3A), and focused on transcripts significantly downregulated in defactinib treatment. As shown in Figure 3B–J, strikingly, several target genes were substantially enriched in same functional sets, such as chemoresistance (Figure 3B), cell cycle (Figure 3C), growth factors, cytokines, and chemokines (Figure 3D), malignant progression (Figure 3E), cell plasticity (Figure 3F), heat shock protein family (Figure 3G), metabolism (Figure 3H), oncogene (Figure 3I), or transcriptional factors (Figure 3J), after 4 or 24 h defactinib treatment. A detailed analysis of representative genes involved in these functional sets suggested that many of these genes were sustained inhibition by defactinib after 4 or 24 h treatment (Table S1). |
| ln Vivo |
At three hours, defactinib (VS-6063) doses of 25 mg/kg twice a day or higher statistically significantly inhibit pFAK (Tyr397); by the 24-hour mark, expression has returned. Consequently, the dosage regimen for the next round of therapeutic trials will be Defactinib administered at a rate of 25 mg/kg twice day. Female naked mice with peritoneal HeyA8 tumors are randomly assigned to 4 groups (n = 10 per group) for the purpose of therapy experiments. 1) weekly PTX intraperitoneal injection; 2) twice-daily oral administration of vehicle; 3) twice-daily oral administration of phosphate-buffered saline (control); 4) simultaneous administration of VDefactinib 25 mg/kg and PTX intraperitoneally weekly. In the HeyA8 model, PTX monotherapy reduced tumor weight by 87.4%; combination therapy produced the largest reduction in tumor weight, at 97.9% (P=0.05 compared with PTX). When comparing the combination group to PTX, there is a 92.7% tumor weight reduction in the SKOV3ip1 model (P<0.001)[1].
In Vivo Effects of VS-6063/Defactinib [1] Defactinib/VS-6063 doses of 25mg/kg twice a day or greater statistically significantly inhibited pFAK (Tyr397) at 3 hours, with return of expression noted by 24 hours (Supplementary Figure 5, available online). Therefore, administration of Defactinib/VS-6063 at 25mg/kg twice a day was selected as the dosing schedule for subsequent therapy experiments. For therapy experiments, female nude mice bearing HeyA8 tumors in the peritoneal cavity were randomly divided into 4 groups (n = 10 per group): 1) vehicle orally twice daily and phosphate-buffered saline intraperitoneally weekly (control); 2) VS-6063/Defactinib 25mg/kg orally twice daily; 3) PTX intraperitoneally weekly; and 4) both VS-6063 25mg/kg orally twice daily and PTX intraperitoneally weekly. There was an 87.4% reduction in tumor weight by PTX monotherapy in the HeyA8 model, and combination therapy resulted in the greatest tumor weight reduction, with a 97.9% reduction (P = .05 compared with PTX) (Figure 2, A and B). In the SKOV3ip1 model, a 92.7% tumor weight reduction was observed in the combination group compared with PTX (P < .001). Defactinib is efficient for inhibition of tumor malignancy in vivo [2] We further used three xenografted models, including subcutaneous tumor cell inoculation model (evaluation of tumor growth), popliteal lymph node metastasis model (evaluation of the lymph node metastatic ability of tumor cells), or lung colonization model (evaluation of metastatic ability of tumor cells), to comprehensively observe Defactinib-mediated antitumor effect in vivo. We subcutaneously inoculated KYSE410 and KYSE510 cells into the right flank of BALB/c mice. When xenografts reached approximately 100 mm3, we treated animals with defactinib (25 mg/kg/day, p.o.) for approximately 3 consecutive weeks and observed tumor growth. As shown in Figure 5A, after 2 weeks of treatment, tumor growth in defactinib group was significantly delayed, compared with control group. The tumor growth regression-mediated by defactinib was sustained to Week 3. Furthermore, after 3 weeks treatment, defactinib effectively blocked the activation of FAK in indicated tumor tissues (Figure S4). Results of popliteal lymph node metastasis model showed that FAK inhibition dramatically reduced the volume of ESCC cells in the lymph nodes from defactinib treatment group, compared with control group (Figure 5B). We further evaluated the effect of Defactinib on ESCC progression in a lung colonization model. Animals were intravenously injected with the indicated ESCC cells. As shown in Figure 5C, THE number of tumor nests in lungs from defactinib-treated group was significantly lower than that of control group. Results of Figure 5D showed that defactinib significantly extended the survival period of ESCC tumor-beared animals, compared with control treatment. IHC assay showed that dafactinib significantly decreased the expression of proliferation biomarker-Ki-67 (Figure 5E) and angiogenic biomarker-CD31 (Figure 5F) in indicated tumors. Importantly, histological analysis of heart, liver, spleen and kidney tissues showed no obvious alterations between control group and Defactinib treatment group, suggesting that defactinib did not produce significantly toxic effects on normal tissues (Figure 6A). Body weight between defactinib and control treatment groups was no apparent difference (Figure 6B). |
| Enzyme Assay |
In a manner that was dependent on both time and dosage, defactinib prevented FAK phosphorylation at the Tyr397 site. Defactinib and paclitaxel together significantly reduced proliferation and enhanced apoptosis, leading to tumor weight reductions of 92.7% to 97.9%. Data from RPPA indicated that in taxane-resistant cell lines, defactinib lowered AKT and YB-1 levels. In taxane-resistant cells, FAK inhibition improved chemosensitivity through a mechanism that was dependent on AKT and reduced YB-1 phosphorylation and, consequently, CD44. Increased nuclear YB-1 expression (χ²) = 37.7; P <.001) was correlated with nuclear FAK expression in human ovarian cancer samples. A statistically significant worse median overall survival (24.9 vs 67.3 months; hazard ratio = 2.64; 95% confidence interval = 1.38 to 5.05; P =.006) was linked to the coexpression of nuclear FAK and YB-1.
Enzyme-linked immunosorbent assay analysis of nulcear factor-κB transcriptional activity [2] Human nulcear factor-κB (NF-κB) p65 transcription factor activity assay kit was applied to evaluate the activity of NF-κB, according to the manufacturer's instructions. Briefly, nuclear extracts, DNA binding buffer, DTT, and transcriptional factor-activity assay reagent were added to 96-well (100 μl/well) for 2 h at room temperature. After washing four times with washing buffer, the 96-well plate was added with NF-κB p65 primary antibody solution (100 μl/well) for 1 h at room temperature. Then, horseradish peroxidase-conjugated secondary antibody, TMB one-step substrate reagent and stop solution were sequentially added. The optical denstiy value of NF-κB p65 activity was read at 450 nm using a microplate reader. The experiment was repeated five times. Microarray assay [2] The Agilent SurePrint G3 human gene expression v3 8 × 60 K microarray was used to evaluate the changes of downstream genes. Briefly, total ribonucleic acid (RNA) of KYSE410 cells was extracted and transcribed to complementary deoxyribonucleic acid (cDNA). Then, cDNA was labeled with cyanine-3-CTP, hybridized with microarray, which was washed and scanned using the Agilent Scanner G2505C. The fluorescent intensity data were extracted with Feature Extraction software, and then Genespring as used to obtain the raw data. The threshold set for upregulated or downregulated genes was a fold change of ≥2 and a p value of ≤.05. |
| Cell Assay |
The MTT assay is performed after 96 hours of treatment with increasing concentrations of defactinib for ovarian cancer cells. Experiments carried out in triplicate validate the results.
In Vitro Gene Silencing [1] YB-1 small interfering RNA (siRNA) 1 (target sequence 5′-CCUAUGGGCGUCGACCACA-3′) and siRNA2 (target sequence 5′-GUUCCAGUUCAAGGCAGUA-3′) and used to silence YB-1 expression in the ovarian cancer cell lines. A nonsilencing siRNA that did not share sequence homology with any known human mRNA from a Basic Local Alignment Search Tool (BLAST) search was used as control, as previously described. Western Blot Analysis [1] Lysates from cultured cells was prepared as previously described. Typically, 30 μg of protein was fractionated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Additional details for immunoblotting are provided in the Supplementary Methods (available online). Cell Viability, Proliferation, and Apoptosis Assays [1] Viability [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)], proliferation (5-ethynil-2’-deoxyuridine), and apoptosis (annexin-V phycoerythrin [PE]/7AAD staining) assays were performed as previously described. Cell proliferation/viability assay [2] The antiproliferative effect of Defactinib on ESCC cells was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) method. Briefly, 3 × 103 indicated cells in 100 μl of RPMI 1640 medium were seeded in 96-well plates. Twenty-four hours later, cells were attached and treated with different doses of Defactinib (0–25 μM) for 72 h. Then, the medium was discarded, and cells were incubated with MTS solution for 1 h. Plates were scanned spectrophotometrically at 490 nm, and the number of viable cells was positively correlated with the formazan product. Transwell migration/invasion assays [2] Migration of cells was assayed using Transwell chamber systems with polycarbonate membrane inserts containing 8-μm pore size. The indicated ESCC cells (~1 × 105 cells) were seeded in the upper chamber with 200 μl FBS-free RPMI 1640 medium, and the lower chamber was added with 1 ml of RPMI 1640 medium with 20% FBS. After 24 h, the upper chambers were fixed in methanol for 10 min, stained with 2% crystal violet solution for 30 min, and the nonmigratory cells in the upper chamber were removed with cotton swabs. The migratory cells were then photographed under a microscope. The experiment was repeated five times. For the transwell invasion assay, the membrane of the upper chamber was precoated with 50 μl of 2.5 mg/ml matrigel solution. Other experimental conditions were similar to the migration assay. |
| Animal Protocol |
Mice: The antitumor effects of defactinib are assessed by intraperitoneal injection of SKOV3ip1, SKOV3-TR, HeyA8, and HeyA8-MDR cells. Mice are randomized into four groups of ten (control, PTX alone, Defactinib alone, and PTX plus Defactinib) one week after the tumor cell injection. Three to four weeks later, treatment is started. Weekly intraperitoneal injections of PTX (2 mg/kg for SKOV3ip1 and SKOV3-TR) or 2.5 mg/kg for HeyA8 and HeyA8-MDR) are administered; twice-daily oral administration of defactinib (25 mg/kg) is recommended. HBSS was administered intraperitoneally once a week to control mice, while vehicle was given twice daily. Day 35 (SKOV3ip1 or SKOV3-TR), Day 28 (HeyA8 or HeyA8-MDR), or when any mouse appears moribund are the dates at which the mice are killed after being observed every day for side effects of the therapy. The total weight of the body, the mass and incidence of the tumor, and the quantity of tumor nodules are noted. The three methods used to treat tumors are formalin fixation, paraffin embedding, and snap freezing in a liquid nitrogen-based optimal cutting temperature (OCT) compound.
Female BALB/c nude mice aged 4 weeks old were purchased from Beijing Vital River Laboratories and maintained under standard pathogen-free conditions. All animal experiment was conducted in accordance with the protocol approved by the Institutional Review Board of Peking University Cancer Hospital & Institute. For evaluating the antitumor growth ability of Defactinib, the indicated ESCC cells (1 × 106/100 μl PBS/mouse) were subcutaneously inoculated into the right flank of each mouse (n = 5/group). When tumors reached approximately 100 mm3, animals were treated with defactinib (25 mg/kg/day, p.o.) for 3 weeks. The selected dosage and administration of defactinib were referred to previous report.17 For evaluation of FAK activity in tumor tissues, the human phospho-FAK (Tyr397) kit was used. The exact protocol was according to the manufacturer's instruction. Briefly, tumor lysates were incubated in wells of enzyme-linked immunosorbent assay plate for 2 h at 37°C. Then, lysates were discarded and detection antibody solutions were added into the appropriated well to measure the expression of phosphorylated FAK (Tyr397).[2] The antilymphatic metastasis effect of Defactinib on ESCC cells was examined using a popliteal lymph node metastasis model, which was established in mice by injecting the foot pads with the indicated ESCC cells (1 × 106/100 μl PBS/mouse, n = 5/group). Tumor and lymph node volumes were measured and calculated using the formula: length × width2 × 0.5.[2] The effect of Defactinib on ESCC metastasis was evaluated using a lung colonization model. Animals were intravenously injected with indicated ESCC cells (1 × 105/100 μl PBS/mouse, n = 5/group) and treated with defactinib. 4 weeks after injection, the mice were killed and the lungs were harvested for hematoxylin and eosin (H&E) staining.[2] For survival assay, treatments were consistent with above subcutaneous xenografted model. Survival event was recorded when tumor burden reached more than 1 cm3 in diameter or per absolute survival events. For immunohistochemical (IHC) staining evaluation of Ki-67 and CD31 expressions in tumor tissues, tumors were fixed in 10% neutral-buffered formalin for 24 h. Then, tumors were paraffin-embedded and 5 μm sections were cut. Ki-67 and CD31 antibodies (diluted at 1:500) were used for IHC staining.[2] For evaluation of toxicity of Defactinibon mice, organ tissues, including liver, heart, spleen, and kidney of mice in subcutaneous xenografted model, were collected for H&E staining.[2] |
| ADME/Pharmacokinetics |
Pharmacokinetics [https://pubmed.ncbi.nlm.nih.gov/27025608/]
VS-6063/Defactinib was rapidly absorbed, with median Tmax observed at 2.0 h (range 0.5–4.0 h) postdose following oral doses of 200–600 mg BID. Plasma VS-6063 exposure (Cmax and AUC) increased in a less than dose proportional manner and the mean AUC0-12 and AUC0-24 values remained relatively consistent across the full dose range evaluated (Fig. 1). Doses above 400 mg BID did not result in a significant increase in VS-6063 exposure. A similar Cmax was observed in the 400 and 600 mg BID dose cohorts on both Days 1 and 15 and mean AUC values were relatively consistent across the 200–600 mg BID dose range. Clearance appeared to be increased with dose on both Days 1 and 15, which is consistent with the less than dose proportional increase in exposure. Median half-life values ranged from 2.3 to 4.3 h across all dose regimens and both PK study days. The Day 1 mean CL/F values were 45.6, 105, and 204 L/h for the 200, 400, and 600 mg doses, respectively. The Day 15 mean CL/F values were 32. 1, 70, and 123 L/h for the 200, 400, and 600 mg doses, respectively (Table 3). VS-6063 was detected in the urine of all patients and appeared consistent across all dose cohorts. The mean renal clearance (CLr) values ranged from 0.0855 to 0.179 L/h, and the percent relative to the total dose administered ranged from 0.0439 to 0.356 %. All 9 subjects had systemic concentrations of the 4 metabolites of VS-6063/Defactinib that were evaluated (M2, M3, M4, and M5). Median plasma Tmax values for all metabolites were observed at 2.0–4.0 h postdose administration for all cohorts on both PK sampling days. Based on the relative plasma Cmax and AUC0-12 values for the metabolites compared to VS-6063 values, M2 was the most abundant metabolite, followed by M4, M3, and then M5. Both the M2 and M4 exposures appeared to be >10 % of the parent exposure, while M3 and M5 had exposures that were <10 % of the parent exposure. In the urine, the M2 metabolite was the most abundant and was excreted in amounts greater than the parent compound. |
| Toxicity/Toxicokinetics |
Safety and tolerability [https://pubmed.ncbi.nlm.nih.gov/27025608/]
Treatment-related adverse events (AEs) occurring in at least two subjects are summarized in Table 2. The most commonly reported AEs overall were unconjugated hyperbilirubinemia (7 patients, 78 %), fatigue (6 patients, 67 %), decreased appetite (4 patients, 44 %), and diarrhea (3 patients, 33 %). Only one patient in the 200 mg dose cohort experienced a Grade 3 AE of unconjugated hyperbilirubinemia. All other toxicities were manageable and were predominantly mild in intensity (Grade 1 or Grade 2) in severity. There were no AEs leading to death or SAEs, and no AEs leading to early study withdrawal. No DLTs were reported in any dose cohort. Hyperbilirubinemia was asymptomatic and its onset typically occurred within the first 2 weeks of initiating treatment. Patients with Grade 1 or 2 unconjugated hyperbilirubinemia were able to continue dosing, although bilirubin levels tended to fluctuate during treatment. Hyperbilirubinemia was reported across all dose cohorts, for 3 (100 %) patients in the 200 mg dose cohort, 2 (67 %) patients in the 400 mg dose cohort, and 2 (67 %) patients in the 600 mg dose cohort. One event of hyperbilirubinemia (200 mg cohort) was Grade 3 in severity. This patient had Grade 1–2 hyperbilirubinemia starting on Day 7; the Grade 3 event began on Day 42 and resolved 6 days after onset following interruption of study drug. All reports of hyperbilirubinemia were considered to be related to Defactinib. None of these subjects had concurrent increases in ALT or AST above ULN. The most common events of gastrointestinal disorders were diarrhea reported in 3 (33 %) subjects and nausea reported in 2 (22 %). Diarrhea was reported in 1 (33 %) subject in the 400 mg dose cohort and 2 (67 %) subjects in the 600 mg dose cohort. Nausea was reported in 1 (33 %) subject in the 200 mg dose cohort, and 1 (33 %) subject in the 600 mg dose cohort. Both reports of nausea were mild in severity as were 2 of the 3 reports of diarrhea; 1 subject in the 600 mg group experienced diarrhea of moderate intensity. No clinically meaningful changes in any ECG parameter were observed for any dose cohort and no subject had a QTc interval ≥500 ms or QTc increase from baseline >30 ms. |
| References |
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| Additional Infomation |
Defactinib Hydrochloride is the hydrochloride salt form of defactinib, an orally bioavailable, small-molecule focal adhesion kinase (FAK) inhibitor with potential antiangiogenic and antineoplastic activities. Defactinib inhibits FAK, which may prevent the integrin-mediated activation of several downstream signal transduction pathways, including those involving RAS/MEK/ERK and PI3K/Akt, thus inhibiting tumor cell migration, proliferation, survival, and tumor angiogenesis. The tyrosine kinase FAK, a signal transducer for integrins, is normally activated by binding to integrins in the extracellular matrix (ECM) but may be upregulated and constitutively activated in various tumor cell types.
Defactinib has been investigated for the treatment of Malignant Pleural Mesothelioma. Defactinib is an orally bioavailable, small-molecule focal adhesion kinase (FAK) inhibitor with potential antiangiogenic and antineoplastic activities. Defactinib inhibits FAK, which may prevent the integrin-mediated activation of several downstream signal transduction pathways, including those involving RAS/MEK/ERK and PI3K/Akt, thus inhibiting tumor cell migration, proliferation, survival, and tumor angiogenesis. The tyrosine kinase FAK, a signal transducer for integrins, is normally activated by binding to integrins in the extracellular matrix (ECM) but may be upregulated and constitutively activated in various tumor cell types. See also: Defactinib Hydrochloride (annotation moved to). Background We previously found focal adhesion kinase (FAK) inhibition sensitizes ovarian cancer to taxanes; however, the mechanisms are not well understood. Methods We characterized the biologic response of taxane-resistant and taxane-sensitive ovarian cancer models to a novel FAK inhibitor (VS-6063). We used reverse-phase protein arrays (RPPA) to identify novel downstream targets in taxane-resistant cell lines. Furthermore, we correlated clinical and pathological data with nuclear and cytoplasmic expression of FAK and YB-1 in 105 ovarian cancer samples. Statistical tests were two-sided, and P values were calculated with Student t test or Fisher exact test. Results We found that VS-6063 inhibited FAK phosphorylation at the Tyr397 site in a time- and dose-dependent manner. The combination of VS-6063 and paclitaxel markedly decreased proliferation and increased apoptosis, which resulted in 92.7% to 97.9% reductions in tumor weight. RPPA data showed that VS-6063 reduced levels of AKT and YB-1 in taxane-resistant cell lines. FAK inhibition enhanced chemosensitivity in taxane-resistant cells by decreasing YB-1 phosphorylation and subsequently CD44 in an AKT-dependent manner. In human ovarian cancer samples, nuclear FAK expression was associated with increased nuclear YB-1 expression (χ 2 = 37.7; P < .001). Coexpression of nuclear FAK and YB-1 was associated with statistically significantly worse median overall survival (24.9 vs 67.3 months; hazard ratio = 2.64; 95% confidence interval = 1.38 to 5.05; P = .006). Conclusions We have identified a novel pathway whereby FAK inhibition with VS-6063 overcomes YB-1–mediated paclitaxel resistance by an AKT-dependent pathway. These findings have implications for clinical trials aimed at targeting FAK.[1] The clinical therapeutic efficacy toward esophageal squamous cell carcinoma (ESCC) is undesirable, due to the lack of targeted agents. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in multiple fields of tumorigenesis, recently has been indicated as a promising therapeutic target in ESCC treatment. Here, we revealed that defactinib, a specific FAK inhibitor, effectively suppressed the malignancy of ESCC cells. Mechanistically, defactinib dose and time-dependently induced the dissociation of phosphoinositide-3-kinase (PI3K) from FAK, resultantly led to blockade of protein kinase B (AKT) signaling, and the expression of several oncogenes, such as SOX2, MYC, EGFR, MET, MDM2, or TGFBR2, identified by microarray and real-time polymerase chain reaction assay. Specifically, this FAK inhibition-mediated suppression of PI3K/AKT signaling and downstream ESCC specific biomarkers was maintained to 24 h in in vitro experiments to guarantee the treatment durability and efficacy. Importantly, defactinib suppressed tumor growth, metastatic ability, and increased overall survival of xenografted animals without producing significantly systematic toxicity. Our data suggest that FAK inhibition provides an excellent targeted therapy toward ESCC by effectively inhibiting PI3K/AKT pathway and downstream molecular network.[2] |
| Molecular Formula |
C20H22CLF3N8O3S
|
|---|---|
| Molecular Weight |
546.95
|
| Exact Mass |
546.118
|
| Elemental Analysis |
C, 43.92; H, 4.05; Cl, 6.48; F, 10.42; N, 20.49; O, 8.78; S, 5.86
|
| CAS # |
1073160-26-5
|
| Related CAS # |
Defactinib;1073154-85-4; 1073160-26-5; 1345713-71-4
|
| PubChem CID |
25117347
|
| Appearance |
White to off-white solid powder
|
| LogP |
4.165
|
| Hydrogen Bond Donor Count |
4
|
| Hydrogen Bond Acceptor Count |
13
|
| Rotatable Bond Count |
8
|
| Heavy Atom Count |
36
|
| Complexity |
804
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
Cl[H].S(C([H])([H])[H])(N(C([H])([H])[H])C1C(C([H])([H])N([H])C2C(C(F)(F)F)=C([H])N=C(N([H])C3C([H])=C([H])C(C(N([H])C([H])([H])[H])=O)=C([H])C=3[H])N=2)=NC([H])=C([H])N=1)(=O)=O
|
| InChi Key |
RCHQNUQAHJNRBY-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C20H21F3N8O3S.ClH/c1-24-18(32)12-4-6-13(7-5-12)29-19-28-10-14(20(21,22)23)16(30-19)27-11-15-17(26-9-8-25-15)31(2)35(3,33)34;/h4-10H,11H2,1-3H3,(H,24,32)(H2,27,28,29,30);1H
|
| Chemical Name |
N-methyl-4-[[4-[[3-[methyl(methylsulfonyl)amino]pyrazin-2-yl]methylamino]-5-(trifluoromethyl)pyrimidin-2-yl]amino]benzamide;hydrochloride
|
| Synonyms |
VS-6063 HCl; PF-04554878 HCl; Defactinib hydrochloride; Defactinib HCl; VS6063 HCl; VS 6063 HCl; VS-6063 HCl; PF04554878 HCl; PF 04554878 HCl; PF4554878 HCl; PF-4554878 HCl; PF4554878 HCl; 1073160-26-5; Defactinib hydrochloride [USAN]; UNII-L2S469LM49; VS-6063 HYDROCHLORIDE; L2S469LM49; Defactinib hydrochloride (USAN); DEFACTINIB HYDROCHLORIDE [WHO-DD];
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.67 mg/mL (1.22 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 6.7 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. Solubility in Formulation 2: ≥ 0.67 mg/mL (1.22 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 6.7 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. View More
Solubility in Formulation 3: ≥ 0.67 mg/mL (1.22 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 5% DMSO+50% PEG 300+5% Tween 80+ddH2O: 5mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8283 mL | 9.1416 mL | 18.2832 mL | |
| 5 mM | 0.3657 mL | 1.8283 mL | 3.6566 mL | |
| 10 mM | 0.1828 mL | 0.9142 mL | 1.8283 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT04439331 | Active Recruiting |
Drug: Defactinib Hydrochloride | Refractory Lymphoma Advanced Lymphoma |
National Cancer Institute (NCI) |
August 12, 2015 | Phase 2 |
| NCT04720417 | Active Recruiting |
Drug: Defactinib Hydrochloride Procedure: Biopsy |
Metastatic Uveal Melanoma | Thomas Jefferson University | January 26, 2021 | Phase 2 |
| NCT02465060 | Active Recruiting |
Drug: Defactinib Drug: Defactinib Hydrochloride |
Lymphoma Melanoma |
National Cancer Institute (NCI) |
August 17, 2015 | Phase 2 |
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 In vitro biological effects of VS-6063 on taxane-sensitive and taxane-resistant cell lines.J Natl Cancer Inst.2013 Oct 2;105(19):1485-95. |
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 In vivo effects of VS-6063 combined with paclitaxel (PTX).J Natl Cancer Inst.2013 Oct 2;105(19):1485-95. |
 VS-6063 restores YB-1–mediated paclitaxel (PTX) resistance.J Natl Cancer Inst.2013 Oct 2;105(19):1485-95. |
 VS-6063 downregulated YB-1 phosphorylation and nuclear translocation in taxane-resistant cells by an AKT-dependent pathway.J Natl Cancer Inst.2013 Oct 2;105(19):1485-95. |
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 FAK inhibition or silencing of YB-1 downregulates the expression of CD44.J Natl Cancer Inst.2013 Oct 2;105(19):1485-95. |
Impact of pFAK (Tyr 397) and pYB-1 (Ser 102) expressions on patient survival.J Natl Cancer Inst.2013 Oct 2;105(19):1485-95. |
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