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Backgrounds: The clinical features of autosomal dominant polycystic kidney disease (ADPKD) differ among patients even if they have the same gene mutation in PKD1 or PKD2. This suggests that there is diversity in the expression of other modifier genes or in the underlying molecular mechanisms of ADPKD, but these are not well understood. Methods: We primarily cultured solute carrier family 12 member 3 (SLC12A3)-positive urine-derived distal tubular epithelial cells from 6 ADPKD patients and 4 healthy volunteers and established immortalized cell lines. The diversity in receptor tyrosine kinase (RTK) phosphorylation by phospho-RTK array in immortalized tubular epithelial cells was analyzed. Results: We noted diversity in the activation of several molecules, including Met, a receptor of hepatocyte growth factor (HGF). Administration of golvatinib, a selective Met inhibitor, or transfection of small interfering RNA for Met suppressed cell proliferation and downstream signaling only in the cell lines in which hyperphosphorylation of Met was observed. In three-dimensional culture of Madin-Darby canine kidney (MDCK) cells as a cyst formation model of ADPKD, HGF activated Met, resulting in an increased total cyst number and total cyst volume. Administration of golvatinib inhibited these phenotypes in MDCK cells. Conclusion: Analysis of urine-derived tubular epithelial cells demonstrated diverse RTK phosphorylation in ADPKD, and Met phosphorylation was noted in some patients. Considering the difference in the effects of golvatinib on immortalized tubular epithelial cells among patients, this analysis may aid in selecting suitable drugs for individual ADPKD patients.

Small-cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers, and is characterized as extremely aggressive, often displaying rapid tumor growth and multiple organ metastases. In addition, the clinical outcome of SCLC patients is poor due to early relapse and acquired resistance to standard chemotherapy treatments. Hence, novel therapeutic strategies for the treatment of SCLC are urgently required. Accordingly, several molecular targeted therapies were evaluated in SCLC; however, they failed to improve the clinical outcome. The receptor tyrosine kinase MET is a receptor for hepatocyte growth factor (HGF), and aberrant activation of HGF/MET signaling is known as one of the crucial mechanisms enabling cancer progression and invasion. Here, we found that the HGF/MET signaling was aberrantly activated in chemoresistant or chemorelapsed SCLC cell lines (SBC-5, DMS273, and DMS273-G3H) by the secretion of HGF and/or MET copy number gain. A cell-based in vitro assay revealed that HGF/MET inhibition, induced either by MET inhibitors (crizotinib and golvatinib), or by siRNA-mediated knockdown of HGF or MET, constrained growth of chemoresistant SCLC cells through the inhibition of ERK and AKT signals. Furthermore, treatment with either crizotinib or golvatinib suppressed the systemic metastasis of SBC-5 cell tumors in natural killer cell-depleted SCID mice, predominantly through cell cycle arrest. These findings reveal the therapeutic potential of targeting the HGF/MET pathway for inhibition, to constrain tumor progression of SCLC cells showing aberrant activation of HGF/MET signaling. We suggest that it would be clinically valuable to further investigate HGF/MET-mediated signaling in SCLC cells.

Cancer is a major cause of death worldwide. Small molecule inhibitors have become a major therapeutic treatment for cancer. In this study, a series of novel 4-(pyrrolo[2,3-d]pyrimidine-4-yloxy)benzamide derivatives were designed, synthesized and evaluated for their antitumor activity against the A549, Hela and MCF-7 cell lines. Among them, the optimal compound 35 was found to possess excellent inhibitory activity against the A549, Hela and MCF-7 cell lines with IC50 values of 5.29 ± 0.58, 3.72 ± 0.91, and 9.23 ± 0.56 μM, which were superior to Golvatinib. The structure–activity relationship showed that the introduction of 7H-pyrrolo[2,3-d]pyrimidine along with the F atom of the central and terminal benzene was beneficial to the improvement of inhibitory activity of the target compounds. Besides, we took further study on the combined mode between compound 35 and c-Met kinase through molecular docking.

Almost all cancers show intrinsic and/or evasive resistance to vascular endothelial growth factor (VEGF) inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. From these clinical observations, the Ang2 and Tie2 (its receptor) axis has been focused on as a promising target. Here, we show a novel strategy to circumvent the resistance by combining multi-tyrosine kinase inhibitors lenvatinib (VEGF receptor, fibroblast growth factor receptor, and RET inhibitor) and golvatinib (E7050; c-Met, Tie2, and EphB4 inhibitor). Tie2 identifies a highly pro-angiogenic macrophage subset, Tie2-expressing macrophages (TEM). Angi-Tie2 and EphB4-EphrinB2 signaling plays critical roles in pericyte-mediated vessel stabilization. In vitro analyses suggested that golvatinib combined with lenvatinib inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib and lenvatinib inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable, and no macroscopic change was observed. These preclinical studies suggest that modulation of the tumor microenvironment by a strategic and well-tolerated combination of multi-targeting tyrosine kinase inhibitors may sensitize cancer to VEGF inhibitors.

Purpose: Receptor tyrosine kinases c-Met and Ron transduce signals regulating cell migration and matrix invasion. This phase I dose-escalation trial tested golvatinib, a highly potent, small-molecule, ATP-competitive inhibitor of c-Met and multiple members of the Eph receptor family plus c-Kit and Ron. Experimental Design: Patients with advanced solid tumors received golvatinib orally, once daily, continuously. Using a “3+3” design, dosing started at 100 mg once daily, escalating to the maximum tolerated dose (MTD) defined by dose-limiting toxicities. Pharmacokinetic, pharmacodynamic, and preliminary antitumor activity was assessed during dose escalation and in a MTD expansion cohort. Results: Thirty-four patients were treated at six dose levels. The MTD was determined as 400 mg once daily. Three dose-limiting toxicities were observed: grade 3 increased γ-glutamyltransferase and alkaline phosphatase (200 mg), repeated grade 2 fatigue, and grade 3 fatigue (50.0%). Frequent treatment-related adverse events (with incidence >10%) included diarrhea (58.8%), nausea (50%), vomiting (44.1%), fatigue (41.2%), decreased appetite (32.4%), elevated alanine aminotransferase (32.4%), elevated aspartate aminotransferase (20.6%), dry skin (11.8%), and dysgeusia (11.8%). Best overall response was stable disease (median duration 85 days, range 85–237). Pharmacokinetics demonstrated high variability, although maximum plasma concentration and area under the plasma concentration–time curve increased with dose. Soluble urokinase-type plasminogen activator receptor, VEGFR2, c-Met, and angiopoietin-2 levels increased after dose. Posttreatment decrease in either p-c-Met or p-ERK was observed in 3 of 4 paired biopsies at MTD. Conclusions: Golvatinib at the MTD of 400 mg once daily was well tolerated with pharmacodynamic evidence of c-Met target modulation. Clin Cancer Res; 20(24); 6284–94. ©2014 AACR.

Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted.

Although vascular endothelial growth factor (VEGF) inhibitors provide significant clinical benefit, they often require dose reductions or even withdrawals due to their severe toxicities. Furthermore, almost all cancers show intrinsic and/or evasive resistance to VEGF inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. Response to lenvatinib (E7080; VEGFR1-3 inhibitor) is also reported to correlate with low Ang2 level in differentiated thyroid cancer and endometrial cancer. From these clinical observations, Ang2 and its receptor Tie2 has been focused as promising targets. Here, we demonstrated mechanisms of resistance induced by Ang2 and a novel strategy to circumvent the resistance by combination of multi-tyrosine kinase inhibitors (TKIs), lenvatinib and golvatinib (E7050; c-Met, Tie2, EphB4 inhibitor). Tie2 defines a highly pro-angiogenic macrophage subset, Tie2-expressing macrophage (TEM). Ang-Tie2 and EphB4-EphrinB2 signaling play critical roles in pericyte-mediated vessel stabilization. Ectopic expression of Ang2 in thyroid cancer conferred resistance to lenvatinib and enhanced pericyte-associated endothelial network development and TEM infiltration. In vitro analyses suggested that golvatinib/lenvatinib combination inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib/lenvatinib combination inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable in mice, and no macroscopic change was observed. These results suggest that modulation of tumor microenvironment by strategic and well-tolerated combination of multi-targeting TKIs sensitizes cancer to VEGF inhibitors, which warrants further clinical investigation to determine the clinical benefit of anti-angiogenesis cancer therapy. Citation Format: Youya Nakazawa, Satoshi Kawano, Junji Matsui, Yasuhiro Funahashi, Osamu Tohyama, Hiroki Muto, Takayuki Nakagawa, Tomohiro Matsushima. Maximizing the efficacy of anti-angiogenesis cancer therapy: A multi-targeting strategy by tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2014-2980

Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several types of tumors, however, some tumor shows intrinsic resistance to VEGFR inhibitors. In addition, patients initially responding develop acquired resistance to VEGFR inhibitors after varying periods of time. Thus it is urgent to develop strategy to overcome anti-VEGF therapy resistance. Hepatocyte growth factor (HGF)/Met signaling is activated in various types of tumors. Recently, it is reported that HGF/Met signaling contributes to resistance to tyrosine kinase inhibitors such as EGFR, BRAF, ALK inhibitors. This suggests that Met inhibitors may be promising in treating HGF/Met activated anti-VEGF therapy-resistant tumors. We have identified lenvatinib (E7080) as a highly-potent VEGFR inhibitor and phase III trial is ongoing in thyroid cancer. Lenvatinib inhibited the growth of HUVEC induced by VEGF, however, addition of HGF to HUVEC conferred resistance to lenvatinib. This resistance was cancelled by the addition of golvatinib (E7050) which is identified as a potent Met/EphB4 inhibitor and phase I trial of the lenvatinib/golvatinib combination therapy is ongoing. In mouse models bearing various tumor cell lines expressing VEGF and HGF, treatment with lenvatinib alone demonstrated only moderate antitumor activity, however, combination with golvatinib demonstrated synergistic antitumor activity with decrease of tumor vessel density. Furthermore, it is reported that EphB4 has critical roles in tumor vessel development and maturation. Our unique in vitro assay systems, endothelial cells/pericyte 2D co-culture assay and endothelial cells/pericyte 3D co-culture sprouting assay, suggested that EphB4 inhibitory activity of golvatinib disrupted anti-VEGF therapy-resistant tumor vascular in the combination with lenvatinib. Together, these data demonstrated that combination therapy with golvatinib may be promising to overcome anti-VEGF therapy resistance. Citation Format: Satoshi Kawano, Takayuki Nakagawa, Yuya Nakazawa, Yoshiaki Sato, Tomohiro Matsushima, Yasuhiro Funahashi. Combination of VEGFR inhibitor lenvatinib (E7080) and Met/EphB4 inhibitor golvatinib (E7050) overcomes VEGFR inhibitor-resistant tumor vascular. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1619. doi:10.1158/1538-7445.AM2013-1619

294 Background: Golvatinib (G) is highly potent, small molecule ATP-competitive inhibitor of the c-Met receptor tyrosine kinase and multiple members of the Eph receptor family as well as c-Kit and Ron, based on isolated kinase assays. G plus sorafenib (S) had an additive cell killing effect in a HepG2 hepatocellular cell viability assay, thus warranting further evaluation of the combination in HCC. Methods: The study is an ongoing open-label phase I/II study. Eligible pts have advanced HCC, Child-Pugh (CP) A or B, up to 2 prior regimens, including S. A 3+3 dose escalation design was used to determine the maximum tolerated dose (MTD) with planned doses of G (200 mg, 300 mg, 400 mg) PO qd each in combination with S 400 mg bid in 28 day cycles.The dose limiting toxicity (DLT) evaluation period was the first 28 days. Treatment continued until disease progression or development of unmanageable toxicities. Response was assessed by RECIST 1.1. Phase I is complete and preliminary data are presented. Results: 13 pts (med age: 60; M/F: 83%/17%, CP A/B: 92%/8%) were enrolled in 2 cohorts and the MTD was declared as G 200 mg qd and S 400 mg bid. Median treatment duration, 112 days. Confirmed partial responses (PRs) were observed in 2/12 (17%) pts and durable SD was observed in 4/13 (31%) pts based on investigator assessment. Conclusions: G in combination with S appeared to demonstrate manageable toxicity in advanced HCC pts. The PRs and durable SD observed in this pretreated pt population appear favourable and support continued evaluation in phase II. Clinical trial information: NCT01271504. [Table: see text]

Background Golvatinib is a highly potent, small-molecule ATP-competitive inhibitor of the c-Met receptor tyrosine kinase and multiple members of the Eph receptor family as well as c-Kit and Ron, based on isolated kinase assays. Golvatinib showed preclinical evidence of anti-tumor activity. Two phase 1 studies were conducted: a once daily (QD) dosing study in UK and a twice daily (BID) dosing study in JP to evaluate the MTD, safety, PK, PD and preliminary activity. Methods Golvatinib was administered orally, QD or BID, continuously to patients with advanced solid tumors. Blood samples for PK and PD analysis were collected at multiple time-points. Tumor tissues were collected pre- and post-cycle 1 for PD analysis in an expanded MTD cohort. Results 34 patients were treated at 6 QD doses levels (100, 200, 270, 360, 450 and 400 mg QD) and 16 patients were treated at 4 BID doses levels (50, 100, 200 and 300 mg BID). In the QD dosing, 3 DLTs occurred: Gr3 GGT and alkaline phosphatase (n = 1; 200 mg QD) and repeated Gr2 (n = 1) and Gr3 (n = 1) fatigue, both at 450 mg QD. In the BID dosing, one DLT (Gr3 ALT) and one equivalent DLT (Gr2 vomiting, nausea and anorexia) occurred, both at 300 mg BID. The MTDs were determined to be 400 mg QD and 200 mg BID. Frequently occurring AEs (of which, all ≤G3) across both dosing regimens (n = 50) were fatigue 58% (G3: 10%), nausea 56% (G3: 2%), diarrhea 54%, vomiting 48%, decreased appetite 48% (G3: 8%), ALT increase 44% (G3: 2%) and AST increase 32%. Best response included 6 SDs (>84 days) and 4 SDs (>84 days) in the QD and the BID dosing, respectively. PK showed high variability and plasma concentration increased with dose. The Cmax was reached at a median time of 2–4 h, and declined with a t1/2 of ∼40 h. Plasma PD analysis showed a significant increase in soluble c-Met levels after golvatinib treatment. Tumour tissue PD analysis in five patients at 400 mg QD demonstrated a baseline elevated MET gene copy number, with c-Met over-expression and post-treatment decline phospho (p)-c-Met expression in 1 patient, post-treatment decline in p-c-Met in the second patient, and post-treatment decline in p-ERK in the third patient. Conclusions The MTD of golvatinib was achieved at 400 mg QD and 200 mg BID. Preliminary PD analysis demonstrated evidence of c-Met target modulation. QD dosing was selected for further evaluation of golvatinib in phase 2 studies.

3030 Background: Golvatinib is a highly potent, small molecule ATP-competitive inhibitor of the c-Met receptor tyrosine kinase and multiple members of the Eph receptor family as well as c-Kit and Ron, based on isolated kinase assays. Golvatinib showed preclinical evidence of anti-tumor activity. This phase 1 study was performed to determine the MTD, safety, PK, PD and preliminary activity of golvatinib. Methods: Patients (pts) with advanced solid tumors, ECOG PS 0-1, ≥ 18 years (yrs) and adequate organ function were eligible. Golvatinib was administered orally, once daily (QD), continuously. Blood samples for PK and PD analysis were collected at multiple time-points. Mandatory tumor biopsies for PD analysis were taken pre and post Cycle 1 in an expanded MTD cohort. Results: 34 pts (M/F: 21/13; median age 63.5yrs [range 32-78]) were treated at 6 dose levels: 100, 200, 270, 360, 450 and 400 mg. Tumor types were colorectal (n=15), lung (n=4), renal (n=4), esophageal (n=2), melanoma (n=2) and others (n=7). Three DLTs were observed: Gr3 GGT and alkaline phosphatase (n=1; 200mg) and repeated Gr2 (n=1) and Gr3 (n=1) fatigue, both at 450mg. The MTD was determined to be 400 mg QD. Frequently occurring adverse events ([AEs]; all grades) were fatigue: 68% (Gr3: 15%), diarrhea: 65%, nausea: 62%, vomiting: 53%, decreased appetite: 47% (Gr3: 9%), ALT increase: 38% and AST increase: 23%. No Gr4 AEs were observed. Best response was stable disease in 6 pts lasting >84 days. PK showed high variability and plasma concentration increased with dose. The Cmax was reached within a median time of 4 hours. Plasma PD analysis showed an increase in soluble c-Met and Ang 2 levels after golvatinib treatment. Tumor PD analysis in 5 pts at 400 mg demonstrated a baseline elevated MET gene copy number, with c-Met overexpression and post treatment decline in phospho(p)-c-Met expression in 1 pt; post-treatment decline in p-c-Met in a 2nd pt, and post-treatment decline in p-ERK in a 3rd pt. Conclusions: Golvatinib at an MTD of 400 mg QD has manageable toxicity. Preliminary PD analysis demonstrated evidence of c-Met target modulation. Further evaluation will continue in phase 2 combination studies.

3079 Background: Golvatinib is a highly potent, small molecule ATP-competitive inhibitor of the c-Met receptor tyrosine kinase and multiple members of the Eph receptor family as well as c-Kit and Ron, based on isolated kinase assays. Golvatinib showed antiproliferative activity in human cancer xenograft models, warranting further investigation in the clinical setting. Methods: Japanese patients (pts) with advanced solid tumors that had progressed after approved therapy received oral golvatinib at escalating doses of 50 to 100, 200 and 300 mg administered twice daily (BID) in conventional 3 pt cohorts. The primary objective was to determine the MTD of golvatinib when administered continually BID. The secondary objectives were to evaluate the safety, PK, PD biomarkers, and efficacy. Results: 16pts were enrolled (8 M, 8 F; med age 59 years; ECOG 0-1). Tumor types were colorectal (5), gastric (4) and others (7). One DLT (Gr 3 ALT increase) and 1 equivalent DLT (Gr 2 vomiting, nausea and anorexia) appeared in 2 pts in the 300 mg BID cohort and the dose escalation was terminated. The MTD was determined to be 200 mg BID. Frequently observed adverse drug reactions were proteinuria [50.0% (8/16)], ALT increased [43.8% (7/16)], AST increased, nausea, vomiting [37.5% (6/16)], and blood alkaline phosphatase increased, decreased appetite, diarrhea, leukopenia [31.3% (5/16)], which were all mild to moderate in severity with ≤ Gr 2. No related severe adverse event was observed. Responses included 4 stable diseases (84-113 days) in 3 pts with gastric cancer and 1 patient with NSCLC, 11 progressive diseases, and 1 non evaluable. The golvatinib plasma concentration after first-dose reached Cmax at a median time of 2 to 4 hours, and declined bi-exponentially with a t½ of approximately 40 hours. Cmax and AUC0-inf increased with dose. Analysis of blood PD biomarker showed a significant increase in soluble c-Met levels after golvatinib treatment. Conclusions: The MTD of golvatinib was achieved at 200 mg BID. Golvatinib was well tolerated with manageable toxicities at dose levels up to and including the MTD. 4 of 16 pts showed stable disease after golvatinib treatment.