Multitargeted Tyrosine Kinase Inhibitors Sorafenib Research Articles

Novel 2-phenyloxypyrimidine derivative induces apoptosis and autophagy via inhibiting PI3K pathway and activating MAPK/ERK signaling in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality globally. Because most patients are diagnosed at advanced stages of the disease, multi-targeted tyrosine kinase inhibitor sorafenib is the only available drug to show limited effectiveness. Novel and effective therapies are unmet medical need for advanced HCC patients. Given that the aberrant expression and activity of platelet-derived growth factor receptor α (PDGFRα) are closely associated with the pathogenesis of HCC, here we present the discovery and identification of a novel PDGFRα inhibitor, N-(3-((4-(benzofuran-2-yl)pyrimidin-2-yl)oxy)-4-methylphenyl)-4-((4-methylpiperazin-1-yl)methyl)benzamide (E5) after comparison of different derivatives. We found that E5 inhibited proliferation and induced apoptosis in HCC cells. Since the pan-caspase inhibitor Z-VAD-FMK partially rescued HCC cells from E5-reduced cell viability, autophagic cell death triggered by E5 was subsequently investigated. E5 could induce the conversion of LC3-I to LC3-II, increase the expression of Atg5 and restore the autophagy flux blocked by chloroquine. Meanwhile, E5 was able to downregulate the PDGFRα/PI3K/AKT/mTOR pathway and to activate MAPK/ERK signaling pathway. Taken together, in addition to the possibility of E5 as a valuable drug candidate, the present study further supports the notion that targeted inhibition of PDGFRα is a promising therapeutic strategy for HCC.

Adjuvant Sorafenib Fails to Improve Outcome in Hepatocellular Cancer

The only approved therapy for metastatic or unresectable hepatocellular cancer (HCC) is the multitargeted tyrosine kinase inhibitor sorafenib. Now,

Second-Line Everolimus Fails to Improve Survival in Hepatocellular Cancer

For patients with metastatic or unresectable hepatocellular cancer (HCC), the multitargeted tyrosine-kinase inhibitor sorafenib is the only therapy shown to improve survival. However, no known treatment is effective for patients with disease progression after sorafenib. Investigators now report the results of an industry-sponsored, international, phase III, randomized, double-blind, placebo-controlled trial (EVOLVE-1) evaluating the mTOR inhibitor everolimus in patients progressing …

Antitumor effect of vascular endothelial growth factor inhibitor sunitinib in preclinical models of hepatocellular carcinoma

Tumor recurrence and metastasis is the most common cause of mortality in hepatocellular carcinoma (HCC) patients. Despite positive results with vascular endothelial growth factor (VEGF) inhibitors in preclinical studies using HCC xenograft models, the clinical outcome in HCC patients has been disappointing. So far, only the multitargeted tyrosine kinase inhibitor sorafenib has been shown to significantly improve survival in HCC patients, suggesting that this class of agents could be effective against HCC. Recently, another VEGF inhibitor, sunitinib, showed survival benefits in HCC hepatitis B-positive patients, but failed to improve survival in HCC hepatitis C-positive patients. Obviously, concomitant liver disease, liver function in general, and the local liver environment have a huge impact on treatment outcomes. In this study, we aimed to examine the antiproliferative effect of sunitinib in different HCC cell lines in vitro, and then in xenograft and orthotopic models of HCC in order to assess the effect of the local liver vasculature on drug efficacy. Human cancer cell lines Huh7.5, Hep3B, and SK-Hep-1 were used for in-vitro studies. In in-vivo studies, each mouse carried Huh7.5 cells in both the subcutaneous and the intrahepatic compartment; therefore, drug exposure and treatment regimen were identical in both tumors. Sunitinib has the potential to moderately inhibit proliferation in the Huh7.5 cell line, induce p53 in the p53-wild-type cell line SK-hep-1, and to increase the S-phase and the sub-G1 component of the cell cycle in the Hep3B cell line. Diverse responses to sunitinib in HCC cell lines emphasize the heterogeneity of HCC tumors and may further explain the discrepancy between preclinical and clinical results. The in-vivo results show that sunitinib treatment was far less effective against intrahepatic tumors compared with xenografts. Histological data indicate that large solid intrahepatic tumors are severely affected by sunitinib as shown by large areas of necrosis and diminished number of viable tumor cells. The real problem when treating intrahepatic tumors with sunitanib and/or other VEGF inhibitors seems to arise from unopposed local growth of the small tumors and perhaps the development of distant micrometastases. Even though both xenograft and orthotopic models have limitations, these models add value to our understanding of tumor biology and help to better design treatment paradigms for patients with HCC. In comparison with xenograft models, the orthotopic HCC model allows for a more realistic assessment of drug efficacy in patients, in particular by enhancing our knowledge of the role that organ vasculature plays in the development of local metastasis and tumor resistance to antiangiogenic treatments.

Editorial Comment

For decades response rate has been a major source of determining efficacy in human malignancies. The palliation and gratification achieved on witnessing tumor shrinkage is remarkable. However, systematic clinical trials and experience have revealed that response rate is not always a surrogate for clinical outcomes. For instance, sorafenib and temsirolimus revealed only 10% and 8.6% response rates, but a significant improvement in progression-free survival (PFS) and overall survival (OS,) respectively. 1 Escudier B. Eisen T. Stadler W.M. et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med. 2007; 356: 125-134 Crossref PubMed Scopus (4291) Google Scholar , 2 Hudes G. Carducci M. Tomczak P. et al. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med. 2007; 356: 2271-2281 Crossref PubMed Scopus (3237) Google Scholar However, the issues of ongoing toxicity and cost force clinicians to make decisions regarding continuing therapy, based on response. The changes in response determination imaging and the use of targeted therapies in kidney cancer suggest the need for a contemporary and clinically relevant response measurement technique in this disease. The latter also has to be easily and uniformly reproducible. The Loss of Radiographic Enhancement in Primary Renal Cell Carcinoma Tumors Following Multitargeted Receptor Tyrosine Kinase Therapy is an Additional Indicator of ResponseUrologyVol. 75Issue 5PreviewTo characterize radiographic intratumoral contrast enhancement in the primary tumor of patients with renal cell carcinoma treated with either sorafenib or sunitinib, and to compare the relationship between primary tumor response and loss of enhancement. Use of the antiangiogenic multitargeted tyrosine kinase inhibitors sorafenib and sunitinib in renal cell carcinoma often results in stabilization of tumor size based on measurement of external tumor diameter; however, internal tumor changes in enhancement have been occasionally noted. Full-Text PDF

Neoadjuvant Clinical Trial With Sorafenib for Patients With Stage II or Higher Renal Cell Carcinoma

The multitargeted tyrosine kinase inhibitor sorafenib is used for the treatment of advanced-stage renal cell carcinoma. However, the safety and efficacy of this agent have yet to be evaluated in the preoperative period, where there may be potential advantages including tumor downstaging. This prospective trial evaluates the safety and feasibility of sorafenib in the preoperative setting. Thirty patients with clinical stage II or higher renal masses, selected based on their candidacy for nephrectomy, underwent preoperative treatment with sorafenib. Toxicities, surgical complications, and tumor responses were monitored. Of the thirty patients enrolled, 17 patients had localized disease and 13 had metastatic disease. After a course of sorafenib therapy (median duration, 33 days), a decrease in primary tumor size (median, 9.6%) and radiographic evidence of loss of intratumoral enhancement, quantified using a methodology similar to Choi criteria (median, 13%), was also observed. According to Response Evaluation Criteria in Solid Tumors, of the 28 patients evaluable for response, two patients had a partial response and 26 had stable disease, with no patients progressing on therapy. Toxicities from sorafenib were similar to that expected with this class of medication. All patients were able to proceed with nephrectomy and no surgical complications related to sorafenib administration were observed. The administration of preoperative sorafenib therapy can impact the size and density of the primary tumor and appears safe and feasible. Further studies are required to determine if preoperative systemic therapy improves outcomes in patients undergoing nephrectomy for renal cell carcinoma.

Evaluation of Response in Malignant Tumors Treated With the Multitargeted Tyrosine Kinase Inhibitor Sorafenib: A Multitechnique Imaging Assessment

The purpose of this article is to illustrate the characteristic changes induced in different tumor types by the multitargeted tyrosine kinase inhibitor sorafenib. Sorafenib reduces tumor perfusion and thereby induces necrosis and often hemorrhage. Malignant tumors treated with sorafenib undergo both morphologic and functional changes; however, the morphologic changes are less frequent and inadequate for early evaluation of response. Therefore, imaging tools accurately assessing hemorrhage and decrease in tumor perfusion with subsequent necrosis should be the mainstay in monitoring targeted therapy agents.

The Loss of Radiographic Enhancement in Primary Renal Cell Carcinoma Tumors Following Multitargeted Receptor Tyrosine Kinase Therapy is an Additional Indicator of Response

To characterize radiographic intratumoral contrast enhancement in the primary tumor of patients with renal cell carcinoma treated with either sorafenib or sunitinib, and to compare the relationship between primary tumor response and loss of enhancement. Use of the antiangiogenic multitargeted tyrosine kinase inhibitors sorafenib and sunitinib in renal cell carcinoma often results in stabilization of tumor size based on measurement of external tumor diameter; however, internal tumor changes in enhancement have been occasionally noted. Thirty patients who received sunitinib or sorafenib therapy were evaluated for primary tumor response with contrast-enhanced computed tomography images before and after at least 1 cycle of treatment. Evaluation of intratumoral contrast enhancement was quantified using a workstation that allowed for three-dimensional renderings of the kidney and measurement of density in Hounsfield units (HU). The relationship between loss of intratumoral enhancement and other outcome variables was examined. A loss of enhancement within the primary tumor, following therapy with tyrosine kinase inhibitors, was positively associated with primary tumor response (P = .0053). Additionally, the degree of post-treatment tumor enhancement was positively associated with tumor response to tyrosine kinase inhibition (P = .045). Intratumoral changes in computed tomography enhancement after receptor tyrosine kinase inhibition correlate with primary tumor response, and may be a useful adjunct to the standard response evaluation criteria in solid tumors (RECIST criteria) in assessing response to therapy. Prospective studies evaluating antiangiogenic agents should explore intratumoral changes in contrast enhancement as part of response criteria, and examine the effect of intratumoral changes on survival-based outcomes.

Preclinical Evaluation of Sorafenib in Combination with Cytarabine and Clofarabine in Acute Myeloid Leukemia (AML).

Deregulation of receptor tyrosine kinases and downstream signal transduction pathways may promote leukemogenesis by conferring cell proliferation and survival advantages in AML. The multitargeted tyrosine kinase inhibitor sorafenib (with activity against FLT3, c-kit, PDGFR, VEGFR, RAF) has been shown to have potent antitumor effects in AML cell lines with c-kit and FLT3 mutations, possibly by inducing G0/G1 cell cycle block and apoptosis and inhibiting STAT and/or MAPK signaling (S. Hu Proc AACR 2007). Sorafenib has shown single-agent activity in AML, but clinical responses have not been durable, suggesting that sorafenib is likely to be more clinically effective if combined with other antileukemic agents. In this study, we evaluated the antitumor activity of sorafenib in combination with the nucleoside analogues clofarabine and cytarabine in three AML cell lines (MV4-11, THP-1 and U937), which represent different FAB types and variable alterations in tyrosine kinases. Three sequences of administration including simultaneous, pre-chemotherapy, and pre-sorafenib were evaluated with a MTT cellular proliferation assay using 24- to 72-hr exposures (duplicate experiments performed with 8 replicates each). Combined drug effects were analyzed using the computer software CalcuSyn. The combination of clofarabine with sorafenib at a fixed ratio of 5:1 was synergistic to additive or antagonistic in MV4-11 cells (with FLT-3 ITD) depending on sequence; simultaneous > pre-sorafenib > pre-clofarabine with combination index (CI) values at ≥ ED50 of 0.53–0.80, 0.54–1.12, and 0.93–1.20, respectively. The combination of sorafenib and clofarabine was antagonistic in THP-1 and U937 cells with all three sequences evaluated (CI values at ≥ ED50 of 1.25–1.96). The combination of cytarabine with sorafenib at a fixed ratio of 250:1 was synergistic in MV4-11 cells regardless of the sequence of administration with CI values at ≥ ED50 of 0.28–0.77 in all 3 cell lines, representing an approximate 3–4- fold dose reduction index for both drugs. Although studies evaluating cytarabine and sorafenib in THP-1 and U937 cells are pending, results in MV4-11 cells indicate that cytarabine with sorafenib may be more effective than the combination of clofarabine with sorafenib. Combination studies of clofarabine or cytarabine with sorafenib in blasts from children with AML are ongoing. To understand potential differences between the nucleoside analogues when combined with sorafenib, studies were performed to determine the effect of sorafenib 5 μM (pre-treatment for 15 min) on the cellular accumulation (1-hr exposure) of clofarabine and cytarabine in MV4-11 cells (2 to 3 independent experiments performed in duplicate). Sorafenib decreased the intracellular accumulation of clofarabine by about 2-fold, but increased the intracellular accumulation of cytarabine by approximately 2-fold; transporter studies are ongoing to identify the mechanism(s) of the observed differential drug interaction. In conclusion, sorafenib in combination with cytarabine may represent a promising treatment strategy for AML, where simultaneous or sequential administration appears to be equally effective.

Activity of Tyrosine Kinase Inhibitors in Multiple Myeloma.

Tyrosine kinase inhibitors (TKIs) have been successfully introduced for the treatment of cancer. Imatinib, dasatinib and nilotinib target bcr/abl and were found to induce molecular remissions in chronic myeloid leukemia. Imatinib has also been found to be active in other malignancies like gastrointestinal stromal tumors. Sunitinib and sorafenib are multi-targeted tyrosine kinase inhibitors and so far, have shown activity against renal cell carcinoma and other cancers. Gefitinib targets the tyrosine kinase of epidermal growth factor receptor and has been found to be active against some cases of non-small cell lung cancer. There is circumstantial evidence that tyrosine kinases and their receptors (e.g. VEGF, IGF-1 and FGFR3) are active in multiple myeloma. In order to develop new treatments for multiple myeloma (MM), we tested several currently available TKIs for their activity against MM cell lines.Materials and methods: The a cell lines MC/CAR, ARH77, RPMI 8226, ARP1, JJN3, MM1S, and INA-6 were treated with various concentrations of TKIs and analyzed for cell growth in liquid culture, proliferation, apoptosis, and gene expression pattern screening 14,500 genes using U133A_2 arrays.Results: Imatinib, nilotinib, dasatinib, gefitinib induced cytotoxicity in most cases at high concentrations (50% inhibitory concentration ≥ 100 μMol), whereas sunitinib and sorafenib were active at lower concentrations (50% IC 1– 5 μMol). The cytoxicity was observed early (within 4 to 24 hours of exposure) and involves apoptosis. Interleukin-6 did not offer protection against the cytotoxicity of sorafenib or sunitinib, however the inhibition of proliferation was more pronounced in low fetal calf serum (2.5 versus 10%). A short-term exposure of the myeloma cell line MM1S to 10 μMol sorafenib resulted in more than 2 fold changes in 283 genes or sequences (175 up, 108 down). If only 10 fold changes are considered, 21 genes or sequences were upregulated (mainly enzymes, regulators and ligands) and 11 downregulated (mainly regulatory proteins, among them IL6 signal transducer).Conclusion: We found that the multitargeted TKIs sorafenib and sunitinib are active in vitro against multiple myeloma. We plan to investigate patient samples, and to elucidate the targets and the mechanisms of action. Our data will support clinical trials both as single agent and in combination with other drugs like bortezomib, thalidomide, alkylators and ionizing radiation.

399 POSTER Synergistic antitumor activity of the combination of the multi-targeted tyrosine kinase inhibitor sorafenib and of EGFR inhibitors in human colon and lung cancer cell lines

Tyrosine kinase receptors of the ErbB family have become promising targets for anti-neoplastic drugs, but mechanisms of resistance are incompletely understood. To investigate such pathways, we applied a small-molecule, selective EGFR inhibitor, OSI-774, to three well-characterized colon cancer cell lines and studied the alterations of expression and activation of receptors in the erbB family.MTT assays were performed to determine the IC50s of GEO, FET, and HCT 116 human colorectal cancer cell lines treated with OSI-774. Plated cells were then exposed to either DMSO control or 7 μm of OSI-774 for treatment durations of 1, 3, 5, 7, 10, 14, 28, and 56 days. Cell lysates were evaluated by Western blotting, evaluating both total and phosphorylated levels of EGFR, Her-2/neu, and erbB-3.IC50 values for GEO, FET, and HCT 116 cell lines exposed to OSI-774 were 12.0, 16.0, and greater than 100 μm, respectively. In all treated cell lines, OSI-774 diminished EGFR activation but did not affect total expression compared with controls. In contrast, Her-2/neu activation was increased in all cell lines. These changes in EGFR and Her-2/neu were identified within 24 h but peaked later in the treatment cycle. ErbB-3 expression and activation did not follow a consistent pattern between cell lines.Inhibition of EGFR led to increased activation of Her-2/neu. This result suggests a possible mechanism by which cells might escape the proapoptotic signals resulting from EGFR blockade. Our findings suggest concurrent inhibition of multiple members of the erbB family may yield stronger apoptotic responses than single receptor blockade alone.