Small Molecular Tyrosine Kinase Inhibitors Research Articles

BLUE-BLACK DISCOLORATION OF THE NAILS ASSOCIATED WITH GEFITINIB

Blue-black discoloration appeared on all fingernails in a 57-year-old woman after 3 weeks of gefitinib 250mg once daily for her locally advanced lung cancer (Figure 1 & 2), and it could not be removed by solvents, such as alcohol. The nails did not become thickened or atrophic, and adjacent skin was normal. During the period of treatment, no exposure of other significant drug insult was noted. Gefitinib is a small molecular tyrosine kinase inhibitor blocking the epidermal growth factor receptor (EGFR) pathway (1, 2). The most frequent cutaneous manifestation is a papulopustular eruption varying from 24% to 62% of patients on gefitinib in clinical trials, followed by nail changes, including painful paronychial inflammation, ingrown nails and xerosis. Approximately 10% to 15% of patients have nail toxicity between 4 and 8 weeks after initiation of treatment (1). However, blue discoloration or pigmentation of nails is rarely associated with gefitinib in previous literature. In contrast to skin rash, resolving within 4 weeks after cessation of EGFR pathway inhibitors, nail toxicity can be persistent for several months after withdrawal (2). Nevertheless, the mechanism is still unknown (1, 2). Blue-black discoloration can occur in the nail plate, vascular nail bed or the lunula, and pigmentation is usually related to drugs, such as minocycline, zidovudine and antimalarial drugs. In certain circumstances, however, we have to pay attention, as blue lunula can also be a marker of more serious diseases, such as argyria, Wilson’s disease and congenital diseases including haemoglobin M disease and hereditary acrolabial telangectasia (3).

Denaturing capillary electrophoresis for automated detection of L858R mutation in exon 21 of the epidermal growth factor receptor gene in prediction of the outcome of lung cancer therapy

The presence of activating mutations within the tyrosine kinase domain of the epidermal growth factor receptor gene has been attributed to a positive response to biological therapy of lung cancer by small-molecular tyrosine kinase inhibitors, gefitinib and erlotinib. Among the two most significant mutation types are deletions in exon 19 and a single point substitution in exon 21 (termed L858R). The exon 19 deletions can readily be examined by fragment analysis, due to the characteristic length difference between the normal and mutated PCR product. Analysis of the L858R point mutation, however, presents a greater challenge. The current paper is aimed at developing a sensitive, yet simple, low-cost mutation detection assay directed at the L858R mutation using a method based on CE of heteroduplexes under partial denaturing conditions. We perform optimization of separation conditions on different commercial instruments including ones equipped with 8, 16 and 96 capillaries. We present normalized migration reproducibility in the range from 1 (8 and 16) to 5% (96) RSD. A reliable distinction of the R836R silent polymorphism from a potential presence of the L858R mutation is also demonstrated. In its implementation, the presented assay is just another application running on a conventional CE platform without the need of dedicated instrumentation.

Role of antiapoptotic STAT3 and BCL-2 signaling in promoting lung tumor cells to survive against combined MET-/EGFR-targeted inhibitors.

10644 Background: Epidermal growth factor receptor (EGFR) inhibitors in lung cancer are largely limited by the development of resistant disease that ultimately limits their clinical utility. Combined targeted therapy strategies using both MET and EGFR small molecular tyrosine kinase inhibitors (TKIs) are under preclinical and clinical investigation to enhance the therapeutic efficacy and to circumvent acquired EGFR TKI resistance in lung cancer. These combined therapeutic approaches are likely to have unique mechanisms of resistance. Methods: We investigated the effects of combined MET/EGFR TKI treatment on signal transducer and activator of transcription 3 (STAT3) and B-cell lymphoma protein 2 (BCL-2) family members in lung cancer cell lines and xenografts models that are resistant to EGFR inhibitors. Results: Activated STAT3 and BCL-2/BCL-XL induction were seen in lung tumor cells that survived targeted inhibition by MET kinase inhibitors (SU11274 and PHA665752), either alone or in combination with EGFR inhibitors. Using in vivo xenograft model with luciferase-expressing H1975 cells for bioluminescence imaging (BLI), we show that while the H1975 xenograft exhibited excellent imaging tumor response when treated with combined SU11274 (MET TKI) and erlotinib (EGFR TKI), activated P-STAT3[Y705] expression was evident in the residual microscopic tumor survivor cells within the treated xenograft. Interestingly, the resistant survivor tumor cells were located predominantly along the peripheral rind juxtaposing the host stroma. BCL-2 expression was also found induced in the tumor survivor cells during the course of targeted combined MET/EGFR inhibition in vivo. Conclusions: Results of our study support the role of activated STAT3, and its downstream transcriptional targets of the antiapoptotic BCL-2 family members as potential markers for tumor resistance against combined MET/EGFR targeted inhibitors in lung cancer. Further investigations to target the intrinsic apoptotic pathway with BCL-2 family inhibitors, which may overcome or prevent resistance against combined MET/EGFR TKIs, are warranted to impact on the long term outcome in lung cancer therapy. No significant financial relationships to disclose.

Inhibition of IGF-1R-Dependent PI3K Activation Sensitizes Colon Cancer Cells Specifically to DR5-Mediated Apoptosis But Not to rhTRAIL

Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) initiates apoptosis in tumor cells upon binding to its cognate agonistic receptors, death receptors 4 and 5 (DR4 and DR5). The activity of the insulin-like growth factor 1 (IGF-1) survival pathway is often increased in cancer, influencing both cell proliferation and apoptosis. We hypothesized that inhibiting the IGF-1 receptor (IGF-1R) using NVP-AEW541, a small molecular weight tyrosine kinase inhibitor of the IGF-1R, could increase death receptor (DR)-mediated apoptosis in colon cancer cells.Methods: The analyses were performed by caspase assay, flow cytometry, Western blotting, immunoprecipitation and fluorescent microscopy.Results: Preincubation with NVP-AEW541 surprisingly decreased apoptosis induced by recombinant human TRAIL (rhTRAIL) or an agonistic DR4 antibody while sensitivity to an agonistic DR5 antibody was increased. NVP-AEW541 could inhibit IGF-1-induced activation of the phosphatidylinositol 3-kinase (PI3K) pathway. The effects of the PI3K inhibitor LY294002 on TRAIL-induced apoptosis were similar to those of NVP-AEW541, further supporting a role for IGF-1R-mediated activation of PI3K. We show that PI3K inhibition enhances DR5-mediated caspase 8 processing but also lowers DR4 membrane expression and DR4-mediated caspase 8 processing. Inhibition of PI3K reduced rhTRAIL sensitivity independently of the cell line preference for either DR4- or DR5-mediated apoptosis signaling.Conclusion: Our study indicates that individual effects on DR4 and DR5 apoptosis signaling should be taken into consideration when combining DR-ligands with PI3K inhibition.

Synthesis and Characterization of Her-2 Targetable N-(2-Hydroxypropyl)methacrylamide Copolymer Conjugates.

Abstract Background: Current Her-2 overexpressing cancer therapies are limited by rapid drug resistance development from Trastuzumab (TRZ) treatment or low selectivity and non specific side effects of hepatotoxicity with small molecular tyrosine kinase inhibitors such as lapatinib. N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-drug conjugates with tailored side-chain composition have demonstrated their clinical potential as tumor targetable chemotherapeutic delivery systems. The present study evaluates the potential of novel HPMA copolymer-TRZ conjugates to enhance the therapeutic index of Her-2 targeted therapy.Material and Methods: HPMA copolymer-TRZ conjugates were synthesized in two steps. In the first step, an HPMA copolymer precursor containing reactive p-nitrophenyl ester (ONp) side chains was synthesized by free radical precipitation polymerization. In the second step a series of polymer precursors were conjugated to varying amounts of TRZ by aminolysis. The feed ratio of TRZ to HPMA copolymer was calculated based on the number of primary amine groups available on TRZ surface as determined spectrophotometrically using the 2,3,6-Tri-nitrobenzenesulfonic acid (TNBS) assay. The copolymer conjugates were characterized by correlating the feed ratio with the degree of conjugation (TNBS assay) and molecular weight distribution (size exclusion chromatography). The antiproliferative action of HPMA copolymer-TRZ conjugates was evaluated on a Her-2 overexpressing BT474 breast cancer cell line. Cell proliferation was quantified by MTT assay. Activation of Her-2 after treatment with polymer conjugate was measured by western immunoblotting.Results and Discussion: A series of three HPMA copolymer-TRZ conjugates were successfully synthesized and characterized. It was observed that an increase in the feed ratio of HPMA copolymer precursor to TRZ resulted in a corresponding increase in the degree of TRZ conjugation. The size exclusion chromatography profile of the conjugates was also consistent with the degree of conjugation. As HPMA copolymer precursor contents increased, the elution time decreased suggesting an increase in the hydrodynamic volume of conjugates. The growth inhibition effects of all HPMA copolymer-TRZ conjugates were concentration dependant. The half maximal inhibitory concentrations (IC50) of the conjugates with TRZ/HPMA=1/1, 2/1 and 4/1 were 10, 29, and 20 nM respectively, which were comparable to that of free TRZ (8 nM) suggesting that the antibody retained bioactivity against Her-2 overexpressing cancer cells upon conjugation to the polymer. Western immunoblotting showed that polymer conjugated TRZ effectively downregulated activated Her-2 (phospho-Her2) to a greater extent than free TRZ.Conclusion: Here we report for the first time, the successful synthesis, characterization and in vitro evaluation of HPMA copolymer-anti-Her-2 drug (TRZ) conjugates for active targeting to Her-2 overexpressing breast cancer cells. The conjugate compositions were consistent with their molar feed ratios and inhibited model cancer cell growth comparable to the free drug. Preliminary studies indicate improved efficacy of TRZ against Her-2 positive breast cancer cells when administered as polymeric conjugate. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5065.

EGFR-Targeted Therapies in Lung Cancer: Predictors of Response and Toxicity

The EGFR pathway has emerged as a key target in non-small-cell lung cancer. EGF receptor (EGFR) inhibition in non-small-cell lung cancer is achieved via small molecular tyrosine kinase inhibitors, such as erlotinib or gefitinib, or monoclonal antibodies such as cetuximab. A growing body of evidence is identifying potential molecular predictors of response and toxicity. This includes tumor-related molecular markers, such as EGFR mutation and copy number, as well as germline markers such as polymorphisms in EGFR or EGFR pathway-related genes. This review focuses on the current state of knowledge of predictors of response and toxicity to EGFR inhibitors in lung cancer.

Sunitinib: From Rational Design to Clinical Efficacy

Sunitinib (SU011248) is an oral small molecular tyrosine kinase inhibitor that exhibits potent antiangiogenic and antitumor activity. Tyrosine kinase inhibitors such as SU6668 and SU5416 (semaxanib) demonstrated poor pharmacologic properties and limited efficacy; therefore, sunitinib was rationally designed and chosen for its high bioavailability and its nanomolar-range potency against the antiangiogenic receptor tyrosine kinases (RTKs)--vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR). Sunitinib inhibits other tyrosine kinases including, KIT, FLT3, colony-stimulating factor 1 (CSF-1), and RET, which are involved in a number of malignancies including small-cell lung cancer, GI stromal tumors (GISTs), breast cancer, acute myelogenous leukemia, multiple endocrine neoplasia types 2A and 2B, and familial medullary thyroid carcinoma. Sunitinib demonstrated robust antitumor activity in preclinical studies resulting not only in tumor growth inhibition, but tumor regression in models of colon cancer, non-small-cell lung cancer, melanoma, renal carcinoma, and squamous cell carcinoma, which were associated with inhibition of VEGFR and PDGFR phosphorylation. Clinical activity was demonstrated in neuroendocrine, colon, and breast cancers in phase II studies, whereas definitive efficacy has been demonstrated in advanced renal cell carcinoma and in imatinib-refractory GISTs, leading to US Food and Drug Administration approval of sunitinib for treatment of these two diseases. Studies investigating sunitinib alone in various tumor types and in combination with chemotherapy are ongoing. The clinical benchmarking of this small-molecule inhibitor of members of the split-kinase domain family of RTKs will lead to additional insights regarding the biology, potential biomarkers, and clinical utility of agents that target multiple signaling pathways in tumor, stromal, and endothelial compartments.

The epidermal growth factor receptor (EGFR) in head and neck cancer: its role and treatment implications

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptors. Its stimulation by endogenous ligands, EGF or transforming growth factor-alpha (TGF-α) results in activation of intracellular tyrosine kinase, therefore, cell cycle progression. High levels of EGFR expression are correlated with poor prognosis and resistance to radiation therapy in a variety of cancers, mostly in squamous-cell carcinoma of the head and neck (SCCHN). Blocking the EGFR by a monoclonal antibody results in inhibition of the stimulation of the receptor, therefore, in inhibition of cell proliferation, enhanced apoptosis, and reduced angiogenesis, invasiveness and metastases. The EGFR is a prime target for new anticancer therapy in SCCHN, and other agents in development include small molecular tyrosine kinase inhibitors and antisense therapies.

Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis.

The multifunctional growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its receptor tyrosine kinase c-Met have emerged as key determinants of brain tumor growth and angiogenesis. SF/HGF and c-Met are expressed in brain tumors, the expression levels frequently correlating with tumor grade, tumor blood vessel density, and poor prognosis. Overexpression of SF/HGF and/or c-Met in brain tumor cells enhances their tumorigenicity, tumor growth, and tumor-associated angiogenesis. Conversely, inhibition of SF/HGF and c-Met in experimental tumor xenografts leads to inhibition of tumor growth and tumor angiogenesis. SF/HGF is expressed and secreted mainly by tumor cells and acts on c-Met receptors that are expressed in tumor cells and vascular endothelial cells. Activation of c-Met leads to induction of proliferation, migration, and invasion and to inhibition of apoptosis in tumor cells as well as in tumor vascular endothelial cells. Activation of tumor endothelial c-Met also induces extracellular matrix degradation, tubule formation, and angiogenesis in vivo. SF/HGF induces brain tumor angiogenesis directly through only partly known mechanisms and indirectly by regulating other angiogenic pathways such as VEGF. Different approaches to inhibiting SF/HGF and c-Met have been recently developed. These include receptor antagonism with SF/HGF fragments such as NK4, SF/HGF, and c-Met expression inhibition with U1snRNA/ribozymes; competitive ligand binding with soluble Met receptors; neutralizing antibodies to SF/HGF; and small molecular tyrosine kinase inhibitors. Use of these inhibitors in experimental tumor models leads to inhibition of tumor growth and angiogenesis. In this review, we summarize current knowledge of how the SF/HGF:c-Met pathway contributes to brain tumor malignancy with a focus on glioma angiogenesis.

Cetuximab: An epidermal growth factor receptor chimeric human-murine monoclonal antibody

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptors. It is composed of extracellular domains, including a ligand-binding domain, a hydrophobic transmembrane region and a tyrosine kinase-containing cytoplasmic region. Stimulation of the EGFR by endogenous ligands, EGF or transforming growth factor-alpha (TGF-alpha), results in a conformational change in the receptor, permitting it to enter into dimers and other oligomers. Dimerization results in activation of intracellular tyrosine kinase, protein phosphorylation and stimulation of various cell signaling pathways that mediate gene transcription and cell cycle progression. The EGFR is expressed on normal human cells but higher levels of expression of the receptor have also been shown to be correlated with malignancy in a variety of cancers. In addition, expression of the EGFR by malignant cells is associated with poor prognosis and resistance to therapy. Cetuximab is a chimeric human-murine monoclonal antibody that binds competitively and with high affinity to the EGFR. Binding of the antibody to the EGFR prevents stimulation of the receptor by endogenous ligands and results in inhibition of cell proliferation, enhanced apoptosis, and reduced angiogenesis, invasiveness and metastasis. Binding of cetuximab to the receptor also results in internalization of the antibody-receptor complex which leads to an overall downregulation of EGFR expression. The EGFR is a prime target for new anticancer therapy, and other agents in development include small molecular tyrosine kinase inhibitors and antisense therapies. Preclinical studies have demonstrated that cetuximab reduces chemotherapy and radiotherapy resistance in human tumor cell lines in vitro and in nude mice bearing xenografts of human tumors. In clinical and preclinical studies cetuximab has been shown to induce response to treatment when used in combination with chemotherapy in patients previously refractory to chemotherapy. Based on these studies, cetuximab can be added to regimens using docetaxel, cisplatin, carboplatin, irinotecan, paclitaxel and fluorouracil and may add to treatment efficacy. Phase I dose-finding studies showed that saturation of cetuximab clearance occurred after administration of 400 mg/m2 as a loading dose followed by weekly infusions of 250 mg/m2. The most commonly reported adverse event associated with cetuximab treatment is an acneiform rash that occurs in 70-80% of patients treated with cetuximab. The rash is rarely dose- or treatment-limiting, and may diminish in intensity with continued exposure to cetuximab. Improvement may be seen after treatment with topical antibiotic preparations, topical steroids or topical retinoids. The rash resolves fully after discontinuation of cetuximab treatment. EGFR is widely expressed in skin and skin biopsies in areas involved with the characteristic cetuximab eruption demonstrate neutrophilic folliculitis. In fact, analysis of four phase II clinical trials of cetuximab in combination with chemotherapy in patients with colorectal cancer, squamous cell carcinoma of the head and neck, or pancreatic cancer showed that development of the acneiform rash was significantly correlated with response to treatment; grade 3 rash may be especially predictive of response. It is possible that development of acneiform rash may become an important clinical prognostic marker. Serious cetuximab-related toxicities include hypersensitivity, infusion reactions and interstitial lung disease. Results of a large phase II study have shown response when used in combination with irinotecan in 22.9% of patients with EGFR-expressing, irinotecan-refractory, colorectal cancer. Cetuximab has recently been approved for this indication in the United States, Switzerland, Iceland, Norway and the 25 member states of the European Union. Other phase II and III studies show significant response to treatment in variable proportions of patients with squamous cell carcinoma of the head and neck, non-small cell lung cancer and pancreatic cancer when cetuximab is used first or second line in combination with chemotherapy. Thus, cetuximab is emerging as a very promising new therapy to be used in conjunction with existing therapies for the treatment of a spectrum of solid tumors.