pyrimidinones and regorafenib

A sensitive and selective method of high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) has been developed for the simultaneous quantification of six anticancer protein kinase inhibitors (PKIs), dabrafenib, trametinib, vemurafenib, cobimetinib, pazopanib, regorafenib, and two active metabolites (regorafenib-M2 and regorafenib-M5) in human plasma. Plasma protein precipitation with methanol enables the sample extraction of 100 μL aliquot of plasma. Analytes are detected by electrospray triple-stage quadrupole mass spectrometry and quantified using the calibration curves with stable isotope-labeled internal standards. The method was validated based on FDA recommendations, including assessment of extraction yield (74-104%), matrix effects, analytical recovery (94-104%) with low variability (<15%). The method is sensitive (lower limits of quantification within 1 to 200 ng/mL), accurate (intra- and inter-assay bias: -0.3% to +12.7%, and -3.2% to +6.3%, respectively) and precise (intra- and inter-assay CVs within 0.7-7.3% and 2.5-8.0%, respectively) over the clinically relevant concentration range (upper limits of quantification 500 to 100,000 ng/mL). This method is applied in our laboratory for both clinical research programs and routine therapeutic drug monitoring service of PKIs.

Topics: Administration, Oral; Antineoplastic Agents; Azetidines; Child; Chromatography, High Pressure Liquid; Humans; Imidazoles; Indazoles; Indoles; Limit of Detection; Linear Models; Oximes; Phenylurea Compounds; Piperidines; Pyridines; Pyridones; Pyrimidines; Pyrimidinones; Reproducibility of Results; Sulfonamides; Tandem Mass Spectrometry; Vemurafenib

Advances in therapies targeting proteins and pathways affected by genetic alterations has raised the possibility of personalized cancer treatments.. The efficacy of targeting molecular aberrations was determined in the pancreatic ductal adenocarcinoma (PDAC) cell line, CAPAN2. Two mutations were targeted, KRAS (p.G12V) and ABL1 (p.G1060D), and cells were treated with regorafenib and trametinib, individually and in combination.. Exposure to either drug significantly increased cell death compared to the current standard of care, gemcitabine. Treatment with combinations of the drugs led to significant increases in cell death compared to either monotherapy. Strong additive/synergistic interactions were observed across a range of dosages and ratios, reducing dose requirements with potential clinical relevance.. The data obtained in this PDAC cell model: i) support the use of matched monotherapies; ii) indicate the effectiveness of matched combination therapies; and iii) provide potential proof-of-concept for precision medicine approach to cancer treatment.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Deoxycytidine; Drug Synergism; Gemcitabine; Humans; Pancreatic Neoplasms; Phenylurea Compounds; Piperazines; Protein Kinase Inhibitors; Pyridines; Pyridones; Pyrimidinones

Modulation of the stroma response is considered a promising approach for the treatment of chronic pancreatitis and pancreatic cancer. The aim of this study was to evaluate the effects of three clinically available small molecule kinase inhibitors, regorafenib, trametinib and dactolisib, on effector functions of activated pancreatic stellate cells (PSCs), which play a key role in pancreatic fibrosis.. Cultured rat PSCs were exposed to small molecule kinase inhibitors. Proliferation and cell death were assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine and cytotoxicity, respectively. Levels of mRNA were determined by real-time PCR, while protein expression and phosphorylation were analyzed by immunoblotting. Interleukin-6 levels in culture supernatants were quantified by ELISA. Zymography assays were performed to monitor collagenase activity in culture supernatants.. The MEK inhibitor trametinib and the dual phosphatidylinositol 3-kinase/mTOR inhibitor dactolisib, but not the multi-kinase inhibitor regorafenib, efficiently inhibited PSC proliferation. Trametinib as well as regorafenib suppressed the expression of two autocrine mediators of PSC activation, interleukin-6 and transforming growth factor-beta1. Dactolisib-treated cells expressed less alpha1 type I collagen and lower levels of alpha-smooth muscle actin, a marker of the myofibroblastic PSC phenotype. Simultaneous application of dactolisib and trametinib displayed additive inhibitory effects on cell growth without statistically significant cytotoxicity. Activity of matrix metalloproteinase-2 was not affected by any of the drugs.. We suggest the combination of two drugs, that specifically target two key signaling pathways in PSC, Ras-Raf-MEK-ERK (trametinib) and phosphatidylinositol 3-kinase-AKT-mTOR (dactolisib), as a concept to modulate the activation state of the cells in the context of fibrosis.

Topics: Actins; Animals; Cell Proliferation; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Dose-Response Relationship, Drug; Fibrosis; Imidazoles; Interleukin-6; Male; Mitogen-Activated Protein Kinase Kinases; Pancreatic Stellate Cells; Pancreatitis, Chronic; Phenylurea Compounds; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Pyridines; Pyridones; Pyrimidinones; Quinolines; Rats, Inbred Lew; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1