ABSTRACT
BACKGROUND: Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS. METHODS: Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively. RESULTS: Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti-cancer therapy.
Subject(s)
Bone Neoplasms , Nicotinamide N-Methyltransferase , Osteosarcoma , Adolescent , Adult , Child , Female , Humans , Male , Young Adult , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Bone Neoplasms/drug therapy , Cell Line, Tumor , Cell Movement , Cell Proliferation , Drug Resistance, Neoplasm/genetics , Nicotinamide N-Methyltransferase/metabolism , Nicotinamide N-Methyltransferase/genetics , Osteosarcoma/genetics , Osteosarcoma/pathology , Osteosarcoma/metabolism , Osteosarcoma/drug therapy , RNA, Small Interfering/geneticsABSTRACT
Osimertinib is a "third-generation'' oral, irreversible, tyrosine kinase inhibitor. It is used in the treatment of non-small cellular lung carcinoma and spares wild-type EGFR. Due to its reactive nature, osimertinib is, in addition to oxidative routes, metabolized through GSH coupling and subsequent further metabolism of these conjugates. The extent of the non-oxidative metabolism of osimertinib is unknown, and methods to quantify this metabolic route have not been reported yet. To gain insight into this metabolic route, a sensitive bioanalytical assay was developed for osimertinib, the active desmethyl metabolite AZ5104, and the thio-metabolites osimertinibs glutathione, cysteinylglycine, and cysteine conjugates was developed. The ease of synthesis of these metabolites was a key-part in the development of this assay. This was done through simple one-step synthesis and subsequent LC-purification. The compounds were characterized by NMR and high-resolution mass spectrometry. Sample preparation was done by a simple protein crash with acetonitrile containing the stable isotopically labeled internal standards for osimertinib and the thio-metabolites, partial evaporation of solvents, and reconstitution in eluent, followed by UHPLC-MS/MS quantification. The assay was successfully validated in a 2-2000â¯nM calibration range for all compounds except the glutathione metabolite, where the LLOQ was set at 6â¯nM due to low accuracy at 2â¯nM. Limited stability was observed for osimertinib, AZ5104, and the glutathione metabolite. The clinical applicability of the assay was demonstrated in samples of patients treated with 80â¯mg osimertinib once daily, containing all investigated compounds at detectable and quantifiable levels.
Subject(s)
Acrylamides/pharmacokinetics , Aniline Compounds/pharmacokinetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Monitoring/methods , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacokinetics , Acrylamides/administration & dosage , Acrylamides/blood , Acrylamides/metabolism , Administration, Oral , Aged , Aged, 80 and over , Aniline Compounds/administration & dosage , Aniline Compounds/blood , Aniline Compounds/metabolism , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/genetics , Chromatography, High Pressure Liquid/methods , Dipeptides/blood , Dipeptides/chemical synthesis , Dipeptides/metabolism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Glutathione/blood , Glutathione/chemical synthesis , Glutathione/metabolism , Humans , Lung Neoplasms/blood , Lung Neoplasms/genetics , Middle Aged , Mutation , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/blood , Protein Kinase Inhibitors/metabolism , Proton Magnetic Resonance Spectroscopy , Sulfhydryl Compounds/blood , Sulfhydryl Compounds/chemical synthesis , Sulfhydryl Compounds/metabolism , Tandem Mass Spectrometry/methodsABSTRACT
Ibrutinib is a targeted covalent inhibitor frequently used for the treatment of various lymphomas. In addition to oxidative metabolism, it is metabolized through glutathione coupling. The quantitative insight into this kind of metabolism is scarce, and tools for quantitation are lacking. The non-oxidative metabolism could prove a more prominent role when oxidative metabolism is impaired. Also, in-vitro studies could over-estimate the effect of CYP450-inhibition. To gain quantitative insight into this relatively unknown biotransformation pathway of the drug we have developed a validated simple, fast and sensitive bio-analytical assay for ibrutinib, dihydrodiol-ibrutinib, and the glutathione, cysteinylglycine and cysteine conjugates of ibrutinib in human plasma. The method emphasizes on simplicity, the thiol-conjugates were synthesized by a simple one step synthesis, followed by LC-purification. Sample preparation was done by a simple protein crash with acetonitrile containing labeled internal standards, evaporation of solvents, and reconstitution in eluent. Finally, the compounds were quantified using UHPLC-MS/MS. The assay was successfully validated in a 0.5-500nM calibration range for all compounds, and also a lower range of 0.05-50â¯nM was demonstrated for ibrutinib to accommodate for even the lowest trough levels. This assay has a considerably higher sensitivity than previous published assays, with the previous lowest LLOQ being 1.14â¯nM. Both, ibrutinib, dihydrodiol-ibrutinib and the cysteine conjugate were deemed stable under refrigerated or frozen storage conditions. At room temperature, the glutathione conjugate showed rapid degradation into the cysteinylglycine conjugate in plasma. Finally, the applicability of the assay was demonstrated in patient samples.