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Introduction: CEP-37440 was synthesized and supplied by the research and development division of Teva Branded Pharmaceutical Products (West Chester, PA, United States). CEP-37440 represents a newly developed compound that exhibits selectivity inhibition of Focal Adhesion Kinase and Anaplastic Lymphoma Kinase FAK/ALK receptors, demonstrating novel characteristics as an orally active inhibitor. The simultaneous inhibition of ALK and FAK can effectively address resistance and enhance the therapeutic efficacy against tumors through a synergistic mechanism. Methods: The objective of this research was to create an LC-MS/MS method that is precise, efficient, environmentally friendly, and possesses a high level of sensitivity for the quantification of CEP-37440 in human liver microsomes (HLMs). The aforementioned approach was subsequently employed to evaluate the metabolic stability of CEP-37440 in HLMs in an in vitro setting. The validation procedures for the LC-MS/MS analytical method in the HLMs were performed following the bio-analytical method validation guidelines set out by the US-FDA. The AGREE program was utilized to assess the ecological impacts of the current LC-MS/MS methodology. Results and Discussion: The calibration curve linearity was seen in the range of 1-3000 ng/mL. The inter-day accuracy (% RE) exhibited a range of -2.33% to 3.22%, whilst the intra-day accuracy demonstrated a range of -4.33% to 1.39%. The inter-day precision (% RSD) exhibited a range of 0.38% to 3.60%, whilst the intra-day precision demonstrated a range of 0.16% to 6.28%. The determination of the in vitro half-life (t1/2) and moderate intrinsic clearance (Clint) of CEP-37440 yielded values of 23.24 min and 34.74 mL/min/kg, respectively. The current manuscript is considered the first analytical study for CEP-37440 quantification with the application to metabolic stability assessment. These results suggest that CEP-37440 can be categorized as a pharmaceutical agent with a moderate extraction ratio. Consequently, it is postulated that the administration of CEP37440 to patients may not lead to the accrual of dosages within the human organs. According to in silico P450 metabolic and DEREK software, minor structural alterations to the ethanolamine moiety or substitution of the group in drug design have the potential to enhance the metabolic stability and safety profile of novel derivatives in comparison to CEP-37440. [ABSTRACT FROM AUTHOR]

Background and Objectives: Dovitinib (DVB) is a pan-tyrosine kinase inhibitor (TKI) that can be administered orally. In September 2023, the FDA granted Oncoheroes approval to proceed with an Investigational New Drug (IND) application for dovitinib. This application is intended for the treatment of relapsed or advanced juvenile solid tumors, namely, osteosarcoma. Materials and Methods: The target of the present study was to develop a rapid, green, accurate, and sensitive UHPLC-MS/MS method for measuring DVB levels in human liver microsomes (HLMs). The validations of the HLMs were performed via the established UHPLC-MS/MS approach, as stated in the US FDA reported guidelines for the standards of bioanalytical method validation protocol. The StarDrop in silico software package (version 6.6), which involves the DEREK and WhichP450 in silico modules, was used to check the DVB structure for hazardous alerts and metabolic instability. The DVB and encorafenib (EFB), internal standard, and chromatographic peaks were successfully separated using a reversed phase column (an Eclipse Plus Agilent C8 column) and an isocratic mobile phase. The production of DVB parent ions was accomplished by utilizing the positive ionization mode of an ESI source. The identification and measurement of DVB daughter ions were conducted using the MRM mode. Results: The inter-day accuracy and precision exhibited a spectrum of values in the range of −0.56% to 9.33%, while the intra-day accuracy and precision showcased a range of scores between 0.28% and 7.28%. The DVB calibration curve showed a linear relationship that ranged from 1 to 3000 ng/mL. The usefulness of the currently validated UHPLC-MS/MS method was approved by the lower limit of quantification (LLOQ) of 1 ng/mL. The AGREE findings demonstrate that the UHPLC-MS/MS method had a noteworthy degree of ecological greenness. The in vitro half-life (t1/2) and intrinsic clearance (Clint) of DVB were calculated to be 15.48 min and 52.39 mL/min/kg, respectively, which aligned with the findings from the WhichP450 software (version 6.6). Conclusions: Via the usage of in silico software, it has been observed that making small changes to the structure of the aryl piperazine ring and quinolinone moieties, or replacing these groups in the drug design process, shows potential for enhancing the metabolic safety and stability of newly developed derivatives compared to DVB. [ABSTRACT FROM AUTHOR]

Palbociclib (Ibrance; Pfizer) was approved for the management of metastatic breast cancer characterized by hormone receptor‐positive/human epidermal growth factor receptor 2 negative status. The objective of this study was to create a fast, precise, environmentally friendly, and highly sensitive ultra‐high‐performance liquid chromatography‐tandem mass spectrometry approach for quantifying palbociclib (PAB) in human liver microsomes with the application for assessing metabolic stability. The validation features were performed in agreement with the bioanalytical method validation standards outlined by the US Food and Drug Administration. The StarDrop software (WhichP450 and DEREK modules) was used in screening the metabolic lability and structural alerts of PAB. The separation of PAB and encorafenib (as an internal standard) was achieved on a C8 column, employing an isocratic mobile phase. The inter‐day and intra‐day accuracy and precision ranged from ‐6.00% to 4.64% and from ‐2.33% to 3.13%, respectively. The constructed calibration curve displayed a linearity in the range of 1–3000 ng/mL. The sensitivity of the established approach was proven by the lower limit of quantification of 0.73 ng/mL. The Analytical GREEness calculator results revealed the high level of greenness of the developed method. The PAB's metabolic stability (t1/2 of 18.5 min and a moderate clearance (Clint) of 44.8 mL/min/kg) suggests a high extraction ratio medication that matched the WhichP450 software results. [ABSTRACT FROM AUTHOR]

Zotizalkib (ZTK, TPX‐0131) is a fourth‐generation highly effective inhibitor of wild‐type anaplastic lymphoma kinase (ALK) and ALK‐resistant mutations that can penetrate the central nervous system. It exhibited greater potency compared to all five officially approved ALK inhibitors. The aim of this study was to develop a rapid, accurate, eco‐friendly, and highly sensitive ultra‐high‐performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS) method for measuring the concentration of ZTK in human liver microsomes (HLMs). The validation aspects of the current UHPLC‐MS/MS methodology in the HLMs were conducted in accordance with the bioanalytical method validation standards specified by the US Food and Drug Administration. ZTK and encorafenib were separated using an Agilent C8 column (Eclipse Plus) and an isocratic mobile phase. The calibration curve for the developed ZTK exhibited a linear relationship within the concentration range of 1–3000 ng/mL. The results from the Analytical Green‐ness Metric Approach program (0.76) suggested that the created method demonstrated a significant degree of environmental sustainability. The in vitro half‐life (t1/2) and intrinsic clearance (Clint) of ZTK were determined to be 15.79 min and 51.35 mL/min/kg, respectively that suggests the ZTK exhibits characteristics similar to those of a medication with a high extraction ratio. These approaches are crucial for the progress of novel pharmaceutical development, especially in improving metabolic stability. [ABSTRACT FROM AUTHOR]

Clopidogrel (CLOP) is widely used worldwide for cardiovascular complications. CLOP is highly metabolized in the liver to its active metabolite by cytochrome P450 enzymes. Studies have shown that khat, an addictive substance, is a powerful inhibitor of cytochrome P450 enzymes and can influence the metabolism of drugs that are concomitantly used. Therefore, this study was designed to evaluate the effects of khat on the pharmacokinetics of CLOP in rats. In this study, rats were administered either CLOP alone or CLOP combined with khat and their plasma were obtained at different time intervals and analyzed using the newly developed and validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method using foretinib (FTB) as the internal standard. The corresponding peak area of the analyte versus FTB was used for calculating the peak ratio. The validated LC-MS/MS method resulted in the separation of the well-defined quantifiable peaks of CLOP, FTB, and CLOP metabolite within 7 min. Results showed a significant influence of khat on the peak ratio of CLOP metabolite, which was found to be significantly decreased (P < 0.05) in comparison to CLOP alone, suggesting significant decrease in the conversion of CLOP to its active metabolite due to the inhibition of CYP450 enzymes by khat. Therefore, there might be a need for dose adjustment for regular khat chewers using CLOP.

The fragmentation pattern of certain isatin-based compounds was carried out using collision-induced dissociation inside the triple quadrupole mass analyzer. These data were used as a clue for the identification of metabolites of the recently reported isatin-based antiproliferative agent, namely, N′-[5-bromo-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide (1) in rat liver microsomes (RLMs) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Prediction of the vulnerable sites for metabolic pathways in compound 1 was performed by WhichP450 module of StarDrop software. In vitro metabolites for compound 1 were identified with the aid of rat liver microsomes. The in silico data were utilized as a guide for the practical work. Compound 1 was metabolized into three (hydroxylated, reduced and O-demethylated) metabolites in RLMs in the presence of NADPH. The chemical structures of those metabolites were elucidated, and the metabolic pathways were proposed by comparing the fragmentation pattern of the isatin–indole conjugates 1–7. The data presented in this paper provided useful information on the effect of different substituents on the ionization/fragmentation processes and can be used in the characterization of isatin derivatives. In silico toxicity assessments for the title compounds 1–7 and for the metabolites of compound 1 were conducted utilizing the deductive estimation of risk from existing knowledge (DEREK) module of StarDrop software.

This study investigates the crystal structure, physicochemical properties, and pharmacokinetic profile of Ethyl 2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate (EAMT) as a potential therapeutic agent. The crystal structure was analyzed using Hirshfeld surface analysis in conjunction with the quantum theory of atoms in molecules (QT-AIM). Non-covalent interactions were evaluated through reduced-density gradient reduction, revealing that the EAMT crystal is stabilized by hydrogen bonds between EAMT molecules in the crystal and between EAMT molecules and water molecules. The molecular electrostatic nature of interactions was examined using MESP, while global and local descriptors were calculated to assess the compound's reactivity. Molecular docking with the Adenosine A1 receptor was performed and validated through a 50 ns molecular dynamics simulation (MDS). Results suggest that EAMT influences protein structure, potentially stabilizing specific secondary structure elements. The compactness analysis showed a slightly more compact protein conformation and a marginally increased solvent exposure in the presence of the EAMT ligand, as indicated by Rg and SASA values. The total binding free energy (ΔG total) was determined to be −114.56 kcal/mol. ADMET predictions demonstrated EAMT's compliance with Lipinski's and Pfizer's rule of five, indicating good oral availability. The compound may exhibit low-potency endocrine activity. In conclusion, EAMT presents potential as a therapeutic candidate, warranting further exploration of its molecular interactions, pharmacokinetics, and potential safety concerns. [ABSTRACT FROM AUTHOR]

Dubermatinib (DMB, TP-0903), a benzenesulfonamide, is an inhibitor of the tyrosine kinase AXL, which is a member of the TAM family and can prevent GAS6-mediated activation of AXL in cancer cells. Patients with previously treated chronic lymphocytic leukemia are being studied in phase I/II clinical trials to determine its antineoplastic potential (CLL). In the current work, the Xenosite web predictor tool was employed to predict the vulnerable sites of metabolism and the reactivity pathways (cyanide and GSH) of DMB. Subsequently, we present the analysis and identification of in vitro and reactive intermediates of DMB using liquid chromatography ion trap mass spectrometry (LC–ITMS). Human liver microsomes (HLMs) were exposed to dimethylbenzene in a laboratory setting, and the resulting metabolites were collected through protein precipitation. Intense reactivity toward nucleophilic macromolecules was seen in the metabolites of the piperazine and pyrimidine rings in DMB, iminium, and 2,5-quinone-imine, respectively. To assess the toxicities of the possibly reactive metabolites, DMB was incubated with HLMs in the presence of 1.0 mM KCN and 1.0 mM glutathione. The DMB metabolites found by LC–MS/MS were seven in vitro phase I metabolites, three cyano adducts, and two GSH conjugates. Phase I in vitro metabolic reactions included N-demethylation, hydroxylation, and dechlorination. DMB and its metabolites have not been investigated for their metabolism in vitro. [ABSTRACT FROM AUTHOR]