Ion Trap LC/MS reveals the generation of reactive intermediates in acalabrutinib metabolism: phase I metabolic profiling and bioactivation pathways elucidation.

Acalabrutinib (CALQUENCE; ACB) is a Bruton tyrosine kinase inhibitor (BTKI) used to treat mantle cell lymphoma, small lymphocytic lymphoma (SLL), and chronic lymphocytic leukemia (CLL). On 21 November 2019, ACB was approved by the U.S. FDA for the use as a single therapy for the treatment of CLL/SLL. In silico studies were first done to propose vulnerable sites of metabolism and reactivity pathways by StarDrop software and Xenosite online software; respectively. ACB metabolites and stable adducts were characterized in vitro from rat liver microsomes (RLMs) using Ion Trap LC/MS. Generation of reactive intermediates (RIs) in the in vitro metabolism of ACB was investigated using glutathione, potassium cyanide, and methoxylamine as trapping nucleophiles for the RIs including iminopyridinone, iminium, and aldehyde, respectively, to form stable adducts that can be identified and characterized by Ion Trap LC/MS. Five phase I metabolites, seven 6-iminopyridin-3(6H)-one and five aldehyde RIs of ACB were identified. Based on literature reviews, the generation of RIs of ACB, and the subsequent drug-induced organ toxicity (DIOT) reactions may provide an explanation of ACB ADRs. Additional drug discovery investigations can be performed to facilitate the creation of novel medications with improved safety characteristics.

Ponatinib: A comprehensive drug profile.

Ponatinib is a prescription medication used to treat a rare form of blood cancer called Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (CML) that is resistant to other treatments. It belongs to a class of drugs called tyrosine kinase inhibitors, which work by blocking abnormal proteins that promote the growth of cancer cells. In this chapter, the synthesis methods and physicochemical properties of ponatinib were reviewed, besides the characterization of the ponatinib structure using different techniques such as elemental analysis, IR, UV, (1H and 13C) NMR, MS, and XRD. Furthermore, the compendial method for analysis of ponatinib was not found, while the literature review of a non-compendial method for analysis of ponatinib, such as spectroscopic, chromatographic, and immunoassay methods, was covered. Moreover, pharmacology and biochemistry were surveyed in the pharmacokinetic and pharmacodynamic studies.

An Ultrafast UPLC-MS/MS Method for Characterizing the In Vitro Metabolic Stability of Acalabrutinib.

Acalabrutinib, commercially known as Calquence®, is a pharmacological molecule that has robust inhibitory activity against Bruton tyrosine kinase. The medicine in question was carefully developed by the esteemed pharmaceutical company AstraZeneca. The FDA granted authorization on 21 November 2019 for the utilization of acalabrutinib (ACB) in the treatment of small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL) in adult patients. The aim of this study was to develop a UPLC-MS/MS method that is effective, accurate, environmentally sustainable, and has a high degree of sensitivity. The methodology was specifically developed with the intention of quantifying ACB in human liver microsomes (HLMs). The methodology described above was subsequently utilized to assess the metabolic stability of ACB in HLMs in an in vitro environment. The validation procedures for the UPLC-MS/MS method in the HLMs were conducted in accordance with the bioanalytical method validation criteria established by the U.S.- DA. The utilization of the StarDrop software (version 6.6), which integrates the P450 metabolic module and DEREK software (KB 2018 1.1), was employed for the purpose of evaluating the metabolic stability and identifying potential hazardous alarms associated with the chemical structure of ACB. The calibration curve, as established by the ACB, demonstrated a linear correlation across the concentration range of 1 to 3000 ng/mL in the matrix of HLMs. The present study conducted an assessment of the accuracy and precision of the UPLC-MS/MS method in quantifying inter-day and intra-day fluctuations. The inter-day accuracy demonstrated a spectrum of values ranging from -1.00% to 8.36%, whilst the intra-day accuracy presented a range of values spanning from -2.87% to 4.11%. The t1/2 and intrinsic clearance (Clint) of ACB were determined through in vitro testing to be 20.45 min and 39.65 mL/min/kg, respectively. The analysis concluded that the extraction ratio of ACB demonstrated a moderate level, thus supporting the recommended dosage of ACB (100 mg) to be administered twice daily for the therapeutic treatment of persons suffering from B-cell malignancies. Several computational tools have suggested that introducing minor structural alterations to the butynoyl group, particularly the alpha, beta-unsaturated amide moiety, or substituting this group during the drug design procedure, could potentially enhance the metabolic stability and safety properties of novel derivatives in comparison to ACB.

Evaluation of Alectinib Metabolic Stability in HLMs Using Fast LC-MS/MS Method: In Silico ADME Profile, P450 Metabolic Lability, and Toxic Alerts Screening.

Alectinib, also known as Alecensa®, is prescribed for the therapeutic treatment of individuals diagnosed with metastatic non-small cell lung cancer (NSCLC) who have a specific genetic mutation referred to as anaplastic lymphoma kinase (ALK) positivity. The Food and Drug Administration granted regular approval to alectinib, a drug developed by Hoffmann-La Roche, Inc. (Basel, Switzerland)/Genentech, Inc. (South San Francisco, CA, USA), on 6 November 2017. The screening of the metabolic stability and identification of hazardous alarms within the chemical structure of ALC was conducted using the StarDrop software package (version 6.6), which incorporates the P450 metabolic module and DEREK software (KB 2018 1.1). The primary aim of this investigation was to develop a high-throughput and accurate LC-MS/MS technique for the quantification of ALC in the metabolic matrix (human liver microsomes; HLMs). The aforementioned methodology was subsequently employed to assess the metabolic stability of ALC in HLMs through in vitro tests, with the obtained results further validated using in silico software. The calibration curve of the ALC showed a linear correlation that exists within the concentration range from 1 to 3000 ng/mL. The LC-MS/MS approach that was recommended exhibited accuracy and precision levels for both inter-day and intra-day measurements. Specifically, the accuracy values ranged from -2.56% to 3.45%, while the precision values ranged from -3.78% to 4.33%. The sensitivity of the established approach was proved by its ability to adhere to an LLOQ of 0.82 ng/mL. The half-life (t1/2) and intrinsic clearance (Clint) of ALC were estimated to be 22.28 min and 36.37 mL/min/kg, correspondingly, using in vitro experiments. The ALC exhibited a moderate extraction ratio. The metabolic stability and safety properties of newly created derivatives can be enhanced by making modest adjustments to the morpholine and piperidine rings or by substituting the substituent, as per computational software. In in silico ADME prediction, ALC was shown to have poor water solubility and high gastrointestinal absorption along with inhibition of some cytochrome P450s (CYP2C19 and CYP2C9) without inhibition of others (CYP1A2, CYP3A4, and CYP2D6) and P-glycoprotein substrate. The study design that involves using both laboratory experiments and different in silico software demonstrates a novel and groundbreaking approach in the establishment and uniformization of LC-MS/MS techniques for the estimation of ALC concentrations, identifying structural alerts and the assessment of its metabolic stability. The utilization of this study strategy has the potential to be employed in the screening and optimization of prospective compounds during the drug creation process. This strategy may also facilitate the development of novel derivatives of the medicine that maintain the same biological action by targeted structural modifications, based on an understanding of the structural alerts included within the chemical structure of ALC.

Reactive intermediates formation and bioactivation pathways of spebrutinib revealed by LC-MS/MS: In vitro and in silico metabolic study.

Spebrutinib is a new Bruton tyrosine kinase inhibitor developed by Avila Therapeutics and Celgene. Spebrutinib (SPB) is currently in phase Ib clinical trials for the treatment of lymphoma in the United States. Preliminary in-silico studies were first performed to predict susceptible sites of metabolism, reactivity pathways and structural alerts for toxicities by StarDrop WhichP450™ module, Xenosite web predictor tool and DEREK software; respectively. SPB metabolites and adducts were characterized in vitro from rat liver microsomes (RLM) using LC-MS/MS. Formation of reactive intermediates was investigated using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles for the unstable and reactive iminium, iminoquinone and aldehyde intermediates, respectively, with the aim to produce stable adducts that can be detected and characterized using mass spectrometry. Fourteen phase I metabolites, four cyanide adducts, six GSH adducts and three methoxylamine adducts of SPB were identified and characterized. The proposed metabolic pathways involved in generation of phase I metabolites of SPB are oxidation, hydroxylation, o-dealkylation, epoxidation, defluorination and reduction. Several in vitro reactive intermediates were identified and characterized, the formation of which can aid in explaining the adverse drug reactions of SPB. Several iminium, 2-iminopyrimidin-5(2H)-one and aldehyde intermediates of SPB were revealed. Acrylamide is identified as a structural alert for toxicity by DEREK report and was found to be involved in the formation of several glycidamide and aldehyde reactive intermediates.

Investigation of Fenebrutinib Metabolism and Bioactivation Using MS3 Methodology in Ion Trap LC/MS.

Fenebrutinib is an orally available Bruton tyrosine kinase inhibitor. It is currently in multiple phase III clinical trials for the management of B-cell tumors and autoimmune disorders. Elementary in-silico studies were first performed to predict susceptible sites of metabolism and structural alerts for toxicities by StarDrop WhichP450™ module and DEREK software; respectively. Fenebrutinib metabolites and adducts were characterized in-vitro in rat liver microsomes (RLM) using MS3 method in Ion Trap LC-MS/MS. Formation of reactive and unstable intermediates was explored using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles to capture the transient and unstable iminium, 6-iminopyridin-3(6H)-one and aldehyde intermediates, respectively, to generate a stable adducts that can be investigated and analyzed using mass spectrometry. Ten phase I metabolites, four cyanide adducts, five GSH adducts and six methoxylamine adducts of fenebrutinib were identified. The proposed metabolic reactions involved in formation of these metabolites are hydroxylation, oxidation of primary alcohol to aldehyde, n-oxidation, and n-dealkylation. The mechanism of reactive intermediate formation of fenebrutinib can provide a justification of the cause of its adverse effects. Formation of iminium, iminoquinone and aldehyde intermediates of fenebrutinib was characterized. N-dealkylation followed by hydroxylation of the piperazine ring is proposed to cause the bioactivation to iminium intermediates captured by cyanide. Oxidation of the hydroxymethyl group on the pyridine moiety is proposed to cause the generation of reactive aldehyde intermediates captures by methoxylamine. N-dealkylation and hydroxylation of the pyridine ring is proposed to cause formation of iminoquinone reactive intermediates captured by glutathione. FBB and several phase I metabolites are bioactivated to fifteen reactive intermediates which might be the cause of adverse effects. In the future, drug discovery experiments utilizing this information could be performed, permitting the synthesis of new drugs with better safety profile. Overall, in silico software and in vitro metabolic incubation experiments were able to characterize the FBB metabolites and reactive intermediates using the multistep fragmentation capability of ion trap mass spectrometry.

Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method.

Selpercatinib (SLP; brand name Retevmo®) is a selective and potent RE arranged during transfection (RET) inhibitor. On 21 September 2022, the FDA granted regular approval to SLP (Retevmo, Eli Lilly, and Company). It is considered the only and first RET inhibitor for adults with metastatic or locally advanced solid tumors with RET gene fusion. In the current experiment, a highly specific, sensitive, and fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying SLP in human liver microsomes (HLMs) was developed and applied to the metabolic stability evaluation of SLP. The LC-MS/MS method was validated following the bioanalytical methodology validation guidelines outlined by the FDA (linearity, selectivity, matrix effect, accuracy, precision, carryover, and extraction recovery). SLP was detected by a triple quadrupole detector (TQD) using a positive ESI source and multiple reaction monitoring (MRM) mode for mass spectrometric analysis and estimation of analytes ions. The IS-normalized matrix effect and extraction recovery were acceptable according to the FDA guidelines for the bioanalysis of SLP. The SLP calibration standards were linear from 1 to 3000 ng/mL HLMs matrix, with a regression equation (y = 1.7298x + 3.62941) and coefficient of variation (r2 = 0.9949). The intra-batch and inter-batch precision and accuracy of the developed LC-MS/MS method were -6.56-5.22% and 5.08-3.15%, respectively. SLP and filgotinib (FLG) (internal standard; IS) were chromatographically separated using a Luna 3 µm PFP (2) stationary phase (150 × 4.6 mm) with an isocratic mobile phase at 23 ± 1 °C. The limit of quantification (LOQ) was 0.78 ng/mL, revealing the LC-MS/MS method sensitivity. The intrinsic clearance and in vitro t1/2 (metabolic stability) of SLP in the HLMs matrix were 34 mL/min/kg and 23.82 min, respectively, which proposed an intermediate metabolic clearance rate of SLP, confirming the great value of this type of kinetic experiment for more accurate metabolic stability predictions. The literature review approved that the established LC-MS/MS method is the first developed and reported method for quantifying SLP metabolic stability.

Development of an LC-MS/MS Method for Quantification of Sapitinib in Human Liver Microsomes: In Silico and In Vitro Metabolic Stability Evaluation.

Sapitinib (AZD8931, SPT) is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) family (pan-erbB). In multiple tumor cell lines, STP has been shown to be a much more potent inhibitor of EGF-driven cellular proliferation than gefitinib. In the current study, a highly sensitive, rapid, and specific LC-MS/MS analytical method for the estimation of SPT in human liver microsomes (HLMs) was established with application to metabolic stability assessment. The LC-MS/MS analytical method was validated in terms of linearity, selectivity, precision, accuracy, matrix effect, extraction recovery, carryover, and stability following the FDA guidelines for bioanalytical method validation. SPT was detected using electrospray ionization (ESI) as an ionization source under multiple reaction monitoring (MRM) in the positive ion mode. The IS-normalized matrix factor and extraction recovery were acceptable for the bioanalysis of SPT. The SPT calibration curve was linear, from 1 ng/mL to 3000 ng/mL HLM matrix samples, with a linear regression equation of y = 1.7298x + 3.62941 (r2 = 0.9949). The intraday and interday accuracy and precision values of the LC-MS/MS method were -1.45-7.25% and 0.29-6.31%, respectively. SPT and filgotinib (FGT) (internal standard; IS) were separated through the use of an isocratic mobile phase system with a Luna 3 µm PFP(2) column (150 × 4.6 mm) stationary phase column. The limit of quantification (LOQ) was 0.88 ng/mL, confirming the LC-MS/MS method sensitivity. The intrinsic clearance and in vitro half-life of STP were 38.48 mL/min/kg and 21.07 min, respectively. STP exhibited a moderate extraction ratio that revealed good bioavailability. The literature review demonstrated that the current analytical method is the first developed LC-MS/MS method for the quantification of SPT in an HLM matrix with application to SPT metabolic stability evaluation.

Development and Validation of a Rapid LC-MS/MS Method for Quantifying Alvocidib: In Silico and In Vitro Metabolic Stability Estimation in Human Liver Microsomes.

Alvocidib (AVC; flavopiridol) is a potent cyclin-dependent kinase inhibitor used in patients with acute myeloid leukemia (AML). The FDA has approved orphan drug designation to AVC for treating patients with AML. In the current work, the in silico calculation of AVC metabolic lability was done using the P450 metabolism module of the StarDrop software package, that is expressed as a composite site lability (CSL). This was followed by establishing an LC-MS/MS analytical method for AVC estimation in human liver microsomes (HLMs) to assess metabolic stability. AVC and glasdegib (GSB), used as internal standards (IS), were separated utilizing a C18 column (reversed chromatography) with an isocratic mobile phase. The lower limit of quantification (LLOQ) was 5.0 ng/mL, revealing the sensitivity of the established LC-MS/MS analytical method that exhibited a linearity in the range 5-500 ng/mL in the HLMs matrix with correlation coefficient (R2 = 0.9995). The interday and intraday accuracy and precision of the established LC-MS/MS analytical method were -1.4% to 6.7% and -0.8% to 6.4%, respectively, confirming the reproducibility of the LC-MS/MS analytical method. The calculated metabolic stability parameters were intrinsic clearance (CLint) and in vitro half-life (t1/2) of AVC at 26.9 µL/min/mg and 25.8 min, respectively. The in silico results from the P450 metabolism model matched the results generated from in vitro metabolic incubations; therefore, the in silico software can be used to predict the metabolic stability of the drugs, saving time and resources. AVC exhibits a moderate extraction ratio, indicating reasonable in vivo bioavailability. The established chromatographic methodology was the first LC-MS/MS method designed for AVC estimation in HLMs matrix that was applied for AVC metabolic stability estimation.

Piperazine ring toxicity in three novel anti-breast cancer drugs: an in silico and in vitro metabolic bioactivation approach using olaparib as a case study.

The metabolic activation of small-molecule drugs into electrophilic reactive metabolites is widely recognized as an indicator of idiosyncratic adverse drug reactions (IADRs). Three novel anti-breast cancer drugs containing piperazine rings, ribociclib (Kisqali®, RCB), abemaciclib (Verzenio®, ABC), and olaparib (Lynparza®, OLP), were selected to study the effect of different chemical environment on the piperazine ring activation using in silico and in vitro metabolic experiments. ABC and RCB were previously studied and we noticed the piperazine ring in ABC could be strongly bioactivated generating more reactive intermediates than piperazine ring in RCB. OLP was further used as a case study to show the power of in silico software to predict the piperazine ring activation that was approved using in vitro experiments. Initially, predictions of susceptible sites in the metabolism and reactivity pathways were performed using the StarDrop P450 model and XenoSite reactivity tool, respectively. Later, in vitro OLP metabolites were characterized based on rat liver microsomes (RLMs) using KCN (trapping agent) using LC-MS/MS. The main goal of the current study was to answer the question of whether the presence of a piperazine ring in the chemical structure should be always considered a structural alert. Piperazine ring in RBC and ABC was bioactivated through a metabolic sequence that involves the hydroxylation of α-carbon to the tertiary nitrogen atoms of the piperazine ring. In the case of OLP, no cyano adduct was formed due to the presence of two carbonyl groups attached to the two nitrogen atoms of the piperazine ring (neutral amide groups). From the results, piperazine ring in certain cases should not be considered as a structural alert as in the case of OLP due to the presence of two electron withdrawing group that stops the proposed toxicity. Also blocking the bioactive center (α-carbon) using steric hindrances such as methyl group, also the isosteric replacement of α-carbon hydrogen with fluoro atom can aid in reducing the toxic side effects of ABC and RCB. These experiments were done in vitro through incubation with RLMs in the presence of KCN. Also, the results are supported by data generated from in silico software. In the future, drug discovery studies using this concept could be undertaken, allowing for the development of new drugs with reasonable safety profiles. Overall, in vitro RLMs incubations and in silico experiments were able to predict successfully that the piperazine ring should not always be considered a structural alert.

Profiling of in vivo, in vitro and reactive zorifertinib metabolites using liquid chromatography ion trap mass spectrometry.

Zorifertinib (AZD-3759; ZFB) is a potent, novel, oral, small molecule used for the treatment of non-small cell lung cancer (NSCLC). ZFB is Epidermal Growth Factor Receptor (EGFR) inhibitor that is characterized by good permeability of the blood-brain barrier for (NSCLC) patients with EGFR mutations. The present research reports the profiling of in vitro, in vivo and reactive metabolites of ZFB. Prediction of vulnerable metabolic sites and reactivity pathways (cyanide and GSH) of ZFB were performed by WhichP450™ module (StarDrop software package) and XenoSite reactivity model (XenoSite Web Predictor-Home), respectively. ZFB in vitro metabolites were done by incubation with isolated perfused rat liver hepatocytes and rat liver microsomes (RLMs). Extraction of ZFB and its related metabolites from the incubation matrix was done by protein precipitation. In vivo metabolism was performed by giving ZFB (10 mg kg-1) through oral gavage to Sprague Dawley rats that were housed in metabolic cages. Urine was collected at specific time intervals (0, 6, 12, 18, 24, 48, 72, 96 and 120 h) from ZFB dosing. The collected urine samples were filtered then stored at -70 °C. N-Methyl piperazine ring of ZFB undergoes phase I metabolism forming iminium intermediates that were stabilized using potassium cyanide as a trapping agent. Incubation of ZFB with RLMs were performed in the presence of 1.0 mM KCN and 1.0 mM glutathione to check reactive intermediates as it is may be responsible for toxicities associated with ZFB usage. For in vitro metabolites there were six in vitro phase I metabolites, three in vitro phase II metabolites, seven reactive intermediates (four GSH conjugates and three cyano adducts) of ZFB were detected by LC-IT-MS. For in vivo metabolites there were six in vivo phase I and three in vivo phase II metabolites of ZFB were detected by LC-IT-MS. In vitro and in vivo phase I metabolic pathways were N-demethylation, O-demethylation, hydroxylation, reduction, defluorination and dechlorination. In vivo phase II metabolic reaction was direct sulphate and glucuronic acid conjugation with ZFB.

A validated LC-MS/MS analytical method for the quantification of pemigatinib: metabolic stability evaluation in human liver microsomes.

Pemigatinib (PMB) is a small molecule inhibitor of fibroblast growth factor receptor 1 (FGFR1), FGFR2 and FGFR3. On April 17, 2020, the US Food and Drug Administration granted accelerated approval for PMB for the treatment of adults with previously treated, unresectable metastatic or locally advanced cholangiocarcinoma with a fibroblast growth factor receptor 2 (FGFR2) fusion or other rearrangement. PMB is considered the first targeted treatment for cholangiocarcinoma approved in the US. In this study, in silico prediction of PMB metabolic stability was done using the WhichP450 module of the StarDrop software package. Further, an LC-MS/MS analytical method was developed for PMB quantification in human liver microsomes (HLM) to experimentally assess metabolic stability. PMB and flavopiridol (FVL), used as an internal standard IS, were resolved using an isocratic mobile phase and a C18 stationary phase. The LC-MS/MS method showed linearity in the range of 5 to 500 ng mL-1 in an HLM matrix (R 2 = 0.9995). The lower limit of quantification (LLOQ) was 5 ng mL-1, indicating sensitivity. The inter- and intra-day accuracy and precision were within a variability of 10, confirming the reproducibility of the method. The measured in vitro half-life and intrinsic clearance of PMB were 27.29 min and 25.40 µL min-1 mg-1, respectively. PMB showed a moderate extraction ratio suggesting good bioavailability. The developed analytical method is the first LC-MS/MS method specific for PMB quantification with application to metabolic stability assessment.

Estimation of zorifertinib metabolic stability in human liver microsomes using LC-MS/MS.

Zorifertinib (AZD-3759; ZFB) is a novel, potent, oral, small molecule used to treat non-small cell lung cancer. ZFB is an epidermal growth factor receptor inhibitor that is capable of crossing blood-brain barrier. The in silico metabolic software used for ZFB metabolic stability prediction was the StarDrop software package (WhichP450 module). An LC-MS/MS analytical method (fast and accurate) was established for ZFB quantification in human liver microsomes (HLMs) in order to estimate its metabolic stability. ZFB and encorafenib (ENF) (internal standard; IS) were separated through the use of an isocratic mobile phase system with a C8 stationary phase column. The LC-MS/MS method for ZFB exhibited linearity in the range of 5 ng/mL to 500 ng/mL in HLMs matrix with a linear regression equation: y = 0.2438x - 0.341 (R² = 0.9992). The limit of quantification (LOQ) was 3.78 ng/mL confirming the LC-MS/MS method sensitivity. The inter- and intraday accuracy and precision were less than 9.56% confirming the reproducibility of the LC-MS/MS method. The intrinsic clearance and in vitro half-life of ZFB were 32.5 µL/min/mg and 21.33 min, respectively. ZFB exhibited a moderate extraction ratio that revealed good bioavailability. Literature review demonstrated that the developed analytical method is the first developed LC-MS/MS method for determining ZFB metabolic stability.

LC-MS/MS Estimation of Rociletinib Levels in Human Liver Microsomes: Application to Metabolic Stability Estimation.

BACKGROUND: Rociletinib (CO-1686; RLC) is a new, small molecule that is orally administered to inhibit mutant-selective covalent inhibitor of most epidermal growth factor receptor (EGFR)-mutated forms, including T790M, L858R, and exon 19 deletions, but not exon 20 insertions. Non-small-cell lung cancer (NSCLC) with a gene mutation that encodes EGFR is sensitive to approved EGFR inhibitors, but usually resistance develops, which is frequently mediated by T790M EGFR mutation. RLC is an EGFR inhibitor found to be active in preclinical models of EGFR-mutated NSCLC with or without T790M. METHODS: In silico drug metabolism prediction of RLC was executed with the aid of the WhichP450 module (StarDrop software package) to verify its metabolic liability. Second, a fast, accurate, and competent LC-MS/MS assay was developed for RLC quantification to determine its metabolic stability. RLC and bosutinib (BOS) (internal standard; IS) were separated using an isocratic elution system with a C18 column (reversed stationary phase). RESULTS: The developed LC-MS/MS analytical method showed linearity of 5-500 ng/mL with r2 ≥ 0.9998 in the human liver microsomes (HLMs) matrix. A limit of quantification of 4.6 ng/mL revealed the sensitivity of the analytical method, while the acquired inter- and intra-day accuracy and precision values below 4.63% inferred the method reproducibility. RLC metabolic stability estimation was calculated using intrinsic clearance (20.15 µL/min/mg) and in vitro half-life (34.39 min) values. CONCLUSION: RLC exhibited a moderate extraction ratio indicative of good bioavailability. The developed analytical method herein is the first LC-MS/MS assay for RLC metabolic stability.

LC-MS/MS method for the quantification of the anti-cancer agent infigratinib: Application for estimation of metabolic stability in human liver microsomes.

Infigratinib (INF) is a novel small molecule, administered orally, which acts as a human fibroblast growth factor receptors (FGFRs) inhibitor. FGFRs are a family of receptor tyrosine kinases (RTK) reported to be upregulated in various tumor cell types. In 1 December 2020, BridgeBio Pharma Inc. announced FDA approval of INF as a New Drug Application, granting it Priority Review for the treatment of cholangiocarcinoma (CCA). Thus, the current study aimed to establish a validated LC-MS/MS method to estimate the INF concentration in the HLM matrix. In silico prediction of INF metabolism was done using the StarDrop® WhichP450™ module to verify its metabolic stability. An accurate and efficient LC-MS/MS analytical method was developed for INF metabolic stability evaluation. INF and duvelisib (DVB) (internal standard; IS) were eluted using an isocratic mobile phase with a C18 column as a stationary reversed phase. The established LC-MS/MS method showed a linear range over 5-500 ng/mL (r2 ≥ 0.9998) in human liver microsomes (HLMs). The sensitivity of the method was confirmed at its limit of quantification (4.71 ng/mL), and reproducibility was indicated by inter- and intra-day accuracy and precision (within 7.3%). The evaluation of INF metabolic stability was assessed, which reflected an intrinsic clearance of 23.6 µL/min/mg and in vitro half-life of 29.4 min. The developed approach in the current study is the first LC-MS/MS method for INF metabolic stability assessment. Application of the developed method in HLM in vitro studies suggests that INF has a moderate extraction ratio, indicating relatively good predicted oral bioavailability.

A Validated LC-MS/MS Assay for the Simultaneous Quantification of the FDA-Approved Anticancer Mixture (Encorafenib and Binimetinib): Metabolic Stability Estimation.

The concurrent use of oral encorafenib (Braftovi, ENF) and binimetinib (Mektovi, BNB) is a combination anticancer therapy approved by the United States Food and Drug Administration (USFDA) for patients with BRAFV600E/V600K mutations suffering from metastatic or unresectable melanoma. Metabolism is considered one of the main pathways of drug elimination from the body (responsible for elimination of about 75% of known drugs), it is important to understand and study drug metabolic stability. Metabolically unstable compounds are not good as they required repetitive dosages during therapy, while very stable drugs may result in increasing the risk of adverse drug reactions. Metabolic stability of compounds could be examined using in vitro or in silico experiments. First, in silico metabolic vulnerability for ENF and BNB was investigated using the StarDrop WhichP450 module to confirm the lability of the drugs under study to liver metabolism. Second, we established an LC-MS/MS method for the simultaneous quantification of ENF and BNB applied to metabolic stability assessment. Third, in silico toxicity assessment of ENF and BNB was performed using the StarDrop DEREK module. Chromatographic separation of ENF, BNB, and avitinib (an internal standard) was achieved using an isocratic mobile phase on a Hypersil BDS C18 column. The linear range for ENF and BNB in the human liver microsome (HLM) matrix was 5-500 ng/mL (R2 ≥ 0.999). The metabolic stabilities were calculated using intrinsic clearance and in vitro half-life. Furthermore, ENF and BNB did not significantly influence each other's metabolic stability or metabolic disposition when used concurrently. These results indicate that ENF and BNB will slowly bioaccumulate after multiple doses.

Identification and characterization of in vitro, in vivo, and reactive metabolites of tandutinib using liquid chromatography ion trap mass spectrometry.

Tandutinib (TND) is a novel, oral small molecule designed for treating acute myeloid leukemia (AML) by inhibiting type III receptor tyrosine kinases. This study reports the use of in silico, in vivo, and in vitro methods to investigate the metabolism and possible metabolic bioactivation of TND. First, in silico metabolism of TND was assessed using the WhichP450™ module of the StarDrop® software to determine labile sites of metabolism in the TND chemical structure. Second, the XenoSite reactivity model, a web-based metabolism prediction software, was used to determine probable bioactive centers. Based on the in silico outcomes, a list of predicted metabolites and reactive intermediates were prepared. Third, in vitro and in vivo experiments were performed. In vitro TND metabolites were generated through incubation of TND with rat liver microsomes (RLMs). Another incubation of TND with RLMs was separately performed in the presence of GSH and KCN to check for the generation of reactive intermediates (soft and hard electrophiles). In vitro phase II metabolism was assessed by incubation of TND with isolated perfused rat hepatocytes. In vivo metabolism was investigated by oral gavage of TND (37 mg kg-1) in Sprague Dawley rats. Five in vitro phase I metabolites, one in vitro phase II and five reactive iminium intermediates (cyano adducts), six in vivo phase I, and one in vivo phase II metabolites of TND were characterized. The in vitro and in vivo metabolic pathways involved were O-dealkylation, α-hydroxylation, α-carbonyl formation, reduction, glucuronide, and sulfate conjugation. No GSH conjugate or its catabolic products were detected either in vitro or in vivo. Two cyclic tertiary rings of TND (piperazine and piperidine) were metabolically bioactivated to generate reactive iminium intermediates forming cyano adducts with KCN. The formed reactive intermediates may be the reason behind TND toxicity. In silico toxicological studies were performed for TND and its related (in vitro and in vivo) metabolites were evaluated using the DEREK software tool.

Galeon: A Biologically Active Molecule with In Silico Metabolite Prediction, In Vitro Metabolic Profiling in Rat Liver Microsomes, and In Silico Binding Mechanisms with CYP450 Isoforms.

Galeon, a natural cyclic-diarylheptanoid (CDH), which was first isolated from Myrica gale L., is known to have potent cytotoxicity against A549 cell lines, anti-tubercular activity against Mycobacterium tuberculosis H37Rv, chemo-preventive potential, and moderate topoisomerase inhibitory activity. Here, in silico metabolism and toxicity prediction of galeon by CYP450, in vitro metabolic profiling study in rat liver microsomes (RLMs), and molecular interactions of galeon-CYP450 isoforms were performed. An in silico metabolic prediction study showed demethyl and mono-hydroxy galeon were the metabolites with the highest predictability. Among the predicted metabolites, mono-hydroxy galeon was found to have plausible toxicities such as skin sensitization, thyroid toxicity, chromosome damage, and carcinogenicity. An in vitro metabolism study of galeon, incubated in RLMs, revealed eighteen Phase-I metabolites, nine methoxylamine, and three glutathione conjugates. Identification of possible metabolites and confirmation of their structures were carried out using ion-trap tandem mass spectrometry. In silico docking analysis of galeon demonstrated significant interactions with active site residues of almost all CYP450 isoforms.

Characterization of Stable and Reactive Metabolites of the Anticancer Drug, Ensartinib, in Human Liver Microsomes Using LC-MS/MS: An in silico and Practical Bioactivation Approach.

BACKGROUND: Ensartinib (ESB) is a novel anaplastic lymphoma kinase inhibitor (ALK) with additional activity against Abelson murine leukemia (ABL), met proto-oncogene (MET), receptor tyrosine kinase (AXL), and v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1) and is considered a safer alternative for other ALK inhibitors. ESB chemical structure contains a dichloro-fluorophenyl ring and cyclic tertiary amine rings (piperazine) that can be bioactivated generating reactive intermediates. METHODS: In vitro metabolic study of ESB with human liver microsomes (HLMs) was performed and the hypothesis of generating reactive intermediates during metabolism was tested utilizing trapping agents to capture and stabilize reactive intermediates to facilitate their LC-MS/MS detection. Reduced glutathione (GSH) and potassium cyanide (KCN) were utilized as trapping agents for quinone methide and iminium intermediates, respectively. RESULTS: Four in vitro ESB phase I metabolites were characterized. Three reactive intermediates including one epoxide and one iminium intermediates were characterized. ESB bioactivation is proposed to occur through unexpected metabolic pathways. The piperazine ring was bioactivated through iminium ions intermediates generation, while the dichloro-phenyl group was bioactivated through a special mechanism that was revealed by LC-MS/MS. CONCLUSION: These findings lay the foundations for additional work on ESB toxicity. Substituents to the bioactive centers (piperazine ring), either for blocking or isosteric replacement, would likely block or interrupt hydroxylation reaction that will end the bioactivation sequence.

Identification of Iminium Intermediates Generation in the Metabolism of Tepotinib Using LC-MS/MS: In Silico and Practical Approaches to Bioactivation Pathway Elucidation.

Tepotinib (Tepmetko™, Merck) is a potent inhibitor of c-Met (mesenchymal-epithelial transition factor). In March 2020, tepotinib (TEP) was approved for use in Japan for the treatment of patients who suffered from non-small cell lung cancers (NSCLC) harboring an MET exon 14 skipping alteration and have progressed after platinum-based therapy. Practical and in silico experiments were used to screen for the metabolic profile and reactive intermediates of TEP. Knowing the bioactive center and structural alerts in the TEP structure helped in making targeted modifications to improve its safety. First, the prediction of metabolism vulnerable sites and reactivity metabolic pathways was performed using the StarDrop WhichP450™ module and the online Xenosite reactivity predictor tool, respectively. Subsequently, in silico data were used as a guide for the in vitro practical work. Second, in vitro phase I metabolites of TEP were generated from human liver microsome (HLM) incubations. Testing for the generation of unstable reactive intermediates was performed using potassium cyanide as a capturing agent forming stable cyano adduct that can be characterized and identified using liquid chromatography tandem mass spectrometry (LC-MS/MS). Third, in silico toxicity assessment of TEP metabolites was performed, and structural modification was proposed to decrease their side effects and to validate the proposed bioactivation pathway using the DEREK software. Four TEP phase I metabolites and four cyano adducts were characterized. The reactive intermediate generation mechanism of TEP may provide an explanation of its adverse reactions. The piperidine ring is considered a structural alert for toxicity as proposed by the DEREK software and a Xenosite reactivity model, which was confirmed by practical experiments. Steric hindrance or isosteric replacement at α-carbon of the piperidine ring stop the bioactivation sequence that was confirmed using the DEREK software. More drug discovery studies can be performed using this perception permitting the design of new drugs with an increased safety profile. To our knowledge, this is the first study for the identification of in vitro phase I metabolites and reactive intermediates in addition to toxicological properties of the metabolites for TEP that will be helpful for the evaluation of TEP side effects and drug-drug interactions in TEP-treated patients.