Rapid LC-MS/MS Bosutinib Quantification with Applications in Metabolic Stability Estimation.

Bosutinib (BOS) is FDA approved drug for the treatment of chronic phase (CP) Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML). We report a fast, sensitive, and simple LC-MS/MS method, validated for the determination of BOS in human liver microsomes, utilizing tofacitinib (TOF) as the internal standard. The separation of BOS and TOF was done using a 1.8 µm C18 column (2.1 × 50 mm) at room temperature using the isocratic elution system of acetonitrile-water (30:70, v/v) containing 0.1 M formic acid at a flow rate of 0.15 mL/min, and a triple-quadrupole tandem mass spectrometer (TQD-MS) with an electrospray ionization (ESI) source that was operated in the positive ion mode. The method was validated according to the European Medicines Agency, and the rapid and specific quantification of BOS in human liver microsomes was achieved in the range of 5-200 ng/mL, with a determination coefficient of 0.999. Intra- and inter-day accuracy and precision values were <4% in all cases. The procedure is rapid, specific, reliable, and can be applied in metabolic stability evaluations since it is the first LC-MS/MS method specific to BOS quantification. The metabolic stability assessment of BOS showed high CLint (34.3 µL/min/mg) and short in vitro t1/2 values of 20.21 min, indicating that BOS may be rapidly eliminated from the blood by the liver.

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.

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.

Selective Stability Indicating Liquid Chromatographic Method Based on Quality by Design Framework and In Silico Toxicity Assessment for Infigratinib and Its Degradation Products.

Infigratinib, a protein kinase inhibitor employed in the therapeutic management of cholangiocarcinoma, was subjected to various stress conditions, including hydrolytic (acidic and alkaline), oxidative, photolytic, and thermal stress, in accordance with the rules established by the International Council for Harmonization. A cumulative count of five degradation products was observed. The application of the Quality by Design principle was utilized in the development of a rapid and specific separation method for Infigratinib and its degradation products. The methodology employed in this study was derived from an experimental design approach, which was utilized to examine the critical process parameters associated with chromatographic systems. The reversed-phase high-performance liquid chromatography technique, employing a C18 column and a mobile phase composed of a gradient mixture of 25 mM ammonium acetate buffer at pH 6.0 and acetonitrile, successfully facilitated the chromatographic separation. The methodology was expanded to include the utilization of UPLC-quadrupole tandem mass spectrometry in order to conduct a comprehensive analysis of the structural properties and characterize the degradation products. Overall, five degradation products were found in different stress conditions. The method was verified at certain working points, wherein a linearity range (5.0-200.0 µg/mL) was developed and other parameters such as accuracy, repeatability, selectivity, and system suitability were evaluated. Finally, the toxicity and mutagenicity of Infigratinib and its degradation products were predicted using in silico software, namely DEREK Nexus® (version 6.2.1) and SARAH Nexus® (version 3.2.1). Various toxicity endpoints, including chromosomal damage, were predicted. Additionally, two degradation products were also predicted to be mutagenic.

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.

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.

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.

Ionophore-Based Polymeric Sensors for Potentiometric Assay of the Anticancer Drug Gemcitabine in Pharmaceutical Formulation: A Comparative Study.

Gemcitabine is a chemotherapeutic agent used to treat various malignancies, including breast and bladder cancer. In the current study, three innovative selective gemcitabine hydrochloride sensors are developed using 4-tert-butylcalix-[8]-arene (sensor 1), ß-cyclodextrin (sensor 2), and γ-cyclodextrin (sensor 3) as ionophores. The three sensors were prepared by incorporating the ionophores with o-nitrophenyl octyl ether as plasticizer and potassium tetrakis(4-chlorophenyl) borate as ionic additive into a polyvinyl chloride polymer matrix. These sensors are considered environmentally friendly systems in the analytical research. The linear responses of gemcitabine hydrochloride were in the concentration range of 6.0 × 10-6 to 1.0 × 10-2 mol L-1 and 9.0 × 10-6 to 1.0 × 10-2 mol L-1 and 8.0 × 10-6 to 1.0 × 10-2 mol L-1 for sensors 1, 2, and 3, respectively. Over the pH range of 6-9, fast-Nernst slopes of 52 ± 0.6, 56 ± 0.3, and 55 ± 0.8 mV/decade were found in the same order with correlation regressions of 0.998, 0.999, and 0.998, respectively. The lower limits of detection for the prepared sensors were 2.5 × 10-6, 2.2 × 10-6, and 2.7 × 10-6 mol L-1. The sensors showed high selectivity and sensitivity for gemcitabine. Validation of the sensors was carried out in accordance with the requirements established by the IUPAC, while being inexpensive and easy to use in drug formulation. A statistical analysis of the methods in comparison with the official method showed that there was no significant difference in accuracy or precision between them. It was shown that the new sensors could selectively and accurately find gemcitabine hydrochloride in bulk powder, pharmaceutical formulations, and quality control tests. The ionophore-based sensor shows several advantages over conventional PVC membrane sensor sensors regrading the lower limit of detection, and higher selectivity towards the target ion.

A Liquid Chromatography Tandem Mass Spectrometry Method for the Simultaneous Estimation of the Dopamine Receptor Antagonist LE300 and Its N-methyl Metabolite in Plasma: Application to a Pharmacokinetic Study.

LE300 is a novel dopamine receptor antagonist used to treat cocaine addiction. In the current study, a sensitive and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been established and validated for the simultaneous analysis of LE300 and its N-methyl metabolite, MLE300, in rat plasma with an application in a pharmacokinetic study. The chromatographic elution of LE300, MLE300, and Ponatinib (IS, internal standard), was carried out on a 50 mm C18 analytical column (ID: 2.1 mm and particle size: 1.8 µm) maintained at 22 ± 2 °C. The run time was 5 min at a flow rate of 0.3 mL/min. The mobile phase consisted of 42% aqueous solvent (10 mM ammonium formate, pH: 4.2 with formic acid) and 58% organic solvent (acetonitrile). Plasma samples were pretreated using protein precipitation with acetonitrile. The electrospray ionization (ESI) source was used to generate an ion-utilizing positive mode. A multiple reaction monitoring mass analyzer mode was utilized for the quantification of analytes. The linearity of the calibration curves in rat plasma ranged from 1 to 200 ng/mL (r2 = 0.9997) and from 2 to 200 ng/mL (r2 = 0.9984) for LE300 and MLE300, respectively. The lower limits of detection (LLOD) were 0.3 ng/mL and 0.7 ng/mL in rat plasma for LE300 and MLE300, respectively. Accuracy (RE%) ranged from -1.71% to -0.07% and -4.18% to -1.48% (inter-day), and from -3.3% to -1.47% and -4.89% to -2.15% (intra-day) for LE300 and MLE300, respectively. The precision (RSD%) was less than 2.43% and 1.77% for the inter-day, and 2.77% and 1.73% for intra-day of LE300 and MLE300, respectively. These results are in agreement with FDA guidelines. The developed LC-MS/MS method was applied in a pharmacokinetic study in Wistar rats. Tmax and Cmax were 2 h and 151.12 ± 12.5 ng/mL for LE300, and 3 h and 170.4 ± 23.3 ng/mL for MLE300.

LC-MS/MS method for the quantitation of decitabine and venetoclax in rat plasma after SPE: Application to pharmacokinetic study.

This study developed a novel, sensitive and selective LC-MS/MS method for the concurrent determination of DCB and VTX in rat plasma using encorafenib as internal standard (IS). To identify DCB, VTX, and IS, the positive multiple reaction monitoring (MRM) mode was used. Chromatographic separation was carried out using a reversed-phase Agilent Eclipse plus C18 column (100 mm × 2.1 mm, 3.5 µm) and an isocratic mobile phase made up of water with 0.1% formic acid and acetonitrile (50:50, v/v, pH 3.2) at a flow rate of 0.30 mL/min for 3.0 min. Prior to analysis, the DCB and VTX with the IS were extracted from plasma using the solid-phase extraction (SPE) method. High recovery rates for DCB, VTX and IS were achieved using the C18 cartridge without interference from plasma endogenous. The developed method was validated as per the FDA guidelines over a linear concentration range in rat plasma from 5-3000 and 5-1000 ng/mL for DCB and VTX, respectively with r2 ≥ 0.998. For both drugs, the lower limits of detection (LLOD) were 2.0 ng/mL. After the HLOQ sample was injected, less than 20% of the LLOQ of DCB, VTX, and less than 5% of the IS carry-over in the blank sample was attained. The overall recoveries of DCB and VTX from rat plasma were in the range of 90.68-97.56%, and the mean RSD of accuracy and precision results was ≤6.84%. For the first time, the newly developed approach was effectively used in a pharmacokinetic study on the simultaneous oral administration of DCB and VTX in rats that received 15.0 mg/kg of DCB and 100.0 mg/kg of VTX.

A Novel Green Micellar HPLC-UV Method for the Estimation of Vandetanib in Pure Form, Human Urine, Human Plasma and Human Liver Microsomes Matrices with Application to Metabolic Stability Evaluation.

Vandetanib (Caprelsa®; VNB) is a prescription medicine that is used for the treatment of medullary thyroid cancer that has disrupted other body parts or that cannot be removed by surgery. It is considered a tyrosine kinase inhibitor (TKI). Fast, sensitive and validated HPLC-UV was established for VNB quantification in pure human biological fluids (urine and plasma) and human liver microsomes (HLMs). This analytical methodology was applied also to the metabolic stability assessment of VNB. This method was performed using a phenyl column (250 mm × 4.6 mm id, 5 µm particle size). A sodium dodecyl sulphate solution (0.05 M, pH 3.0 using 0.02 M orthophosphoric acid) containing 0.3% triethylamine and 10% n-butanol was used as a mobile phase and was pumped isocratically at a flow rate of 0.7 mL/min and at a 260 nm detection wavelength. The total elution time was 6 min with an injection volume of 20 µL. The linearity of the established methodology ranged from 30 to 500 ng/mL in pure form and 50 to 500 ng/mL (r2 ≥ 0.9994) in human biological fluids and HLMs. No significant interference from the matrix components was observed. The proposed methodology revealed the benefits of being green, reliable and economic.

A Rapid and Sensitive Liquid Chromatography-Tandem Mass Spectrometry Bioanalytical Method for the Quantification of Encorafenib and Binimetinib as a First-Line Treatment for Advanced (Unresectable or Metastatic) Melanoma-Application to a Pharmacokinetic Study.

The combination regimen targeting BRAF and MEK inhibition, for instance, encorafenib (Braftovi™, ENF) plus binimetinib (Mektovi®, BNB), are now recommended as first-line treatment in patients with unresectable or metastatic melanoma with a BRAF V600-activating mutation. Patients treated with combination therapy of ENF and BNB demonstrated a delay in resistance development, increases in antitumor activity, and attenuation of toxicities compared with the activity of either agent alone. However, the pharmacokinetic profile of the FDA-approved ENF and BNB is still unclear. In this study, a rapid and sensitive LC-MS/MS bioanalytical method for simultaneous quantification of ENF and BNB in rat plasma was developed and validated. Chromatography was performed on an Agilent Eclipse plus C18 column (50 mm × 2.1 mm, 1.8 µm), with an isocratic mobile phase composed of 0.1% formic acid in water/acetonitrile (67:33, v/v, pH 3.2) at a flow rate of 0.35 mL/min. A positive multiple reaction monitoring (MRM) mode was chosen for detection and the process of analysis was run for 2 min. Plasma samples were pre-treated using protein precipitation with acetonitrile containing spebrutinib as the internal standard (IS). Method validation was assessed as per the FDA guidelines for the determination of ENF and BNB over concentration ranges of 0.5-3000 ng/mL (r2 ≥ 0.997) for each drug (plasma). The lower limits of detection (LLOD) for both drugs were 0.2 ng/mL. The mean relative standard deviation (RSD) of the results for accuracy and precision was ≤ 7.52%, and the overall recoveries of ENF and BNB from rat plasma were in the range of 92.88-102.28%. The newly developed approach is the first LC-MS/MS bioanalytical method that can perform simultaneous quantification of ENF and BNB in rat plasma and its application to a pharmacokinetic study. The mean result for Cmax for BNB and ENF was found to be 3.43 ± 0.46 and 16.42 ± 1.47 µg/mL achieved at 1.0 h for both drugs, respectively. The AUC0-∞ for BNB and ENF was found to be 18.16 ± 1.31 and 36.52 ± 3.92 µg/mL.h, respectively. On the other hand, the elimination half-life (t1/2kel) parameters for BNB and ENF in the rat plasma were found to be 3.39 ± 0.43 h and 2.48 ± 0.24 h, and these results are consistent with previously reported values.

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.

EGFR Inhibitor Gefitinib Induces Cardiotoxicity through the Modulation of Cardiac PTEN/Akt/FoxO3a Pathway and Reactive Metabolites Formation: In Vivo and in Vitro Rat Studies.

Gefitinib (GEF) is a selective inhibitor of the epidermal growth factor receptor (EGFR) used to treat non-small cell lung cancer. Yet, few cases of cardiotoxicity have been reported. However, the role of the PTEN/Akt/FoxO3a pathway, which mediates GEF anticancer activity, in GEF cardiotoxicity remains unclear. For this purpose, in vitro H9c2 cells and in vivo rat cardiomyocytes were utilized as study models. Treatment of H9c2 cells and Sprague-Dawley rats with GEF significantly induced the expression of hypertrophic and apoptotic markers at mRNA and protein levels with an increased plasma level of troponin. This was accompanied by induction of autophagy and mitochondrial dysfunction in H9c2 cells. Inhibition of cardiac EGFR activity and Akt cellular content of in vitro and in vivo rat cardiomyocytes by GEF increased PTEN and FoxO3a gene expression and cellular content. Importantly, treatment of H9c2 cells with PI3K/Akt inhibitor increased PTEN and FoxO3a mRNA expression associated with potentiation of GEF cardiotoxicity. In addition, by using LC-MS/MS, we showed that GEF is metabolized in the rat heart microsomes into one cyanide- and two methoxylamine-adduct reactive metabolites, where their formation was entirely blocked by CYP1A1 inhibitor, α-naphthoflavone. The current study concludes that GEF induces cardiotoxicity through modulating the expression and function of the cardiac PTEN/AKT/FoxO3a pathway and the formation of CYP1A1-mediated reactive metabolites.

Detection and characterization of olmutinib reactive metabolites by LC-MS/MS: Elucidation of bioactivation pathways.

Olmutinib (Olita™) is an orally bioavailable third generation epidermal growth factor receptor tyrosine kinase inhibitor. Olmutinib was approved in South Korea in May 2016 for the treatment of patients suffering from locally advanced or metastatic epidermal growth factor receptor T790M mutation-positive non-small cell lung cancer. Reactive olmutinib intermediates may be responsible for the severe side effects associated with the treatment. However, literature review revealed no previous reports on the structural identification of reactive olmutinib metabolites. In this work, the formation of reactive olmutinib metabolites in rat liver microsomes was investigated. Methoxylamine, glutathione, and potassium cyanide were used as capturing agents for aldehyde, iminoquinones, and iminium intermediates, respectively. The stable complexes formed were identified using liquid chromatography-tandem mass spectrometry. The major phase I metabolic pathway observed in vitro was hydroxylation of the piperazine ring. Seven potential reactive intermediates were characterized, including three iminium ions, three iminoquinones, and one aldehyde. Based on the findings, various bioactivation pathways were postulated. Hence, identifying the reactive intermediates of olmutinib that may be the cause of severe side effects can provide new insights, leading to improved treatments for patients.

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.

A reliable and stable method for the determination of foretinib in human plasma by LC-MS/MS: Application to metabolic stability investigation and excretion rate.

Foretinib (GSK1363089) is a multiple receptor tyrosine kinases inhibitor. In this study, a reliable, fast liquid chromatography-tandem mass spectrometric method was described for assaying foretinib in plasma, urine, and rat liver microsome samples. Simple extraction procedure by protein preciptation with acetonitrile was implemented for foretinib and brigatinib (internal standard) analysis. Chromatographic resolution of analytes was achieved on C18 column with the help of isocratic mobile phase. The binary mobile phase consisted of 60% ammonium formate (10 mM, pH 4.2) and 40% acetonitrile at a flow rate of 0.25 mL/min. Run time was 3 min, and both foretinib and brigatinib were eluted within 0.74 and 1.95 min; they were detected in positive ion mode utilizing multiple reactions monitoring mode. Linearity of the proposed method ranged from 5 to 500 ng/mL (r2 ≥ 0.9993) in the human plasma. Lower limit of quantification and detection were 6.0 and 1.8 ng/mL, respectively. Intraday and interday precision and accuracy were 0.16 to 1.67 % and -2.39 to -0.52 %. In vitro half-life and intrinsic clearance were 24.93 min and 6.56 mL/min/kg, respectively. Literature review showed that no previous studies have been proposed for the analytical quantification of foretinib in human plasma or its metabolic stability. The established method was also applied to estimate the rate of foretinib excretion in rat urine. The developed method can be used for foretinib pharmacokinetic applications.

An LC-MS/MS method for rapid and sensitive high-throughput simultaneous determination of various protein kinase inhibitors in human plasma.

A reliable, high-throughput and sensitive LC-MS/MS procedure was developed and validated for the determination of five tyrosine kinase inhibitors in human plasma. Following their extraction from human plasma, samples were eluted on a RP Luna®-PFP 100 Å column using a mobile phase system composed of acetonitrile and 0.01 m ammonium formate in water (pH ~4.1) with a ratio of (50:50, v/v) flowing at 0.3 mL min-1 . The mass spectrometer was operating with electrospray ionization in the positive ion multiple reaction monitoring mode. The proposed methodology resulted in linear calibration plots with correlation coefficients values of r2 = 0.9995-0.9999 from concentration ranges of 2.5-100 ng mL-1 for imatinib, 5.0-100 ng mL-1 for sorafenib, tofacitinib and afatinib, and 1.0-100 ng mL-1 for cabozantinib. The procedure was validated in terms of its specificity, limit of detection (0.32-1.71 ng mL-1 ), lower limit of quantification (0.97-5.07 ng mL-1 ), intra- and inter assay accuracy (-3.83 to +2.40%) and precision (<3.37%), matrix effect and recovery and stability. Our results demonstrated that the proposed method is highly reliable for routine quantification of the investigated tyrosine kinase inhibitors in human plasma and can be efficiently applied in the rapid and sensitive analysis of their clinical samples.

Development and validation of HPLC-MS/MS method for the determination of lixivaptan in mouse plasma and its application in a pharmacokinetic study.

The aim of the present study was to develop a simple, sensitive and accurate liquid chromatography-electrospray ionization tandem mass spectrometry (ESI-MS/MS) method for the determination of lixivaptan (LIX) in mouse plasma using vildagliptin as the internal standard (IS). A precipitation procedure was used for the extraction of LIX and vildagliptin from mouse plasma. Chromatographic separation of LIX was achieved using a C18 analytical column (50 × 2.1 mm, 1.8 µm) at 25°C. The mobile phase comprised acetonitrile and ammonium formate (10 mm, pH 3.1; 40:60, v/v) pumped at a flow rate of 0.3 mL min-1 . A tandem mass spectrometer with an electrospray ionization source was used to perform the assay. Quantification of LIX at m/z 290 → 137 and IS at 154 → 97 was attained through multiple reaction monitoring. The investigated method was authenticated following the bio-analytical method of validation guidelines of the US Food and Drug Administration. The developed method showed a good linearity over the concentration range from 5 to 500 ng mL-1 , and the calibration curve was linear (r = 0.9998). The mean recovery of LIX from mouse plasma was 99.2 ± 0.68%. All validation parameters for LIX were within the levels required for acceptance. The proposed method was effectively used for a pharmacokinetic study of LIX in mouse plasma.

A highly efficient and sensitive LC-MS/MS method for the determination of afatinib in human plasma: application to a metabolic stability study.

Afatinib (AFT) is a new tyrosine kinase inhibitor approved for the treatment of nonsmall cell lung cancer. In the present study, a simple, specific, rapid and sensitive liquid chromatography tandem mass-spectrometric method for the quantification of AFT in human plasma, was developed and validated. Chromatographic separation of the analytes was accomplished on a reversed-phase Luna(®) -PFP 100 Å column (50 × 2.0 mm; 3.0 µm) maintained at ambient temperature. Isocratic elution was carried out using acetonitrile-water (40:60, v/v) containing 10 mm ammonium formate buffer (pH 4.5) adjusted with formic acid at a flow rate of 0.4 mL min(-1) . The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring mode. The method yields a linear calibration plot (r(2) = 0.9997) from a quantification range of 0.5-500 ng mL(-1) with the lower limit of quantification and lower limit of detection of 1.29 and 0.42 ng mL(-1) , respectively. The intra- and inter-day precision and accuracy were estimated and found to be in the ranges of 1.53-4.11% for precision and -2.80-0.38% for accuracy. Finally, quantification of afatinib in a metabolic stability study in rat liver microsomes was achieved through the proposed method. Copyright © 2016 John Wiley & Sons, Ltd.