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.

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.

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.

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.

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.

Enhanced Codelivery of Gefitinib and Azacitidine for Treatment of Metastatic-Resistant Lung Cancer Using Biodegradable Lipid Nanoparticles.

Lung cancer is a formidable challenge in clinical practice owing to its metastatic nature and resistance to conventional treatments. The codelivery of anticancer agents offers a potential solution to overcome resistance and minimize systemic toxicity. The encapsulation of these agents within nanostructured lipid carriers (NLCs) provides a promising strategy to enhance lymphatic delivery and reduce the risk of relapse. This study aimed to develop an NLC formulation loaded with Gefitinib and Azacitidine (GEF-AZT-NLC) for the treatment of metastatic-resistant lung cancer. The physicochemical properties of the formulations were characterized, and in vitro drug release was evaluated using the dialysis bag method. The cytotoxic activity of the GEF-AZT-NLC formulations was assessed on a lung cancer cell line, and hemocompatibility was evaluated using suspended red blood cells. The prepared formulations exhibited nanoscale size (235-272 nm) and negative zeta potential values (-15 to -31 mV). In vitro study revealed that the GEF-AZT-NLC formulation retained more than 20% and 60% of GEF and AZT, respectively, at the end of the experiment. Hemocompatibility study demonstrated the safety of the formulation for therapeutic use, while cytotoxicity studies suggested that the encapsulation of both anticancer agents within NLCs could be advantageous in treating resistant cancer cells. In conclusion, the GEF-AZT-NLC formulation developed in this study holds promise as a potential therapeutic tool for treating metastatic-resistant lung cancer.

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.

Vandetanib.

Vandetanib is an anti-cancer drug called an antineoplastic kinase inhibitor. The FDA authorized vandetanib on April6, 2011 for the treatment of nonresectable, locally progressed, or metastatic medullary thyroid carcinoma in adults. Because Vandetanib can make the Q-T interval last longer, it shouldn't be given to people with serious heart problems like congenital long QT syndrome or heart failure that hasn't been fixed yet. This chapter provides an overview of Vandetanib's physical and molecular properties, mode of action, pharmacokinetics, and common applications. In furthermore, a detailed summary of the reported techniques of Vandetanib measurement will be provided to assist analysts in selecting the most practical approach for its estimation in routine analysis. This chapter will also explain the synthesis methods developed in the preparation of vandetanib as well as pharmacology of its. In addition, this section summarizes the analytical and characterization techniques utilized to characterize vandetanib row material.

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 and validation of an UPLC-ESI-MS/MS method for quantification of duvelisib in plasma: application to pharmacokinetic study in rats.

Duvelisib (DUV) is a new oral phosphoinositide-3-kinase (PI3K)-δ and PI3K-γ inhibitor. It is used for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). This study describes the development and validation of a new highly sensitive and efficient UPLC-ESI-MS/MS method for quantitation of DUV in plasma samples and its application to the pharmacokinetic study of DUV in rats. The method employed a very simple step for plasma sample pretreatment via precipitation of protein using methanol. DUV and ceritinib (CRB) as an internal standard (IS) were separated on a porous Hypersil BDS-C18 column (125 mm × 2 mm, 3 µm) using a mobile phase consisting of ammonium formate (10 mM, pH 4.2):acetonitrile (42 : 58, v/v), pumped isocratically at a flow rate of 0.3 mL min-1. DUV and CRB were eluted at 0.58 and 1.10 min, respectively. The mass spectrometric analysis was performed using an ESI in positive mode with multiple reaction monitoring (MRM). The technique was validated in accordance with the standards for validating bioanalytical methods established by the International Conference on Harmonization (ICH). The method's linear range was 5-500 ng mL-1, and its correlation coefficient was satisfactory as it is almost unity (0.9999). The limit of quantitation (LOQ) was 5 ng mL-1, while the limit of detection (LOD) was 1.7 ng mL-1. The recovery of the spiking DUV was between 94.95 and 102.21%, and the relative standard deviation (RSD) was less than 2.70%, confirming the method's accuracy and precision. The specificity/carryover of the method was proved. The robustness and ruggedness of the method was proved as the recovery values were 97.6-101.96% (±01.17-2.20%) and 98.74-102.00 (±1.18-4.02%) for robustness and ruggedness, respectively. The stability of DUV under the different analytical conditions were documented as the recovery values were in the range of 95.89-103.28% and the RSD values did not exceed 7.36%. The method was efficiently used to analyze DUV in human plasma samples that had been spiked with DUV and to conduct pharmacokinetic investigations of DUV in rats after giving them a single oral dosage of 25 mg kg-1 of the drug. The methodology is distinguished by excellent sensitivity, accuracy, and ease of sample pretreatment. Furthermore, it is efficient and has a short run time, which makes it high throughput and accordingly enables faster processing of many samples in clinical laboratories.

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.

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.

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.

Crosstalk between aryl hydrocarbon receptor (AhR) and BCL-2 pathways suggests the use of AhR antagonist to maintain normal differentiation state of mammary epithelial cells during BCL-2 inhibition therapy.

INTRODUCTION: Activating the aryl hydrocarbon receptor upon exposure to environmental pollutants promotes development of breast cancer stem cell (CSCs). BCL-2 family proteins protect cancer cells from the apoptotic effects of chemotherapeutic drugs. However, the crosstalk between AhR and the BCL-2 family in CSC development remains uninvestigated. OBJECTIVES: This study explored the interaction mechanisms between AhR and BCL-2 in CSC development and chemoresistance. METHODS: A quantitative proteomic analysis study was performed as a tool for comparative expression analysis of breast cancer cells treated by AhR agonist. The basal and inducible levels of BCL-2, AhR, and CYP1A1 in vitro breast cancer and epithelial cell lines and in vivo mice animal models were determined by RT-PCR, Western blot analysis, immunofluorescence, flow cytometry, silencing of the target, and immunohistochemistry. In addition, an in silico toxicity study was conducted using DEREK software. RESULTS: Activation of the AhR/CYP1A1 pathway in mice increased EpCAMHigh/CD49fLow CD61+ luminal progenitor-like cells in early tumor formation but not in advanced tumors. In parallel, a chemoproteomic study on breast cancer MCF-7 cells revealed that the BCL-2 protein expression was the most upregulated upon AhR activation. The crosstalk between the AhR and BCL-2 pathways in vitro and in vivo modulated the CSCs features and chemoresistance. Interestingly, inhibition of BCL-2 in mice by venetoclax (VCX) increased EpCAMHigh/CD49fLow CD61+ luminal progenitor-like cells, causing inhibition of epithelial lineage markers, disruption of mammary gland branching and induced the epithelial-mesenchymal transition in mammary epithelial cells (MECs). The combined treatment of VCX and AhR antagonists in mice corrected the abnormal differentiation in MECs and protected mammary gland branching and cell identity. CONCLUSIONS: This is the first study to report crosstalk between AhR and BCL-2 in breast CSCs and provides the rationale for using a combined treatment of BCL-2 inhibitor and AhR antagonist for more effective cancer prevention and treatment.

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.

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.