A Quality by Design (QbD) driven gradient high performance liquid chromatography method development for the simultaneous estimation of dasatinib and nilotinib in lipid nanocarriers.

Tyrosine kinase inhibitors (TKIs) are effective as a targeted treatment for chronic myeloid leukemia (CML), which can selectively suppress BCR-ABL1 kinase activity. CML therapy with TKIs combination has been supported by in-vitro, in-vivo, and patient-based data where the nilotinib-dasatinib co-administration has exerted superior anticancer efficacy with greater cellular uptake, less resistance to chemotherapy, and no additive adverse events encountered. Therefore, it is essential to develop a suitable analytical method for the simultaneous estimation of these drugs in the developed novel lipid nanocarriers like liposomes. Design of Experiment (DoE) has been implemented as a tool of QbD to systematically investigate the relation between the HPLC method attributes and analytical responses, i.e., chromatographic detection, quantification, and peak properties for dasatinib and nilotinib. An Ishikawa diagram is constructed to delineate possible influencing variables to the analytical performances. Afterward, 4 factors 2 level full factorial design (FFD) was employed to model and identify the main effects and interaction effects between the factors selected after the initial risk assessment. The suggested design space for optimized chromatographic conditions by QbD analysis is linear within the selected range of drug concentrations, accurate and precise, sensitive, and robust according to the ICH guidelines. The optimal method is comprised of a 1 mL/min flow rate of mobile phase (ACN and 20 mM KH2PO4 of pH 7.00) in gradient mode at 25 °C column temperature for 20 µL sample injection volume and detection wavelength fixed at 297 nm. Most importantly, this novel HPLC method is simple and selective enough to evaluate dasatinib and nilotinib content in the lipid nanocarriers.

Polarity Directed Appraisal of Pharmacological Potential and HPLC-DAD Based Phytochemical Profiling of Polygonum glabrum Willd.

The present study was designed to evaluate polarity-dependent extraction efficiency and pharmacological profiling of Polygonum glabrum Willd. Crude extracts of leaves, roots, stems, and seeds, prepared from solvents of varying polarities, were subjected to phytochemical, antioxidant, antibacterial, antifungal, antidiabetic, and cytotoxicity assays. Maximum extraction yield (20.0% w/w) was observed in the case of an acetone:methanol (AC:M) root extract. Distilled water:methanol (W:M) leaves extract showed maximum phenolic contents. Maximum flavonoid content and free radical scavenging potential were found in methanolic (M) seed extract. HPLC-DAD quantification displayed the manifestation of substantial quantities of quercetin, rutin, gallic acid, quercetin, catechin, and kaempferol in various extracts. The highest ascorbic acid equivalent total antioxidant capacity and reducing power potential was found in distilled water roots and W:M leaf extracts, respectively. Chloroform (C) seeds extract produced a maximum zone of inhibition against Salmonella typhimurium. Promising protein kinase inhibition and antifungal activity against Mucor sp. were demonstrated by C leaf extract. AC:M leaves extract exhibited significant cytotoxic capability against brine shrimp larvae and α-amylase inhibition. Present results suggest that the nature of pharmacological responses depends upon the polarity of extraction solvents and parts of the plant used. P. glabrum can be considered as a potential candidate for the isolation of bioactive compounds with profound therapeutic importance.

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.

Synthesis, Anticancer Screening of Some Novel Trimethoxy Quinazolines and VEGFR2, EGFR Tyrosine Kinase Inhibitors Assay; Molecular Docking Studies.

A new series of 8-methoxy-2-trimethoxyphenyl-3-substituted quinazoline-4(3)-one compounds were designed, synthesized, and screened for antitumor activity against three cell lines, namely, Hela, A549, and MDA compared to docetaxel as reference drug. The molecular docking was performed using Autodock Vina program and 20 ns molecular dynamics (MD) simulation was performed using GROMACS 2018.1 software. Compound 6 was the most potent antitumor of the new synthesized compounds and was evaluated as a VEGFR2 and EGFR inhibitor with (IC50, 98.1 and 106 nM respectively) compared to docetaxel (IC50, 89.3 and 56.1 nM respectively). Compounds 2, 6, 10, and 8 showed strong cytotoxic activities against the Hela cell line with IC50 of, 2.13, 2.8, 3.98, and 4.94 µM, respectively, relative to docetaxel (IC50, 9.65 µM). Compound 11 showed strong cytotoxic activity against A549 cell line (IC50, 4.03 µM) relative to docetaxel (IC50, 10.8 µM). Whereas compounds 6 and 9 showed strong cytotoxic activity against MDA cell line (IC50, 0.79, 3.42 µM, respectively) as compared to docetaxel (IC50, 3.98 µM).

Pharmacokinetics of genistein distribution in blood and retinas of diabetic and non-diabetic rats.

Genistein, a natural tyrosine kinase inhibitor, may act as an intraocular antiangiogenic agent. Its therapeutical use, however, is limited by its nonlinear pharmacokinetics. We aimed to determine genistein's kinetics and retinal tissue distributions in normal and diabetic rats. We developed an isocratic, reverse-phase C18 HPLC system to measure genistein concentration in blood and retinas of streptozotocin (65 mg/kg IV)-diabetic and non-diabetic rats receiving two types of genistein-rich diet (150 and 300 mg/kg) for ten days. Genistein's decay exhibited a two-compartmental open model. Half-lives of distribution and elimination were 2.09 and 71.79 min, with no difference between groups. Genistein steady-state concentration in blood for 150 and 300 mg/kg diet did not differ between diabetic (0.259 ± 0.07 and 0.26 ± 0.06 µg/ml) and non-diabetic rats (0.192 ± 0.05 and 0.183 ± 0.09 µg/ml). In retina, genistein concentration was significantly higher in diabetic rats (1.05 ± 0.47 and 0.997 ± 0.47 µg/gm wt. vs. 0.087 ± 0.11 and 0.314 ± 0.18 µg/gm wt., p < 0.05). The study determined that increasing genistein dose did not change its bioavailability, perhaps due to the poor aqueous solubility. The retina's increased genistein could be due to increased permeability of blood-retinal barrier that occurs early in diabetes.

Simultaneous Quantification of BCR-ABL and Bruton Tyrosine Kinase Inhibitors in Dried Plasma Spots and Its Application to Clinical Sample Analysis.

BACKGROUND: Recent reports highlight the importance of therapeutic drug monitoring (TDM) of BCR-ABL and Bruton tyrosine kinase inhibitors (TKIs); thus, large-scale studies are needed to determine the target concentrations of these drugs. TDM using dried plasma spots (DPS) instead of conventional plasma samples is a promising approach. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of BCR-ABL and Bruton TKIs for further TDM studies. METHODS: A 20-µL aliquot of plasma was spotted onto a filter paper and dried completely. Analytes were extracted from 2 DPS using 250 µL of solvent. After cleanup by supported liquid extraction, the sample was analyzed by LC-MS/MS. Applicability of the method was examined using samples of patients' DPS transported by regular mail as a proof-of-concept study. The constant bias and proportional error between plasma and DPS concentrations were assessed by Passing-Bablok regression analysis, and systematic errors were evaluated by Bland-Altman analysis. RESULTS: The method was successfully validated over the following calibration ranges: 1-200 ng/mL for dasatinib and ponatinib, 2-400 ng/mL for ibrutinib, 5-1000 ng/mL for bosutinib, and 20-4000 ng/mL for imatinib and nilotinib. TKI concentrations were successfully determined for 93 of 96 DPS from clinical samples. No constant bias between plasma and DPS concentrations was observed for bosutinib, dasatinib, nilotinib, and ponatinib, whereas there were proportional errors between the plasma and DPS concentrations of nilotinib and ponatinib. Bland-Altman plots revealed that significant systematic errors existed between both methods for bosutinib, nilotinib, and ponatinib. CONCLUSIONS: An LC-MS/MS method for the simultaneous quantification of 6 TKIs in DPS was developed and validated. Further large-scale studies should be conducted to assess the consistency of concentration measurements obtained from plasma and DPS.

Discovery of new small-molecule cyclin-dependent kinase 6 inhibitors through computational approaches.

Excessive cell proliferation due to cell cycle disorders is one of the hallmarks of breast cancer. Cyclin-dependent kinases (CDKs), which are involved in the transition of the cell cycle from G1 phase to S phase by combining CDKs with cyclin, are considered promising targets with broad therapeutic potential based on their critical role in cell cycle regulation. Pharmacological evidence has shown that abnormal cell cycle due to the overexpression of CDK6 is responsible for the hyperproliferation of cancer cells. Blocking CDK6 expression inhibits tumour survival and growth. Therefore, CDK6 can be regarded as a potential target for anticancer therapeutics. Thus, small molecules that can be considered CDK inhibitors have been developed into promising anticancer drugs. In this study, combined structure-based and ligand-based in silicon models were created to identify new chemical entities against CDK6 with the appropriate pharmacokinetic properties. The database used to screen drug-like compounds in this thesis was based on the best E-pharmacophore hypothesis and the best ligand-based drug hypothesis. As a result, 147 common compounds were identified by further molecular docking. Surprisingly, the in vitro evaluation results of 20 of those compounds showed that the two had good CDK6 inhibitory effects. The best compound was subjected to kinase panel screening, followed by molecular dynamic simulations. The 50-ns MD studies revealed the pivotal role of VAL101 in the binding of inhibitors to CDK6. Overall, the identification of two new chemical entities with CDK6 inhibitory activity demonstrated the feasibility and potential of the new method.

Determination of intracellular anlotinib, osimertinib, afatinib and gefitinib accumulations in human brain microvascular endothelial cells by liquid chromatography/tandem mass spectrometry.

RATIONALE: Brain metastases are a common complication in patients with non-small-cell lung cancer (NSCLC). Anlotinib hydrochloride is a novel multi-target tyrosine kinase inhibitor (TKI) exhibiting a superior overall response rate for brain metastases from NSCLC. The penetrability of anlotinib and three generations of epidermal growth factor receptor (EGFR) TKIs (osimertinib, afatinib and gefitinib) into brain microvascular endothelial cells (HBMECs) was compared. METHODS: A sensitive quantification method for the four TKIs was developed using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). Anlotinib and the three EGFR TKIs were separated on an ACQUITY BEH C18 column after a direct protein precipitation, and then analyzed using electrospray ionization in positive ion mode. The linearity, accuracy, precision, limit of quantification, specificity and stability were assessed. RESULTS: The four analytes could be efficiently quantified in a single run of 3.8 min. The validation parameters of all analytes satisfy the acceptance criteria of bioanalytical method guidelines. The calibration range was 0.2-200 ng mL-1 for anlotinib and gefitinib, 1-500 ng mL-1 for osimertinib and 1-200 ng mL-1 for afatinib. The penetration of anlotinib across HBMECs was comparable with that of afatinib and gefitinib but less than that of osimertinib. CONCLUSIONS: A sensitive LC/MS/MS method to simultaneously measure anlotinib, osimertinib, afatinib and gefitinib in cell extracts was successfully validated and applied to determine their uptake inside HBMECs, which could pave the way for future research on the role of anlotinib in NSCLC brain metastases.

Chromatographic bioanalytical assays for targeted covalent kinase inhibitors and their metabolites.

Deriving from targeted kinase inhibitors (TKIs), targeted covalent kinase inhibitors (TCKIs) are a new class of TKIs that are covalently bound to their target residue of kinase receptors. Currently, there are many new TCKIs under clinical development besides afatinib, ibrutinib, osimertinib, neratinib, acalabrutinib, dacomitinib, and zanubrutinib that are already approved by the FDA. Subsequently, there is an increasing demand for bioanalytical methods to qualitatively and quantitively investigate those compounds, leading to a number of papers reporting the development, validation, and use of bioanalytical methods for TCKIs. Most publications describe the technological set up of analytical methods that allow quantification of TCKIs in various biomatrices such as plasma, cerebrospinal fluid, urine, tissue, and liver microsomes. In addition, the identification of metabolites and biotransformation pathways of new TCKIs has gained more interest in recent years. We provide an overview of bioanalytical methods of this new class of TCKIs. The included issues are sample pretreatment, chromatographic separation, detection, and method validation. In the scope of bioanalysis of TCKIs, protein precipitation is mostly applied to treat the biological matrices sample. Liquid chromatographic in reversed-phase mode (RPLC) and mass detection with triple quadrupole (QqQ) are the most often utilized separation and quantitative detection modes, respectively. There may be a possibility of increased use of the high-resolution mass spectrometry (HRMS) for qualitative investigation purposes in the future. We also found that US FDA and EMA guidelines are the most common guidelines employed as validation framework for the bioanalytical methods of TCKIs.

DBS Assay with LC-MS/MS for the Determination of Idelalisib, A Selective PI3K-δ Inhibitor in Mice Blood and Its Application to a Pharmacokinetic Study.

Idelalisib is a selective and second-generation PI3K-δ inhibitor, approved for the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this paper, we present a fully validated dried blood spot (DBS) method for the quantitation of idelalisib from mice blood using an LC-MS/MS, which was operated under multiple reaction monitoring mode. To the punched DBS discs, acidified methanol enriched with internal standard (IS; larotrectinib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of idelalisib and the IS was achieved on an Atlantis dC18 column using a mixture of 10 mM ammonium formate:acetonitrile (25:75, v/v). The flow-rate and injection volume were 0.80 mL/min and 2.0 µL, respectively. Idelalisib and the IS were eluted at ~0.98 and 0.93 min, respectively and the total run time was 2.00 min. Idelalisib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 416.1→176.1 and 429.1→342.1, respectively was used for the quantitation. The calibration range was 1.01-4 797 ng/mL. No matrix effect and carry over were observed. Haematocrit did not influence DBS idelalisib concentrations. All the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mice pharmacokinetic study.

LC-MS/MS Estimation of the Anti-Cancer Agent Tandutinib Levels in Human Liver Microsomes: Metabolic Stability Evaluation Assay.

PURPOSE: Tandutinib (MLN518 or CT 53518) (TND) is a novel, oral, small-molecule inhibitor of type III receptor tyrosine kinases utilized for the treatment of acute myeloid leukemia (AML). MATERIALS AND METHODS: In silico prediction of hepatic drug metabolism for TND was determined using the StarDrop® WhichP450™ module to confirm its metabolic liability. Second, an efficient and accurate LC-MS/MS method was established for TND quantification to evaluate metabolic stability. TND and entrectinib (ENC) (internal standard; IS) were resolved using an isocratic elution system with a reversed stationary phase (C8 column). RESULTS: The established LC-MS/MS method exhibited linearity (5-500 ng/mL) with r2 ≥0.9999 in the human liver microsomes matrix. The method sensitivity was indicated by the limit of quantification (3.8 ng/mL), and reproducibility was revealed by inter- and intraday precision and accuracy (below 10.5%). TND metabolic stability estimation was calculated using intrinsic clearance (22.03 µL/min/mg) and in vitro half-life (29.0 min) values. CONCLUSION: TND exhibited a moderate extraction ratio indicative of good bioavailability. According to the literature, the approach developed in the present study is the first established LC-MS/MS method for assessing TND metabolic stability.

Validated HPLC Method for Quantification of a Novel Trk Inhibitor, Larotrectinib in Mice Plasma: Application to a Pharmacokinetic Study.

Larotrectinib, is an orally active novel small molecule approved for the treatment of solid tumors in pediatrics and adult patients. It acts by inhibiting tropomyosin receptor kinase. In this paper, we report the development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of larotrectinib in mice plasma as per the FDA regulatory guideline. Plasma samples processing was accomplished through simple protein precipitation using acetonitrile enriched with internal standard (IS, enasidenib). The chromatographic analysis was performed using a gradient mobile phase comprising 10 mM ammonium acetate and acetonitrile at a flow-rate of 0.8 mL/min on an X-Terra Phenyl column. The UV detection wave length was set at λmax 262 nm. Larotrectinib and the IS eluted at 3.85 and 6.60 min, respectively with a total run time of 8.0 min. The calibration curve was linear over a concentration range of 0.20-5.00 µg/mL (r2=≥0.992). The intra- and inter-day precision and accuracy results were within the acceptable limits. Results of stability studies indicated that larotrectinib was stable on bench-top, in auto-sampler, up to three freeze/thaw cycles and long-term storage at -80°C. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

Identification of novel CDK2 inhibitors by a multistage virtual screening method based on SVM, pharmacophore and docking model.

Cyclin-dependent kinase 2 (CDK2) is the family of Ser/Thr protein kinases that has emerged as a highly selective with low toxic cancer therapy target. A multistage virtual screening method combined by SVM, protein-ligand interaction fingerprints (PLIF) pharmacophore and docking was utilised for screening the CDK2 inhibitors. The evaluation of the validation set indicated that this method can be used to screen large chemical databases because it has a high hit-rate and enrichment factor (80.1% and 332.83 respectively). Six compounds were screened out from NCI, Enamine and Pubchem database. After molecular dynamics and binding free energy calculation, two compounds had great potential as novel CDK2 inhibitors and they also showed selective inhibition against CDK2 in the kinase activity assay.

Quantification of the tyrosine kinase inhibitor erlotinib in human scalp hair by liquid chromatography-tandem mass spectrometry: Pitfalls for clinical application.

An LC-MS/MS method was developed and validated to quantify the tyrosine kinase inhibitor erlotinib in human scalp hair, as alternative matrix to monitor long-term erlotinib exposure. Hair samples from 10 lung cancer patients were measured and correlated with plasma concentrations. Hair segments of 1 ± 0.1 cm each were pulverized and for at least 18 h incubated in methanol at ambient temperature. A liquid-liquid extraction purified the extracts and they were analyzed with LC-MS/MS, using erlotinib-d6 as internal standard. The procedure method was validated for selectivity, sensitivity, precision, lower limit of detection, linearity and accuracy. The within and between run precisions including the lower limit of quantification did not exceed 12.5%, while the accuracy ranged from 103 to 106%. A weak correlation between hair and plasma concentration was found (R2 = 0.48). Furthermore, a large inter-individual variability was noted in the disposition of both plasma and hair samples. The highest hair concentrations were observed in black hair compared with other (grey and brown) hair colors. Generally, a linear reduction in hair concentration was found from proximal to distal hair segments. Additional in vitro experiments suggest an accelerated degradation of erlotinib in hair by artificial UV light and also wash-out by shampoo mixtures pretreatment compared with control samples. In conclusion, a reliable and robust LC-MS/MS method was developed to quantify erlotinib in hair. However, clinical and in vitro evaluations showed that the method is not suitable for monitoring long-term erlotinib exposure. The pitfalls of this application outweigh the current benefits.

Single-Molecule Force Measurement Guides the Design of Multivalent Ligands with Picomolar Affinity.

Interaction of multiple entities and receptors, or multivalency is widely applied to achieve high affinity ligands for diagnostic and therapeutic purposes. However, lack of knowledge on receptor distribution in living subjects remains a challenge for rational structure design. Herein, we develop a force measurement platform to probe the distribution and separation of the cell surface vascular endothelial growth factor receptors (VEGFR) in live cells, and use this to assess the geometry of appropriate linkers for distinct multivalent binding modes. A tetravalent lead ZD-4, which was developed from an antitumor drug ZD6474 (Vandetanib) with combined hybrid binding effects, yielded a 2000-fold improvement in the binding affinity to VEGFR with IC50 value of 25 pm. We confirmed the improved affinity by the associated increase of tumor uptake in the VEGFR-targeting positron emission tomography (PET) imaging using U87 tumor xenograft mouse model.

Development of a validated LC-MS/MS method for the in vitro and in vivo quantitation of sunitinib in glioblastoma cells and cancer patients.

Sunitinib is a multi-targeted tyrosine kinase inhibitor approved for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumor and is currently being investigated against other forms of malignant tumors. Recently great interest has emerged for the application of sunitinib to glioblastoma treatment. In order to have a method with broad applicability it will be of importance to have access to a method that could be applied both in human plasma and cell uptake studies. No method has been reported thus far for the estimation of sunitinib uptake in glioma cells. We therefore set out to develop a method that could be applied for quantifying sunitinib in human plasma and in cell uptake studies. The method was validated and accredited according to ISO 17025:2005 guideline in human plasma and successfully applied to cancer patient plasma. Also, the method was effectively recruited to establish a protocol for the evaluation of sunitinib accumulation into M095K glioma cells. This method could significantly contribute to developmental phases in repurposing this drug in different cancer types.

Discovery of Affinity-Based Probes for Btk Occupancy Assays.

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.

Dual-mixed/CMC model for screening target components from traditional Chinese medicines simultaneously acting on EGFR & FGFR4 receptors.

Radix Salviae Miltiorrhiae (also known as DanShen (DS) in China), a popular herbal drug in traditional Chinese medicine (TCM) for promoting blood circulation and treating blood stasis, has been reported to possess potential anti-tumor effects. The aim of the study was to develop an effective and practical method for screening and identifying bioactive compounds from Radix Salviae Miltiorrhiae. In this work, the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptors 4 (FGFR4) dual-mixed/cell membrane chromatography (CMC) coupled with high performance liquid chromatography-electrospray ionization-ion trap-time of flight-multistage mass spectrum (HPLC-ESI-IT-TOF-MSn) was established and successfully used to identify the active components from Radix Salviae Miltiorrhiae. Salvianolic acid C (SAC), tanshinone I (Tan-I), tanshinone IIA (Tan-IIA), and cryptotanshinone (C-Tan) were identified as bioactive components with EGFR and FGFR4 activities. MTT and kinase assay were performed to investigate inhibitory effects of these compounds against EGFR and FGFR4 cells growth in vitro. Both cell viability and kinase activity showed that cryptotanshinone acting on EGFR receptor and tanshinone IIA acting on FGFR4 receptor. In conclusion, the EGFR & FGFR4 dual-mixed/CMC can simultaneously screen the bioactive components from TCMs that act on both EGFR and FGFR4 receptors, which significantly improve the efficiency of specific bioactive components identification from a complex system.

Bioavailability, Biotransformation, and Excretion of the Covalent Bruton Tyrosine Kinase Inhibitor Acalabrutinib in Rats, Dogs, and Humans.

Acalabrutinib is a targeted, covalent inhibitor of Bruton tyrosine kinase (BTK) with a unique 2-butynamide warhead that has relatively lower reactivity than other marketed acrylamide covalent inhibitors. A human [14C] microtracer bioavailability study in healthy subjects revealed moderate intravenous clearance (39.4 l/h) and an absolute bioavailability of 25.3% ± 14.3% (n = 8). Absorption and elimination of acalabrutinib after a 100 mg [14C] microtracer acalabrutinib oral dose was rapid, with the maximum concentration reached in <1 hour and elimination half-life values of <2 hours. Low concentrations of radioactivity persisted longer in the blood cell fraction and a peripheral blood mononuclear cell subfraction (enriched in target BTK) relative to plasma. [14C]Acalabrutinib was metabolized to more than three dozen metabolites detectable by liquid chromatography-tandem mass spectrometry, with primary metabolism by CYP3A-mediated oxidation of the pyrrolidine ring, thiol conjugation of the butynamide warhead, and amide hydrolysis. A major active, circulating, pyrrolidine ring-opened metabolite, ACP-5862 (4-[8-amino-3-[4-(but-2-ynoylamino)butanoyl]imidazo[1,5-a]pyrazin-1-yl]-N-(2-pyridyl)benzamide), was produced by CYP3A oxidation.Novel enol thioethers from the 2-butynamide warhead arose from glutathione and/or cysteine Michael additions and were subject to hydrolysis to a ß-ketoamide. Total radioactivity recovery was 95.7% ± 4.6% (n = 6), with 12.0% of dose in urine and 83.5% in feces. Excretion and metabolism characteristics were generally similar in rats and dogs. Acalabrutinib's highly selective, covalent mechanism of action, coupled with rapid absorption and elimination, enables high and sustained BTK target occupancy after twice-daily administration.

Assessment of DNA-PKcs kinase activity by quantum dot-based microarray.

Therapeutic efficacy against cancer is often based on a variety of DNA lesions, including DNA double-strand breaks (DSBs) which are repaired by homologous recombination and non-homologous end joining (NHEJ) pathways. In the past decade, the functions of the DNA repair proteins have been described as a potential mechanism of resistance in tumor cells. Therefore, the DNA repair proteins have become targets to improve the efficacy of anticancer therapy. Given the central role of DNA-PKcs in NHEJ, the therapeutic efficacy of targeting DNA-PKcs is frequently described as a strategy to prevent repair of treatment-induced DNA damage in cancer cells. The screening of a new inhibitor acting as a sensitizer requires the development of a high-throughput tool in order to identify and assess the most effective molecule. Here, we describe the elaboration of an antibody microarray dedicated to the NHEJ pathway that we used to evaluate the DNA-PKcs kinase activity in response to DNA damage. By combining a protein microarray with Quantum-Dot detection, we show that it is possible to follow the modification of phosphoproteomic cellular profiles induced by inhibitors during the response to DNA damage. Finally, we discuss the promising tool for screening kinase inhibitors and targeting DSB repair to improve cancer treatment.