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.

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.

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).

Identification of the metabolites of tofacitinib in liver microsomes by liquid chromatography combined with high resolution mass spectrometry.

Tofacitinib is an orally available Janus kinase inhibitor. The aim of this study was to investigate the metabolism of tofacitinib in mouse, rat, monkey, and human liver microsomes fortified with ß-nicotinamide adenine dinucleotide phosphate tetrasodium salt and uridine diphosphate glucuronic acid. The biotransformation was executed at a temperature of 37°C for 60 min, and the samples were analyzed by ultra-high performance liquid chromatography combined with high-resolution mass spectrometry (UHPLC-HRMS) operated in positive electrospray ionization mode. The structures of the metabolites were elucidated according to their retention times, accurate masses, and MS/MS spectra. Under the current conditions, a total of 13 metabolites, including 1 glucuronide conjugate, were detected and structurally proposed. Oxygenation of the pyrrolopyrimidine ring, oxygenation of piperidine ring, N-demethylation, oxygenation of piperidine ring side chain, and glucuronidation were the primary metabolic pathways of tofacitinib. Among the tested species, tofacitinib showed significant species difference. Compared with other species, rat showed similar metabolic profiles to those of humans. The present study provides some new information regarding the metabolism of tofacitinib in animals and humans, which would bring us considerable benefits for the subsequent studies focusing on the pharmacological effect and toxicity of this drug.

High-resolution mass spectrometry-based approach for the identification and profiling of the metabolites of taletrectinib formed in liver microsomes.

Taletrectinib is a potent, orally active, and selective ROS1/NTRK kinase inhibitor. The aim of this study was to study the metabolism of taletrectinib in rat, dog, and human liver microsomes. The biotransformation of taletrectinib was carried out using rat, dog, and human liver microsomes supplemented with nicotinamide adenine dinucleotide phosphate tetrasodium salt (NADPH) and uridine diphosphate glucuronic acid (UDPGA). The microsomal incubations were conducted at 37°C for 60 min. The formed metabolites were identified by ultrahigh performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS) using electrospray ionization in the positive ion mode. They were identified by accurate masses and MS/MS spectra and based on their fragmentation pathways. With UHPLC-HRMS, a total of 10 metabolites including one glucuronide conjugate (M7) were structurally identified. M9 and M10 were unambiguously identified as taletrectinib alcohol and taletrectinib ketone, respectively, using reference standards. The phase I metabolic pathways of taletrectinib involved N-dealkylation, O-dealkylation, oxidative deamination, and oxygenation; the phase II metabolic pathways referred to glucuronidation. The current study investigated the in vitro metabolic fate of taletrectinib in animals and human species, which would bring us considerable benefits for the subsequent studies focusing on the pharmacological effect and toxicity of this drug.

Identification of novel compounds that inhibit SnRK2 kinase activity by high-throughput screening.

Abscisic acid (ABA) is a major phytohormone that regulates abiotic stress responses and development. SNF1-rerated protein kinase 2 (SnRK2) is a key regulator of ABA signaling. To isolate compounds which directly affect SnRK2 activity, we optimized a fluorescence-based system for high-throughput screening (HTS) of SnRK2 kinase regulators. Using this system, we screened a chemical library consisting of 16,000 compounds and identified ten compounds (INH1-10) as potential SnRK2 inhibitors. Further characterization of these compounds by in vitro phosphorylation assays confirmed that three of the ten compounds were SnRK2-specific kinase inhibitors. In contrast, seven of ten compounds inhibited ABA-responsive gene expression in Arabidopsis cells. From these results, INH1 was identified as a SnRK2-specific inhibitor in vitro and in vivo. We propose that INH1 could be a lead compound of chemical tools for studying ABA responses in various plant species.

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.

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.

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.

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.

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.

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.

Advancing stereoisomeric separation of an atropisomeric Bruton's tyrosine kinase inhibitor by using sub-2 µm immobilized polysaccharide-based chiral columns in supercritical fluid chromatography.

BMS-986142 is a Bruton's tyrosine kinase inhibitor under development to treat several disease types. The compound contains three chiral elements: one chiral center and two chiral axes, resulting in three potential atropisomeric impurities in its drug substance and drug products. Separation of BMS-986142 atropisomers has been successfully achieved on an achiral polar-embedded C18 column in reversed-phase liquid chromatography (RPLC) and on polysaccharide-based chiral columns in RPLC and supercritical fluid chromatography (SFC). Compared to the RPLC chiral separation, the SFC atropisomeric separation on a sub-2 µm immobilized cellulose-based column is much more efficient and environmentally friendly. The analysis time in SFC was reduced by 8-fold compared to that in RPLC, and the method sensitivity in SFC on the sub-2 µm chiral column in 3.0 mm I.D. was 2 to 4-fold better than that on 3 µm chiral columns in 4.6 mm I.D.. Furthermore, our study suggests that the contribution to band broadening from the extra column volume (ECV) of modern commercial SFC instrument was not negligible for a 3.0 mm I.D. × 100 mm column packed with 1.6 µm particles. This result reaffirms that there is a great need for further improvement of SFC instrument design in order to realize the full theoretical efficiency of both sub-2 µm achiral and chiral columns.

JAK2 regulates Nav1.6 channel function via FGF14Y158 phosphorylation.

BACKGROUND: Protein interactions between voltage-gated sodium (Nav) channels and accessory proteins play an essential role in neuronal firing and plasticity. However, a surprisingly limited number of kinases have been identified as regulators of these molecular complexes. We hypothesized that numerous as-of-yet unidentified kinases indirectly regulate the Nav channel via modulation of the intracellular fibroblast growth factor 14 (FGF14), an accessory protein with numerous unexplored phosphomotifs and required for channel function in neurons. METHODS: Here we present results from an in-cell high-throughput screening (HTS) against the FGF14: Nav1.6 complex using >3000 diverse compounds targeting an extensive range of signaling pathways. Regulation by top kinase targets was then explored using in vitro phosphorylation, biophysics, mass-spectrometry and patch-clamp electrophysiology. RESULTS: Compounds targeting Janus kinase 2 (JAK2) were over-represented among HTS hits. Phosphomotif scans supported by mass spectrometry revealed FGF14Y158, a site previously shown to mediate both FGF14 homodimerization and interactions with Nav1.6, as a JAK2 phosphorylation site. Following inhibition of JAK2, FGF14 homodimerization increased in a manner directly inverse to FGF14:Nav1.6 complex formation, but not in the presence of the FGF14Y158A mutant. Patch-clamp electrophysiology revealed that through Y158, JAK2 controls FGF14-dependent modulation of Nav1.6 channels. In hippocampal CA1 pyramidal neurons, the JAK2 inhibitor Fedratinib reduced firing by a mechanism that is dependent upon expression of FGF14. CONCLUSIONS: These studies point toward a novel mechanism by which levels of JAK2 in neurons could directly influence firing and plasticity by controlling the FGF14 dimerization equilibrium, and thereby the availability of monomeric species for interaction with Nav1.6.

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 human tau-tubulin kinase 1 inhibitors: an integrated e-pharmacophore-based virtual screening and molecular dynamics simulation.

Tau-tubulin kinase 1 inhibitors inhibit tau protein phosphorylation on Ser198, Ser199, Ser202, Ser422, and also in paired helical filaments. We developed receptor-based pharmacophore models by exploiting three TTBK1 protein structures, i.e., 4NFN, 4BTM, and 4BTK. The integrated e-pharmacophore based virtual screening and molecular dynamics simulation recognized four hits viz. ZINC14644839, ZINC00012956, ZINC91332506, and ZINC69775110 as TTBK1 inhibitors. The Glide XP docking energies (-8.48 to -10.71 kcal.mol-1) of hits were better than cocrystal ligand of 4NFN protein structure (-8.37 kcal.mol-1). Among the hits, ZINC14644839 possessed best binding energy with four hydrogen bonding interactions. The inhibitors showed acceptable calculated ADME and blood-brain barrier permeability properties and could be potential TTBK1 inhibitors for neurodegenerative diseases.Communicated by Ramaswamy H. Sarma.

Metabolic profiling of tyrosine kinase inhibitor nintedanib using metabolomics.

Nintedanib is a promising tyrosine kinase inhibitor for clinically treating idiopathic pulmonary fibrosis (IPF). Some clinical cases reported that nintedanib treatment can cause hepatotoxicity and myocardial toxicity. U. S. FDA warns the potential drug-drug interaction when it is co-administrated with other drugs. In order to understand the potential toxicity of nintedanib and avoid drug-drug interaction, the metabolism of nintedanib was systematically investigated in human liver microsomes and mice using metabolomics approach, and the toxicity of metabolites was predicted by ADMET lab. Nineteen metabolites were detected in vivo and in vitro metabolism, and 8 of them were undescribed. Calculated partition coefficients (Clog P) were used to distinguish the isomers of nintedanib metabolites in this study. The major metabolic pathways of nintedanib majorly included hydroxylation, demethylation, glucuronidation, and acetylation reactions. The ADMET prediction indicated that nintedanib was a substrate of the cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp). And nintedanib and most of its metabolites might possess potential hepatotoxicity and cardiotoxicity. This study provided a global view of nintedanib metabolism, which could be used to understand the mechanism of adverse effects related to nintedanib and its potential drug-drug interaction.

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.

Safety, pharmacokinetics and pharmacodynamics of the selective glucocorticoid receptor modulator AZD7594, following inhalation in healthy Japanese volunteers.

INTRODUCTION: AZD7594 is a non-steroidal, selective, glucocorticoid receptor modulator (SGRM), currently in development for the treatment of asthma and chronic obstructive pulmonary disease. This paper reports a randomized placebo-controlled dose escalation study in healthy Japanese male subjects. METHODS: Inhaled AZD7594 was administered as one single dose at day 1 (day 1-4), with subsequent multiple daily doses (day 5-16) via a multiple-dose dry powder inhaler for 12 days of once-daily treatment. At each dose level, subjects were randomized to AZD7594 (n=7) or placebo (n=2). The safety, pharmacokinetics (PK) and pharmacodynamics (PD) of AZD7594 were evaluated. RESULTS: Inhaled AZD7594 was safe and well tolerated up to and including the highest dose 1600 µg tested. Plasma exposure suggested dose-proportional PK. The urinary excretion of AZD7594 was negligible (<0.02%). Dose-related effects were observed for 24 hrs plasma cortisol; however, significant cortisol suppression (25%) was only seen at the highest dose level following multiple doses. There were no or only marginal effects on other biomarkers tested (dehydroepiandrosterone sulfate [DHEA-S] and osteocalcin). CONCLUSION: In conclusion, the early clinical evaluation of inhaled AZD7594 suggests that this novel SGRM is well tolerated in the dose range investigated and also in a Japanese population. It shows dose-proportional plasma exposure, moderate accumulation and has limited impact on systemic markers of glucocorticoid activity.