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Objectives: Pharmacokinetic issues were the leading cause of drug attrition, accounting for approximately 40% of all cases before the turn of the century. To this end, several high-throughput in vitro assays like microsomal stability have been developed to evaluate the pharmacokinetic profiles of compounds in the early stages of drug discovery. At NCATS, a single-point rat liver microsomal (RLM) stability assay is used as a Tier I assay, while human liver microsomal (HLM) stability is employed as a Tier II assay. We experimentally screened and collected data on over 30,000 compounds for RLM stability and over 7000 compounds for HLM stability. Although HLM stability screening provides valuable insights, the increasing number of hits generated, along with the time- and resource-intensive nature of the assay, highlights the need for alternative strategies. One promising approach is leveraging in silico models trained on these experimental datasets. Methods: We describe the development of an HLM stability prediction model using our in-house HLM stability dataset. Results: Employing both classical machine learning methods and advanced techniques, such as neural networks, we achieved model accuracies exceeding 80%. Moreover, we validated our model using external test sets and found that our models are comparable to some of the best models in literature. Additionally, the strong correlation observed between our RLM and HLM data was further reinforced by the fact that our HLM model performance improved when using RLM stability predictions as an input descriptor. Conclusions: The best model along with a subset of our dataset (PubChem AID: 1963597) has been made publicly accessible on the ADME@NCATS website for the benefit of the greater drug discovery community. To the best of our knowledge, it is the largest open-source model of its kind and the first to leverage cross-species data. [ABSTRACT FROM AUTHOR]

Kokusaginine is a bioactive ingredient extracted from Ruta graveolens L., which has a range of biological activities. Its pharmacokinetic (PK) properties are particularly important for clinical applications; however, they have not been fully elucidated. In addition, the effect of sex differences on drug metabolism is increasingly being recognized, but most studies have ignored this important factor. This study aims to fill this knowledge gap by taking an in-depth look at the PK properties of kokusaginine and how gender affects its metabolism and distribution in the body. It also lays the foundation for clinical drug development. In this study, a sensitive ultra-high-performance liquid chromatography (UPLC) method was developed and validated for quantifying kokusaginine in Sprague Dawley (SD) rat plasma and tissue homogenates. Metabolic stability was evaluated in vitro using gender-specific liver microsomes. Innovatively, we incorporated sex as a variable into both in vitro and in vivo PK studies in SD rats, analyzing key parameters with Phoenix 8.3.5 software. The developed UPLC method demonstrated high sensitivity and precision, essential for PK analysis. Notably, in vitro studies revealed a pronounced sex-dependent metabolic variability (p < 0.05). In vivo, gender significantly affected the Area Under the Moment Curve (AUMC)(0-∞) of the plasma PK parameter (p < 0.05) and the AUMC(0-t) of brain tissue (p < 0.0001), underscoring the necessity of sex-specific PK assessments. The calculated absolute bioavailability of 71.13 ± 12.75% confirmed the favorable oral absorption of kokusaginine. Additionally, our innovative tissue-plasma partition coefficient (Kp) analysis highlighted a rapid and uniform tissue distribution pattern. This study presents a sex-inclusive PK evaluation of kokusaginine, offering novel insights into its metabolic profile and distribution. These findings are instrumental for informing clinical medication practices, dosage optimization, and a nuanced understanding of drug efficacy and safety in a sex-specific context. [ABSTRACT FROM AUTHOR]

Objectives: Pharmacokinetic issues were the leading cause of drug attrition, accounting for approximately 40% of all cases before the turn of the century. To this end, several high-throughput in vitro assays like microsomal stability have been developed to evaluate the pharmacokinetic profiles of compounds in the early stages of drug discovery. At NCATS, a single-point rat liver microsomal (RLM) stability assay is used as a Tier I assay, while human liver microsomal (HLM) stability is employed as a Tier II assay. We experimentally screened and collected data on over 30,000 compounds for RLM stability and over 7000 compounds for HLM stability. Although HLM stability screening provides valuable insights, the increasing number of hits generated, along with the time- and resource-intensive nature of the assay, highlights the need for alternative strategies. One promising approach is leveraging in silico models trained on these experimental datasets. Methods: We describe the development of an HLM stability prediction model using our in-house HLM stability dataset. Results: Employing both classical machine learning methods and advanced techniques, such as neural networks, we achieved model accuracies exceeding 80%. Moreover, we validated our model using external test sets and found that our models are comparable to some of the best models in literature. Additionally, the strong correlation observed between our RLM and HLM data was further reinforced by the fact that our HLM model performance improved when using RLM stability predictions as an input descriptor. Conclusions: The best model along with a subset of our dataset (PubChem AID: 1963597) has been made publicly accessible on the ADME@NCATS website for the benefit of the greater drug discovery community. To the best of our knowledge, it is the largest open-source model of its kind and the first to leverage cross-species data.

Kokusaginine is a bioactive ingredient extracted from Ruta graveolens L., which has a range of biological activities. Its pharmacokinetic (PK) properties are particularly important for clinical applications; however, they have not been fully elucidated. In addition, the effect of sex differences on drug metabolism is increasingly being recognized, but most studies have ignored this important factor. This study aims to fill this knowledge gap by taking an in-depth look at the PK properties of kokusaginine and how gender affects its metabolism and distribution in the body. It also lays the foundation for clinical drug development. In this study, a sensitive ultra-high-performance liquid chromatography (UPLC) method was developed and validated for quantifying kokusaginine in Sprague Dawley (SD) rat plasma and tissue homogenates. Metabolic stability was evaluated in vitro using gender-specific liver microsomes. Innovatively, we incorporated sex as a variable into both in vitro and in vivo PK studies in SD rats, analyzing key parameters with Phoenix 8.3.5 software. The developed UPLC method demonstrated high sensitivity and precision, essential for PK analysis. Notably, in vitro studies revealed a pronounced sex-dependent metabolic variability (p < 0.05). In vivo, gender significantly affected the Area Under the Moment Curve (AUMC)(0-∞) of the plasma PK parameter (p < 0.05) and the AUMC(0-t) of brain tissue (p < 0.0001), underscoring the necessity of sex-specific PK assessments. The calculated absolute bioavailability of 71.13 ± 12.75% confirmed the favorable oral absorption of kokusaginine. Additionally, our innovative tissue-plasma partition coefficient (Kp) analysis highlighted a rapid and uniform tissue distribution pattern. This study presents a sex-inclusive PK evaluation of kokusaginine, offering novel insights into its metabolic profile and distribution. These findings are instrumental for informing clinical medication practices, dosage optimization, and a nuanced understanding of drug efficacy and safety in a sex-specific context.

We have investigated the effects of methanol, acetonitrile, dimethyl sulphoxide or dioxane on enzyme kinetic parameters (Km and Vmax) of p-nitrophenol hydroxylation, phenacetin deethylation and metoprolol metabolism in rat liver microsomes, at different solvent concentrations. In the case of p-nitrophenol hydroxylation, methanol (0.1 %-0.75 % v/v) and dimethyl sulphoxide (0.01 %-0.075 % v/v) showed 1.3 and 2.5-fold increase respectively, in the Km with a less marked effect on the Vmax. In contrast, dimethyl sulphoxide (0.1 %-0.75% v/v) showed a 1.96-fold decrease in the Vmax. Unlike, methanol and dimethyl sulphoxide, acetonitrile showed activation of the p-nitrophenol hydroxylation activity as indicated by a 0.7-fold decrease in the Km. In the case of metoprolol metabolism, dioxane resulted in a 1.5 to 3.1-fold increase in the Km of all the three metabolite formation pathways in concentration dependent manner, with less marked effect on Vmax of the reaction. Interestingly, while acetonitrile did not affect the Kmfor metabolite 1 and 2 formation, the Km of metabolite 3 formation was decreased in a concentration dependent manner again with little effect on all the three Vmax values. Further, methanol had little to no effect on the Vmax of metabolite 1 and 2 formation while metabolite 3 formation was increased by 2-fold. The Km of all three metabolite formations was found to be decreased in presence of methanol. The phenacetin deethylation activity in rat liver microsomes followed atypical kinetics. Methanol and dimethyl sulphoxide did not affect the auto activation kinetics at concentration range studied. Dimethyl sulphoxide and dioxane appeared to be unsuitable for characterizing the Cytochrome450 mediated reactions because they showed a very significant effect on the Vmax/Km ratio, starting at concentrations of 0.025 % v/v and 0.25 % v/v, respectively. Methanol and acetonitrile at concentration <0.5 % v/v appeared to be acceptable solvents for solubilization of substrates metabolized by Cytochrome450s. [ABSTRACT FROM AUTHOR]

Yun Hong,1,&ast; Da-Peng Dai,2,&ast; Jian-Ping Cai,2 Shuang-Hu Wang,3 Yi-Ran Wang,1,4 Fang-Ling Zhao,2,4 Shan Zhou,2 Quan Zhou,3 Pei-Wu Geng,3 Yun-Fang Zhou,3 Xue Xu,1 Ji-Hua Shi,1 Qing-Feng Luo1 1Department of Gastroenterology, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, 100730, People’s Republic of China; 2The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology of National Health Commission, Beijing, 100730, People’s Republic of China; 3Laboratory of Clinical Pharmacy, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, 323020, People’s Republic of China; 4Peking University Fifth School of Clinical Medicine, Beijing, 100730, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Qing-Feng Luo, Department of Gastroenterology, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, 100730, People’s Republic of China, Tel + 86 138 1151 9095, Email luoqf2000@126.comPurpose: To study the potential drug–drug interactions between simvastatin and vonoprazan and to provide the scientific basis for rational use of them in clinical practice.Methods: An incubation system was established with rat liver microsomes, and the main metabolite of vonoprazan M-I was detected by UPLC-MS/MS. The IC50 value of simvastatin was then calculated and its inhibitory mechanism against vonoprazan was also analyzed. Twelve SD rats were randomly divided into 2 groups, then they were given simvastatin or saline for 2 weeks continuously. On the day of the experiment, both groups were intragastrically administered with vonoprazan once, followed by the collection of blood at different time points. Then the plasma concentration of vonoprazan and M-I in rats were detected by UPLC-MS/MS.Results: In vitro experiments revealed that simvastatin could inhibit the metabolism of vonoprazan, and its inhibition type belonged to the mixed non-competitive and competitive inhibition model. In vivo experiments in rats demonstrated that the area under concentration time curve (AUC) of vonoprazan was decreased but the clearance (CLz/F) of it was increased in the simvastatin administrated group, as compared to those of the control group. However, M-I in simvastatin treated group exhibited the higher AUC and lower CLz/F values compared to those in the control group. These data indicated that multiple doses of simvastatin administration could reduce the plasma concentration of vonoprazan and accelerate its metabolic rate in rats.Conclusion: Simvastatin could inhibit the metabolism of vonoprazan in vitro but multiple doses of simvastatin exhibited the opposite effect In vivo. Altogether, our data indicated that an interaction existed between simvastatin and vonoprazan and additional cares might be taken when they were co-administrated in clinic.Keywords: vonoprazan, simvastatin, drug–drug interactions, liquid chromatography-tandem mass spectrometry, rat liver microsomes

The cytochrome P450 (CYP) enzymes play a pivotal role in drug metabolism. LC-MS/MS-based targeting technology has been applied to the analysis of CYP enzymes, promoting drug development and drug-drug interaction studies. Rat is one of the most commonly used models for drug metabolism assessment, but LC-MS/MS assay quantifying the abundance of CYP enzymes in rats is rarely reported. Herein, an accurate and stable LC-MS/MS based method was developed and validated for the simultaneous quantification of seven major rat CYP isoforms (CYP1A2, 2B1, 2C6, 2C11, 2D1, 2E1, and 3A1) in liver microsomes. The careful optimization of trypsin digestion and chromatography combined with isotope-labeled peptide as internal standard improved the efficiency and accuracy of the analysis. Highly specific surrogate peptides were obtained by a procedure including trypsin digestion for six hours and separated on a Hypersil Gold C18 column (100 × 2.1 mm, 3 µm) using gradient elution for 15 min with a mobile phase of water containing 0.1% formic acid and acetonitrile. In the method validation, linearity, matrix effect, recovery, stability, accuracy, and precision all meet the requirements. Subsequently, this method was applied to detect seven enzymes in rat liver microsomes from four different sources, and the correlation between the abundance and activity of CYP enzymes was further analyzed. The high-throughput detection method provided in this study will provide support for pertinent pharmaceutical research based on rat models. [ABSTRACT FROM AUTHOR]

Drug metabolism is very important in determining the safety and efficacy of the drug. We worked towards this direction and designed a new strategy to study the in vitro metabolites of Food and Drug Administration approved anticancer drug, ibrutinib which is known to inhibit the Bruton’s tyrosine kinase. The metabolites were formed by incubating the drug, ibrutinib with rat liver microsomes, at 37° for 24 h. The newly formed metabolites were identified and characterized with the help of ultra-high performance liquid chromatography which is interlinked with tandem quadrupole time-of-flight mass spectrometry. We then elucidated the structures of our new metabolites based on liquid chromatography with tandem mass spectrometry data which included accurate masses, the fragmentation of ions and chromatographic retention times. This study described the detailed in vitro metabolite profiling of ibrutinib which will be further helpful to predict the in vivo metabolism of ibrutinib in the human body. Enzyme kinetic parameters (Km and Vmax) for both the metabolites were also established using different substrate concentrations. This newly developed biotransformation method will further help in determining the elimination mechanism of ibrutinib which in turn will assist in predicting the effectiveness and toxicity of the drug. [ABSTRACT FROM AUTHOR]

The role of individual cytochrome P450 (CYPs) responsible for the drug metabolism can be determined through their chemical inhibition. During the pandemic, dexamethasone and remdesivir with omeprazole were used for the treatment of COVID-19, while Ibuprofen was taken to treat the symptoms of fever and headache. This study aimed to examine the potency of ibuprofen remdesivir, and omeprazole as inhibitors of cytochrome P450s using rat liver microsomes in vitro. Dexamethasone a corticosteroid, sometimes used to reduce the body's immune response in the treatment of COVID-19, was used as a probe substrate and the three inhibitors were added to the incubation system at different concentrations and analysed by a validated High Performance Liquid Chromatography (HPLC) method. The CYP3A2 isoenzyme is responsible for dexamethasone metabolism in vitro. The results showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with Ki = 224.981 ± 1.854 µM and IC50 = 230.552 ± 2.020 µM, although remdesivir showed a mixed inhibition pattern with a Ki = 22.504 ± 0.008 µM and IC50 = 45.007 ± 0.016 µM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a Ki = 39.175 ± 0.230 µM and IC50 = 78.351 ± 0.460 µM. These results suggest that the tested inhibitors would not exert a significant effect on the CYP3A2 isoenzyme responsible for the co-administered dexamethasone drug's metabolism in vivo. [ABSTRACT FROM AUTHOR]

This work presents the characteristics of the catalytic activity of hydroxylation of benzo[a] pyrene (B[a]P) in rat liver microsomes using plasma lipoproteins (LP) (VLDL, LDL, HDL) as transport forms of B[a]P. The aim of the study was: to study the effect of the LP-component of the LP-B[a]P complexes on the rate of B[a]P hydroxylation in rat liver microsomes. The studies were carried out on the microsomal fraction of rat liver using B[a]P as a substrate, ultracentrifugation of individual fractions of VLDL, LDL, HDL plasma, and spectrofluorimetric determination of the activity of arylhydrocarbonhydroxylase. In work on microsomes of rat liver the rate of hydroxylation of B[a]P in free form and in the form of B[a]P complexes with various fractions of LP was analyzed. The analysis showed that the arylhydrocarbonhydroxylase activity (pmol 3-OH-B[a]P in 1 min per 1 mg of protein) decreases in the complex form B[a]P with LP. So for transport form B[a]P with HDL the decrease was by 22%, for transport form B[a]P with VLDL the decrease was 30%, and for transport form B[a]P with LDL it was 52%. It should be noted that Km for the substrate remained practically unchanged in all forms. The Vmax and Km values indicate that the LP component in complexes with B[a]P can be a noncompetitive inhibitor of B[a]P hydroxylation in rat liver microsomes.

An efficient, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) chiral analysis method was established for determination of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes. Effects of polysaccharide chiral stationary phases and basic additives on chiral separations of two analytes were discussed in detail. Amylose tris(3, 5‐dimethylphenylcarbamate)‐coated chiral stationary phase showed the best separation performance for them with acetonitrile‐diethylamine‐ethanol‐diethylamine mixture (90:0.1:10:0.1, v/v/v/v) among four chiral stationary phases. Then, multiple reaction monitoring mode was selected as the data acquisition for determination of two pairs of enantiomers. The proposed LC–MS/MS chiral analysis method was validated in terms of linearity, accuracy, precision, and specificity. Good linearity with correlation coefficient over 0.998 was obtained in the concentration range of 0.05–5 μM. Limits of quantification for chloroquine and hydroxychloroquine enantiomers were 5.0 and 1.0 nM, respectively. The recoveries ranged from 81.14% to 111.09%. The intra‐day and inter‐day relative standard deviation were less than 6.5%. Moreover, concentrations of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes were determined through the proposed LC–MS/MS analysis method. After incubated with rat liver microsomes for 10 min, the enantiomeric factor of hydroxychloroquine decreased from 0.50 to 0.45 (p < 0.001). In brief, our developed determination method for chloroquine and hydroxychloroquine enantiomers through LC–MS/MS spectrometry showed the characteristics of high‐efficiency, fast speed, and very low detection limit, and would be greatly beneficial for screening and quantitation of them in biological matrices. [ABSTRACT FROM AUTHOR]

Objectives: The objective of the present study is to study the in-vitro metabolites of FDA approved anticancer drug, belinostat which is a histone deacetylase inhibitor. Methods: The metabolites were formed by incubating the drug, belinostat with rat liver microsomes, at 37°C for 24 hr. The newly formed metabolites were identified and characterized with the help of ultra-high performance liquid chromatography which is interlinked with tandem quadrupole time-of-flight mass spectrometry. Results: Two Phase-I metabolites were formed which include a belinostat amide (reductive metabolite) and belinostat acid (deaminated belinostat). We then elucidated the structures of the formed metabolites based on LC-MS/MS data which included accurate masses, the fragmentation of ions and chromatographic retention times. Conclusion: This study describes the detailed in-vitro metabolite profiling of belinostat which will be further helpful to predict the in-vivo metabolism of belinostat in the human body. Enzyme kinetic parameters (Km and Vmax) for both the metabolites were also established using different substrate concentrations. The study will be helpful in determining the safety and efficacy of the drug. This will further help in determining the elimination mechanism of belinostat which in turn will assist in predicting the effectiveness and toxicity of the drug. [ABSTRACT FROM AUTHOR]

Yunfang Zhou,1,* Ailian Hua,2,* Quan Zhou,1 Peiwu Geng,1 Feifei Chen,1 Lianhe Yan,1 Shuanghu Wang,1 Congcong Wen3 1The Laboratory of Clinical Pharmacy, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, Zhejiang 323000, People’s Republic of China; 2Department of Pharmacy, The First People’s Hospital of Yuhang District, Hangzhou, Zhejiang 311100, People’s Republic of China; 3Laboratory Animal Centre, Wenzhou Medical University, Wenzhou, Zhejiang 325027, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shuanghu WangThe Laboratory of Clinical Pharmacy, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui 323000, Zhejiang, People’s Republic of ChinaEmail wangshuanghu@lsu.edu.cnCongcong WenLaboratory Animal Centre, Wenzhou Medical University, Wenzhou, Zhejiang 325027, People’s Republic of ChinaTel/ Fax +865782780081Email bluce494949@163.comPurpose: The aim of the present study was to investigate the interactions of the main components of Lygodium root (ie, p-coumaric acid, acacetin, apigenin, buddleoside and Diosmetin-7-O-β-D-glucopyranoside) with cytochrome P450 3A enzyme activity both in vitro and in vivo.Methods: In vitro inhibition of drugs was assessed by incubating rat liver microsomes (RLMs) with a typical P450 3A enzyme substrate, midazolam, to determine their 50% inhibitory concentration (IC50) values. For the in vivo study, healthy male Sprague Dawley rats were consecutively administered acacetin or apigenin for 7 days at the dosage of 5 mg/kg after being randomly divided into 3 groups: Group A (control group), Group B (acacetin group) and Group C (apigenin group).Results: Among the five main components of Lygodium root, only acacetin and apigenin showed inhibitory effects on the cytochrome P450 3A enzyme in vitro. The IC50 values of acacetin and apigenin were 58.46 μM and 8.20 μM, respectively. Additionally, the in vivo analysis results revealed that acacetin and apigenin could systemically inhibit midazolam metabolism in rats. The Tmax, AUC(0-t) and Cmax of midazolam in group B and group C were significantly increased (P< 0.05), accompanied by a significant decrease in Vz/F and CLz/F (P< 0.05).Conclusion: Acacetin and apigenin could inhibit the activity of the cytochrome P450 3A enzyme in vitro and in vivo, indicating that herbal drug interactions might occur when taking Lygodium root and midazolam synchronously.Keywords: Lygodium root, drug–drug interactions, rat liver microsomes, midazolam, metabolism

Context Rivaroxaban and ticagrelor are two common drugs for the treatment of atrial fibrillation and acute coronary syndrome. However, the drug–drug interaction between them is still unknown. Objective To investigate the effects of ticagrelor on the pharmacokinetics of rivaroxaban in rats both in vivo and in vitro. Materials and methods A sensitive and reliable UPLC-MS/MS method was developed for the determination of rivaroxaban in rat plasma. Ten Sprague-Dawley rats were randomly divided into ticagrelor pre-treated group (10 mg/kg/day for 14 days) and control group. The pharmacokinetics of orally administered rivaroxaban (10 mg/kg, single dose) with or without ticagrelor pre-treatment was investigated with developed UPLC-MS/MS method. Additionally, Sprague-Dawley rat liver microsomes were also used to investigate the drug–drug interaction between these two drugs in vitro. Results The Cmax (221.34 ± 53.33 vs. 691.18 ± 238.31 ng/mL) and the AUC(0–t) (1060.97 ± 291.21 vs. 3483.03 ± 753.83 μg·h/L) of rivaroxaban increased significantly (p

The role of individual cytochrome P450 (CYPs) responsible for the drug metabolism can be determined through their chemical inhibition. During the pandemic, dexamethasone and remdesivir with omeprazole were used for the treatment of COVID-19, while Ibuprofen was taken to treat the symptoms of fever and headache. This study aimed to examine the potency of ibuprofen remdesivir, and omeprazole as inhibitors of cytochrome P450s using rat liver microsomes in vitro. Dexamethasone a corticosteroid, sometimes used to reduce the body’s immune response in the treatment of COVID-19, was used as a probe substrate and the three inhibitors were added to the incubation system at different concentrations and analysed by a validated High Performance Liquid Chromatography (HPLC) method. The CYP3A2 isoenzyme is responsible for dexamethasone metabolism in vitro. The results showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with Ki = 224.981 ± 1.854 µM and IC50 = 230.552 ± 2.020 µM, although remdesivir showed a mixed inhibition pattern with a Ki = 22.504 ± 0.008 µM and IC50 = 45.007 ± 0.016 µM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a Ki = 39.175 ± 0.230 µM and IC50 = 78.351 ± 0.460 µM. These results suggest that the tested inhibitors would not exert a significant effect on the CYP3A2 isoenzyme responsible for the co-administered dexamethasone drug’s metabolism in vivo.

Rivaroxaban and ticagrelor are two common drugs for the treatment of atrial fibrillation and acute coronary syndrome. However, the drug–drug interaction between them is still unknown. To investigate the effects of ticagrelor on the pharmacokinetics of rivaroxaban in rats both in vivo and in vitro. A sensitive and reliable UPLC-MS/MS method was developed for the determination of rivaroxaban in rat plasma. Ten Sprague-Dawley rats were randomly divided into ticagrelor pre-treated group (10 mg/kg/day for 14 days) and control group. The pharmacokinetics of orally administered rivaroxaban (10 mg/kg, single dose) with or without ticagrelor pre-treatment was investigated with developed UPLC-MS/MS method. Additionally, Sprague-Dawley rat liver microsomes were also used to investigate the drug–drug interaction between these two drugs in vitro. The Cmax (221.34 ± 53.33 vs. 691.18 ± 238.31 ng/mL) and the AUC(0–t) (1060.97 ± 291.21 vs. 3483.03 ± 753.83 μg·h/L) of rivaroxaban increased significantly (p < 0.05) with ticagrelor pre-treatment. The MRT(0–∞) of rivaroxaban increased from 4.41 ± 0.79 to 5.97 ± 1.11 h, while the intrinsic clearance decreased from 9.93 ± 2.55 to 2.89 ± 0.63 L/h/kg (both p < 0.05) after pre-treated with ticagrelor. Enzyme kinetic study indicated that ticagrelor decreased rivaroxaban metabolic clearance with the IC50 value of 14.04 μmol/L. Our in vivo and in vitro results demonstrated that there is a drug–drug interaction between ticagrelor and rivaroxaban in rats. Further studies need to be carried out to verify whether similar interactions truly apply in humans and whether these interactions have clinical significance. [ABSTRACT FROM AUTHOR]

Objectives: N‐(2‐hydroxyphenyl)‐2‐propylpentanamide (HO‐AAVPA), a derivative of valproic acid (VPA), has been proposed as a potential anticancer agent due to its improved antiproliferative effects in some cancer cell lines. Although there is evidence that VPA is metabolized by cytochrome P450 2C11 rat isoform, HO‐AAVPA CYP‐mediated metabolism has not yet been fully explored. Therefore, in this work, the biotransformation of HO‐AAVPA by CYP2C11 was investigated. Methods: Kinetic parameters and spectral interaction between HO‐AAVPA and CYP were evaluated using rat liver microsomes. The participation of CYP2C11 in metabolism of HO‐AAVPA was confirmed by cimetidine (CIM) inhibition assay. Docking and molecular dynamics simulations coupled to MMGBSA methods were used in theoretical study. Key findings: HO‐AAVPA is metabolized by CYP enzymes (KM = 38.94 µm), yielding a hydroxylated metabolite according to its HPLC retention time (5.4 min) and MS analysis (252.2 m/z). In addition, CIM inhibition in rat liver microsomes (Ki = 59.23 µm) confirmed that CYP2C11 is mainly involved in HO‐AAVPA metabolism. Furthermore, HO‐AAVPA interacts with CYP2C11 as a type I ligand. HO‐AAVPA is stabilized at the CYP2C11 ligand recognition site through a map of interactions similar to other typical CYP2C11 substrates. Conclusion: Therefore, rat liver CYP2C11 isoform is able to metabolize HO‐AAVPA. [ABSTRACT FROM AUTHOR]

Aripiprazole belongs to the atypical antipsychotic category of drug, frequently prescribed for the cure of Psychosis. Cytochrome2D6 (CYP2D6) is one of the prominent enzymes that play a key role in the metabolism of Aripiprazole and further formation of an active metabolite, Dehydroaripiprazole takes place. Patients under the treatment with this potent moiety have been reported with the high blood glucose level as a side effect. In addition to this, literature suggests that the leaves of Insulin plant (Costus igneus) are usually administered by diabetic patients (2-3 times) to manage the sugar level without concerning to the physicians. There might be probability while concurrent administration of (Aripiprazole and Insulin plant leaves), leaves inhibit the enzyme and ultimately Dehydroaripiprazole exhibit poor pharmacological action. Hence, the present work was done to investigate the inhibitory effect of Insulin plant extract (IPE) on CYP2D6, with the co-administration of Aripiprazole (to examine the changes in a metabolite of Aripiprazole). In order to carry out this protocol firstly, IPE was prepared by the successive extraction method. Methanolic extract of Insulin plant was found enriched with the Quercetin, which was used as a marker to carry out this study. Presence of Quercetin was confirmed with the Ultra-violet spectroscopy (UV) and High-performance liquid chromatography (HPLC) analytical methods. Characterization of Aripiprazole was done with the help of different analytical tools such as: HPLC, melting point, and UV. Aripiprazole alone and with the several dilutions of IPE were incubated using isolated rat liver microsome (RLM) and analyzed using HPLC. HPLC data demonstrated that the, mixture of IPE+Aripiprazole (herb and drug in liver microsomes), in comparison to Aripiprazole+RLM (alone drug in liver microsomes) has not shown any significant inhibition of the enzyme, and inhibitory concentration (IC50) value found to be 4.49µg/ml. Therefore this study concluded that IPE has shown safe results even at the highest clinical dose after oral administration i.e., 20-1000µg/ml and did not show any significant CYP2D6 inhibition. Nevertheless, to confirm these observations, inclusion of in vivo studies will be advantageous. As per our knowledge, this is the first attempt made on the detection of Herb- Drug interactions (HDI'S) between Insulin plant and Aripiprazole [ABSTRACT FROM AUTHOR]

Ginseng and aconite are well‐known couplet medicinals. Ginsenoside Rg1 is the main active ingredient in ginseng, and aconitine (AC), benzoylaconine (BAC) and aconine (ACN) are three representative alkaloids in aconite, which belong to the diester alkaloids, monoester alkaloids and alkanolamine alkaloids respectively. The aim of this study was to investigate the pharmacokinetic effects of ginsenoside Rg1 on the three types of alkaloids and to provide evidences for their compatibility mechanism. In this study, the ginsenoside Rg1 was simultaneously intragastrically administered to rats with AC, BAC and ACN, respectively, and the rat plasma was collected at different time points. The plasma drug concentrations of the three types of alkaloids were determined by UHPLC–MS/MS, and the pharmacokinetic parameters were calculated. The results indicated that the peak concentration and area under the concentration–time curve of BAC were significantly increased (P < 0.05), those for AC were decreased (P < 0.05), and the values for ACN did not change after pretreatment with ginsenoside Rg1. It was inferred that ginsenoside Rg1 may affect the absorption and metabolism of AC and BAC and then change their pharmacokinetic parameters. Subsequently, their absorption and metabolism were further investigated using the Caco‐2 cell monolayer and rat liver microsomes in vitro. The Caco‐2 cell monolayer absorption assay indicated that ginsenoside Rg1 could promote the absorption of AC and BAC, and the rat liver microsomes metabolism assay indicated that ginsenoside Rg1 accelerated the metabolism of AC and did not affect the other two alkaloids. All of the results indicated that ginsenoside Rg1 may reduce the toxicity of aconite and improve its efficacy by promoting the absorption of BAC and accelerating the metabolism of AC. These results could provide evidence for the compatibility mechanism of the traditional Chinese herbal formula Shenfu Decoction. [ABSTRACT FROM AUTHOR]

Abstract Masitinib (MST) is a selective tyrosine kinase inhibitor. Validated liquid chromatography tandem mass spectrometric method (LC–MS/MS) was developed for the quantification of MST in rat liver microsomes (RLMs) matrix. The developed method was applied to metabolic stability and excretion rate studies. Reversed phase liquid chromatography was used for resolution of MST and bosutinib (IS) using C18 (50 mm × 2.1 mm, 1.8 μm). Binary solvent system consisted of 35% solvent A (0.1% formic acid in H2O, pH: 3.2) and 65% solvent B (acetonitrile) used as mobile phase at flow rate of 0.25 mL with a total run time of 5 min. Injection volume was 5 µL. Generation of ions was done in positive ESI source and quantification of MST and IS were done using MRM mode. The developed method showed a linearity in the range of 5–200 ng/mL (r2 ≥ 0.9992) with LOQ and LOD of 0.25 and 0.76 ng/mL in RLMs. The intra- and inter-day precision and accuracy ranged from 0.95 to 1.49 and − 5.22 to 1.13%, respectively in RLMs. Rate of disappearance of MST during incubation with RLMs was almost linear allover incubation time. In vitro t1/2 was 50.38 min and CLin was 3.11 ± 0.2. The developed method was applied also to measure the rate of masitinib excretion in rat urine. The method can used for further pharmacokinetic studies of MST.

Abstract Vandetanib (VNT) is a new oral tyrosine kinase inhibitor that acts mainly by inhibiting vascular endothelial growth factor receptor (VEGFR). Fast, specific, sensitive and validated LC–MS/MS was established for the determination of VNT in two various matrices including rat liver microsomes (RLMs) and human plasma. This method was applied in metabolic stability investigation of VNT. Resolution of two analytes was performed using C18 column and isocratic mobile phase composed of binary system of 10 mM ammonium formate (pH 4.1) and acetonitrile in a ratio of 1:1. The flow rate was set at 0.25 mL/min and total run time was 4 min with injection volume of 5 µL. Ions were generated by ESI source and analyzed by multiple reaction monitoring mode (basis for quantification) in the Agilent 6410 QqQ analyzer. The linearity of the established method ranged from 5 to 500 ng/mL (r2 ≥ 0.9996) in human plasma and RLMs. LOQ and LOD were 2.48 and 7.52 ng/mL, and 2.14 and 6.49 in human plasma and RLMs matrices. The intra-day and inter-day precision and accuracy were 0.66–2.66% and 95.05–99.17% in human plasma matrix while in RLMs matrix, ranged from 0.97 to 3.08% and 95.8 to 100.09%, respectively. In vitro half-life was 39.85 min and intrinsic clearance was 3.92 ± 0.28 mL/min/kg.

Highlights • The metabolites of hinokiflavone were first characterized. • A rapid and sensitive method was developed. • Glutamine conjugation and glycine conjugation were unique metabolic pathways of hinokiflavone. • The metabolic profiling of hinokiflavone was first inferred and summarized. Abstract Hinokiflavone (HF), belonging to biflavonoids, possesses excellent pharmacological activities, including anti-inflammatory, antioxidant and antitumor activity. Nevertheless, its metabolism in vivo (rats) and in vitro (rat liver microsomes and intestinal flora) is presently not characterized. In this study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) based on four-step strategy was a rapid method for the detection of HF metabolites. A total of 41 metabolites in vivo , 49 metabolites in vitro were characterized. It also verified that intestinal tract exceeds the liver in the biotransformation of HF. More significant, the main metabolic pathways for HF were mainly bio-transformed to various mono-flavone resulting from the rupture of connective C O bonds, which exhibited a large distinction with other biflavones. Noteworthily, glutamine conjugation and glycine conjugation were considered as unique metabolic pathways of HF. The information obtained from this study contributes to better understanding of pharmacological mechanism of HF. [ABSTRACT FROM AUTHOR]

Abstract Saroglitazar, a PPAR αҮ agonist, is currently undergoing global development for the treatment of NASH and other indications. Saroglitazar showed CYP2C8 inhibition in human liver microsomes (IC 50 : 2.9 μM). The aim was to carry out drug-drug interaction (DDI) studies in Wistar rats using saroglitazar (perpetrator drug) with five CYP2C8 substrates. Also, the in vitro CYP2C8 inhibitory potential of saroglitazar in rat liver microsomes (RLM) was evaluated to justify use of preclinical model. The oral pharmacokinetics of various CYP2C8 substrates; montelukast, rosiglitazone, pioglitazone, repaglinide and intravenous pharmacokinetics of paclitaxel was assessed in the presence/absence of oral saroglitazar (4 mg/kg) in Wistar rats. A separate study was performed to assess the oral pharmacokinetics of saroglitazar. Serial blood samples were collected from all studies and the harvested plasma were stored frozen until bioanalysis. LC-MS/MS was used for the analysis of various analytes; concentration data was subjected to noncompartmental pharmacokinetic analysis. Statistical tests (unpaired t -test) were employed to judge the level of DDI. Generally, the pharmacokinetics of CYP2C8 substrates was not affected by the concomitant intake of saroglitazar as judged by the overall exposure (AUC 0–last and AUC 0–inf) and elimination half-life. The CYP2C8 IC 50 of 4.5 μM in RLM for saroglitazar, supported the use of rats for this DDI study. In conclusion, pharmacokinetic data of diverse CYP2C8 substrates suggested that coadministration of saroglitazar did not cause clinically relevant DDI. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Abstract Ginkgo biloba leaves extract (GBE), one of the most widely used traditional Chinese medicines worldwide, can be used for the treatment of diabetes mellitus (DM). However, its biotransformation in liver is not fully known under the state of DM. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (q/TOF-MS) was established for the qualification and quantification of the biotransformation of GBE in normal and diabetic rat liver microsomes (RLMs). 6 metabolites were tentatively identified according to the exact molecular weights and the characteristic fragment ions provided by q/TOF-MS data. The results of metabolic stability showed that the metabolic ratio of four target compounds including quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs were significantly enhanced when comparing with normal RLMs. The results of enzyme kinetics showed that compared with normal RLMs, the Michaelis-Menten constant (K m) value of genistein was obvious increased while its maximal velocity (V max) and intrinsic clearance (CL int) values were significantly decreased by diabetic RLMs, and the V max and CL int values of kaempferol and isorhamnetin were notably enhanced while their K m values were markedly reduced. For the half-time (t 1/2) values of four target compounds and the K m , V max and CL int values of quercetin, there were not statistically significant changes between normal and diabetic RLMs. The results suggest that the developed off-line 2D LC-DAD-q/TOF-MS method is an easy and accurate approach for the study of GBE biotransformation in RLMs and may provide the essential data for further pharmacological and clinical studies of GBE. Highlights • The biotransformation of GBE in diabetic rat liver microsomes (RLMs) was first investigated. • An off-line 2D LC-DAD-q/TOF-MS was first developed and optimized for the analysis. • 6 mono-glucuronide conjugation metabolites were tentatively characterized. • The metabolic stability of quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs was obtained. • The enzyme kinetics of quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs was first reported. [ABSTRACT FROM AUTHOR]

Highlights • An LC–MS/MS method for the determination of cligosiban in rat plasma. • Pharmacokinetic profiles were disclosed. • In vitro and in vivo metabolites were identified. • Demethylation and glucuronidation were the predominant metabolic pathways. Abstract Cligosiban is a highly-affinity nonpeptide oxytocin receptor antagonist. In this study, a simple an sensitive LC–MS/MS method was developed and validated for the determination of cligosiban in rat plasma. The plasma samples were pretreated with acetonitrile as precipitant and then separated on an ACQUITY BEH C 18 column (2.1 × 50 mm, 1.7 μm) with 0.1% formic acid in water and acetonitrile as mobile phase. The analytes were monitored using selected reaction monitoring (SRM) mode with transitions at m/z 420.1→248.1 for cligosiban and m/z 304.1→161.1 for IS. The developed method showed good linearity over the concentration range of 1–1000 ng/mL with coefficient of correlation > 0.996. The lower limit of quantification (LLOQ) is 1 ng/mL. The method was validated for selectivity, precision, accuracy, recovery, and stability in accordance with FDA's guidance. The validated assay has been successfully applied to the pharmacokinetic study of cligosiban in rat plasma after intravenous and oral administration. According to the current results, the oral bioavailability of cligosiban was 63.82%. Furthermore, the metabolites present in rat liver microsomes (RLM), human liver microsomes (HLM) and rat plasma were analyzed by UHPLC-LTQ-Orbitrap-MS method, and four metabolites structurally identified based on their accurate masses, and fragment ions. The proposed metabolic pathways of cligosiban were demethylation and glucuronidation. This study is the first report on the pharmacokinetic and metabolic information of cligosiban, which would provide insights into the effectiveness and toxicity of cligosiban. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract The present study was aimed to evaluate the effects of amphiphilic copolymer micelles on six major hepatic cytochrome P450 (CYP) isoforms. A series of mPEG 2k -PCL x polymeric micelles (mPEG 2k -PCL 2k , mPEG 2k -PCL 3.5k , mPEG 2k -PCL 5k and mPEG 2k -PCL 10k) ranging from 20 to 100 nm were prepared to investigate the inhibitory or inductive activities by in vitro incubations of rat liver microsomes and primary rat hepatocytes. Inhibition of these polymeric micelles on CYP1A2, CYP2B1, CYP2C6, CYP2C11, CYP2D2 and CYP3A1/2 isoenzymes were observed above their critical micelle concentrations (>10 μg·mL−1) and in a concentration-dependent manner. The mPEG 2k -PCL 2k micelles showed the strongest inhibition of CYP1A2, followed by CYP2C11. The micelles with lower molecular weight PCL segment exhibited more potent inhibitory potential. Induction on CYP1A2, CYP2B1 and CYP3A1/2 activity (2.1–7.2-fold, 1.5–2.4-fold and 1.3–3.0-fold, respectively) were detected at all tested concentrations (0.1–1000 μg·mL−1 or 0.1–100 μg·mL−1). Accordingly, most of the mRNA levels were upregulated. As demonstrated in ex vivo fluorescence imaging results, the mPEG 2k -PCL x micelles mainly accumulated in the liver after intravenous administration. In conclusion, mPEG 2k -PCL x micelles can interfere with the normal metabolic function of CYP450s in vitro , indicating polymeric micelles as promising drug nano-carriers might cause micelle–drug interaction and the in vivo interaction deserves further investigation. [ABSTRACT FROM AUTHOR]

Inhibition of cytochrome P450 (CYP) alters the pharmacokinetic parameters of the drug and causes drug−drug interactions. Salicylic acid been used for the treatment of colorectal cancer (CRC) and chemoprevention in recent decades. Thus, the aim of this study was to examine the in vitro inhibitory effect of salicylic acid on CYP2E1 activity in rat liver microsomes (RLMs) using high-performance liquid chromatography (HPLC). High-performance liquid chromatography analysis of a CYP2E1 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 282 nm using 60% H2O, 25% acetonitrile, and 15% methanol as mobile phase. The CYP2E1 assay showed a good linearity (R2 > 0.999), good reproducibility, intra- and inter-day precision (i (inhibition constant) = 83.56 ± 2.730 µM and concentration of inhibitor causing 50% inhibition of original enzyme activity (IC50) exceeding 100 µM (IC50 = 167.12 ± 5.460 µM) for CYP2E1 enzyme activity. Salicylic acid in rats would have both low and high potential to cause toxicity and drug interactions with other drugs that are substrates for CYP2E1.

To explore metabolism mechanism of paeoniflorin in the liver and further understand intact metabolism process of paeoniflorin, a rapid, convenient and effective assay is described using ultra-performance liquid chromatography coupled with hybrid quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). The strategy was confirmed in the following primary processes: firstly, different concentration of paeoniflorin, rat liver microsomes, coenzymes and different incubated conditions were optimized to build a biotransformation model of rat liver microsomes in vitro by high performance liquid chromatography with diode array detection (HPLC-DAD); secondly, the metabolites of paeoniflorin in rat liver microsomes were detected and screened using UPLC-Q-TOF-MS/MS by comparing the total ion chromatogram (TIC) of the experimental group with those of control groups; finally, the molecular formulae and corresponding chemical structures of paeoniflorin metabolites were identified by comparing the MS and MS/MS spectra with the self-constructed database and simulation software. Based on this analytical strategy, 20 metabolites of paeoniflorin were found and 6 metabolites (including four new compounds) were tentatively identified. It was shown that hydrolysis and oxidation were the major metabolic pathways of paeoniflorin in rat liver microsomes, and the main metabolic sites were the structures of pinane and the ester bond. These findings were significant for a better understanding of the metabolism of paeoniflorin in rat liver microsomes and the proposed metabolic pathways of paeoniflorin might provide fundamental support for the further research in the pharmacological mechanism of Paeoniae Radix Rubra (PRR).

Entrectinib (ENC) is a potent orally available anaplastic lymphoma kinase (ALK) inhibitor. In 10 July 2017, biotechnology company (Ignyta) announced that granted orphan drug designation approval was given by the FDA to ENC for “treatment of NTRK fusion-positive solid tumors”. A validated LC-MS/MS methodology was developed for ENC quantification in human plasma matrix. The supposed method characterized by high speed, specificity and sensitivity. This established method was applied for metabolic degradation assessment of ENC. Reversed stationary phase (C 18 column) and elution mobile phase (48% 10 mM ammonium formate in H 2 O (pH: 4.2 adjusted by adding few drops of formic acid): 52% ACN) were utilized for chromatographic resolution of ENC and lapatinib as internal standard (IS). Total elution time, flow rate and injection volume were 4 min., 0.25 mL/min., and 5 μL, respectively. Electrospray ionization source was used for ions generation, while positive multiple reactions monitoring (MRM) mode was used for ion analysis. The data of calibration curve of ENC in human plasma was linear in the range of 5–500 ng/mL with correlation coefficient (r 2 ) >0.999. LOQ and LOD for ENC were 2.17 ng/mL and 0.71 ng/mL, respectively. Inter-day and intra-day precision and accuracy were 97.52 to 101.83%, and 0.38 to 1.32%, respectively. Intrinsic clearance (Clint) and in vitro half-life (t 1/2 ) were equal to 15.67 mL/min/kg and 9.1 min, respectively. To our knowledge, this is considered the first method for ENC quantification in human plasma and its metabolic degradation assessment. [ABSTRACT FROM AUTHOR]

1. The objective was to investigate the underlying mechanism of the stereoselectivity in the metabolism of rhynchophylline (RIN) and isorhynchophylline (IRN) epimers in rat liver microsomes (RLM). 2. After incubation, eight metabolites of RIN (M1-5) and IRN (M6-8) reacted at A- and C-ring were identified using LC-Q-TOF/MS. Metabolic pathways included oxidation, hydroxylation, N-oxidation and dehydrogenation. In addition, hydroxylation at A-ring was the major metabolic pathway for RIN whereas the oxidation at C-ring was the major one for IRN. 3. Enzyme kinetics showed that the intrinsic clearance (CLint) for IRN elimination was 1.9-fold higher than RIN and the degradation half-life (T1/2) of RIN was 4.7-fold higher than that of IRN, indicating IRN was more favorable to be metabolized than RIN in RLM. 4. Data from chemical inhibition study demonstrated CYP3A was the predominant isoform involved in the metabolic elimination of both epimers, as well as the formation of M1-8. 5. In conclusion, data revealed that due to the spatial configurations at C-7 position, RIN and IRN epimers possessed different hepatic metabolic pathways and elimination rates which were mainly mediated by CYP3A. [ABSTRACT FROM AUTHOR]

Along with the prevalence of drug combination therapies, an increasing number of cases about drug-drug interactions (DDI) have been reported, which has drawn a lot of attention due to the potential toxicity and/or therapeutic failure. Pharmacokinetic interactions based on drug metabolic enzymes should be responsible for a great many of DDI. UDP-glucuronosyltransferases (UGT) as the main phase II metabolic enzymes are involved in the metabolism of many endogenous and exogenous substrates. Herein, we designed and optimized a validated cocktail method for the simultaneous evaluation of drug-mediated inhibition of the main five UGT isoforms using respective specific probe substrates (estradiol for UGT1A1, chenodeoxycholic acid for UGT1A3, serotonin for UGT1A6, propofol for UGT1A9/PROG and zidovudine for UGT2B7/AZTG) in human and rat liver microsomes by liquid chromatography–tandem mass spectrometry (LC MS/MS). Moreover, we investigated the risk of interactions among UGT probe substrates, and validated the cocktail method by known positive inhibitors of UGT isoforms. To minimize the substrates interaction, we developed two cocktail subgroups which were further optimized via exploring the experimental conditions. In particular, the cocktail inhibition assay for rapid assessment of in vitro rat UGTs was firstly reported and the values of K m in the liver microsomes from humans and rats were close to each other in the specific UGT subtype. In conclusion, this study has successfully established the cocktail approach to explore UGT activity, especially for UGT inhibition in a fast and efficient way. [ABSTRACT FROM AUTHOR]

Corydalis saxicola Bunting, a well-known traditional Chinese medicine in south China, has been widely used for the treatment of various hepatic diseases. Its active ingredients are Corydalis saxicola Bunting total alkaloids (CSBTA), which primarily include dehydrocavidine, palmatine, and berberine. These representative alkaloids could be metabolized by hepatic CYP450s. Hence, it is necessary to investigate the potential influences of CSBTA on CYP450s to explore the possibility of herb-drug interactions. In present study, in vitro inhibition and in vivo induction studies were performed to evaluate the potential effects of CSBTA extract on CYP450s in rats. Inhibition assay illustrated that CSBTA exerted inhibitory effects on CYP1A2 (IC50 , 38.08 μg/ml; Ki , 14.3 μg/ml), CYP2D1 (IC50 , 20.89 μg/ml; Ki , 9.34 μg/ml), CYP2C6/11 (IC50 for diclofenac and S-mephenytoin, 56.98 and 31.59 μg/ml; Ki, 39.0 and 23.8 μg/ml), and CYP2B1 (IC50 , 48.49 μg/ml; Ki , 36.3 μg/ml) in a noncompetitive manner. Induction study showed CSBTA had obvious inhibitory rather than inductive effects on CYP1A2 and CYP2C6/11. Interestingly, neither inhibition nor induction on CYP3A was observed for CSBTA. In conclusion, CSBTA-drug interactions might occur through CYP450s inhibition, particularly CYP1A and CYP2D. Further studies are still needed to elucidate the underlying mechanisms of inhibition. [ABSTRACT FROM AUTHOR]

The aim of the current study was to apply different strategies for generation of metabolites of combretastatin A4 (CA4) and subsequent identification of the unknown products of phase I metabolism. CA4 is a potent anti-tubulin agent currently undergoing clinical trials. The multi-tool analytical approach was based on electrochemistry (EC), in silico predictions, and in vitro studies with the use of rat liver microsomes. With the latter, two different analytical sample preparation methods were applied: protein precipitation and solid phase microextraction, both hyphenated to the liquid chromatography-high-resolution mass spectrometry platform (LC-HRMS). The EC was coupled directly to HRMS. Conventional techniques using enzyme fractions pooled from human or animals remain a method of choice for determinations of phase I of drug metabolism, EC and in silico methods, which enable determinations of metabolism patterns, are in turn considered to have great potential as fast alternatives to in vitro assays. While individual findings attained via employment of these four methods showed high similarity in relation to generated metabolic pathways for CA4, each method was found to provide unique features not identified with other approaches. In this paper, these differences are reviewed in view of potential artifacts and true metabolite production via various metabolism patterns under different experimental conditions. In addition, the reliability, applicability, MS compatibility issues, and potential of each of these technologies are discussed. [ABSTRACT FROM AUTHOR]

Ixerin Z and 11,13α-dihydroixerin Z are the important ingredients of sesquiterpene lactones in Kudiezi. In this study, the metabolites of Ixerin Z and 11,13α- dihydroixerin Z in rat liver microsomes were screened and identified using high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ/Orbitrap MS) at negative ion mode. The separation was conducted on Waters Acquity UPLC BEH C18 column (2. 1 mmX50 mmX1. 7 μm) with the gradient elution of 0. 10 formic acid aqueous solution (A) and acetonitrile (B). As a result, Ixerin Z, 11,13α-dihydroixerin Z and 16 metabolites were selected out, among which 15 metabolites were characterized based on the retention time, accurate mass measurements and fragmentation patterns. The results showed that there were two possible metabolic pathways of Ixerin Z in rat liver microsomes： 1) Metabolites 2, 5 and 12 were generated from Ixerin Z after hydroxylation ； 11,13α-dihydroixerin Z was generated from Ixerin Z after hydrogenated on its double bond, then metabolites 8, 14 and 15 were generated from 11, 13α-dihydroixerin Z after hydroxylation. 2) The metabolites were generated from the prototype via addition reaction, such as cysteine combination (1 and 4) , glutathione combination (7 and 9). There were also two possible metabolic pathways of 11, 13α-dihydroixerin Z in rat liver microsomes： 1) Ixerin Z was generated from 11,13α- dihydroixerin Z after hydrogenated on its double bond, then the metabolites 12 was generated from Ixerin Z after hydroxylation. Metabolites 3, 8, 10, 13, 14 and 15 were generated from 11，13α—dihydroixerin Z after hydroxylation, followed by hydrogenation to produce 6 and 11. 2) Metabolite 7 was generated in conjugation reaction. The results demonstrate that they mainly undergo oxidation, reduction, hydrolysis via phase I in rat liver microsomes, which will help to clarify the metabolic pattern of Ixerin Z and 11, 13α-dihydroixerin Z, and can also provide support for further new drug and toxicology research. [ABSTRACT FROM AUTHOR]

Brigatinib (BGB) belongs to a class of drugs called ALK inhibitor. On April 28, 2017, BGB has been approved by U.S. FDA for use in metastatic ALK-positive NSCLC. A fast, specific, sensitive and validated LC-MS/MS method was developed for the quantification of BGB in human plasma matrix. This method was applied successfully to study metabolic stability of BGB. Reversed phase (C18 column) and isocratic binary mobile phase (55% 0.1% formic acid: 45% ACN) were used for chromatographic separation of BGB and ponatinib (IS). The flow rate, total run time and injection volume were fixed at 0.2 mL/min, 4 min, 5 μL respectively. ESI source was utilized for ions formation, while multiple reaction monitoring (MRM) mode was used for ion analysis. In human plasma matrix, the Linearity range of the calibration curve was 5–500 ng/mL (r 2  ≥ 0.9982). LOQ and LOD were found to be 1.89 and 5.72 ng/mL. The precision and accuracy for the intra-day and inter-day were 0.45 to 1.85% and 97.37 to 104.85%. In vitro half-life (t 1/2 ) and intrinsic clearance (CL int ) were equal to 12.0 min and 13.1 ± 0.15 mL/min/kg respectively. The quantification of BGB in human plasma or its metabolic stability has not been studied as seen in literature review. [ABSTRACT FROM AUTHOR]

Nevadensin is major constituents of Lysionotus pauciflorus Maxim . (Chinese name: Shidiaolan), which has a variety of pharmacological effects such as anti-mycobacterium tuberculosis activities, antitussive, anti-inflammatory and anti-hypertensive. In this paper, we investigated the metabolism of nevadensin in vitro and in vivo . A strategy was firstly developed to identify the metabolites of nevadensin by using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). An on-line data acquisition method a multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Furthermore, some assistant tools, such as key fragment ions (KFI), were employed for compound hunting and identification. Based on the proposed method, 23 metabolites were structurally characterized in vivo including 16 phase I and 7 phase II metabolites, and 12 metabolites were detected in vitro containing 10 phase I and 2 phase II metabolites. The results indicated that oxidation, hydrolysis, demethylation, methylation, sulfate conjugation and glucuronide conjugation were main metabolic pathways of nevadensin. In a word, this study maybe can provide reference and valuable evidence for further investigation of the metabolic mechanism of nevadensin. [ABSTRACT FROM AUTHOR]

We previously reported that MDR-1339, an inhibitor of β-amyloid protein aggregation, was likely to be eliminated by biotransformation in rats. The objective of this study was to determine the chemical identity of metabolites derived from this aggregate inhibitor and to characterize the kinetics of formation of these metabolites in rats. Using high performance liquid chromatography coupled with mass spectrometry with a hybrid triple quadrupole-linear ion trap, 7 metabolites and 1 potential metabolic intermediate were identified in RLM incubations containing MDR-1339. In addition to these, 3 glucuronide metabolites were detected in urine samples from rats receiving a 10 mg/kg oral dose of MDR-1339. When the kinetics of the formation of two major metabolites, M1 and M2, were analyzed assuming simple Michaelis-Menten kinetics, the V max and K m values were found to be 0.459 ± 0.0196 nmol/min/mg protein and 28.3 ± 3.07 μM for M1, and 0.101 ± 0.00537 nmol/min/mg protein and 14.7 ± 2.37 μM for M2, respectively. When chemically synthesized M1 and M2 were individually administered to rats intravenously at the dose of 5 mg/kg respectively, the volume of distribution and elimination clearance were determined to be 4590 ± 709 mL/kg and 68.4 ± 5.60 mL/min/kg for M1 and 15300 ± 8110 mL/kg and 98.0 ± 19.5 mL/min/kg for M2, respectively. When MDR-1339 was intravenously administered to rats at a dose of 5 mg/kg, the parent drug and M1 were readily detected for periods of up to 6 h after the administration, but M2 was observed only from 2 to 4 h. A standard moment analysis indicates that the formation clearance of M1 is 6.01 mL/min/kg, suggesting that 19.7% of the MDR-1339 dose was eliminated in rats. These observations indicate that the hepatic biotransformation of MDR-1339 results in the formation of at least 10 metabolites and that M1 is the major metabolite derived from this aggregation inhibitor in rats. [ABSTRACT FROM AUTHOR]

1-Chloro-2-hydroxy-3-butene (CHB) is an in vitro metabolite of 1,3-butadiene, a rodent/human carcinogen. To search for an approach detecting CHB in vivo, it is vital to obtain a full understanding of CHB metabolism. Previously, we demonstrated that CHB was bioactivated to 1-chloro-3-buten-2-one (CBO) by alcohol dehydrogenase. However, CHB metabolism by cytochrome P450s has not been reported. Thus, in the present study, CHB metabolism by rat liver microsomes was investigated. The results showed that CHB was converted to 1-chloro-3,4-epoxy-2-butanol (CEB) and CBO. 4-Methylpyrazole, a cytochrome P450 2E1-specific inhibitor, inhibited the formation of both CEB and CBO, while 1-benzylimidazole, a generic cytochrome P450 inhibitor, completely abolished the formation of CEB and CBO, suggesting that CHB metabolism was mediated by cytochrome P450s. Because the molecules have two chiral centers, CEB was detected as two stereoisomers, which were designated D-CEB and M-CEB, and were characterized as (2 S ,3 R )-/(2 R ,3 S )-CEB and (2 R ,3 R )-/(2 S ,3 S )-CEB, respectively. The amounts of M-CEB were more than those of D-CEB by 50–80%. The amounts of CEB and CBO increased linearly over time from 10 (or 20 min for CBO) to 50 min. CHB metabolism followed Michaelis-Menten kinetics; the K m and V max values were determined to be 6.4 ± 0.7 mM and 0.10 ± 0.01 nmol/min/mg protein for D-CEB, 4.2 ± 0.5 mM and 0.16 ± 0.01 nmol/min/mg protein for M-CEB, and 4.0 ± 0.5 mM and 4.6 ± 0.5 nmol/min/mg protein for CBO, respectively. Thus, CBO was the dominant product of CHB metabolism. Moreover, CEB was genotoxic at ≥ 50 μM as evaluated by the comet assay. Collectively, the data showed that CHB could be bioactivated to CEB and CBO by cytochrome P450s with CBO being the predominant product. Thus, the formation of CEB and CBO can be used as evidence of CHB production. The products may also play a role in toxicity of CHB. [ABSTRACT FROM AUTHOR]

Masitinib (MST) is a selective tyrosine kinase inhibitor. Validated liquid chromatography tandem mass spectrometric method (LC-MS/MS) was developed for the quantification of MST in rat liver microsomes (RLMs) matrix. The developed method was applied to metabolic stability and excretion rate studies. Reversed phase liquid chromatography was used for resolution of MST and bosutinib (IS) using C (50 mm × 2.1 mm, 1.8 μm). Binary solvent system consisted of 35% solvent A (0.1% formic acid in HO, pH: 3.2) and 65% solvent B (acetonitrile) used as mobile phase at flow rate of 0.25 mL with a total run time of 5 min. Injection volume was 5 µL. Generation of ions was done in positive ESI source and quantification of MST and IS were done using MRM mode. The developed method showed a linearity in the range of 5-200 ng/mL (r ≥ 0.9992) with LOQ and LOD of 0.25 and 0.76 ng/mL in RLMs. The intra- and inter-day precision and accuracy ranged from 0.95 to 1.49 and − 5.22 to 1.13%, respectively in RLMs. Rate of disappearance of MST during incubation with RLMs was almost linear allover incubation time. In vitro t was 50.38 min and CL was 3.11 ± 0.2. The developed method was applied also to measure the rate of masitinib excretion in rat urine. The method can used for further pharmacokinetic studies of MST. [ABSTRACT FROM AUTHOR]

Purpose: To characterize and identify metabolites of (-)-epicatechin in microsomal fraction of rat hepatocytes (MFRHs). Methods: A single incubation of (-)-epicatechin (1 mL, 50 ìg/mL) in MFRH (0.5 mg/mL) was used for the generation of metabolites. Thereafter, the sample was subjected to protein precipitation prior to analysis with ultra-high performance liquid chromatography coupled to linear ion-trap orbitrap mass spectrometry (UHPLC-LTQ-Orbitap MS). Results: Nine metabolites of (-)-epicatechin were characterized on the basis of high resolution mass measurement, MS spectra and literature data. Based on their structures, the major metabolic routes of (-)-epicatechin in MFRHs were identified as hydroxylation, dihydroxylation and glycosylation. Conclusion: This is the first report on metabolites of (-)-epicatechin in MFRHs, and it is helpful in gaining deeper insight into the metabolism of (-)-epicatechin in vivo. The results will also provide guidance in research on the pharmacokinetics of new drugs. [ABSTRACT FROM AUTHOR]

• The liver microsome sensor was successfully developed for rapid screening of AFB1. • The electrochemical signal was based on the RLM metabolic conversion pathway. • The Au@MXene nanocomposite significantly improved the electro-performance. Cytochrome P450 enzymes (CYPs) catalyze the production of aflatoxin B1 (AFB1) metabolites, which play an important role in carcinogenesis. In this study, we report a simple electrochemical liver-microsome-based biosensor using a composite of gold nanoparticles adsorbed on MXene (Au@MXene) for rapid screening of AFB1. Rat liver microsomes (RLMs) were directly adsorbed on the Au@MXene nanocomposite. The high conductivity, large specific surface area, and good biocompatibility of the Au@MXene nanocomposite enabled the direct electron transfer between the RLMs and the electrode and maintained the biological activity of the enzyme in the RLMs to a large extent. The metabolic behavior of the RLM biosensor that was developed for the electrocatalyst of AFB1 to its hydroxylation metabolite aflatoxin M1 (AFM1) was confirmed. Based on the change in the electrical signal generated by this metabolic behavior, we established the relationship between AFB1 content and amperometric (I-t) current signal. When the AFB1 concentration ranged from 0.01 μM to 50 μM, the AFB1 concentration was linearly related to the electrical signal with a limit of detection of 2.8 nM. The results of the recovery experiments for corn samples showed that the recovery and accuracy of the sensor were consistent with the UPLC-MS/MS method. [ABSTRACT FROM AUTHOR]

Eupatorin is the major bioactive component of Java tea (Orthosiphon stamineus), exhibiting strong anticancer and anti-inflammatory activities. However, no research on the metabolism of eupatorin has been reported to date. In the present study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combined with an efficient online data acquisition and a multiple data processing method were developed for metabolite identification in vivo (rat plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora). A total of 51 metabolites in vivo, 60 metabolites in vitro were structurally characterized. The loss of CH2, CH2O, O, CO, oxidation, methylation, glucuronidation, sulfate conjugation, N-acetylation, hydrogenation, ketone formation, glycine conjugation, glutamine conjugation and glucose conjugation were the main metabolic pathways of eupatorin. This was the first identification of metabolites of eupatorin in vivo and in vitro and it will provide reference and valuable evidence for further development of new pharmaceuticals and pharmacological mechanisms.

Ochratoxin A (OTA), one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), UHPLC-Orbitrap-high resolution mass spectrometry (HRMS) and liquid chromatography-multiple stage mass spectrometry (LC-MSn) were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with β-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.

Background/Aims: The aim of this study was to investigate the drug-drug interaction between carvedilol and citalopram based on carvedilol metabolism in vitro and his pharmacokinetics (PKs) in vivo after the oral administration of the single drug and both drugs, and reveal citalopram effects on the PKs of carvedilol. Methods: Each rat was cannulated on the femoral vein, prior to being connected to BASi Culex ABC ® . Carvedilol was orally administrated in rats (3.57 mg/ kg body weight [b.w.]) in the absence of citalopram or after a pre-treatment with multiple oral doses of citalopram (1.42 mg/kg b.w.). Plasma concentrations of carvedilol were determined using high-performance liquid chromatography-MS at the designated time points after drug administration, and the main PK parameters were calculated by noncompartmental analysis. In addition, effects of citalopram on the metabolic rate of carvedilol were investigated using ratpooled liver microsome incubation systems. Results: During co-administration, significant increases of the area under the plasma concentration-time curve as well as of the peak plasma concentration were observed. The rat-pooled liver microsome incubation experiment indicated that citalopram could decrease the metabolic rate of carvedilol. Conclusion: Citalopram co-administration led to a significant alteration of carvedilol's PK profile in rats; it also demonstrated, in vitro, these effects could be explained by the existence of a drugdrug interaction mediated by CYP2D6 inhibition. [ABSTRACT FROM AUTHOR]

1. The aim of this study was to identify the hepatic metabolic enzymes, which involved in the biotransformation of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) in rat and humanin vitro. 2. The parent drug of W-1 was incubated with rat liver microsomes (RLMs) or recombinant CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, respectively) in the presence or absence of nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system. The metabolites of W-1 were analyzed with liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). 3. The parent drug of W-1 was metabolized in a NADPH-dependent manner in RLMs. The kinetic parameters of prototype W-1 including Km, Vmax, and CLintwere 2.3 μM, 3.3 nmol/min/mg protein, and 1.4 mL/min/mg protein, respectively. Two metabolites M1 and M2 were observed in shorter retention times (2.988 and 3.188 min) with a higher molecular ion at m/z 463.0160 (both M1 and M2) than that of the W-1 parent drug (6.158 min with m/z 447.0218). The CYP selective inhibition and recombinant enzymes also showed that two hydroxyl metabolites M1 and M2 are mainly mediated by CYP2C19 and CYP3A4. 4. The identification of CYPs involved in W-1 biotransformation is important to understand and minimize, if possible, the potential of drug–drug interactions. [ABSTRACT FROM PUBLISHER]

Dextran sulfate sodium (DSS) induced experimental colitis presents a histologic resemblance to human ulcerative colitis (UC). Altered cytochrome P450s (CYPs) have been reported in this model and patients with UC. In this study, six CYPs activities were quantitatively determined in microsomes of liver (RLMs), kidney (RRMs) and intestine (RIMs) from rats with colitis at acute (5% DSS for 7 days, UCA) and remission (7-day DSS treatment followed by 7-day cessation, UCR) phases and compared with normal rats. Generally, CYPs activities varied with isoform, organ, and disease status. Hepatic CYP1A2, 2B1, 2C6/11, 2E1 and 3A1/2 activities were reduced by acute colitis and completely or partially restored after DSS was halted. Although DSS treatment decreased the V max of renal CYP2C6/11 and increased that of CYP2D2, their CL int, in vitro were comparable among normal, acute and remission stages. DSS treatment changed the kinetics of CYP3A1/2-mediated nifedipine metabolism in RRMs from biphasic to classical kinetics. Notably, CYP2D2 activity was elevated in liver and kidney in acute UC, while enhanced in liver and decreased in kidney in remission. In intestine, CYP3A1/2 activity was increased in UCA and further enhanced after DSS withdrawal. These findings highlight the necessity of quantifying enzyme activity for precision drug therapy. [ABSTRACT FROM AUTHOR]