Rat Liver Microsomal Metabolism Research Articles

The impacts of natural product miltirone and the CYP2D6 pharmacogenetic phenotype on fluoxetine metabolism.

Introduction: To study the effects of drug-induced CYP2D6 activity inhibition and genetic polymorphisms on fluoxetine metabolism, rat liver microsomes (RLMs) and SD rats were used to investigate the potential drug‒drug interactions (DDIs), and CYP2D6 http://muchong.com/t-10728934-1 recombinant baculosomes were prepared and subjected to catalytic reactivity studies. Methods and Results: All analytes were detected by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC‒MS/MS). After screening for 27 targeted natural products, miltirone was identified as having obvious inhibitory effect on fluoxetine metabolism in RLMs. In vivo, the concentration of fluoxetine in rat blood increased markedly after miltirone administration. The molecular docking results showed that miltirone bound more strongly to CYP2D6 than fluoxetine, and PHE120 may be the key residue leading to the inhibition of CYP2D6-mediated fluoxetine N-demethylation by miltirone. In terms of the genetic polymorphism of CYP2D6 on fluoxetine metabolism, the intrinsic clearance values of most variants were significantly altered. Among these variants, CYP2D6*92 and CYP2D6*96/Q424X were found to be catalytically inactive for fluoxetine metabolism, five variants (CYP2D6*89/L142S, *97/F457L, *R497, *V342M and *R344Q) exhibited markedly increased clearance values (>125.07%) and seven variants (CYP2D6*2, *10, *87/A5V, *93/T249P, *E215K, *R25Q and *R440C) exhibited significantly decreased clearance values (from 6.62% to 66.79%) compared to those of the wild-type. Conclusion: Our results suggest that more attention should be given to subjects in the clinic who take fluoxetine and also carry one of these infrequent CYP2D6 alleles or are coadministered drugs containing miltirone.

Comparative Analysis of the Gelsemium Alkaloids Metabolism in Human, Pig, Goat, and Rat Liver Microsomes.

The aim of this study was to investigate the metabolism of Gelsemium elegans in human, pig, goat and rat liver microsomes and to elucidate the metabolic pathways and cleavage patterns of the Gelsemium alkaloids among different species. A human, goat, pig and rat liver microparticles were incubated in vitro. After incubating at 37°C for 1 hour and centrifuging, the processed samples were detected by HPLC/Qq-TOFMS was used to detect alcohol extract of Gelsemium elegans and its metabolites. Forty-six natural products were characterized from alcohol extract of Gelsemium elegans and 13 metabolites were identified. These 13 metabolites belong to the gelsemine, koumine, gelsedine, humantenine, yohimbane, and sarpagine classes of alkaloids. The metabolic pathways included oxidation, demethylation and dehydrogenation. After preliminary identification, the metabolites detected in the four species were different. All 13 metabolites were detected in pig and rat microsomes, but no oxidative metabolites of Gelsedine-type alkaloids were detected in goat and human microsomes. In this study, Gelsemium elegans metabolic patterns in different species are clarified and the in vitro metabolism of Gelsemium elegans is investigated. It is of great significance for its clinical development and rational application.

Simultaneous Estimation of Quercetin and trans-Resveratrol in Cissus quadrangularis Extract in Rat Serum Using Validated LC-MS/MS Method: Application to Pharmacokinetic and Stability Studies.

Cissus quadrangularis is a nutrient-rich plant with a history of use in traditional medicine. It boasts a diverse range of polyphenols, including quercetin, resveratrol, β-sitosterol, myricetin, and other compounds. We developed and validated a sensitive LC-MS/MS method to quantify quercetin and t-res biomarkers in rat serum and applied this method to pharmacokinetic and stability studies. The mass spectrometer was set to negative ionization mode for the quantification of quercetin and t-res. Phenomenex Luna (C18(2), 100 A, 75 × 4.6 mm, 3 µ) column was utilized to separate the analytes using an isocratic mobile phase consisting of methanol and 0.1% formic acid in water (82:18). Validation of the method was performed using various parameters, including linearity, specificity, accuracy, stability, intra-day, inter-day precision, and the matrix effect. There was no observed significant endogenous interference from the blank serum. The analysis was completed within 5.0 min for each run, and the lower limit of quantification was 5 ng/mL. The calibration curves showed a linear range with a high correlation coefficient (r2 > 0.99). The precision for intra- and inter-day assays showed relative standard deviations from 3.32% to 8.86% and 4.35% to 9.61%, respectively. The analytes in rat serum were stable during bench-top, freeze-thaw, and autosampler (-4 °C) stability studies. After oral administration, the analytes showed rapid absorption but underwent metabolism in rat liver microsomes despite being stable in simulated gastric and intestinal fluids. Intragastric administration resulted in higher absorption of quercetin and t-res, with greater Cmax, shorter half-life, and improved elimination. No prior research has been conducted on the oral pharmacokinetics and stability of anti-diabetic compounds in the Ethanolic extract of Cissus quadrangularis EECQ, making this the first report. Our findings can provide the knowledge of EECQ's bioanalysis and pharmacokinetic properties which is useful for future clinical trials.

Effects of dietary flavonoids on the metabolism of vortioxetine and its potential mechanism.

Quercetin and apigenin are two common dietary flavonoids widely found in foods and fruits. Quercetin and apigenin can act as the inhibitors of CYP450 enzymes, which may affect the pharmacokinetics of clinical drugs. Vortioxetine (VOR), approved for marketing by the Food and Drug Administration (FDA) in 2013, is a novel clinical drug for treating major depressive disorder (MDD). This study aimed to evaluate the effects of quercetin and apigenin on the metabolism of VOR in in vivo and in vitro experiments. Firstly, 18 Sprague-Dawley rats were randomly divided into three groups: control group (VOR), group A (VOR + 30 mg/kg quercetin) and group B (VOR + 20 mg/kg apigenin). We collected the blood samples at different time points before and after the final oral administration of 2 mg/kg VOR. Subsequently, we further used rat liver microsomes (RLMs) to investigate the half-maximal inhibitory concentration (IC50) of the metabolism of vortioxetine. Finally, we evaluated the inhibitory mechanism of two dietary flavonoids on VOR metabolism in RLMs. In animal experiments, we found AUC (0-∞) (area under the curve from 0 to infinity) and CLz/F (clearance) to be obviously changed. Compared to controls, AUC (0-∞) of VOR in group A and group B was 2.22 and 3.54 times higher, respectively, while CLz/F of VOR in group A and group B was significantly decreased down to nearly two-fifth and one-third. In in vitro studies, the IC50 value of quercetin and apigenin in the metabolic rate of vortioxetine was 5.322 μM and 3.319 μM, respectively. Ki value of quercetin and apigenin was found to be 0.279 and 2.741, respectively, and the αKi value of quercetin and apigenin was 0.066 and 3.051 μM, respectively. Quercetin and apigenin exhibited inhibitory effects on the metabolism of vortioxetine in vivo and in vitro. Moreover, quercetin and apigenin non-competitively inhibited the metabolism of VOR in RLMs. Thus, we should pay more attention to the combination between these dietary flavonoids and VOR in the future clinical use.

Evaluation of metabolism and cytochrome P450 mediated interaction liabilities of naringenin

Naringenin is one of the major components of grapefruit juice. It has a broad spectrum of pharmacological activities, and many studies report that grapefruit juice inhibits cytochrome P450 (CYP) 3A4 leading to drug interactions. Naringenin was profiled through various in vitro studies like metabolic stability and glucuronidation in rat and human liver microsomes while, CYP inhibition using human liver microsomes. In addition, pharmacokinetic profiling was conducted upon intravenous (i.v.) and oral administration in rats. Naringenin undergoes both phase I and phase II metabolism in rat liver microsomes, and in human liver microsomes, it is predominantly metabolized by phase II. Glucuronidation which is addition (conjugation) of glucuronic acid to various functional groups is one of the major metabolic pathways of Naringenin. Naringenin, at 1.0 μM and 10.0 μM, did not elicit any appreciable inhibition of the 5 major CYP isoforms (CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6). Oral pharmacokinetic studies at 100, 300,and 1000 mg/kg dose and intravenous pharmacokinetic studies at 1 mg/kg dose were performed in male SD rats. Naringenin exhibited very short half-life (0.27 h) and rapid elimination (Clearance=110.65 mL/min/kg) after i.v. administration. There was saturation in Cmax and exposure beyond 100 mg/kg, and the absolute bioavailability was found to be ≤ 5% at the tested oral doses. This present experiment suggests that naringenin does not substantially inhibit CYP3A4 (or any of the tested five isoforms) isoforms per se. Given the minimal involvement of CYP enzymes in the metabolism of naringenin and minimal inhibition of CYP enzymes (IC50> 10 μM), the potential for drug-drug interactions involving CYP substrates and inhibitors is very minimal in humans.

Formation of DHP-DNA Adducts from Rat Liver Microsomal Metabolism of 1,2-Unsaturated Pyrrolizidine Alkaloid-Containing Plant Extracts and Dietary Supplements.

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals. We previously determined that carcinogenic PAs and PA N-oxides commonly form a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts, namely, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4. This set of DHP-DNA adducts has been implicated as a potential biomarker of PA-induced liver tumor initiation from metabolism of individual carcinogenic PAs. To date, it is not known whether this generality occurs from metabolism of PA-containing plant extracts. In this study, we investigate the rat liver microsomal metabolism of nine PA-containing plant extracts and two PA-containing dietary supplements in the presence of calf thymus DNA. The presence of carcinogenic PAs and PA N-oxides in plant extracts was first confirmed by LC-MS/MS analysis with selected reaction monitoring mode. Upon rat liver microsomal metabolism of these PA-containing plant extracts and dietary supplements, the formation of this set of DHP-DNA adducts was confirmed. Thus, these results indicate that metabolism of PA-containing plant extracts and dietary supplements can generate DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts, thereby potentially initiating liver tumor formation.

Effects of Water Miscible Organic Co-solvents on Enzyme Kinetic Parameters of Drug Metabolizing Enzymes in Rat Liver Microsomes

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 Km for 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.

Physiologically based pharmacokinetic modelling and simulation to predict the plasma concentration profile of schaftoside after oral administration of total flavonoids of Desmodium styracifolium.

Introduction: The total flavonoids of Desmodium styracifolium (TFDS) are the flavonoid extracts purified from Desmodii Styracifolii Herba. The capsule of TFDS was approved for the treatment of urolithiasis by NMPA in 2022. Schaftoside is the representative compound of TFDS that possesses antilithic and antioxidant effects. The aim of this study was to develop a physiologically based pharmacokinetic (PBPK) model of schaftoside to simulate its plasma concentration profile in rat and human after oral administration of the total flavonoids of Desmodium styracifolium. Methods: The physiologically based pharmacokinetic model of schaftoside was firstly developed and verified by the pharmacokinetic data in rats following intravenous injection and oral administration of the total flavonoids of Desmodium styracifolium. Then the PBPK model was extrapolated to human with PK-Sim® software. In order to assess the accuracy of the extrapolation, a preliminary multiple-dose clinical study was performed in four healthy volunteers aged 18-45years old. The predictive performance of PBPK model was mainly evaluated by visual predictive checks and fold error of Cmax and AUC0-t of schaftoside (the ratio of predicted to observed). Finally, the adult PBPK model was scaled to several subpopulations including elderly and renally impaired patients. Results: Schaftoside underwent poor metabolism in rat and human liver microsomes in vitro, and in vivo it was extensively excreted into urine and bile as an unchanged form. By utilizing literature and experimental data, the PBPK model of schaftoside was well established in rat and human. The predicted plasma concentration profiles of schaftoside were consistent with the corresponding observed data, and the fold error values were within the 2-fold acceptance criterion. No significant pharmacokinetic differences were observed after extrapolation from adult (18-40 years old) to elderly populations (71-80years) in PK-Sim®. However, the plasma concentration of schaftoside was predicted to be much higher in renally impaired patients. The maximum steady-state plasma concentrations in patients with chronic kidney disease stage 3, 4 and 5 were 3.41, 12.32 and 23.77 times higher, respectively, than those in healthy people. Conclusion: The established PBPK model of schaftoside provided useful insight for dose selection of the total flavonoids of Desmodium styracifolium in different populations. This study provided a feasible way for the assessment of efficacy and safety of herbal medicines.

Evaluation of commonly used cardiovascular drugs in inhibiting vonoprazan metabolism in vitro and in vivo.

As a novel acid-suppressing drug, vonoprazan shows the potential to replace traditional proton-pump inhibitors. With its widespread use, some adverse effects that require further study have emerged due to drug–drug interactions. Our study is the first experiment that evaluated the drug–drug interactions of eleven common cardiovascular drugs that inhibit vonoprazan metabolism in vitro and in vivo. Rat liver microsome incubation and molecular simulation docking were applied to explore the inhibition mechanism. Amlodipine and nifedipine showed inhibitory effects on vonoprazan metabolism in both rat and human liver microsomes in the first evaluation part in vitro. The inhibition mechanism analysis results demonstrated that amlodipine and nifedipine might inhibit the metabolism of vonoprazan by a mixed type of competitive and non-competitive inhibition. However, the pharmacokinetic data of the vonoprazan prototype revealed that amlodipine affected vonoprazan in vivo while nifedipine did not. Thus, more attention should be paid when amlodipine is prescribed with vonoprazan. Furthermore, the changes in its carboxylic acid metabolites MI hinted at a complex situation. Molecular simulation suggested the CYP2B6 enzyme may contribute more to this than CYP3A4, and further inhibitory experiments preliminarily verified this speculation. In conclusion, the use of vonoprazan with cardiovascular drugs, especially amlodipine, should receive particular attention in clinical prescriptions.

An HPLC-UV validated bioanalytical method for measurement of in vitro phase 1 kinetics of α-synuclein binding bifunctional compounds

Aggregates of the protein α-synuclein are associated with pathophysiology of Parkinson’s disease and are present in Lewy Bodies found in the brains of Parkinson’s patients. We previously demonstrated that bifunctional compounds composed of caffeine linked via a six carbon chain to either 1-aminoindan (C8-6-I) or nicotine (C8-6-N) bind α-synuclein and protect yeast cells from α-synuclein mediated toxicity. A critical step in development of positron emission tomography (PET) probes for neurodegenerative diseases is evaluation of their metabolic stability. We determined that C8-6-I, and C8-6-N both undergo phase 1 P450 metabolism in mouse, rat, and human liver microsomes. We utilised this metabolic information to guide the design of fluorinated analogues for use as PET probes and determined that the fluorine in 19F-C8-6-I and 19F-C8-6-N is stable to P450 enzymes. We have developed and validated an analytical HPLC-UV method following FDA and EMA guidelines to measure in vitro phase 1 kinetics of these compounds and determine their Vmax, KM and CLint,u in mouse liver microsomes. We found that C8-6-I and 19F-C8-6-I have a two- to fourfold lower CLint,u than C8-6-N, and 19F-C8-6-N. Our approach shows a simple, specific, and effective system to design and develop compounds as PET probes.

Evaluation of Herb-Drug Interaction Between Danshen and Rivaroxaban in Rat and Human Liver Microsomes.

The combination of Salvia miltiorrhiza (Danshen) and rivaroxaban is a promising treatment option in clinical practice in China, but the herb–drug interaction between Danshen and rivaroxaban remains unclear. Therefore, this study aims to reveal the interaction between Danshen and rivaroxaban. We not only investigated the inhibitory properties of Danshen tablet on rivaroxaban metabolism in rat and human liver microsomes but also evaluated the inhibitory effects of Danshen tablet and its eight active components (dihydrotanshinone I, tanshinone I, tanshinone IIA, cryptotanshinone, danshensu, salvianolic acid A, salvianolic acid B, and salvianolic acid C) on cytochrome P450 (CYP) enzymes. The results showed that Danshen tablet potently inhibited the metabolism of rivaroxaban in rat and human liver microsomes. In the CYP inhibition study, we found that dihydrotanshinone I, the active component of Danshen tablet, potently inhibited the activities of rat CYP3A and CYP2J, with IC50 values at 13.85 and 6.39 μM, respectively. In further inhibition kinetic study, we found that Danshen tablet is a mixed inhibitor in rivaroxaban metabolism in rat and human liver microsomes, with the K i value at 0.72 and 0.25 mg/ml, respectively. In conclusion, there is a potential interaction between Danshen tablet and rivaroxaban. Danshen tablet inhibits the metabolism of rivaroxaban, which may be because its lipid-soluble components such as dihydrotanshinone I strongly inhibit the activities of CYP enzymes, especially CYP3A and CYP2J. Therefore, when Danshen tablet and rivaroxaban are used simultaneously in the clinic, it is necessary to strengthen the drug monitoring of rivaroxaban and adjust the dosage.

Inhibitory effects of traditional Chinese medicine colquhounia root tablet on the pharmacokinetics of tacrolimus in rats

Ethnopharmacological relevanceTacrolimus (TAC) was widely used in various renal diseases while high recurrence rate and high expense restricted its applications. Traditional herbal medicine has become increasingly popular as an adjuvant therapy to minimize the adverse effects of TAC. Colquhounia root tablet (CRT), a prescribed drug prepared from the water extract of the peeled root of Tripterygium hypoglaucum (H. Lév.) Hutch., showed excellent anti-inflammatory, analgesic and immunosuppressive pharmacological properties. TAC used in combination with CRT was substantially more efficacious and safer than the monotherapy for the treatment of nephrotic syndrome. However, studies on their herb-drug interaction were scanty. Aim of the studyThe study was proposed to examine the effect of CRT on the pharmacokinetics of TAC in rats and identify the key natural constituents in CRT that affected the metabolism of TAC. Materials and methodsTAC was orally and intravenously administered to rats alone or in combination with CRT and the pharmacokinetic parameters of TAC were compared. After pretreatment with CRT for 15 d, the expressions of the drug-metabolizing enzymes (DMEs), drug transporters (DTs) and nuclear receptors (NRs) were determined by polymerase chain reaction and western blotting and compared with the control group. The hepatic microsomal incubation system was employed to confirm the inhibitory effects of CRT and its major components on rat cytochrome P450 (CYP) 3A2. The roles of the primary components in the regulation of human CYP3A4 and mouse P-gp activities were evaluated by using docking analysis. ResultsThe blood concentrations of TAC were significantly increased in a dose- and pretreatment time-dependent manner after combined administration of CRT. The maximal effect was found at 300 mg/kg (43.70 ± 8.77 ng/mL and 141.45 ± 21.58 h·ng/mL) in a single dose run and the pharmacokinetic parameters gradually returned to the normal levels at 24 h interval of long-term CRT pretreatment. In contrast, CRT had no effect on the pharmacokinetics of intravenous TAC. Further study indicated that the mRNA and protein expressions of DMEs and DTs, such as CYP3A1, CYP3A2, P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 in rat intestine and liver were down-regulated, whereas the expressions of NRs like constitutive androstane receptor and pregnane X receptor were up-regulated after multiple oral doses of CRT. Molecular docking showed the binding potency of five CRT major constituents with both human CYP3A4 and mouse P-gp. Celastrol, wilforgine and wilforine were the strongest inhibitors towards midazolam metabolism in rat liver microsomes, with the 50% inhibition concentrations being at 8.33 μM, 22.18 μM and 22.22 μM, respectively. ConclusionsOur results revealed that co-dosing of CRT could lead to a significant increase in blood concentration of TAC and this effect could be ascribed to the resultant co-regulation of DMEs, DTs and NRs. Our study provided an experimental basis for the combination use of CRT and TAC in clinical practice.

Potential Effects of Ibuprofen, Remdesivir and Omeprazole on Dexamethasone Metabolism in Control Sprague Dawley Male Rat Liver Microsomes (Drugs Often Used Together Alongside COVID-19 Treatment).

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.

Stereochemistry of chiral pesticide uniconazole and enantioselective metabolism in rat liver microsomes

In this work, stereochemistry of uniconazole enantiomers and their metabolism behaviors in rat liver microsomes have been researched. Significance analysis has been applied in data processing. Absolute configurations of uniconazole enantiomers were identified through vibrational circular dichroism spectroscopy. According to their elution order from the chiral column using the CO2-methanol (80:20, v/v) mixture, two eluted fractions were determined to be (R)-uniconazole and (S)-uniconazole, respectively. A high-efficient and sensitive LC-MS/MS chiral analysis method was established for investigating the metabolism of uniconazole enantiomers in rat liver microsomes. The metabolic half-life of (R)-uniconazole (38.7 min) in rat liver microsomes was half that of (S)-enantiomer (74.5 min), and maximum velocity of metabolism, Michaelis constant of metabolism as well as the intrinsic metabolic clearance of (R)-uniconazole were significantly higher than (S)-enantiomer (p < 0.05), which indicated that (R)-uniconazole was preferentially metabolized in rat liver microsomes. By the virtue of molecular docking, (R)-uniconazole exhibited a higher binding affinity to cytochrome CYP2D2 than (S)-enantiomer, which corroborated well with the metabolism results. This work will shed light on the risk assessment of uniconazole toward human health and the ecological environment.

Inhibitory effect of propafenone on vortioxetine metabolism in vitro and in vivo

Vortioxetine, as a new therapeutic drug of major depressive disorder (MDD), was approved for MDD in the USA. The significance of our study in this paper was to explore the inhibitory effect of propafenone on the metabolism of vortioxetine through the in vivo and in vitro experiments. In the vitro experiments, we added a series of concentrations of propafenone into an incubation system as the inhibitors and calculated the half-maximal inhibitory concentration (IC50) of propafenone on vortioxetine metabolism in human liver microsomes (HLMs) and rat liver microsomes (RLMs). Twelve male Sprague-Dawley (SD) rats were included in the vivo experiments. We randomly divided them into Group A (control group) and Group B (90 mg/kg propafenone). 30 min later, a single oral dose of 4.0 mg/kg vortioxetine was administrated to each rat. Then, we used ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to determine the concentrations of vortioxetine and its metabolite Lu AA34443. From our results, it indicated that propafenone inhibited the metabolic rate of vortioxetine in the vitro studies with the IC50 of 0.48 μM and 16.5 μM for HLMs and RLMs, respectively. And, propafenone could competitively inhibit vortioxetine in both HLMs and RLMs for the inhibitory mechanisms. Moreover, a single oral dose of 90 mg/kg propafenone obviously enchanced the exposure of vortioxetine in rats, but not Lu AA34443. Combined with the vitro and vivo data, propafenone showed the inhibitory effect on vortioxetine metabolism. Thus, more attention to the safety of vortioxetine in clinic should be paid when taking it with propafenone in combination for the therapy.

Exploring 1-adamantanamine as an alternative amine moiety for metabolically labile azepane ring in newly synthesized benzo[d]thiazol-2(3H)one σ receptor ligands.

In this work we report the structure-activity relationships, binding properties, and metabolic stability studies of a series of benzo[d]thiazol-2(3H)one as sigma receptors (σRs) ligands. Specifically, to improve the metabolic stability of the cyclic amine fragment of our lead compound (SN56), the metabolically unstable azepane ring was replaced with a 1-adatamantamine moiety. Within the synthesized analogs, compound 12 had low nanomolar affinity for the σ1R (K i = 7.2 nM) and moderate preference (61-fold) over the σ2R. In vitro metabolic stability studies showed a slight improvement of the metabolic stability for 7-12, even though an extensive metabolism in rat liver microsomes is being observed. Furthermore, metabolic soft spot identification of 12 suggested that the N-methyl group of the adamantyl moiety is a major site of metabolism.

Stereoselective environmental behavior and biological effects of the chiral bitertanol

Bitertanol is a widely used chiral triazole fungicide. The stereoselective environmental behavior and biological effects of bitertanol are not clear. The present study evaluated the stereoselectivity of bitertanol, including its degradation in five typical soils (under laboratory controlled aerobic, anaerobic and sterilization conditions), metabolism in rat liver microsomes (RLM; in vitro), and the endocrine disruption effects on the estrogen receptor (ER) and thyroid hormone receptor (TR) using reporter gene assays. The results indicated that (1S,2R)-bitertanol and (1R,2S)-bitertanol had faster degradation rates in soil than the other stereoisomers. The half-lives of four bitertanol stereoisomers ranged from 9.1 d to 86.6 d in different soils under different conditions. (1S,2R)-bitertanol was preferentially metabolized in RLM. The molecular docking results confirmed the in vitro experiments that (1S,2R)-bitertanol had shortest binding distances and lowest energies with cytochrome P450 enzymes (CYPs). Four bitertanol stereoisomers showed stereoselective antagonistic effects on ER. Additionally, (1S,2R)-bitertanol and (1R,2S)-bitertanol exhibited antagonistic effects on TR. These results suggest that the use of pure (1S,2R)-bitertanol instead of the commercial stereoisomer mix, may help reduce environmental pollution and biological toxicity.

Role of Cell Death in Toxicology: Does It Matter How Cells Die?

My research activity started with studies on drug metabolism in rat liver microsomes in the early 1960s. The CO-binding pigment (cytochrome P450) had been discovered a few years earlier and was subsequently found to be involved in steroid hydroxylation in adrenal cortex microsomes. Our early studies suggested that it also participated in the oxidative demethylation of drugs catalyzed by liver microsomes, and that prior treatment of the animals with phenobarbital caused increased levels of the hemoprotein in the liver, and similarly enhanced rates of drug metabolism. Subsequent studies of cytochrome P450-mediated metabolism of toxic drugs in freshly isolated rat hepatocytes characterized critical cellular defense systems and identified mechanisms by which accumulating toxic metabolites could damage and kill the cells. These studies revealed that multiple types of cell death could result from the toxic injury, and that it is important to know which type of cell death results from the toxic injury.

The Cytochrome P450-Mediated Metabolism Alternation of Four Effective Lignans From Schisandra chinensis in Carbon Tetrachloride-Intoxicated Rats and Patients With Advanced Hepatocellular Carcinoma.

It is highly valuable to study the pharmacokinetics of herbal components under the pathological condition of liver dysfunction for safe and rational use of herbal medicines. In this study, the pharmacokinetic profiles of four effective lignans from Schisandra chinensis (SC), schisandrin, schisantherin A, deoxyshisandrin and γ-schisandrin, were investigated in carbon tetrachloride (CCl4)-intoxicated rats. The metabolism of the four lignans was also studied using microsomes from patients with advanced hepatocellular carcinoma. In situ intestinal and hepatic perfusions were conducted to clarify the contributions from impairments of gut and liver on the pharmacokinetics of the four schisandra lignans in CCl4-intoxicated rats. The metabolism in rat and human liver microsomes and transport in Caco-2 monolayer cell model were studied to reveal the key factors for the in vivo disposition of the four lignans. When SC alcoholic extract was orally administrated to CCl4-intoxicated rat for a short term (4 days), the pharmacokinetics of four active SC lignans was significantly changed while its hepatotherapeutic effect was not obviously observed. The plasma concentrations of the four schisandra lignans were dramatically elevated compared with the control. The Cmax, AUC and MRT were all increased or prolonged significantly while parameter CLz/F was obviously reduced in rat pretreated with CCl4. In hepatic perfusion study and liver microsomes incubation, it was found that the hepatic metabolism of the four lignans was markedly decreased mainly due to the activity reduction of multiple CYP450 isoenzymes involved the metabolism, which, eventually, might lead to the alternation of their pharmacokinetic profiles in CCl4-intoxicated rats or patients with advanced hepatocellular carcinoma. The pharmacokinetic studies of SC components in pathological situation of liver dysfunction are expected to provide useful data for rational and safe application of SC preparations in clinic or further pharmacological and toxicological research.

Preclinical evaluation of saroglitazar magnesium, a dual PPAR-α/γ agonist for treatment of dyslipidemia and metabolic disorders

1. Saroglitazar, a novel peroxisome proliferator-activated receptor (PPAR) agonist, regulates lipid and glucose metabolism. The objective of this report is to provide a preclinical evaluation (in vitro/in vivo) of ADME properties of saroglitazar. In vitro studies included determination of permeability, metabolic stability, plasma protein binding, CYP reaction phenotyping and CYP inhibitory liability. In vivo studies included oral bioavailability and pharmacokinetic assessment in mouse, rat and dog. The excretion of saroglitazar was determined in rats. Exploratory metabolism of saroglitazar was evaluated using in vitro and in vivo samples.2. Saroglitazar was metabolically more stable in human liver microsomes as compared to rat and dog liver microsomes, highly protein bound (98–99.6%) with high Caco2 permeability (104 nm/s) with <2 efflux ratio. In vitro metabolism in rat, dog and human liver microsomes revealed three putative metabolites corresponding to di-hydroxylation, mono-oxygenation and dehydrogenation moieties.3. Oral bioavailability was 100%, 72% and 47% in mouse, rat and dog, respectively. The intravenous clearance and volume of distribution of saroglitazar were 3.6, 8.5 and 6.9 mL/min/kg and 1.3, 4.8 and 1.8 L/kg for mouse, rat and dog, respectively. The elimination half-life of saroglitazar ranged between 6 and 15 h. Saroglitazar appeared to be eliminated via hepatobiliary route with negligible renal excretion.