Liver Microsomal System Research Articles

Characterization of CYP3A5 Selective Inhibitors for Reaction Phenotyping of Drug Candidates.

CYP3A is one of the most important classes of enzymes and is involved in the metabolism of over 70% drugs. While several selective CYP3A4 inhibitors have been identified, the search for a selective CYP3A5 inhibitor has turned out to be rather challenging. Recently, several selective CYP3A5 inhibitors have been identified through high-throughput screening of ~ 11,000 compounds and hit expansion using human recombinant enzymes. We set forth to characterize the three most selective CYP3A5 inhibitors in a more physiologically relevant system of human liver microsomes to understand if these inhibitors can be used for reaction phenotyping studies in drug discovery settings. Gomisin A and T-5 were used as selective substrate reactions for CYP3A4 and CYP3A5 to determine IC50 values of the two enzymes. The results showed that clobetasol propionate and loteprednol etabonate were potent and selective CYP3A5 reversible inhibitors with selectivity of 24-fold against CYP3A4 and 39-fold or more against the other major CYPs. The selectivity of difluprednate in HLM is much weaker than that in the recombinant enzymes due to hydrolysis of the acetate group in HLM. Based on the selectivity data, loteprednol etabonate can be utilized as an orthogonal approach, when experimental fraction metabolized of CYP3A5 is greater than 0.5, to understand CYP3A5 contribution to drug metabolism and its clinical significance. Future endeavors to identify even more selective CYP3A5 inhibitors are warranted to enable accurate determination of CYP3A5 contribution to metabolism versus CYP3A4.

Reducing Hepatotoxicity Mechanism of Radix Wikstroemia Indica by Processing with “Sweat Soaking Method” Using UPLC-MS/MS and a Cocktail Probe Substrate

Background: Radix Wikstroemia indica is a traditional Chinese medicine (TCM) used as anti-inflammatory and anti-tumor drug. However, it has serious hepatotoxicity, "Sweat soaking method" processed could effectively decrease its hepatotoxicity. Objective: The objective of this study is to study the effects of Radix Wikstroemia indica on six kinds of cytochrome P450(CYP450) isozymes of rat liver microsomes before and after processing, and to study the mechanism of Radix Wikstroemia indica processed by the "Sweat soaking method" to reduce liver toxicity in rats. Methods: In this study, the effects of Radix Wikstroemia indica and processed Radix Wikstroemia indica on the six main CYP450 isoforms (2E1, 1A2, 2C6, 2D1, 2C11, and 3A1) were investigated in vitro. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we carried out in vitro enzymatic studies in liver microsomal incubation systems via UPLC-MS/MS. Results: The results showed that the established UPLC-MS/MS method was precise and reliable. Compared with the blank group, the activities of six enzymes in the RWI and PRWI groups were higher than those in the blank group. At the same dose, the enzyme activities of CYP2E1, CYP1A2, CYP2C6, CYP2C11, and CYP3A1 increased with the increase in dose, and the enzyme activities of the RWI group were higher than those of the PRWI group. The enzyme activities of CYP2E1 and CYP1A2 in the Radix Wikstroemia indica group were significantly increased compared with the blank group, CYP3A1 in the RWI high-dose group was higher than that in the blank group and PRWI group with statistical differences (p<0.05 or p<0.01). Conclusion: The processed Radix Wikstroemia indica could reduce liver injury, and its detoxication mechanism might be related to the decrease in enzyme activity of CYP1A2, CYP2E1 and CYP3A1.

Identification of the Hepatic Metabolites of Flumazenil and their Kinetic Application in Neuroimaging.

Studies of the neurobiological causes of anxiety disorders have suggested that the γ-aminobutyric acid (GABA) system increases synaptic concentrations and enhances the affinity of GABAA (type A) receptors for benzodiazepine ligands. Flumazenil antagonizes the benzodiazepine-binding site of the GABA/benzodiazepine receptor (BZR) complex in the central nervous system (CNS). The investigation of flumazenil metabolites using liquid chromatography (LC)-tandem mass spectrometry will provide a complete understanding of the in vivo metabolism of flumazenil and accelerate radiopharmaceutical inspection and registration. The main goal of this study was to investigate the use of reversed-phase high performance liquid chromatography (PR-HPLC), coupled with electrospray ionization triple-quadrupole tandem mass spectrometry (ESI-QqQ MS), to identify flumazenil and its metabolites in the hepatic matrix. Carrier-free nucleophilic fluorination with an automatic synthesizer for [18F]flumazenil, combined with nano-positron emission tomography (NanoPET)/computed tomography (CT) imaging, was used to predict the biodistribution in normal rats. The study showed that 50% of the flumazenil was biotransformed by the rat liver homogenate in 60 min, whereas one metabolite (M1) was a methyl transesterification product of flumazenil. In the rat liver microsomal system, two metabolites were identified (M2 and M3), as their carboxylic acid and hydroxylated ethyl ester forms between 10 and 120 min, respectively. A total of 10-30 min post-injection of [18F]flumazenil showed an immediate decreased in the distribution ratio observed in the plasma. Nevertheless, a higher ratio of the complete [18F]flumazenil compound could be used for subsequent animal studies. [18F] According to in vivo nanoPET/CT imaging and ex vivo biodistribution assays, flumazenil also showed significant effects on GABAA receptor availability in the amygdala, prefrontal cortex, cortex, and hippocampus in the rat brain, indicating the formation of metabolites. We reported the completion of the biotransformation of flumazenil by the hepatic system, as well as [18F]flumazenil's potential as an ideal ligand and PET agent for the determination of the GABAA/BZR complex for multiplex neurological syndromes at the clinical stage.

LC-Q-TOF/MS-based Fragmentation Behavior Study and In vitro Metabolites Identification of Nine Benzodiazepines.

Benzodiazepines (BZDs) are compounds that contain one diazepine ring and two benzene rings, and are widely used to treat central nervous system diseases. However, drug abuse and BZDs' illegal addition may affect normal life and even lead to grave social harm. As BZDs may be metabolized and eliminated quickly, it is of great theoretical and practical significance to clarify their metabolic profile. In this paper, LC-Q-TOF/MS-based fragmentation behavior has been investigated for nine benzodiazepine drugs available and widely used in clinical treatment (diazepam, nitrazepam, clonazepam, oxazepam, lorazepam, alprazolam, estazolam, triazolam, and midazolam), and their metabolic profile has been studied by using in vitro human liver microsomal incubation. A regular human liver microsomal system was used to investigate the potential biotransformation of the nine benzodiazepines in vitro, and an LC-Q/TOF-MS was used to perform fragmentation behavior studies and metabolite identification. As a result, characteristic fragmentation pathway and diagnostic fragment ions of the nine BZDs were analyzed, and 19 metabolites of the 9 benzodiazepines were found and identified, with glucuronidation and hydroxylation considered as their most important metabolic pathways. These experimental data add to our knowledge of the nine benzodiazepine drugs and their metabolism study, which could provide useful information and evidence of their in vivo metabolic profile prediction and help promote their monitoring in both clinical use and social/illegal abuse.

Biotransformation of Penindolone, an Influenza A Virus Inhibitor.

Penindolone (PND) is a novel broad-spectrum anti-Influenza A Virus (IAV) agent blocking hemagglutinin-mediated adsorption and membrane fusion. The goal of this work was to reveal the metabolic route of PND in rats. Ultra-high-performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS) was used for metabolite identification in rat bile, feces and urine after administration of PND. A total of 25 metabolites, including 9 phase I metabolites and 16 phase II metabolites, were characterized. The metabolic pathways were proposed, and metabolites were visualized via Global Natural Product Social Molecular Networking (GNPS). It was found that 65.24-80.44% of the PND presented in the formation of glucuronide conjugate products in bile, and more than 51% of prototype was excreted through feces. In in vitro metabolism of PND by rat, mouse and human liver microsomes (LMs) system, PND was discovered to be eliminated in LMs to different extents with significant species differences. The effects of chemical inhibitors of isozymes on the metabolism of PND in vitro indicated that CYP2E1/2C9/3A4 and UGT1A1/1A6/1A9 were the metabolic enzymes responsible for PND metabolism. PND metabolism in vivo could be blocked by UGTs inhibitor (ibrutinib) to a certain extent. These findings provided a basis for further research and development of PND.

Effect of polyethylene glycol 400 on the pharmacokinetics and tissue distribution of baicalin by intravenous injection based on the enzyme activity of UGT1A8/1A9

Baicalin (BG) is a bioactive flavonoid extracted from the dried root of the medicinal plant, Scutellaria radix (SR) (dicotyledonous family, Labiatae), and has several biological activities. Polyethylene glycol 400 (PEG400) has been used as a suitable solvent for several traditional Chinese medicines (TCM) and is often used as an excipient for the compound preparation of SR. However, the drug–excipient interactions between BG and PEG400 are still unknown. Herein, we evaluated the effect of a single intravenous PEG400 administration on the BG levels of rats using pharmacokinetic and tissue distribution studies. A liver microsome and recombinant enzyme incubation system were used to further confirm the interaction mechanism between PEG400 and UDP-glucuronosyltransferases (UGTs) (UGT1A8 and UGT1A9). The pharmacokinetic study demonstrated that following the co-intravenous administration of PEG400 and BG, the total clearance (CLz) of BG in the rat plasma decreased by 101.60% (p < 0.05), whereas the area under the plasma concentration-time curve (AUC)0-t and AUC0-inf increased by 144.59% (p < 0.05) and 140.05% (p < 0.05), respectively. Additionally, the tissue distribution study showed that the concentration of BG and baicalein-6-O-β-D-glucuronide (B6G) in the tissues increased, whereas baicalein (B) in the tissues decreased, and the total amount of BG and its metabolites in tissues altered following the intravenous administration of PEG400. We further found that PEG400 induced the UGT1A8 and UGT1A9 enzyme activities by affecting the maximum enzymatic velocity (Vmax) and Michaelis–Menten constant (Km) values of UGT1A8 and UGT1A9. In conclusion, our results demonstrated that PEG400 interaction with UGTs altered the pharmacokinetic behaviors and tissue distribution characteristics of BG and its metabolites in rats.

Novel biomimetic nanocomposite for investigation of drug metabolism

In vitro mimicking of hepatic drug metabolism is a key issue in early-stage drug discovery. Synthetic metalloporphyrins show structural similarity with the heme type prosthetic group of cytochrome P450 as primary hepatic enzyme in oxidative drug biotransformation. Therefore, they can catalyze these oxidations. Concerning economical aspects and the poor stability of metalloporphyrin, their immobilization onto or into solid carriers can be promising solution. This study presents a novel immobilized metalloporphyrin nanocomposite system and its potential use as biomimetic catalysts. The developed two-step immobilization procedure consists of two main steps. First, the ionic binding of meso-tetra(parasulphonatophenyl) iron porphyrin onto functionalized magnetic nanoparticles is established, followed by embedding the nanoparticles into polylactic acid nanofibers by electrospinning technique. Due to the synergistic morphological and chemo-structural advantages of binding onto nanoparticles and embedding in polymeric matrices the biomimetic efficiency of metalloporphyrin can be remarkably enhanced, while substrate conversion value was tenfold larger than which could be achieved with classic human liver microsomal system.

In Vitro Metabolite Identification Studies for the New Psychoactive Substances Furanylfentanyl, TFMPP, and 5-MeO-DALT in Human Liver Microsomes

Aims: Understand the metabolic behavior of new psychoactive substances, furanyl fentanyl, TFMPP, and 5-MeO-DALT. Background: New psychoactive substances (NPS) are associated with several health and social harms on both the individual and societal levels. Many are not regulated and have become increasingly popular among drug users worldwide. The lack of clinical studies on the effects and toxicity of these drugs has made the interpretation of their toxicological symptoms difficult. Objective: Perform an in vitro metabolism study of new psychoactive substances furanyl fentanyl, TFMPP, and 5-MeO-DALT, revealing their possible metabolites and metabolic pathways in the human liver microsome. Method: A regular human liver microsomal system was used to investigate the potential biotransformation of furanyl fentanyl, TFMPP, and 5-MeO-DALT in vitro, and high-resolution mass spectrometry (LC-Q/TOF-MS) was used to perform metabolite detection and identification. Result: The three components were substantially metabolized in 4 hours with varied metabolic pathways, and most of the metabolites were generated through phase I metabolic reactions. Furanyl fentanyl underwent the metabolic pathways of epoxidation and hydration, furanyl ring-opening and oxidation, hydroxylation, hydrolysis of the amide group, and N-dealkylation; TFMPP underwent the metabolic pathways of hydroxylation, and the successive piperazidine ring scission; while 5-MeO-DALT underwent the metabolic pathways of O-demethylation and glucuronidation, dihydroxylation, hydroxylation, oxidation, O-demethylation, N-dealkylation and methylation and N-dealkylation. Conclusion: Our data would contribute to a better understanding of furanyl fentanyl, TFMPP, and 5- MeO-DALT in their in vitro metabolism study, which was beneficial to predicting their metabolic behavior in vivo, and promoting their drug monitoring in both clinically used and socially/illegally abused.

Comprehensive Analysis of Pterostilbene Metabolites In Vivo and In Vitro Using a UHPLC-Q-Exactive Plus Mass Spectrometer with Multiple Data-Mining Methods

Pterostilbene, a stilbene phytoalexin, is mainly obtained from blueberries and grape vines; however, its metabolic mechanisms were unclear in vivo. In the present study, three different methods were used to prepare biological samples, and then, an efficient strategy based on ultrahigh-performance liquid chromatography coupled with mass spectrometry was developed to screen and identify pterostilbene metabolites in rat urine, plasma, liver, and feces. In order to elucidate pterostilbene or its metabolites involved in vitro, this study was assessed by the liver microsome system. As a result, a total of 88 pterostilbene metabolites were characterized. Among them, 77 metabolites in vivo and 14 metabolites in vitro were found; 50 and 38 metabolites were observed in rat plasma and urine, while only 4 and 12 metabolites were detected in rat feces and liver, inferring that plasma and urine possessed more diverse types of pterostilbene metabolites; 41 metabolic products were obtained by solid-phase extraction, and 9 and 10 metabolites were screened by methanol precipitation and acetonitrile precipitation, respectively, indicating that solid-phase extraction could be adopted as the most acceptable method for pterostilbene metabolism. The results also demonstrated that pterostilbene mainly underwent glucosylation, dehydrogenation, hydrogenation, demethoxylation, sulfation, NAC binding, methylene ketogenic, acetylation, and methylation. In summary, this research provides an idea for the further study of drug metabolism.

Ontogeny of Pediatric Pharmacogenetics: Celebrating the Past and Vision for the Future.

Ontogeny of Pediatric Pharmacogenetics: Celebrating the Past and Vision for the Future.

Monooxygenase System and NO Metabolism in Liver Microsomes of Rats with Toxic Hepatitis and the Effect of Sesquiterpene Lactones.

We analyzed changes in activities of enzymes of phases I and II of xenobiotic biotransformation and parameters of NO metabolism in liver microsomes of rats with toxic CCl4-induced hepatitis after a 14-day course of sesquiterpene lactones from Artemisia leucodes (10 mg/kg). It was found that toxic hepatitis was associated with significant inhibition of NADPH-cytochrome c-reductase, benzo(a)pyrene hydroxylase, and NADPH-diaphorase, reduced cytochrome P-450 content, and enhanced induction of nitrate/nitrite reductase with accumulation of NO metabolites in the liver. Administration of sesquiterpene lactones stimulated activity of the studied components of the cytochrome P-450 system and promoted recovery of the NOergic system components; the effects were most pronounced in 7 and 14 days after treatment.

Interaction of Cp6-his and Cp6 with bovine serum albumin and liver microsomes: Spectroscopic and molecular docking studies

The conjugation of photosensitizer with a molecule specific to receptors over-expressed on cancer cells is a promising approach for improving tumor selectivity in photodynamic therapy (PDT). Since chemical nature of the photosensitizer is also affected by conjugation, this can have significant influence on its transport by serum proteins and clearance via liver microsomal enzyme system. In this report, the interaction of chlorin p6-histamine conjugate (Cp6-his) and free chlorin p6 (Cp6) with bovine serum albumin (BSA) and mouse liver microsomes was assessed by measuring the quenching of intrinsic tryptophan (Trp) fluorescence of proteins. The effect of photosensitizer binding on the activity of microsomal NADPH-cytochrome P-450 and NADH-cytochrome b5 reductase was also determined. The formation of protein carbonyl and lipid peroxides was measured to assess the photodynamic damage to proteins and lipids respectively. Further, the binding interaction between Cp6-his conjugate and parent Cp6 with BSA and cytochrome P-450 was validated using in-silico molecular docking approach. Results show that as compared to free Cp6, the affinity of conjugate to bind with serum albumin is 4 orders of magnitude less, which was also corroborated with finding of docking studies. In contrast, with microsomal proteins the binding affinity of Cp6-his was observed to be lower by 2 orders of magnitude as compared to that for Cp6. Further, the inhibition of the activities of microsomal enzymes induced by Cp6 conjugate and free Cp6 was nearly same (~15-20%). Measurements on quenching of photosensitizer fluorescence by iodide ions revealed that conjugate localized slightly deeper in microsomal membrane as compared to free Cp6 consistent with the slight difference in lipophilicity of the two photosensitizers. The amount of photodynamic damage to BSA induced by Cp6-his was significantly less as compared to that for Cp6, whereas no difference was observed in damage induced to microsomal protein and lipid. These results suggest that conjugation of Cp6 to histamine affects its binding to albumin but doses not significantly alters its interaction with microsomal enzyme system.

Detoxification mechanism of Aconiti Lateralis Radix Praeparata combined with dried Rehmanniae Radix based on metabolic enzymes in liver

The enzymes CYP1 A2 and CYP3 A4 were measured by building a &quot;Cocktail&quot; probe drug and the incubation system of liver microsomes. The compatibility of Aconiti Lateralis Radix Praeparata combined with dried Rehmanniae Radix on CYP450 enzyme protein and gene expression was explored from the level of protein and molecular biology. It explored the molecular mechanism of compatibility detoxication of Aconiti Lateralis Radix Praeparata to provide scientific support for clinical safe and effective application of Aconiti Lateralis Radix Praeparata. The CYP450 enzyme activity was determined by using &quot;Cocktail&quot; probe drugs. The content of CYP450 enzyme was measured by CO reduction of differential spectrum method. The mRNA expression of CYP1 A2 and CYP3 A4 enzyme was detected by RT-PCR technology. Compared with the blank group, the CYP1 A2 and CYP3 A4 enzyme activity and mRNA expression were increased in the dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata group with significant differences(P&lt;0.05), while the CYP3 A4 enzyme activity and mRNA expression were no influence in the Aconiti Lateralis Radix Praeparata group. The CYP3 A4 enzyme activity and mRNA expression were increased in the dried Rehmanniae Radix and the dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata group, and there were significant differences(P&lt;0.05). The content of CYP450 enzyme was decreased in the Aconiti Lateralis Radix Praeparata group, contributed to extremely significant difference(P&lt;0.01). The content of CYP450 enzyme was increased in the dried Rehmanniae Radix and the dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata group, and there were significant differences(P&lt;0.05). The CYP1 A2 and CYP3 A4 enzyme activity and gene expression were enhanced after dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata. The metabolism of toxic ingredients of Aconiti Lateralis Radix Praeparata was accelerated to reach an effect of detoxication. The detoxication mechanism of compatibility of Aconiti Lateralis Radix Praeparata was verified from the viewpoint of liver metabolic enzymes.

S(+)-(2E)-N-(2-Hydroxypropyl)-3-Phenylprop-2-Enamide (KM-568): A Novel Cinnamamide Derivative with Anticonvulsant Activity in Animal Models of Seizures and Epilepsy.

Epilepsy is one of the most frequent neurological disorders affecting about 1% of the world’s human population. Despite availability of multiple treatment options including antiseizure drugs, it is estimated that about 30% of seizures still remain resistant to pharmacotherapy. Searching for new antiseizure and antiepileptic agents constitutes an important issue within modern medicinal chemistry. Cinnamamide derivatives were identified in preclinical as well as clinical studies as important drug candidates for the treatment of epilepsy. The cinnamamide derivative presented here: S(+)-(2E)-N-(2-hydroxypropyl)-3-phenylprop-2-enamide (S(+)-N-(2-hydroxypropyl)cinnamamide, compound KM-568) showed anticonvulsant activity in several models of epilepsy and seizures in mice and rats. It was active in a genetic animal model of epilepsy (Frings audiogenic seizure-susceptible mouse model, ED50 = 13.21 mg/kg, i.p.), acute seizures induced electrically (maximal electroshock test ED50 = 44.46 mg/kg mice i.p., ED50 = 86.6 mg/kg mice p.o., ED50 = 27.58 mg/kg rats i.p., ED50 = 30.81 mg/kg rats p.o., 6-Hz psychomotor seizure model 32 mA ED50 = 71.55 mg/kg mice i.p., 44 mA ED50 = 114.4 mg/kg mice i.p.), chronic seizures induced electrically (corneal kindled mouse model ED50 = 79.17 mg/kg i.p., hippocampal kindled rat model ED50 = 24.21 mg/kg i.p., lamotrigine-resistant amygdala kindled seizure model in rats ED50 = 58.59 mg/kg i.p.), acute seizures induced chemically (subcutaneous metrazol seizure threshold test ED50 = 104.29 mg/kg mice i.p., ED50 = 107.27 mg/kg mice p.o., ED50 = 41.72 mg/kg rats i.p., seizures induced by picrotoxin in mice ED50 = 94.11 mg/kg i.p.) and the pilocarpine-induced status epilepticus model in rats (ED50 = 279.45 mg/kg i.p., ED97 = 498.2 mg/kg i.p.). The chemical structure of the compound including configuration of the chiral center was confirmed by NMR spectroscopy, LC/MS spectroscopy, elemental analysis, and crystallography. Compound KM-568 was identified as a moderately stable derivative in an in vitro mouse liver microsome system. According to the Ames microplate format mutagenicity assay performed, KM-568 was not a base substitution or frameshift mutagen. Cytotoxicity evaluation in two cell lines (HepG2 and H9c2) proved the safety of the compound in concentrations up to 100 µM. Based on the results of anticonvulsant activity and safety profile, S(+)-(2E)-N-(2-hydroxypropyl)-3-phenylprop-2-enamide could be proposed as a new lead compound for further preclinical studies on novel treatment options for epilepsy.

Metabolic activation of TM5441 in vitro and in vivo: Formation of reactive metabolites and human enzymes involved

TM5441, a furan-containing drug, is an inhibitor of plasminogen activator inhibitor-1 (PAI-1), which can induce intrinsic apoptosis of human cancer cell lines. The aim of this study was to identify the reactive metabolites of TM5441 and to reveal the bioactivation pathways that are associated with its hepatotoxicity. The reactive metabolites were trapped by using glutathione (GSH) or N-acetyl-lysine (NAL) in rat, dog, and human liver microsomal incubation system after exposure to TM5441. Two metabolic activation pathways were disclosed. The first bioactivation pathway was dominated by Cytochrome P450 enzymes (CYP450s); TM5441 was metabolized into cis-2-butene-1,4-dial derivative dependent on NADPH, which can be trapped in the liver microsomal incubations fortified with GSH or NAL as trapping agents. Five metabolites (M1, M2, M9, M12 and M13) associated with GSH and three metabolites (M4, M7 and M14) associated with NAL were identified by liquid chromatography-high resolution mass spectrometry. The second bioactivation pathway was catalyzed by UDP-glucuronosyltransferases (UGTs); TM5441 was conjugated with glucuronide to form acyl-glucuronide (M10), which further reacted with GSH, resulting in the identification of a TM5441-S-acyl-GSH adduct (M11) in liver microsomal incubations fortified with uridine-5′-diphosphoglucuronidc acid (UDPGA) and GSH. M9, M10, M11, M12 and M13 were also detected in bile samples of rats given TM5441. Compared with rat, dog would display closer bioactivation profiles to human. The CYP450 enzyme responsible for the bioactivation of TM5441 was mainly identified as CYP3A4, using human recombinant CYP450 enzymes and specific inhibitory studies. The UGT enzymes responsible for the bioactivation of TM5441 mainly involved UGT2B7, 1A1 and 1A4. These results facilitate the understanding of the bioactivation of TM5441 and potential toxicological implications.

Pharmacokinetics and pharmacodynamics analysis of XQ-1H and its combination therapy with clopidogrel on cerebral ischemic reperfusion injury in rats

Ischemic stroke is the main cause of disability and mortality worldwide. 10-O-(N N-dimethylaminoethyl)-ginkgolide B methane-sulfonate (XQ-1 H) is a novel drug based on the remedial approach for ischemic stroke. Clopidogrel, a widely used anti-platelet drug, is often co-prescribed in the clinic. In this study, we established an UPLC-MS/MS spectrometry method for the determination of XQ-1H and investigated the pharmacokinetic effect of clopidogrel on XQ-1H in rats subjected to middle cerebral artery occlusion (MCAO). Meanwhile, the anti-apoptotic and neuroprotective effects of XQ-1H and its combination with clopidogrel were also studied. The results revealed that XQ-1H and its combination with clopidogrel abridged brain infarct volume, cerebral edema and alleviated neurological dysfunction caused by cerebral ischemic reperfusion injury. Further study demonstrated that XQ-1H combined with clopidogrel lessened TUNEL positive cells, up-regulated bcl-2 expression notably and down-regulated bax expression as compared to both XQ-1H and clopidogrel individually. In addition, a rapid, sensitive UPLC-MS/MS method was developed to quantify the concentration of XQ-1H in MCAO/R rats. Our pharmacokinetic results showed that clopidogrel significantly increased the exposure of XQ-1H, increased the peak plasma concentration (Cmax), area under the curve (AUC) and slowed elimination of XQ-1H in the co-administered group. Besides, for further exploring which CYP450 isoforms are involved in the XQ-1H metabolism, XQ-1H was incubated in human liver microsomes (HLMs) system with or without P450 isoform-selective inhibitors. Our results revealed that clopidogrel altered pharmacokinetics of XQ-1H potentially and subsequently enhanced the pharmacological effect of XQ-1H. Moreover, XQ-1H could be applied as an efficacious neuroprotective agent for ischemic stroke because of its considerable effect on averting neuronal apoptosis.

Prediction of potential drug interactions of apigenin based on molecular docking and in vitro inhibition experiments

The purpose of this study was to investigate the effect of apigenin on UGT1 A1 enzyme activity and to predict the potential drug-drug interaction of apigenin in clinical use. First,on the basis of previous experiments,the binding targets and binding strength of apigenin to UGT1 A1 enzyme were predicted by computer molecular docking method. Then the inhibitory effect of apigenin on UGT1 A1 enzyme was evaluated by in vitro human liver microsomal incubation system. Molecular docking results showed that apigenin was docked into the active region of UGT1 A1 enzyme protein F,consistent with the active region of bilirubin docking,with moderate affinity. Apigenin flavone mother nucleus mainly interacted with amino acid residues ILE343 and VAL345 to form hydrophobic binding Pi-Alkyl. At the same time,the hydroxyl group on the mother nucleus and the amino acid residue LYS346 formed an additional hydrogen bond,which increased the binding of the molecule to the protein. These results suggested that the flavonoid mother nucleus structure had a special structure binding to the enzyme protein UGT1 A1,and the introduction of hydroxyl groups into the mother nucleus can increase the binding ability. In vitro inhibition experiments showed that apigenin had a moderate inhibitory effect on UGT1 A1 enzyme in a way of competitive inhibition,which was consistent with the results of molecular docking. The results of two experiments showed that apigenin was the substrate of UGT1 A1 enzyme,which could inhibit the activity of UGT1 A1 enzyme competitively,and there was a risk of drug interaction between apigenin and UGT1 A1 enzyme substrate in clinical use.

Demethylbellidifolin isolated from Swertia bimaculate against human carboxylesterase 2: Kinetics and interaction mechanism merged with docking simulations

In this study, forty-nine kinds of traditional Chinese medicines (TCMs) were evaluated for their inhibitory activities against human carboxylesterase 2 (HCE 2) using a human liver microsome (HLM) system. Swertia bimaculata showed significant inhibition on HCE 2 at 10 μg/mL among forty-nine kinds of TCMs. The extract of Swertia bimaculata was separated by preparative HPLC to afford demethylbellidifolin (1) identified by MS, 1H NMR, and 13C NMR spectra. Demethylbellidifolin (1) was assayed for its inhibitory HCE 2 effect by HCE 2-mediated DDAB hydrolysis, and its potential IC50 value was 3.12 ± 0.64 μM. Demethylbellidifolin (1) was assigned as a mixed-type competitive inhibitor with the inhibiton constant Ki value of 6.87 µM by Lineweaver-Burk and slope plots. Living cell imaging was conducted to corroborate its inhibitory HCE 2 activity. Molecular docking indicated potential interactions of demethylbellidifolin (1) with HCE 2 through two hydrogen bonds of the C-3 and C-5 hydroxy groups with amino acid residues Glu227 and Ser228 in the catalytic cavity, respectively.

Pharmacokinetics, tissue distribution, plasma protein binding, and metabolism study of mefunidone, a novel pirfenidone derivative.

Mefunidone (MFD), a novel derivation of pirfenidone (PFD), exhibits promising anti-renal fibrosis activity. To avoid early development failures and attrition in clinical phase 1, an evaluation of the preclinical pharmacokinetic (PK) properties of MFD was conducted in this study. The absolute bioavailability was evaluated in Sprague Dawley rats and cynomolgus monkeys, who received an intragastric or intravenous administration at the dose of 31.25mg/kg and 10mg/kg, respectively. Tissue distribution and plasma protein binding studies were performed to evaluate the distribution characteristics of MFD. The enzymes involved in the metabolism of MFD were assayed by calculating the inhibition rate using the in vitro incubation system of human liver microsomes. The metabolic pathway of MFD was also examined in the in vitro incubation system as well as in rats in vivo. The results revealed satisfactory absorption of MFD in vivo, with an absolute bioavailability of MFD in rats and monkeys of approximately 77.2% and 70.0%, respectively. MFD was rapidly distributed to all tissues and was highly concentrated in the kidney, which was the target organ of MFD. The plasma protein binding rates in rat, monkey, and human plasma were 36.40%-41.68%, 30.88%-63.92%, and 37.75%-57.77%, respectively. MFD was primarily metabolized by the enzymes CYP3A4, CYP2C9, and CYP2C8. The metabolic pathway of MFD is nitrogen demethylation of the pyridine ring of the MFD nucleus, hydroxylation of the aromatic ring, and demethylation of the piperazine ring. In conclusion, MFD exhibited good PK properties in the initial preclinical PK study. The results strongly support further research to investigate the potential of MFD as a clinical anti-renal fibrosis drug.

Metabolic characteristics of active parts of lipid-lowering flavonoid extract of Daidai in liver and intestinal microsomes of rats

The paper studies and compares the metabolic difference of active ingredients of lipid-lowering flavonoid extract of Daidai in rat livers and intestinal microsomes,in order to explore the phase Ⅰ metabolism characteristics of active ingredients in livers and intestines. UPLC-MS/MS was used to establish a quantitative analysis method for active ingredients,neohesperidin and narngin,in a phase Ⅰ metabolism incubation system of liver and intestinal microsomes. Differential centrifugation was used to make liver and intestinal microsomes of rats. A phase Ⅰ metabolism incubation system was established,and the concentrations of the residual at different incubation time points were analyzed. Graphs were plotted to calculate the metabolic elimination half-life of the main active parts,with the natural logarithm residual percentage values ln( X) at different time points as the y axis,and time t as the x axis. The metabolism characteristics of the active ingredients were compared. The established UPLC-MS/MS quantitative analysis method has a good specialization,standard curve and linear range,accuracy and precision,with a satisfactory lower quantitative limit. The method allows quantitative detection of the active ingredients in a phase Ⅰ metabolism incubation system of liver and intestinal microsomes of rats. In the rats liver microsomes incubation system,the metabolic elimination half-life of neohesperidin and narngin were( 2. 20 ± 0. 28) h and( 1. 97±0. 28) h respectively. The elimination half-life of neohesperidin was larger than that of narngin,but with no statistically significant difference. In the rats intestinal microsomes incubation system,the metabolic elimination half-lives of neohesperidin and narngin were( 3. 68±0. 54) h and( 2. 26±0. 13) h respectively. The elimination half-life of neohesperidin was larger than that of narngin,with statistically significant differences( P<0. 05). The elimination half-lives of the active ingredients in liver microsomes were smaller than those in intestinal microsomes. The experiment results showed that the active ingredients of lipid-lowering flavonoid extract of Daidai had different elimination half-lives in phase Ⅰ rats liver and intestinal microsomes incubation system. This implied that they had different metabolic characteristics in rats liver and intestine,and liver may be the main metabolism site of the active ingredients. The phaseⅠ metabolism of narngin was stronger than that of neohesperidin. The differences between their metabolic characteristics may be related to the binding sites of B-ring hydroxyl in flavonoid glycosides and the number of methoxyl group. The results provided an important experimental basis for further development and clinical application of lipid-lowering flavonoid extract preparation of Daidai.