Rat Liver Microsome Incubation Systems Research Articles

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.

Effects of astragaloside IV on the pharmacokinetics of omeprazole in rats

Context: Omeprazole and astragaloside IV (AS-IV) are widely used for the treatment of gastric ulcers in China clinics.Objective: This study investigates the effects of AS-IV on the pharmacokinetics of omeprazole in rats.Materials and methods: The pharmacokinetics of orally administered omeprazole (2 mg/kg), with or without AS-IV (100 mg/kg/day for 7 days) pretreatment, were investigated in male Sprague-Dawley rats (two groups of six animals each) using LC–MS/MS. A Caco-2 cell transwell model and rat liver microsome incubation systems were also used to support the in vivo pharmacokinetic data and investigate its potential mechanism.Results: The results indicated that co-administration of AS-IV could decrease the systemic exposure of omeprazole significantly (p < 0.05), including AUC0–t (717.20 ± 177.63 vs. 1166.25 ± 186.65 ng h/mL) and Cmax (272.35 ± 25.81 vs. 366.34 ± 32.57 ng/mL). The t1/2 of omeprazole also decreased significantly (1.78 ± 0.15 vs. 2.23 ± 0.27 h, p < 0.05). The efflux ratio of omeprazole across the Caco-2 cell transwell model increased significantly from 1.73 to 2.67 (p < 0.05), and the metabolic stability of omeprazole was decreased from 42.6 ± 7.8 to 26.2 ± 5.1 min with the pretreatment of AS-IV (p < 0.05).Discussion and conclusions: AS-IV could decrease the systemic exposure of omeprazole in rats when AS-IV and omeprazole were co-administered, and it might exert these effects through decreasing the absorption of omeprazole by inducing P-gp, or through accelerating the metabolism of omeprazole in rat liver by inducing the activity of CYP3A4.

Effects of glycyrrhizin on the pharmacokinetics of asiatic acid in rats and its potential mechanism

Context: Asiatic acid has been reported to possess a wide range of pharmacological activities.Objective: This study investigates the effects of glycyrrhizin on the pharmacokinetics of asiatic acid in rats and its potential mechanism.Materials and methods: The pharmacokinetics of orally administered asiatic acid (20 mg/kg) with or without glycyrrhizin pretreatment (100 mg/kg/day for seven days) were investigated using a LC–MS method. Additionally, the Caco-2 cell transwell model and rat liver microsome incubation systems were used to investigate the potential mechanism of glycyrrhizin’s effects on the pharmacokinetics of asiatic acid.Results: The results showed that the Cmax (221.33 ± 21.06 vs. 324.67 ± 28.64 ng/mL), AUC0–inf (496.12 ± 109.31 vs. 749.15 ± 163.95 μg·h/L) and the t1/2 (1.21 ± 0.27 vs. 2.04 ± 0.32 h) of asiatic acid decreased significantly (p < 0.05) with the pretreatment of glycyrrhizin. The oral clearance of asiatic acid increased significantly from 27.59 ± 5.34 to 41.57 ± 9.19 L/h/kg (p < 0.05). The Caco-2 cell transwell experiments indicated that glycyrrhizin could increase the efflux ratio of asiatic acid from 1.63 to 2.74, and the rat liver microsome incubation experiments showed that glycyrrhizin could increase the intrinsic clearance rate of asiatic acid from 138.32 ± 11.20 to 221.76 ± 16.85 μL/min/mg protein.Discussion and conclusions: In conclusion, these results indicated that glycyrrhizin could decrease the system exposure of asiatic acid, possibly by inducing the activity of P-gp or CYP450 enzyme.

Investigation of the influence of glycyrrhizin on the pharmacokinetics of celastrol in rats using LC-MS and its potential mechanism

1. The aim of this study was to investigate the effects of glycyrrhizin on the pharmacokinetics of celastrol in rats.2. Twelve male Sprague–Dawley rats were randomly assigned to two groups: control group and test group. Test group was pretreated with glycyrrhizin at a dose of 100 mg/kg/day for 10 days, and then the two groups were orally administered with celastrol at a dose of 1 mg/kg. The concentration of celastrol was determined using a sensitive and reliable LC-MS method.3. The results showed that glycyrrhizin could significantly decrease the plasma concentration (from 64.36 ng/mL to 38.42 ng/mL) and AUC0−t (from 705.39 to 403.43 μg·h/L) of celastrol in rats. To investigate its potential mechanism, the effects of glycyrrhizin on the transport and metabolic stability of celastrol were investigated using Caco-2 cell monolayer transwell model and rat liver microsome incubation systems. The Caco-2 cell monolayer transwell experiments indicated that glycyrrhizin could increase the efflux ratio of celastrol (4.02 versus 6.51). However, the rat liver microsome incubation experiments showed that glycyrrhizin could significantly increase the intrinsic clearance rate of celastrol from 20.3 ± 3.37 to 38.8 ± 4.18 μL/min/mg protein.4. In conclusion, these results indicated that the herb–drug interaction between glycyrrhizin and celastrol might occur when they were coadministered.

Cytochrome P450 Mediated Bioactivation of Saracatinib.

Saracatinib is a highly selective Src kinase inhibitor against all Src kinase family members and has demonstrated anticancer effects in preclinical models. Unfortunately, it has shown multiple adverse effects during its clinical trials, along with time-dependent inhibition of P450 enzymes. The major objective of this study was to identify reactive metabolites of saracatinib in vitro and in vivo. Four oxidative metabolites (M1-M4) were detected in rat and human liver microsomal incubation systems after exposure to saracatinib. An ortho-quinone-derived reactive metabolite existing as a GSH conjugate (M5) was found in microsomes fortified with GSH as a trapping agent. The formation of the metabolites detected was NADPH dependent. Metabolites M2-M4 were also observed in bile and urine of rats given saracatinib, and M5 was only detected in bile. Inhibition and recombinant P450 enzyme incubation studies demonstrated that P450 3A4 was the primary enzyme responsible for the metabolic activation of saracatinib. The metabolism study facilitates the understanding of correlation between saracatinib-induced hepatotoxicity and bioactivation.

Effects of phillyrin and forsythoside A on rat cytochrome P450 activities in vivo and in vitro

1. Phillyrin and forsythoside A are two important active ingredients in Forsythia suspensa. However, the effects of phillyrin and forsythoside A on the activities of cytochrome P450 (CYP450) remain unclear.2. This study aimed to investigate the effects of phillyrin and forsythoside A on the activities of CYP1A2, CYP2C11, CYP2D1 and CYP3A1/2 by cocktail probe drugs in rats both in vivo and in vitro.3. Many pharmacokinetic parameters of caffeine and metoprolol in phillyrin pretreatment group, caffeine and tolbutamide in forsythoside A pretreatment group were affected significantly. In rat liver microsomal incubation system, the concentrations of acetaminophen and dextrophan in the phillyrin pretreatment group are higher than blank control group by 207.69% and 125.00%, however, the concentrations of 4-hydroxytolbutamide and 6β-hydroxytestosterone were not significantly altered. The concentrations of acetaminophen and 4-hydroxytolbutamide in the forsythoside A pretreatment group are higher than blank control group by 223.07% and 154.16%, whereas the concentrations of dextrophan and 6β-hydroxytestosterone were not significantly altered.4. These results indicated that Phillyrin had potential inductive effects on rat CYP1A2 and CYP2D1 activities, without affecting CYP2C11 and CYP3A1/2 activities. Moreover, forsythoside A had inductive effects on the activities of CYP1A2 and CYP2C11, without affecting CYP2D1 and CYP3A1/2 activities.

Drug-drug interactions between moxifloxacin and rifampicin based on pharmacokinetics in vivo in rats.

Moxifloxacin and rifampicin are all the first-line options for the treatment of active tuberculosis, which are often combined for the treatment of multidrug resistance pulmonary tuberculosis in clinic. However, the potential drug-drug interactions between moxifloxacin and rifampicin were unknown. The aim of this study was to investigate the drug-drug interactions between moxifloxacin and rifampicin based on their pharmacokinetics in vivo after oral administration of the single drug and both drugs, and reveal their mutual effects on their pharmacokinetics. Eighteen male Sprague-Dawley rats were randomly assigned to three groups: moxifloxacin group, rifampicin group and moxifloxacin + rifampicin group. Plasma concentrations of moxifloxacin and rifampicin were determined using LC-MS at the designated time points after drug administration, and the main pharmacokinetic parameters were calculated. In addition, effects of moxifloxacin and rifampicin on their metabolic rate and absorption were investigated using rat liver microsome incubation systems and Caco-2 cell transwell model. The main pharmacokinetic parameters of moxifloxacin including Tmax , Cmax , t1/2 and AUC(0-t) increased more in the moxifloxacin + rifampicin group than in the moxifloxacin group, but the difference was not significant (p > 0.05). However, the pharmacokinetic parameters of rifampicin, including peak concentration, area under the concentration-time curve, half-life and the area under the first moment plasma concentration-time curve, increased significantly (p < 0.05) compared with the rifampicin group, and the time to peak concentration decreased significantly (p < 0.05). The mean residence time of rifampicin also increased in moxifloxacin + rifampicin group compared with the rifampicin group, but the difference was not significant (p > 0.05). The rat liver microsome incubation experiment indicated that moxifloxacin could increase the metabolic rate of rifampicin from 23.7 to 38.7 min. However, the Caco-2 cell transwell experiment showed that moxifloxacin could not affect the absorption rate of rifampicin. These changes could enhance the drug efficacy, but they could also cause drug accumulation, which might induce adverse effect, so it was suggested that the drug dosage should be adjusted and the drug concentration in plasma should be monitored if moxifloxacin and rifampicin are co-administered. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro and In vivo effects of six Coptidis alkaloids on liver microsomes UGTs and UGT1A1 activities in rats and mice

In the present study, the effects of six Coptidis alkaloids (berberine, epiberberine, coptisine, jatrorrhizine, palmatine and magnoflorine) on liver microsomes UGTs and UGT1A1 activities in rats and mice were investigated in vitro and in vivo to study the mechanism of metabolic drug-drug interactions of Coptidis Rhizoma with other drugs. In vitro rat and mice liver microsomal incubation systems combined with UDPGA were applied, as well as mice liver microsomes after administration of six Coptidis alkaloids. 4-Nitrophenol and β-estradiol were selected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC, respectively. According to the in vitro rat study, berberine, epiberberine, coptisine and jatrorrhizine significantly inhibited rat liver microsome UGTs activity, particularly epiberberine showed the strongest inhibition. UGT1A1 activity was lowly inhibited by jatrorrhizine, with IC₅₀ at about 227 μmol•L⁻¹, whereas coptisine and magnoflorine significantly activated UGT1A1. According to the in vitro mice study, berberine, coptisine, jatrorrhizine and palmatine significantly inhibited mice liver microsome UGTs activity, and the six alkaloids all significantly activated UGT1A1. According to the in vivo mice study, UGTs activity was significantly activated only in berberine group, while UGT1A1 activity was significantly activated only in jatrorrhizine group. In conclusion, the effects of Coptidis alkaloids on UGT activity showed significant differences in species and between in vitro and in vivo. Meanwhile, the changes in structures of Coptidis alkaloids also have a big impact on UGT activity, which may be one of the causes for the drug-drug interactions between Coptidis Rhizoma and other drugs.

Cytochrome p450-mediated metabolic activation of diosbulbin B.

Diosbulbin B (DIOB), a furan-containing diterpenoid lactone, is the most abundant component of Dioscorea bulbifera L. (DB), a traditional Chinese medicine herb. Administration of purified DIOB or DB extracts has been reported to cause liver injury in animals. The mechanisms of DIOB-induced hepatotoxicity remain unknown. The major objective of this study was to identify reactive metabolites of DIOB. A DIOB-derived cis-enedial was trapped by N-acetyl lysine (NAL) and glutathione (GSH) or N-acetyl cysteine (NAC) in rat and human liver microsomal incubation systems after exposure to DIOB. Four metabolites (M1-M4) associated with GSH were detected by liquid chromatography coupled to tandem mass spectrometry. Apparently, M1 was derived from both NAL and GSH. M2 and M3 resulted from the reaction of GSH without the involvement of NAL. Two molecules of GSH participated in the formation of M4. M2 and M3 were also detected in bile and urine of rats given DIOB. M5, a DIOB-derived NAC/NAL conjugate, was detected in microsomal incubations with DIOB fortified with NAC and NAL as trapping agents. Biomimetic M1-M5 were prepared by oxidation of DIOB with Oxone for metabolite identification. Microsomal incubation study demonstrated that ketoconazole inhibited the production of the enedial in a concentration-dependent manner, and CYP3A4 was found to be the enzyme responsible for the metabolic activation of DIOB. The metabolism study facilitates the understanding of the role of bioactivation of DIOB in its hepatotoxicity.

Copper-Metallothionein from the Toxic Milk Mutant Mouse Enhances Lipid Peroxidation Initiated by an Organic Hydroperoxide

The toxic milk mutation is an autosomal recessive mutation found in an inbred C57BL/6J strain of mice which results in an excessive hepatic accumulation of copper (Cu), mostly associated with metallothionein (MT). The possible toxicological significance of elevated levels of hepatic copper-metallothionein (Cu-MT) was assessed. The effects of Cu-MT on lipid peroxidation (LP) initiated by an organic peroxide were investigated in an in vitro rat liver microsomal incubation system. Addition of Cu-MT (3 μM) could significantly enhance (100%) LP induced by addition of tertiary-butyl hydroperoxide (t-BHP, 0.1 mM). Similar incubations of Cu-MT in the absence of t-BHP showed negligible LP. Addition of copper sulfate at high concentrations (100 μM) also increased t-BHP induced LP, but the enhancement was less pronounced than observed with Cu-MT addition. Chelation of copper with bovine serum albumin and triethylenetetramine tetrahydrochloride eliminated the enhancement of LP by Cu-MT. Evidence is provided on degradation of MT and release of free Cu in the incubation system. Additions of deferoxamine were also found to prevent LP. Therefore, chelatable Cu, released from Cu-MT, may be responsible for the enhancement of the iron-dependent LP in this system but Cu-MT alone in the absence of iron cannot initiate LP. These in vitro results suggest that conditions resulting in high cellular levels of Cu-MT may exhibit a predisposition to oxidative stress.

Formation of glutathione adducts of carbon tetrachloride metabolites in a rat liver microsomal incubation system

Metabolism of CCl 4 by rat liver microsomes causes damage to the membrane. GSH diminishes that damage. One mechanism of GSH protection has been characterized. It involves formation of diglutathione carbonate from two molecules of GSH and one molecule of phosgene, an oxygenated metabolite of CCl 4. The present studies were done to seek other GSH adducts of CCl 4 metabolites and to examine the effect of oxygen tension on their formation. Incubations were carried out in sealed flasks under oxygen concentrations ranging from 0.14 to 21% at 37°. The CCl 4 concentration was 72 μM. 14CCl 4 and 35S-GSH were used to label metabolites. High pressure liquid chromatographic analysis of the aqueous phase demonstrated two GSH adducts of CCl 4 metabolites. One adduct was oxygendependent and was identified as diglutathione carbonate by its co-elution with a diglutathione carbonate standard. Its formation showed no evidence of saturation when GSH concentrations as high as 10 mM were used, indicating that the overall process was nonenzymatic. Formation of the other adduct was greatest under the lowest oxygen concentration studied and none occurred at oxygen tensions of 5% or greater. Based on experiments with radiolabeled CCl 4 and GSH, this metabolite appeared to be a product of one molecule each of CCl 4 and GSH. Formation of this adduct had enzymatic characteristics. It was saturable with respect to GSH with an apparent K m of 70 μM, and other thiol compounds that were tested could not substitute for GSH. The adduct was unstable during isolation attempts and was not characterized further. Formation of these two GSH adducts could account for some of the protection by GSH against CCl 4 injury.

Confirmation of assignment of the trichloromethyl radical spin adduct detected by spin trapping during 13C-carbon tetrachloride metabolism [formula omitted

Rat liver microsomal incubation systems containing the free radical spin trap, phenyl-t-butyl nitrone, as well as an NADPH generating system and [ 13C]CCl 4 (90 atom % 13C) produce electron spin resonance spectra consistent with that expected for a trichloromethyl-phenyl-t-butyl nitrone adduct. This same spectrum is observed in a lipid extract of the liver from a rat orally administered [ 13C]CCl 4 as well as in a solution of phenyl-t-butyl nitrone and [ 13C]CCl 4 irradiated with ultraviolet light.

4-Hydroxy-5-oxo-4,5-dihydrobenzo(a)pyrene, a new metabolite of benzo(a)pyrene.

The detection and the isolation in phenobarbital-induced rat liver microsomal incubation systems of a new metabolite of benzo(a)pyrene are described. The structure of this metabolite and the way in which it is formed are described.

Studies with N-methyl emetine

N-methyl Emetine, like the parent compound, emetine, has different effects on liver microsomal protein-synthesizing activity in the rat depending on whether experiments with this compound are performed in vivo or in vitro. Thus, the pretreatment of animals with N-methyl emetine in vivo at a dose level of 144 μmoles/kg stimulates the incorporation of labeled amino acid into protein in vitro by liver microsomal preparations from 24-hr pretreated rats. However, at the same dosage level, N-methyl emetine pretreatment for 2 hr inhibits the uptake of labeled amino acid into hepatic protein in vivo. In a third type of experiment, the addition of N-methyl emetine directly to a rat liver microsomal incubation system in vitro can completely inhibit the incorporation of l-( 14C)-phenylalanine into protein. These effects obtained with N-methyl emetine are less pronounced than corresponding effects observed with emetine. N-methyl Emetine is not ordinarily metabolized to emetine by N-demethylation in a rat liver microsomal drug-metabolizing system in vitro. However, liver microsomal preparations from phenobarbital-pretreated rats are capable of metabolizing N-methyl emetine by N-demethylation in vitro. Microsomal preparations from rats pretreated with N-methyl emetine at 144 μmole/ kg for 24 hr have a lower capacity for metabolizing drugs in vitro than do the controls. In corresponding experiments with the parent compound, greater inhibition of the drug-metabolizing enzymes in vitro occurs at a much lower pretreatment level with emetine. Substitution of emetine by alkylation of the secondary amine at position N-2′ therefore tends to diminish biological activity with respect to effects observable in liver microsomal preparations of the rat.