Rat Liver Microsomal Activity Research Articles

The influence of drug-induced metabolic enzyme activity inhibition and CYP3A4 gene polymorphism on aumolertinib metabolism

The purpose of this study is to clarify the drug interaction profile of aumolertinib, and the influence of CYP3A4 genetic polymorphism on aumolertinib metabolic characteristics. Through microsomal enzyme reactions, we screened 153 drugs and identified 15 that significantly inhibited the metabolism of aumolertinib. Among them, telmisartan and carvedilol exhibited potent inhibitory activities in rat liver microsomes (RLM) and human liver microsomes (HLM). In vivo, the pharmacokinetic parameters of aumolertinib, including AUC and Cmax, were significantly altered when co-administered with carvedilol, with a notable decrease in the clearance rate CLz/F. Interestingly, the pharmacokinetic parameters of the metabolite HAS-719 exhibited a similar trend as aumolertinib when co-administered. Mechanistically, both telmisartan and carvedilol exhibited a mixed-type inhibition on the metabolism of aumolertinib. Additionally, we used a baculovirus-insect cell expression system to prepare 24 recombinant CYP3A4 microsomes and obtained enzymatic kinetic parameters using aumolertinib as a substrate. Enzyme kinetic studies obtained the kinetic parameters of various CYP3A4 variant-mediated metabolism of aumolertinib. Based on the relative clearance rates, CYP3A4.4, 5, 7, 8, 9, 12, 13, 14, 17, 18, 19, 23, 24, 33, and 34 showed significantly lower clearance rates compared to the wild-type. Among the different CYP3A4 variants, the inhibitory potency of telmisartan and carvedilol on the metabolism of aumolertinib also varied. The IC50 values of telmisartan and carvedilol in CYP3A4.1 were 6.68 ± 1.76 μM and 0.60 ± 0.25 μM, respectively, whereas in CYP3A4.12, the IC50 exceeded 100 μM. Finally, we utilized adeno-associated virus to achieve liver-specific high expression of CYP3A4*1 and CYP3A4*12. In the group with high expression of the less active CYP3A4*12, the magnitude of the drug-drug interaction was significantly attenuated. In conclusion, CYP3A4 genetic polymorphism not only influences the pharmacokinetic characteristics of aumolertinib, but also the inhibitory potency of telmisartan and carvedilol on it.

Effect of P. corylifolia on the pharmacokinetic profile of tofacitinib and the underlying mechanism.

This work aimed to explore the mechanisms underlying the interaction of the active furanocoumarins in P. corylifolia on tofacitinib both in vivo and in vitro. The concentration of tofacitinib and its metabolite M8 was determined using UPLC-MS/MS. The peak area ratio of M8 to tofacitinib was calculated to compare the inhibitory ability of furanocoumarin contained in the traditional Chinese medicine P. corylifolia in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP3A4 (rCYP3A4). We found that bergapten and isopsoralen exhibited more significant inhibitory activity in RLMs than other furanocoumarins. Bergapten and isopsoralen were selected to investigate tofacitinib drug interactions in vitro and in vivo. Thirty rats were randomly allocated into 5 groups (n = 6): control (0.5% CMC-Na), low-dose bergapten (20mg/kg), high-dose bergapten (50mg/kg), low-dose isopsoralen (20mg/kg) and ketoconazole. 10mg/kg of tofacitinib was orally intervented to each rat and the concentration level of tofacitinib in the rats were determined by UPLC-MS/MS. More imporrantly, the results showed that bergapten and isopsoralen significantly inhibited the metabolism of tofacitinib metabolism. The AUC(0-t), AUC(0-∞), MRT(0-t), MRT(0-∞) and Cmax of tofacitinib increased in varying degrees compared with the control group (all p < 0.05), but CLz/F decreased in varying degrees (p < 0.05) in the different dose bergapten group and isopsoralen group. Bergapten, isopsoralen and tofacitinib exhibit similar binding capacities with CYP3A4 by AutoDock 4.2 software, confirming that they compete for tofacitinib metabolism. P. corylifolia may considerably impact the metabolism of tofacitinib, which can provide essential information for the accurate therapeutic application of tofacitinib.

Piper auritum ethanol extract is a potent antimutagen against food-borne aromatic amines. Mechanisms of action and chemical composition.

An ethanol extract of Piper auritum leaves (PAEE) inhibits the mutagenic effect of three food-borne aromatic amines (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) in the TA98 Salmonella typhimurium strain. Preincubation with MeIQx demonstrated in mutagenesis experiments that inhibition of Cytochrome P450 (CYP), as well as direct interaction between component(s) of the plant extract with mutagens, might account for the antimutagenic observed effect. Gas chromatography/mass spectrometry analysis revealed that safrole (50.7%), α-copaene (7.7%), caryophyllene (7.2%), β-pinene (4.2%), γ-terpinene (4.1%) and pentadecane (4.1%) as the main components of PAEE. Piper extract and safrole were able to inhibit the rat liver microsomal CYP1A1 activity that participates in the amines metabolism, leading to the formation of the ultimate mutagenic/ molecules. According to this, safrole and PAEE inhibited MeIQx mutagenicity but not that of the direct mutagen 2-nitrofluorene. No mutagenicity of plant extract or safrole was detected. This study show that PAEE and its main component safrole are associate with the inhibition of heterocyclic amines activation due in part to the inhibition of CYP1A subfamily activity.

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.

Effect of chronic unpredicted mild stress-induced depression on clopidogrel pharmacokinetics in rats.

BackgroundClopidogrel is widely used to prevent and treat cardiovascular atherosclerosis and thrombosis. However, disturbance in the expression and activity of liver cytochrome metabolic enzymes significantly changes clopidogrel efficacy. Therefore, the effect of chronic unpredictable mild stress (CUMS)-induced depression on the expression of liver cytochrome metabolic enzymes and clopidogrel pharmacokinetics in rats were explored.MethodsNine different CUMSs were selected to establish a rat model of depression. Open field experiment and sucrose preference test were applied to explore the depressive behaviors. The concentration of serotonin in the cortex of depressed rats was determined using enzyme linked immunosorbent assay (ELISA). All rats were given 10 mg/kg clopidogrel orally after 12 weeks, and blood samples were collected at different time points. The clopidogrel concentration and CYP2C19/ CYP2C9 activity in rat liver microsomes were assayed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The rat liver drug enzymes expression was determined by Real-Time Quantitative Reverse Transcription PCR (RT-qPCR).ResultsOpen field experiment and sucrose preference test indicated the successful construction of the CUMS-induced depression model. The concentration of serotonin in the cortex of depressed rats decreased by 42.56% (∗∗p < 0.01). The area under the curve of clopidogrel pharmacokinetics decreased by 33.13% (∗p < 0.05) in the depression rats, while distribution volume and clearance increased significantly (∗∗p < 0.01). The half-time and distribution volume did not significantly differ. The CYP2C19 and CYP2C9 activity of liver microsomes in the CUMS-induced depression group were significantly higher than that in the control group (∗∗p < 0.01). CYP2C11 and CYP1A2 mRNA expression up-regulated approximately 1.3 - fold in the depressed rat livers compared with that in the control, whereas that of CYP2C13 was down-regulated by 27.43% (∗∗p < 0.01). CYP3A1 and CYP2C12 expression were slightly up-regulated, and that of CES1 did not change.ConclusionsThese results indicated that CUMS-induced depression altered clopidogrel pharmacokinetics, and the change in CYP450 activity and expression in depressed rat livers might contribute to the disturbance of clopidogrel pharmacokinetics.

Adenine-related compounds modulate UDP-glucuronosyltransferase (UGT) activity in mouse liver microsomes

ABSTRACTAdenine-related compounds are allosteric inhibitors of UDP-glucuronosyltransferase (UGT) in rat liver microsomes (RLM) and human UGT isoforms treated with detergent or pore-forming peptide, alamethicin.To clarify whether the same is true beyond species, the effects of adenine-related compounds on 4-methylumbelliferone (4-MU) glucuronidation were examined using detergent-treated mouse liver microsomes (MLM).Brij-58 treatment of MLM increased the Vmax and the Michaelis constant, Km, of 4-MU. This study was performed using Brij-58-treated MLM as an enzyme source. ATP- and ADP-inhibited 4-MU glucuronidation. In contrast, AMP caused a 1.5-fold increase in glucuronidation. Oxidised forms, NAD+ and NADP+, potently inhibited 4-MU glucuronidation, whereas the reduced forms, NADH and NADPH, did not. Furthermore, the IC50 values of ATP, ADP, NAD+, and NADP+ were approximately 15 μM.In our previous study, ATP was the strongest inhibitor of UGT activity in RLM. However, in this study, the above-mentioned compounds inhibited 4-MU UGT in a comparable and noncompetitive manner. Furthermore, AMP antagonised the inhibitory effects of ATP and ADP.These results suggest that ATP, ADP, NAD+, and NADP+ are common endogenous inhibitors of UGT beyond species.

Assessing CYP2C8-Mediated Pharmaceutical Excipient-Drug Interaction Potential: A Case Study of Tween 80 and Cremophor EL-35.

Pharmaceutical excipients (PEs) are substances included in drug formulations. Recent studies have revealed that some PEs can affect the activity of metabolic enzymes and drug transporters; however, the effects of PEs on CYP2C8 and its interaction potential with drugs remain unclear. In this study, we evaluated the effects of Tween 80 and EL−35 on CYP2C8 in vitro and further investigated their impacts on the PK of paclitaxel (PTX) in rats after single or multiple doses. The in vitro study indicated that Tween 80 and EL−35 inhibited CYP2C8 activity in human and rat liver microsomes. EL−35 also decreased the expression of CYP2C8 in HepG2 cells. In the in vivo study, Tween 80 did not alter the PK of PTX after single or multiple doses, whereas EL−35 administered for 14 days significantly increased the AUC and MRT of PTX. Further analysis indicated that multiple-dose EL−35 reduced the expression of Cyp2c22 and production of 6-OH-PTX in the rat liver. Our study suggested that short-term exposure to both PEs did not affect the PK of PTX in rats, but multiple doses of EL−35 increased the AUC and MRT of PTX by downregulating the hepatic expression of Cyp2c22. Such effects should be taken into consideration during drug formulation and administration.

Tissue and interspecies comparison of catechol-O-methyltransferase mediated catalysis of 6-O-methylation of esculetin to scopoletin and its inhibition by entacapone and tolcapone

Catechol-O-methyltransferase (COMT) methylates both endogenous and exogenous catechol compounds to inactive and safe metabolites. We first optimised conditions for a convenient and sensitive continuous fluorescence-based 6-O-methylation assay of esculetin, which we used for investigating the COMT activity in human, mouse, rat, dog, rabbit, and sheep liver cytosols and microsomes and in ten different rat tissues. Furthermore, we compared the inhibition potencies and mechanisms of two clinically used COMT inhibitors, entacapone and tolcapone, in these species. In most tissues, the COMT activity was at least three times higher in cytosol than in microsomes. In the rat, the highest COMT activity was found in the liver, followed by kidney, ileum, thymus, spleen, lung, pancreas, heart, brain, and finally, skeletal muscle. Entacapone and tolcapone were characterised as highly potent mixed type tight-binding inhibitors. The competitive inhibition type dominated over the uncompetitive inhibition with entacapone, whereas uncompetitive inhibition dominated with tolcapone. Rats, dogs, pigs, and sheep are high COMT activity species, in contrast to humans, mice, and rabbits; COMT activity is highest in the liver. Both entacapone and tolcapone are potent COMT inhibitors, but their inhibition mechanisms differ.

Modulation of rat hepatic CYP3A4 activity by Brassica oleracea, Hibiscus rosa sinensis, and Tradescantia zebrina

One of the major cytochrome P450s, CYP 3A4, is known to be involved in herb-drug and food-drug interactions. The objective of this study was to examine the in vitro effect of Brassica oleracea L. var. pekinensis (Chinese cabbage) juice, methanol extract of Hibiscus rosa sinensis (Hibiscus) yellow flower and Tradescantia zebrina pendula leaves, respectively on CYP 3A4 activity in rat liver microsomes. Differential centrifugation method was used to isolate microsomes from the rat livers. The concentration of formaldehyde released from N-demethylation of aminopyrine in rat liver microsomes was determined. Based on the results obtained, B. oleracea juice at 100 ng/mL and 1000 g/mL significantly reduced CYP3A4 activity when compared to the control group. On the other hand, methanol extract of Hibiscus yellow flower and T. zebrina leaves at 1, 10, 100, and 1000 ng/mL significantly increased the metabolism of aminopyrine by enhancing CYP3A4 activity when compared to the control group. Our findings suggested that Chinese cabbage juice, methanol extract of Hibiscus yellow flower, and T. zebrina leaves could have the potential to interact with the metabolism of aminopyrine in rat liver microsomes by modulating the hepatic CYP activity.

Inhibitory effects of organophosphate esters on carboxylesterase activity of rat liver microsomes

We investigated the inhibitory effects of 13 organophosphate esters (OPEs) and hydrolytic metabolites on the carboxylesterase activity of rat liver microsomes in vitro in order to examine whether there might be a potential impact on human health, and to elucidate the structure activity relationship. Among the test compounds, 2-ethylhexyl diphenyl phosphate (EDPhP) was the most potent inhibitor of carboxylesterase activity, as measured in terms of 4-nitrophenol acetate hydrolase activity, followed by tri-m-cresyl phosphate (TmCP), cresyl diphenyl phosphate (CDPhP) and triphenyl phosphate (TPhP). The IC50 values were as follows: EDPhP (IC50: 0.03 μM) > TmCP (0.4 μM) > CDPhP (0.8 μM) > TPhP (14 μM) > tris(1,3-dichloro-2-propyl) phosphate (17 μM) > tris(2-ethylhexyl) phosphate (77 μM) > tri-n-propyl phosphate (84 μM) > tris(2-chloroethyl) phosphate (104 μM) > tris(2-butoxyethyl) phosphate (124 μM) > tri-n-butyl phosphate (230 μM). The IC50 value of EDPhP was three orders of magnitude lower than that of bis(4-nitrophenyl) phosphate, which is widely used as an inhibitor of carboxylesterase. Trimethyl phosphate, triethyl phosphate and tris(2-chloroisopropyl) phosphate slightly inhibited the carboxylesterase activity; their IC50 values were above 300 μM. Lineweaver-Burk plots indicated that the inhibition by several OPEs was non-competitive. Diphenyl and monophenyl phosphates, which are metabolites of TPhP, showed weaker inhibitory effects than that of TPhP.

Assessment of the inhibition risk of paris saponins, bioactive compounds from Paris polyphylla, on CYP and UGT enzymes via cocktail inhibition assays

Paris saponins, also known as polyphyllins, are natural compounds extracted from Paris polyphylla, which have many pharmacological activities, such as anti-inflammation and anti-cancer. In particular, paris saponin I, II, VII and polyphyllin VI are the components of the quality standard for Paris polyphylla. However, the inhibition risk of polyphyllins on cytochrome P450 (CYP) and UDP-glucuronosyltransferases (UGT) remains unclear. Therefore, this report investigated the potential inhibitory effects of paris saponin I, II, VII and polyphyllin VI on the activities of CYP (CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1 and CYP3A2) and UGT (UGT1A1, UGT1A3, UGT1A6, PROG and AZTG) through cocktail inhibition assays in vitro. In the study of CYP, polyphyllin VI exhibited weak inhibition on CYP2D1 activity in rat liver microsomes with IC50 value at 45.2 μM, while paris saponin VII weakly inhibited CYP2C11 and CYP2E1 activities with IC50 value at 42.0 and 67.7 μM, respectively. In the study of UGT, none of the four steroidal saponins showed significant inhibition risk. In conclusion, paris saponin I, II, VII and polyphyllin VI have very low potential to cause the possible toxicity and drug interactions involving CYP and UGT enzymes, indicating that they are safe enough to take with drugs.

Validation of an HPLC Method for the Simultaneous Quantification of Metabolic Reaction Products Catalysed by CYP2E1 Enzyme Activity: Inhibitory Effect of Cytochrome P450 Enzyme CYP2E1 by Salicylic Acid in Rat Liver Microsomes.

Inhibition of cytochrome P450 (CYP) alters the pharmacokinetic parameters of the drug and causes drug–drug interactions. Salicylic acid been used for the treatment of colorectal cancer (CRC) and chemoprevention in recent decades. Thus, the aim of this study was to examine the in vitro inhibitory effect of salicylic acid on CYP2E1 activity in rat liver microsomes (RLMs) using high-performance liquid chromatography (HPLC). High-performance liquid chromatography analysis of a CYP2E1 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 282 nm using 60% H2O, 25% acetonitrile, and 15% methanol as mobile phase. The CYP2E1 assay showed a good linearity (R2 > 0.999), good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80–120%), and low detection (4.972 µM and 1.997 µM) and quantitation limit values (15.068 µM and 6.052 µM), for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. Salicylic acid acts as a mixed inhibitor (competitive and non-competitive inhibition), with Ki (inhibition constant) = 83.56 ± 2.730 µM and concentration of inhibitor causing 50% inhibition of original enzyme activity (IC50) exceeding 100 µM (IC50 = 167.12 ± 5.460 µM) for CYP2E1 enzyme activity. Salicylic acid in rats would have both low and high potential to cause toxicity and drug interactions with other drugs that are substrates for CYP2E1.

Strain and sex differences in drug hydrolase activities in rodent livers

Carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC) are the major drug hydrolases in humans, and they have different substrate preferences. Because rodents are widely used in preclinical studies, we aimed to clarify the extent of the species, strain, and sex differences in hydrolase activity in rats and mice. Hydrolase activities for 24 compounds were evaluated in Fischer 344, Sprague-Dawley, and Wistar-Imamichi rat liver microsomes (RLM) and Balb/c, C3H/He, C57BL/6J, and ddY mouse liver microsomes (MLM) by comparing the results with the activities in human liver microsomes (HLM). Imidapril hydrolase activities in RLM from all strains were substantially higher than those in MLM and HLM, whereas oseltamivir was hardly hydrolyzed in rodents, although both are specific substrates of CES1 in humans. In rats, males tended to show higher hydrolase activities for most human CES1 substrates than females. Hydrolase activities for irinotecan and procaine, which are CES2 substrates in humans, tended to be higher in RLM and MLM than in HLM. Rifamycins, substrates of human AADAC, were not hydrolyzed in RLM and MLM. The results of this study provide important information about the species, strain, and sex differences in hydrolase activities in rats and mice.

Chemoenzymatic Synthesis and Radical Scavenging of Sulfated Hydroxytyrosol, Tyrosol, and Acetylated Derivatives.

Potential metabolites of bioactive compounds are important for their biological activities and as authentic standards for metabolic studies. The phenolic compounds contained in olive oil are an important part of the human diet, and therefore their potential metabolites are of utmost interest. We developed a convenient, scalable, one-pot chemoenzymatic method using the arylsulfotransferase from Desulfitobacterium hafniense for the sulfation of the natural olive oil phenols tyrosol, hydroxytyrosol, and of their monoacetylated derivatives. Respective monosulfated (tentative) metabolites were fully structurally characterized using LC-MS, NMR, and HRMS. In addition, Folin-Ciocalteu reduction, 1,1-diphenyl-2-picrylhydrazyl radical scavenging, and antilipoperoxidant activity in rat liver microsomes damaged by tert-butylhydroperoxide were measured and compared to the parent compounds. As expected, the sulfation diminished the radical scavenging properties of the prepared compounds. These compounds will serve as authentic standards of phase II metabolites.

Evaluation of the inhibition risk of shikonin on human and rat UDP-glucuronosyltransferases (UGT) through the cocktail approach

Shikonin, a natural red colorant, is widely used for food garnishment and cosmetic ingredient in the world. Shikonin also possesses a variety of pharmacological actions, including anti-inflammation and anti-cancer activities. However, little is known about its effects on the UDP-glucuronosyltransferases (UGT) activity. Therefore, the aim of this study was to evaluate the effect of shikonin on the UGT1A1, UGT1A3, UGT1A6, UGT1A9 and UGT2B7 activities via the human and rat liver microsomal assay and cocktail approach. The results showed shikonin inhibited human and rat liver microsomal UGT activity only in a dose-dependent manner. The further enzyme kinetic studies demonstrated that shikonin was not only a competitive inhibitor of human UGT1A1, UGT1A9, and UGT2B7, but also presented competitive inhibition on rat UGT1A1 and AZTG reactions. In conclusion, shikonin as a reversible inhibitor of UGT enzyme has a high-risk potential to cause the possible toxicity, especially drug-drug or food-drug interactions.

Quantification of the activity of detoxifying enzymes in terrestrial invertebrates: Optimization, evaluation and use of in vitro and ex vivo methods

Abstract The negative effects of pesticides on beneficial terrestrial invertebrates have received much attention, and in this context quantification of the activity of metabolic enzymes provides an important tool. Unfortunately, current methodologies are limited in their use. In this study, the grain beetle, Tenebrio molitor was used to illustrate why in vitro methods are ineffective to quantify metabolic enzymes in some insects. It was further used to optimize an ex vivo assay that can replace in vitro methods when these are ineffective. The ex vivo method can quantify metabolic enzymatic activities of cytochrome P450 mixed function oxidases (CYPs), general esterases (EST) and glutathione S‐transferases (GST). In detail we show, that T. molitor produces inhibitory and/or degrading agents, which are released during tissue homogenization. As these agents inhibit the in vitro CYP activity of rat liver microsomes we suggest, that they are also responsible for the lack of in vitro CYP activity of T. molitor. We tested the effectiveness of broad‐spectrum protease inhibitors as protective agents and found, that they were only effective in concentrations that also interfered directly with the activity of CYP enzymes. As an alternative to in vitro measurements, we optimized an ex vivo method for quantification of CYP, EST and GST on individual gut systems of T. molitor. Our ex vivo method advances previous ex vivo methods, by allowing quantification of three major enzyme families, all in a single individual, and by exploiting real‐time kinetic measurements of the enzymatically produced product. Subsequently, our method was successfully tested for its use on three different life stages: larvae, pupae, and adult insects from T. molitor and Apis mellifera. The method shows promise for expansion to other species and potentially other enzyme families and/or substrates.

Time-dependent inhibition of carbamazepine metabolism by piperine in anti-epileptic treatment

AimsThe first-line anti-epileptic agent carbamazepine has narrow therapeutic index and can potentially interact with piperine, the major component from black pepper. The present study aimed to delineate the mechanism of such interaction for safe usage of carbamazepine during epilepsy control. Materials and methodsThe effect of piperine on carbamazepine hepatic metabolism was examined using rat or human liver microsomes. Mechanistic static model was applied to predict the extent of interaction. In addition, liver microsomal activities, mRNAs and protein expressions of genes regulating carbamazepine metabolism were evaluated after two weeks oral administrations of 3.5 and 35 mg/kg piperine in rats. Moreover, the effect of piperine on the xenobiotic receptor constitutive androstane receptor (CAR) was further accessed. Key findingsTime-dependent inhibitory effect of piperine on carbamazepine metabolism was observed, with kinact and KI of 0.0153 min−1 and 18.34 μM for rat, and 0.0093 min−1 and 9.45 μM for human. Based on such in-vitro metabolic parameters, further estimation using mechanistic static model indicated that piperine could increase the AUC of CBZ by 7% and 11% in rat and human, respectively. Significant inhibition on rat liver microsomal activity, Cyp3a2 mRNA and protein expression, CAR mRNA were demonstrated with piperine at 35 mg/kg. Yet, no direct effect on the activity of CAR for piperine was found. SignificanceWe have demonstrated the time-dependent inhibition by piperine on carbamazepine metabolism as the interaction mechanism. Prolonged use of piperine at high dose could increase carbamazepine concentrations through inhibiting metabolic enzyme activities and their related genes expressions.

Inhibitory effects of ficus deltoidea extracts on UDP-glucuronosyltransferase and glutathione s-transferase drug-metabolizing enzymes

Background: Mas cotek or Ficus deltoidea is conventionally used to treat various diseases and often consumed with other medication and this may give rise to herb–drug interaction. The potential of F. deltoidea for interactions with drug-metabolizing enzymes of UDP-gucuronosytranserase (UGT) and glutathione S-transferase (GST) have not been investigated. Objective: We had evaluated the potential of methanol, ethanol, and aqueous extracts of F. deltoidea to cause UGT- and GST-mediated herb–drug interaction in vitro. Materials and Methods: The total phenolic content and total phenolic content were determined using modified colorimetric method. In the UGT study, para -nitrophenol (p -NP) was employed as a substrate to determine the UGT enzymes activity in rat liver microsomes (RLM) and human liver microsomes (HLM). For the GST study, 1-chloro-2,4-dinitrobenzene was employed as a substrate to determine GST activity in rat liver cytosolic fraction. Results: The total phenolic content in F. deltoidea extracts can be ranked as follows: Methanol extract > aqueous extract > ethanol extract, whereas the content of flavonoid compounds in F. deltoidea extracts can be ranked as: Methanol extract > ethanol extract > aqueous extract. Assessment using the UGT enzymes of RLM (IC50 [Half-maximal inhibitory concentration] = 881.40 ± 1.14 μg/mL) and HLM (IC50 =63.44 ± 1.20 μg/mL) showed that the methanol extract of F. deltoidea significantly inhibited p -NP glucuronidation compared with ethanol and aqueous extracts. For GST inhibition study, methanol extract strongly inhibited GST activity (IC50 =70.73 ± 1.07 μg/mL), whereas no IC50values were determined for ethanol and aqueous extracts. Conclusion: The methanol extract of F. deltoidea containing the highest flavonoid content highlights the possibility of herb–drug interaction through the modulation of p -NP UGT and GST activity.

Inhibition of UGT2B7 Enzyme Activity in Human and Rat Liver Microsomes by Herbal Constituents.

The co-use of conventional drug and herbal medicines may lead to herb-drug interaction via modulation of drug-metabolizing enzymes (DMEs) by herbal constituents. UDP-glucuronosyltransferases (UGTs) catalyzing glucuronidation are the major metabolic enzymes of Phase II DMEs. The in vitro inhibitory effect of several herbal constituents on one of the most important UGT isoforms, UGT2B7, in human liver microsomes (HLM) and rat liver microsomes (RLM) was investigated. Zidovudine (ZDV) was used as the probe substrate to determine UGT2B7 activity. The intrinsic clearance (Vmax/Km) of ZDV in HLM is 1.65 µL/mg/min which is ten times greater than in RLM, which is 0.16 µL/mg/min. Andrographolide, kaempferol-3-rutinoside, mitragynine and zerumbone inhibited ZDV glucuronidation in HLM with IC50 values of 6.18 ± 1.27, 18.56 ± 8.62, 8.11 ± 4.48 and 4.57 ± 0.23 µM, respectively, hence, herb-drug interactions are possible if andrographolide, kaempferol-3-rutinoside, mitragynine and zerumbone are taken together with drugs that are highly metabolized by UGT2B7. Meanwhile, only mitragynine and zerumbone inhibited ZDV glucuronidation in RLM with IC50 values of 51.20 ± 5.95 μM and 8.14 ± 2.12 µM, respectively, indicating a difference between the human and rat microsomal model so caution must be exercised when extrapolating inhibitory metabolic data from rats to humans.

Comparison of xenobiotic metabolism in phase I oxidation and phase II conjugation between rats and bird species

There have been many reports regarding toxic chemicals in birds. Chemicals are mainly metabolized in the liver through phase I oxidation by cytochrome P450 (CYP) and phase II conjugation by conjugated enzymes, such as UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), glutathione-S-transferase (GST), etc. Xenobiotic metabolism differs among bird species, but little detailed information is available. In the present study, the four-ring polycyclic aromatic hydrocarbon (PAH), pyrene, was used as a model xenobiotic to clarify the characteristics of xenobiotic metabolism in birds compared with laboratory animals by in vivo and in vitro studies. Plasma, bile, and excreta (urine and feces) were collected after oral administration of pyrene and analyzed to clarify xenobiotic metabolism ability in chickens and quails. Interestingly, pyrenediol-glucuronide sulfate (PYDOGS) and pyrenediol-diglucuronide (PYDOGG) were present in chickens and quails but not in rats. In addition, the area under the curve (AUC), maximum plasma concentration (Cmax), and time to maximum plasma concentration (Tmax) of pyrene-1-sulfate (PYOS) were higher than those of the parent molecule, pyrene, while the elimination half-life (t1/2) and mean residence time (MRT) were faster than those of the parent pyrene. With regard to sulfation of 1-hydroxypyrene (PYOH), the maximum velocity (Vmax) and Michaelis constant (Km) of rat liver cytosol were greater than those of chicken and quail liver cytosol. Furthermore, Vmax/Km of UGT activity in rat liver microsomes was also greater than those of chicken and quail liver microsomes. Characterization of xenobiotic metabolism revealed species differences between birds and mammals, raising concerns about exposure to various xenobiotics in the environment.