Hydrolytic metabolism of phenyl and benzyl salicylates,fragrances and flavoring agents in foods,by microsomes of rat and human tissues

Salicylates are used as fragrance and flavor ingredients for foods, as UV absorbers and as medicines. Here, we examined the hydrolytic metabolism of phenyl and benzyl salicylates by various tissue microsomes and plasma of rats, and by human liver and small-intestinal microsomes. Both salicylates were readily hydrolyzed by tissue microsomes, predominantly in small intestine, followed by liver, although phenyl salicylate was much more rapidly hydrolyzed than benzyl salicylate. The liver and small-intestinal microsomal hydrolase activities were completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Phenyl salicylate-hydrolyzing activity was co-eluted with carboxylesterase activity by anion exchange column chromatography of the Triton X-100 extracts of liver and small-intestinal microsomes. Expression of rat liver and small-intestinal isoforms of carboxylesterase, Ces1e and Ces2c (AB010632), in COS cells resulted in significant phenyl salicylate-hydrolyzing activities with the same specific activities as those of liver and small-intestinal microsomes, respectively. Human small-intestinal microsomes also exhibited higher hydrolyzing activity than liver microsomes towards these salicylates. Human CES1 and CES2 isozymes expressed in COS cells both readily hydrolyzed phenyl salicylate, but the activity of CES2 was higher than that of CES1. These results indicate that significant amounts of salicylic acid might be formed by microsomal hydrolysis of phenyl and benzyl salicylates in vivo. The possible pharmacological and toxicological effects of salicylic acid released from salicylates present in commercial products should be considered.     

Transesterification of a series of 12 parabens by liver and small-intestinal microsomes of rats and humans

Hydrolytic transformation of parabens (4-hydroxybenzoic acid esters; used as antibacterial agents) to 4-hydroxybenzoic acid and alcohols by tissue microsomes is well-known both in vitro and in vivo. Here, we investigated transesterification reactions of parabens catalyzed by rat and human microsomes, using a series of 12 parabens with C1–C12 alcohol side chains. Transesterification of parabens by rat liver and small-intestinal microsomes occurred in the presence of alcohols in the microsomal incubation mixture. Among the 12 parabens, propylparaben was most effectively transesterified by rat liver microsomes with methanol or ethanol, followed by butylparaben. Relatively low activity was observed with longer-side-chain parabens. In contrast, small-intestinal microsomes exhibited higher activity towards moderately long side-chain parabens, and showed the highest activity toward octylparaben. When parabens were incubated with liver or small-intestinal microsomes in the presence of C1–C12 alcohols, ethanol and decanol were most effectively transferred to parabens by rat liver microsomes and small-intestinal microsomes, respectively. Human liver and small-intestinal microsomes also exhibited significant transesterification activities with different substrate specificities, like rat microsomes. Carboxylesterase isoforms, CES1b and CES1c, and CES2, exhibited significant transesterification activity toward parabens, and showed similar substrate specificity to human liver and small-intestinal microsomes, respectively.     

In-vitro metabolism of glycyrrhetinic acid by human and rat liver microsomes and its interactions with six CYP substrates

Objectives Glycyrrhetinic acid is the main metabolite of glycyrrhizin and the main active component of Licorice root. This study was designed to investigate the in‐vitro metabolism of glycyrrhetinic acid by liver microsomes and to examine possible metabolic interactions that glycyrrhetinic acid may have with other cytochrome P450 (CYP) substrates. Methods Glycyrrhetinic acid was incubated with rat liver microsomes (RLM) and human liver microsomes (HLM). Liquid chromatography tandem mass spectrometry was used for glycyrrhetinic acid or substrates identification and quantification. Key findings The K_m and V_max values for HLM are 33.41 µm and 2.23 nmol/mg protein/min, respectively; for RLM the K_m and V_max were 24.24 µm and 6.86 nmol/mg protein/min, respectively. CYP3A4 is likely to be the major enzyme responsible for glycyrrhetinic acid metabolism in HLM while CYP2C9 and CYP2C19 are considerably less active. Other human CYP isoforms have minimal or no activity toward glycyrrhetinic acid. The interactions of glycyrrhetinic acid and six CYP substrates, such as phenacetin, diclofenac, (S)‐mephenytoin, dextromethorphan, chlorzoxazone and midazolam were also investigated. The inhibitory action of glycyrrhetinic acid was observed in CYP2C9 for 4‐hydroxylation of diclofenac, CYP2C19 for 4′‐hydroxylation of (S)‐mephenytoin and CYP3A4 for 1′‐hydroxylation of midazolam with half maximal inhibitory concentration (IC50) values of 4.3‐fold, 3.8‐fold and 9.6‐fold higher than specific inhibitors in HLM, respectively. However, glycyrrhetinic acid showed relatively little inhibitory effect (IC50 > 400 µm ) on phenacetin O‐deethylation, dextromethorphan O‐demethylation and chlorzoxazone 6‐hydroxylation. Conclusions The study indicated that CYP3A4 is likely to be the major enzyme responsible for glycyrrhetinic acid metabolism in HLM while CYP2C9 and CYP2C19 are considerably less active. The results suggest that glycyrrhetinic acid has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP2C9, CYP2C19 and CYP3A4 substrates.     

2,5-Dihydroxy-4-methoxybenzophenone: a novel major in vitro metabolite of benzophenone-3 formed by rat and human liver microsomes

1.?When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP.

2.?The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole.

3.?The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats.

4.?When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.     

Comparative metabolism of benzo(a)pyrene by ovarian microsomes of various species

Knowledge of the ability of the female reproductive system to metabolize polycyclic aromatic hydrocarbons (PAHs) is critical to the diagnosis and management of female infertility and for risk assessment purposes. The PAHs are a family of widespread pollutants that are released into the environment from automobile exhausts, cigarette smoke, burning of refuse, industrial emissions, and hazardous waste sites. In exposed animals, PAHs become activated to reactive metabolites that interfere with target organ function and as a consequence cause toxicity. The extent of susceptibility to PAH exposure may depend on the ability of animals to metabolize these chemicals. The present study has been undertaken to assess whether any differences exist among mammals in the metabolism of benzo(a)pyrene (BaP), a prototypical PAH compound. Microsomes isolated from the liver and ovaries of rats, mice, goats, sheep, pigs, and cows were incubated with 5 μM BaP. Postincubation, samples were extracted with ethyl acetate and analyzed for BaP/metabolites by reverse‐phase HPLC with fluorescence detection. The rate of metabolism (pmol of metabolite/min/mg protein) was found to be more in liver than in ovary in all the species studied (P < 0.05). The differences in metabolite concentrations were statistically significant (P < 0.0001) among the various species in both organs studied. Multiple species comparison also revealed that the differences were statistically significant (P < 0.001) between rodents (rat and mouse) and higher mammals (ewe, sow, and cow). Even among the higher mammals, in a majority of the cases, the differences in metabolite concentrations were significantly different (P < 0.001) both in ovary and liver. The BaP metabolites identified were 4,5‐diol; 7,8‐diol; 9,10‐diol; 3‐hydroxy BaP; and 9‐hydroxy BaP. The rodent microsomes produced considerably higher proportion of BaP 4,5‐diol and 9,10‐diol than did cow, sow, goat, and sheep. However, microsomes from higher mammals converted a greater proportion of BaP to 3‐hydroxy and 9‐hydroxy BaP, the detoxification products of BaP. Overall, our results revealed a great variation among species to metabolize BaP. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Enantioselective metabolism of camazepam by rat liver microsomes

Camazepam [3-(N,N-dimethyl)carbamoyloxy-7-chloro-1-methyl-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one, CMZ] possesses anxiolytic, anticonvulsant, muscle relaxant and hypnotic properties. CMZ is clinically used as a racemate. The enantioselective metabolism of racemic CMZ by rat liver microsomes was studied. Major metabolites were isolated by normal-phase and reversed-phase liquid chromatography (LC) and further characterized by UV absorption, mass, and circular dichroism spectral analyses, and by chiral stationary phase LC analysis. Following anin vitro incubation of rac-CMZ, the unmetabolized CMZ was found to be enriched in the (S)-CMZ, indicating that the Renantiomer was enantioselectively metabolized. Two of the most abundant metabolites, formed by hydroxylation and demethylation of a methyl group of theN,N-dimethylcarbamyloxy side chain, were found to be enriched in the Renantiomer. The results indicated that the (R)-CMZ was metabolized at a faster rate than (S)-CMZ by rat liver microsomes.     

Comparative metabolism of chrysene and 5-methylchrysene by rat and rainbow trout liver microsomes.

We have investigated the metabolism of chrysene (CHR) and 5-methychyrsene (5-MeCHR) by Shasta rainbow trout (Oncorhyncus mykiss) and Long Evans rat liver microsomes to assess the effect of a non-benzo ring methyl substituent on the reactions involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs). Trout as well as rat liver microsomes metabolized both CHR and 5-MeCHR at essentially similar rates, indicating that the methyl substituent does not alter the substrate specificity of the cytochrome P450(s) involved in the metabolism of the two PAHs. Dihydrodiols were the major CHR metabolites formed by both trout and rat liver microsomes, whereas the trout liver microsomes formed a considerably higher proportion of 5-MeCHR phenols compared to diols, indicating that 5-methyl substitution alters the substrate specificity of trout microsomal epoxide hydrolase for 5-MeCHR epoxides. Unlike trout liver microsomes, rat liver microsomes formed a much greater proportion of 5-MeCHR diols compared to 5-MeCHR phenols, suggesting that 5-MeCHR epoxides are better substrates for the microsomal epoxide hydrolase present in rat liver than for the enzyme in trout liver. Both trout and rat liver microsomes are more efficient at attacking the bay-region bond versus the non-bay-region double bond in chrysene. In contrast the reverse is true in the case of 5-MeCHR, indicating that a non-benzo ring methyl substituent alters the regioselectivity of the enzymes involved in the oxidative metabolism of PAHs.     

In vitro metabolism study of saikosaponin d and its derivatives in rat liver microsomes

1.?Saikosaponins, one of the representative bioactive ingredients in Radix Bupleuri, possess hepatoprotective, anti-inflammatory, antiviral, antitumor, and other pharmacological activities. Up to now, few studies focused on the further metabolism of saikosaponins and their secondary metabolites absorbed into the circulatory system.

2.?To understand the in vivo efficacy of saikosaponin d, the in vitro metabolism of saikosaponin d, and its two derivatives formed in the gastrointestinal tract, prosaikogenin G and saikogenin G was investigated in rat liver microsomes, respectively.

3.?Fifteen metabolites were detected using high-performance liquid chromatography hybrid ion trap and time-of-flight mass spectrometry and triple-quadrupole mass spectrometry, and the predominant metabolic reactions were hydroxylation, carboxylation and combinations of these steps on the aglycone moiety.

4.?The metabolic pathways of saikosaponin d, prosaikogenin G, and saikogenin G were proposed in vitro and the results contribute to the understanding of saikosaponins in vivo metabolism.     

The metabolism of valerylfentanyl using human liver microsomes and zebrafish larvae

Valerylfentanyl, a novel synthetic opioid less potent than fentanyl, has been reported in biological samples, but there are limited studies on its pharmacokinetic properties. The goal of this study was to elucidate the metabolism of valerylfentanyl using an in vitro human liver microsome (HLM) model compared with an in vivo zebrafish model. Nineteen metabolites were detected with N-dealkylation—valeryl norfentanyl and hydroxylation as the major metabolic pathways. The major metabolites in HLMs were also detected in 30 day postfertilization zebrafish. An authentic liver specimen that tested positive for valerylfentanyl, among other opioids and stimulants, revealed the presence of a metabolite that shared transitions and retention time as the hydroxylated metabolite of valerylfentanyl but could not be confirmed without an authentic standard. 4-Anilino-N-phenethylpiperidine (4-ANPP), a common metabolite to other fentanyl analogs, was also detected. In this study, we elucidated the metabolic pathway of valerylfentanyl, confirmed two metabolites using standards, and demonstrated that the zebrafish model produced similar metabolites to the HLM model for opioids.     

In vitro metabolism of nobiletin,a polymethoxy-flavonoid,by human liver microsomes and cytochrome P450

1. Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs.

2. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4′-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4′-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL.

3. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4′-OH-NBL.

4. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

     

Identification of CYP isozymes involved in benzbromarone metabolism in human liver microsomes

Benzbromarone (BBR) is metabolized to 1′‐hydroxy BBR and 6‐hydroxy BBR in the liver. 6‐Hydroxy BBR is further metabolized to 5,6‐dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1′‐hydroxy BBR and 6‐hydroxy BBR and in the metabolism of 6‐hydroxy BBR to 5,6‐dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1′‐hydroxy BBR. The formation rate of 1′‐hydroxy BBR significantly correlated with testosterone 6β‐hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1′‐hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6‐hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6‐hydroxy BBR and diclofenac 4′‐hydroxylation activity in 12 human liver microsomes. The formation of 6‐hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6‐dihydroxy BBR from 6‐hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6‐dihydroxy BBR was significantly correlated with diclofenac 4′‐hydroxylation activity and phenacetin O‐deethylation activity in 12 human liver microsomes. The formation of 5,6‐dihydroxy BBR was inhibited with either tienilic acid or α‐naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1′‐hydroxy BBR and 6‐hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6‐dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolism of deltamethrin and cis- and trans-permethrin by rat and human liver microsomes,liver cytosol and plasma preparations

1. The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague–Dawley rats aged 15, 21 and 90 days and from adult humans.

2. DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes.

3. In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes.

4. The metabolism of pyrethroids by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds.

5. DLM, CPM and TPM were metabolised by rat, but not human, plasma CES enzymes.

6. This study demonstrates that the ability of male rats to metabolise DLM, CPM and TPM by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90?day old rats. As pyrethroid-induced neurotoxicity is due to the parent compound, these results suggest that DLM, CPM and TPM may be more neurotoxic to juvenile than to adult rats.

     

Stereoselective in vitro metabolism of rhynchophylline and isorhynchophylline epimers of Uncaria rhynchophylla in rat liver microsomes

1.?The objective was to investigate the underlying mechanism of the stereoselectivity in the metabolism of rhynchophylline (RIN) and isorhynchophylline (IRN) epimers in rat liver microsomes (RLM).

2.?After incubation, eight metabolites of RIN (M1-5) and IRN (M6-8) reacted at A- and C-ring were identified using LC-Q-TOF/MS. Metabolic pathways included oxidation, hydroxylation, N-oxidation and dehydrogenation. In addition, hydroxylation at A-ring was the major metabolic pathway for RIN whereas the oxidation at C-ring was the major one for IRN.

3.?Enzyme kinetics showed that the intrinsic clearance (CL_int) for IRN elimination was 1.9-fold higher than RIN and the degradation half-life (T_1/2) of RIN was 4.7-fold higher than that of IRN, indicating IRN was more favorable to be metabolized than RIN in RLM.

4.?Data from chemical inhibition study demonstrated CYP3A was the predominant isoform involved in the metabolic elimination of both epimers, as well as the formation of M1-8.

5.?In conclusion, data revealed that due to the spatial configurations at C-7 position, RIN and IRN epimers possessed different hepatic metabolic pathways and elimination rates which were mainly mediated by CYP3A.     

Assay of caffeine metabolism in vitro by human liver microsomes using radio-high-performance liquid chromatography

The low turnover of caffeine in vitro by human liver microsomes makes the study of the metabolic pathways of this compound difficult. Analytical methods with high sensitivity and specificity are needed for the detection of its metabolic products. A method based on the on-line radiometric determination of [8C-³H]caffeine and its principal metabolite (paraxanthine) in man has been developed using reversed-phase high-performance liquid chromatography. The method has been successfully employed in preliminary studies of the kinetics of this reaction.     

Isolation of human hepatic microsomes and their inhibition by cimetidine and ranitidine

Summary Human hepatic microsomes were isolated from wedge biopsies of the liver from 13 patients undergoing abdominal surgery. Ultrasonic homogenisation was used to increase the yield of microsomal monooxygenase activity (7-ethoxycoumarin O-deethylase, NADPH-cytochrome c reductase), resulting in a 30% higher total enzyme activity per g liver than preparation by other techniques. In 4 individual microsomal preparations the influence of cimetidine and ranitidine on Michaelis-Menten kinetics of O-deethylation and of reductase activity were studied. Without the H₂-receptor blocking drugs, enzyme kinetics of O-deethylation with a K_m of 51.0±16.4 µM (n=3) were obtained using Lineweaver-Burke plots. Both, cimetidine and ranitidine inhibited the O-deethylation; cimetidine had a five-fold higher inhibitory affinity (K_i 1.01 and 3.94 mM) to the monooxygenase than ranitidine (K_i 4.96 and 17.70 mM) in the uninduced liver. However, in liver from a patient with induced enzyme activity (K_m=478.0 µM), the K_i of ranitidine was similar to that of cimetidine (K_i ran 3.57 versus K_i cim 2.49 mM). The reductase activity was not inhibited by ranitidine and only marginally so by cimetidine.The results suggest that in human hepatic microsomes oxidative drug metabolism is inhibited by both H₂-receptor antagonists. However, the inhibitory potency of the compounds seems to depend on the individual isozyme pattern of the hepatic microsomes. Thus, while cimetidine is an relatively nonspecific enzyme inhibitor, ranitidine might more selectively inhibit induced drug metabolizing enzymes.     

CYP450 1A2 and multiple UGT1A isoforms are responsible for jatrorrhizine metabolism in human liver microsomes

Jatrorrhizine, one of the protoberberine alkaloids derived from the plant Coptis chinensis, is expected to be developed as a new gastric prokinetic drug, but its metabolic characteristics in humans remain unknown. This study characterized the phase I and phase II metabolites, metabolic kinetics, and cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT) enzymes responsible for the metabolism of jatrorrhizine in human liver microsomes (HLMs). Chemical inhibition in HLMs and metabolism by recombinant human CYP or UGT enzymes were employed to determine the key metabolic enzyme subtypes. In HLMs, demethyleneberberine (demethylated product) and jatrorrhizine glucuronide were identified as the phase I and phase II metabolites, respectively. The enzyme kinetics for both demethylation and glucuronidation were fitted to the Michaelis–Menten equation. Demethylation was inhibited significantly by furafylline and predominantly catalysed by recombinant CYP1A2, whereas glucuronidation was inhibited by silibinin, quercetin, as well as 1‐naphthol and catalysed by recombinant UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9 and UGT1A10. These results showed that jatrorrhizine is metabolized by human CYP1A2 and multiple UGT1A isoforms. Copyright © 2013 John Wiley & Sons, Ltd.     

Assay of caffeine metabolism in vitro by human liver microsomes using radio-high-performance liquid chromatography

The low turnover of caffeine in vitro by human liver microsomes makes the study of the metabolic pathways of this compound difficult. Analytical methods with high sensitivity and specificity are needed for the detection of its metabolic products. A method based on the on-line radiometric determination of [8C-³H]caffeine and its principal metabolite (paraxanthine) in man has been developed using reversed-phase high-performance liquid chromatography. The method has been successfully employed in preliminary studies of the kinetics of this reaction.     

In vitro metabolism of the lignan (−)‐grandisin,an anticancer drug candidate,by human liver microsomes

(?)‐grandisin is a tetrahydrofuran lignan that displays important biological properties, such as trypanocidal, anti‐inflammatory, cytotoxic, and antitumor activities, suggesting its utility as a potential drug candidate. One important step in drug development is metabolic characterization and metabolite identification. To perform a biotransformation study of (?)‐grandisin and to determine its kinetic properties in humans, a high performance liquid chromatography (HPLC) method was developed and validated. After HPLC method validation, the kinetic properties of (?)‐grandisin were determined. (?)‐grandisin metabolism obeyed Michaelis‐Menten kinetics. The maximal reaction rate (V_max) was 3.96 ± 0.18 µmol/mg protein/h, and the Michaelis‐Menten constant (K_m) was 8.23 ± 0.99 μM. In addition, the structures of the metabolites derived from (?)‐grandisin were characterized via gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐mass spectrometry (LC‐MS) analysis. Four metabolites, 4‐O‐demethylgrandisin, 3‐O‐demethylgrandisin, 4,4′‐di‐O‐demethylgrandisin, and a metabolite that may correspond to either 3,4‐di‐O‐demethylgrandisin or 3,5‐di‐O‐demethylgrandisin, were detected. CYP2C9 isoform was the main responsible for the formation of the metabolites. These metabolites have not been previously described, demonstrating the necessity of assessing (?)‐grandisin metabolism using human‐derived materials. Copyright © 2015 John Wiley & Sons, Ltd.     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V_max in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

异鼠李素对人肝微粒体CYPs活性及大鼠原代肝细胞毒性作用研究

目的 研究异鼠李素对肝脏6种CYPs的体外抑制作用,以及对大鼠原代肝细胞的毒性作用。方法 采用人肝微粒体（HLMs）体外温孵法研究异鼠李素对6种细胞色素P450酶（CYPs）——CYP2C19、CYP2D6、CYP3A4、CYP2E1、CYP1A2和CYP2C9的体外抑制作用;使用HPLC-MS/MS法检测异鼠李素和HLMs共同孵育后的代谢产物;利用体外培养的低CYPs活性的大鼠原代肝细胞,考察不同剂量异鼠李素对细胞培养液中乳酸脱氢酶（LDH）、丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）的影响。结果 50 μmol/L的异鼠李素对CYP2E1和CYP1A2有一定的抑制作用,抑制率分别为59.48%和39.91%;异鼠李素和HLMs共同孵育后,产生去甲基化代谢产物3,3'',4'',5,7-五羟基黄酮,转化为极性和水溶性较高的代谢物;30、100、300 μmol/L的异鼠李素会使大鼠原代肝细胞培养液中的ALT和LDH显著上升（P＜0.01）,100、300 μmol/L异鼠李素使AST显著上升（P＜0.05、0.01）,呈浓度相关性。结论 异鼠李素在体外主要经HLMs代谢,同时对CYP2E1和CYP1A2有一定的抑制作用,可能会使CYP2E1和CYP1A2的底物药物在体内的浓度产生变化,导致一系列药物的相互作用;大量使用异鼠李素可能会造成一定程度的肝细胞损伤,且呈现浓度相关性。临床应用应合理设置剂量,并注意潜在的药物之间的相互作用。