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.     

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.     

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.     

Cocktail-substrate assay system for mechanism-based inhibition of CYP2C9, CYP2D6, and CYP3A using human liver microsomes at an early stage of drug development

1. We established a mechanism-based inhibition cocktail-substrate assay system using human liver microsomes and drug–probe substrates that enabled simultaneous estimation of the inactivation of main cytochrome P450 (CYP) enzymes, CYP2C9, CYP2D6, and CYP3A, in drug metabolism.

2. The inactivation kinetic parameters of typical mechanism-based inhibitors, tienilic acid, paroxetine, and erythromycin, for each enzyme in the cocktail-substrate assay were almost in agreement with the values obtained in the single-substrate assay.

3. Using this system, we confirmed that multiple CYP inactivation caused by mechanism-based inhibitors such as isoniazid and amiodarone could be detected simultaneously.

4. Mechanism-based inhibition potency can be estimated by the determination of the observed inactivation rate constants (k_obs) at a single concentration of test compounds because the k_obs of eleven CYP3A inactivators at 10?μM in the assay system nearly corresponded to k_inact/K_I values, an indicator of a compound’s propensity to alter the activity of a CYP in vivo (R²?=?0.97).

5. Therefore, this cocktail-substrate assay is considered to be a powerful tool for evaluating mechanism-based inhibition at an early stage of drug development.

     

大鼠肝微粒体中葛根素的液相色谱-质谱测定法及药物代谢动力学

目的建立大鼠肝微粒体中葛根素及其代谢物的液相色谱质谱测定法,并研究葛根素在大鼠肝微粒体中的药物代谢动力学。方法色谱柱为Waters C18柱(150 mm×4.6 mm,5μm),流动相为甲醇水(体积比为50∶50),通过电喷雾电离源(ESI),以正离子方式进行检测。结果方法的回收率为95.6%~96.8%,其日内、日间的RSD分别为4.9%~7.6%和3.7%~6.2%,葛根素的质量浓度在0.5~20 mg.L-1内与峰面积呈良好的线性关系。在温孵时间0~40 min内、微粒体蛋白质量浓度在0.5~2.0 g.L-1内,葛根素呈线性消除,随着肝微粒体蛋白质量浓度的增大,葛根素呈线性消除,葛根素可被肝微粒体代谢成大豆苷元。其代谢机理为还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)依赖性的氧化代谢。还原型烟酰胺二核苷酸(NADH)对葛根素代谢基本没有催化作用。结论葛根素在大鼠肝微粒体内被迅速代谢,大鼠肝微粒体P450酶参与了葛根素的代谢。     

Influence of the CYP2D6*10 allele on the metabolism of mexiletine by human liver microsomes

AIMS: To study the influence of CYP2D6*10 on the formation of p-hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM) using microsomes from human liver of known genotypes. METHODS: Microsomes from human livers of genotype CYP2D6*1/*1 (n = 5), *1/*10 (n = 6) and *10/*10 (n = 6) were used in this study. The formation of PHM and HMM was determined by high-performance liquid chromatography. RESULTS: The formation rates of PHM and HMM were decreased by more than 50% and 85% in CYP2D6*1/*10 and *10/*10 microsomes, respectively, compared with *1/*1 microsomes. CONCLUSIONS: The metabolism of mexiletine to form PHM and HMM appears to be impaired to a significant extent in human liver microsomes from hetero- and homozygotes of CYP2D6*10.     

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.     

Effects of anticancer drugs on the metabolism of the anticancer drug 5,6-dimethylxanthenone-4-acetic (DMXAA) by human liver microsomes

AIMS: To investigate the effects of various anticancer drugs on the major metabolic pathways (glucuronidation and 6-methylhydroxylation) of DMXAA in human liver microsomes. METHODS: The effects of various anticancer drugs at 100 and 500 microM on the formation of DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) in human liver microsomes were determined by high performance liquid chromatography (h.p.l.c.). For those anticancer drugs showing significant inhibition of DMXAA metabolism, the inhibition constants (Ki) were determined. The resulting in vitro data were extrapolated to predict in vivo changes in DMXAA pharmacokinetics. RESULTS: Vinblastine, vincristine and amsacrine at 500 microM significantly (P < 0.05) inhibited DMXAA glucuronidation (Ki = 319, 350 and 230 microM, respectively), but not 6-methylhydroxylation in human liver microsomes. Daunorubicin and N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA) at 100 and 500 microM showed significant (P < 0.05) inhibition of DMXAA 6-methylhydroxylation (Ki = 131 and 0.59 microM, respectively), but not glucuronidation. Other drugs such as 5-fluoroucacil, paclitaxel, tirapazamine and methotrexate exhibited little or negligible inhibition of the metabolism of DMXAA. Pre-incubation of microsomes with the anticancer drugs (100 and 500 microM) did not enhance their inhibitory effects on DMXAA metabolism. Prediction of DMXAA-drug interactions in vivo based on these in vitro data indicated that all the anticancer drugs investigated except DACA appear unlikely to alter the pharmacokinetics of DMXAA, whereas DACA may increase the plasma AUC of DMXAA by 6%. CONCLUSIONS: These results indicate that alteration of the pharmacokinetics of DMXAA appears unlikely when used in combination with other common anticancer drugs. However, this does not rule out the possibility of pharmacokinetic interactions with other drugs used concurrently with this combination of anticancer drugs.     

氯沙坦与格列美脲在人肝微粒体中的药物相互作用

目的：研究氯沙坦与格列美脲在人肝微粒体中的药物相互作用。方法：200 μL人肝微粒体孵育体系中加入氯沙坦与格列美脲各1~10 μmol·L^-1,于37℃水浴中孵育30 min,终止反应后的样品经处理,应用UPLC-MS/MS法同时检测氯沙坦和格列美脲代谢产物的生成量,采用Dixon作图并计算格列美脲抑制氯沙坦的K_i值以考察格列美脲和氯沙坦的相互抑制作用。结果：在1~10 μmol·L^-1的浓度范围内,格列美脲对氯沙坦表现出明显的抑制作用,相应的K_i值为（0.407 7±0.086 2）μmol·L^-1;氯沙坦仅在格列美脲浓度为1 μmol·L^-1时表现出抑制作用。结论：在人肝微粒体孵育体系中,格列美脲对氯沙坦的抑制作用强于氯沙坦对格列美脲的抑制作用。氯沙坦和格列美脲在人体内的相互作用有待进一步的人体药代动力学研究。     

Stereoselective glucuronidation of formoterol by human liver microsomes

AIMS: Formoterol is a beta2-adrenoceptor agonist marketed as a racemic mixture of the active (R; R)- and inactive (S; S)-enantiomers (rac-formoterol). The drug produces prolonged bronchodilation by inhalation but there is significant interpatient variability in duration of effect. Previous work has shown that in humans formoterol is metabolized by conjugation with glucuronic acid but little is known about the stereoselectivity of this reaction. The aim of the present study was to investigate the glucuronidation of formoterol enantiomers in vitro by human liver microsomes. METHODS: The kinetics of formation of formoterol glucuronides during incubation of racemate and of single formoterol enantiomers with human liver microsomes (n=9) was characterized by chiral h.p.l.c. assay. RESULTS: The kinetics of glucuronidation of the two formoterol enantiomers obeyed the Michaelis-Menten equation. Glucuronidation of formoterol was stereoselective and occurred more than two times faster for (S; S)-formoterol than for (R; R)-formoterol. In incubations with single formoterol enantiomers, the median (n=9) Km values for (R; R)-glucuronide and (S; S)-glucuronide were 827.6 and 840.4 microm, respectively, and the median V max values were 2625 and 4304 pmol min-1 mg-1, respectively. Corresponding values determined in incubations with rac-formoterol were 357.2 and 312.1 microm and 1435 and 2086 pmol min-1 mg-1 for (R; R)- and (S; S)-glucuronide, respectively. Interindividual variation was large with the ratio of V max/Km (S; S/R; R) ranging from 0.57 to 6.90 for incubations with rac-formoterol. CONCLUSIONS: Our study demonstrates that glucuronidation of formoterol by human liver microsomes is stereoselective and subject to high interindividual variability. These findings suggest that clearance of formoterol in humans is subject to variable stereoselectivity which could explain the variation in duration of bronchodilation produced by inhaled formoterol in patients with asthma.     

Flavonoids diosmetin and luteolin inhibit midazolam metabolism by human liver microsomes and recombinant CYP 3A4 and CYP3A5 enzymes

We evaluated the effects of increasing concentrations of the flavonoids salvigenin, diosmetin and luteolin on the in vitro metabolism of midazolam (MDZ), a probe substrate for cytochrome P450 (CYP) 3A enzymes, which is converted into 1'-hydroxy-midazolam (1'-OH-MDZ) and 4-hydroxy-midazolam (4-OH-MDZ) by human liver microsomes. Salvigenin had only a modest effect on MDZ metabolism, whereas diosmetin and luteolin inhibited in a concentration-dependent manner the formation of both 1'-OH-MDZ and 4-OH-MDZ, with apparent K(i) values in the 30-50mumol range. Both diosmetin and luteolin decreased 1'-OH-MDZ formation by human recombinant CYP3A4, but not CYP3A5, whereas they decreased 4-OH-MDZ formation by both recombinant enzymes. To assess whether any relationship exists between the physico-chemical characteristics of flavones and their effects on MDZ metabolism, we tested the effects of three other flavones (flavone, tangeretin, chrysin) on MDZ metabolism by human liver microsomes. Whereas flavones possessing more than two hydroxyl groups (luteolin, diosmetin) inhibited MDZ biotransformation, flavones lacking hydroxyl groups in their A and B rings (flavone, tangeretin) stimulated MDZ metabolism. We also found close relationships between the maximum stimulatory or inhibitory effects of flavones on 1'-OH-MDZ and 4-OH-MDZ formation rates and their log of octanol/water partition coefficients (logP) or their total number of hydroxyl groups. The results of the study may be of clinical relevance since they suggest that luteolin and diosmetin may cause pharmacokinetic interactions with co-administered drugs metabolized via CYP3A.     

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.

     

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.     

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.     

尼莫地平在人肝微粒体内的代谢

:采用人肝微粒体在体外研究尼莫地平 (Nim)在人体内的代谢物及代谢途径 . Nim在人肝微粒体内被迅速代谢成 3个代谢物 ,分别是 Nim二氢吡啶环脱氢代谢物 M1,二氢吡啶环侧链脱甲基代谢物M2 ,二氢吡啶环脱氢及其侧链脱甲基代谢物 M3.Nim在人肝微粒体中的最初的两步代谢反应是其二氢吡啶环脱氢氧化及其侧链脱甲基反应 ,两者的代谢产物可以被进一步代谢为代谢物 M3.CYP3A的特异性抑制剂醋竹桃霉素和酮康唑可以抑制Nim的二氢吡啶环脱氢氧化及其侧链脱甲基反应 ,使 Nim的代谢速率明显下降 ,结果提示 CYP3A参与了 Nim在人肝微粒体内的代谢     

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.     

山冈橐吾碱在雌性大鼠肝微粒体内的代谢(英文)

研究了山冈橐吾碱 (clivorine)在雌性大鼠肝微粒体内的代谢 .山冈橐吾碱在雌性大鼠肝微粒体内的主要代谢物为两个非吡咯代谢物M1和M2 .与雄性大鼠不同 ,生成肝毒性的吡咯代谢物为其次要的代谢途径 .文献报道山冈橐吾碱在雄性大鼠肝微粒体内的主要代谢方式是形成相应的吡咯代谢物 .这提示山冈橐吾碱在雌雄大鼠肝微粒体内的主要代谢方式不同 .CYP4 5 0特异性抑制剂黄胺苯吡唑(CYP2C) ,毛果芸香碱 (CYP2A1) ,二乙基二硫代氨基甲酸钠 (CYP2E1)和酮康唑 (CYP3A)对M1和M2的形成无明显的影响 .黄素单氧化酶的特异性抑制剂甲巯咪唑可以显著地抑制M2 的形成 ,但对M1的形成无明显的抑制作用 ,且M1在肝微粒体中的形成为NADPH非依赖性 ,上述结果提示参与M1和M2代谢的酶分别为肝微粒体中的水解酶和黄素单氧化酶 .另一方面 ,毛果芸香碱 ,黄胺苯吡唑和二乙基二硫代氨基甲酸钠对山冈橐吾碱的吡咯代谢物的形成无明显的影响 ,而CYP3A的特异性抑制剂酮康唑可以显著地抑制吡咯代谢物的生成 ,且山冈橐吾碱在重组的大鼠肝CYP2C12 ,CYP2E1温孵液中无代谢 ,而在重组的大鼠肝CYP3A1和CYP3A2的温孵液中山冈橐吾碱被代谢成相应的吡咯代谢物 .这提示CYP3A作为主要的CYP4 5 0酶参与了山冈橐吾碱的肝毒性吡咯代谢物的形成 .山冈橐吾碱?     

藁本内酯在大鼠肝微粒体中代谢的酶动力学

研究体外大鼠肝微粒体藁本内酯代谢的酶动力学及选择性CYP450酶抑制剂对其代谢的影响。建立测定肝微粒体孵育液中藁本内酯含量的LC-MS法,以尼群地平为内标,二者的定量离子m/z分别选择173和315。考察确定最佳温孵条件,进行藁本内酯代谢的酶促反应动力学研究,通过特异性抑制试验,探讨参与藁本内酯代谢的主要同工酶。结果显示,酮康唑、甲氧苄啶、α-萘黄酮显著抑制藁本内酯的体外代谢,而奥美拉唑、4-甲基吡唑、奎尼丁对其体外代谢影响不大。可见CYP3A4、CYP2C9和CYP1A2是参与藁本内酯代谢的主要代谢酶,CYP2C19、CYP2E1和CYP2D6没有明显参与催化其代谢。

     

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.     

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.