In vitro phase I metabolism of vinclozolin by human liver microsomes

1.?Vinclozolin (Vin) is a fungicide used in agricultural settings and is classified as an endocrine disruptor. Vin is non-enzymatically hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) metabolites. There is no information about Vin biotransformation in humans, therefore, the aim of this study was to characterize its in vitro metabolism using human liver microsomes.

2.?Vin was metabolized to the [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione] (M4) and N-(2,3,4-trihydroxy-2-methyl-1-oxo)-3,5-dichlorophenyl-1-carbamic acid (M7) metabolites, which are unstable and gradually converted to 3′,5′-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (DTMBA, formerly denoted as M5). M4 and DTMBA metabolites co-eluted in the same HPLC peak; this co-elute peak exhibited a Michaelis-Menten kinetic, whereas M7 showed a substrate inhibition kinetics. The K_{M app} for co-eluted M4/DTMBA and M7 was 24.2?±?5.6 and 116.0?±?52.6?μM, the V_{Max app} was 0.280?±?0.015 and 0.180?±?0.060 nmoles/min/mg protein, and the CL_{int app} was 11.5 and 1.5?mL/min/g protein, respectively. The K_i for M7 was 133.2?±?63.9?μM. Cytochrome P450 (CYP) chemical inhibitors furafylline (CYP1A2), ketoconazole (CYP3A4), pilocarpine (CYP2A6) and sulfaphenazole (CYP2C9) inhibited M4/DTMBA and M7 formation, suggesting that Vin is metabolized in humans by CYP.

3.?DTMBA is a stable metabolite and specific of Vin, therefore, it could be used as a biomarker of Vin exposure in humans to perform epidemiological studies.     

In vitro metabolism of cis- and trans-permethrin by rat liver microsomes,and its effect on estrogenic and anti-androgenic activities

Permethrin is a widely applied broad-spectrum pyrethroid insecticide that consists of a mixture of cis- and trans-isomers. We examined the changes of estrogenic and anti-androgenic activities resulting from metabolism of the isomers. Both cis- and trans-permethrin were hydrolyzed to 3-phenoxybenzyl alcohol (PBAlc) by rat liver microsomes, but the extent of hydrolysis of trans-permethrin was much greater than that of the cis-isomer. In the presence of NADPH, PBAlc was further transformed to 4′-hydroxylated PBAlc (4′-OH PBAlc), 3-phenoxybenzaldehyde (PBAld) and 3-phenoxybenzoic acid (PBAcid). cis-Permethrin, but not trans-permethrin, also afforded its 4′-hydroxylated derivative (4′-OH cis-permethrin). trans-Permethrin was an anti-androgen, but also showed weak estrogenic activity, while cis-permethrin was a weak estrogen and a weak anti-androgen. After incubation with rat liver microsomes in the presence of NADPH, cis-permethrin but not trans-permethrin was metabolically activated for estrogenic activity. On the other hand, estrogenic activity of trans-permethrin was not changed, but its anti-androgenic activity was enhanced after incubation. 4′-OH PBAlc and PBAlc showed estrogenic activity, while PBAld and PBAlc showed anti-androgenic activity. PBAcid showed neither activity. 4′-OH cis-permethrin showed both estrogenic and anti-androgenic activities. Overall, our results indicate that permethrin is metabolically activated for estrogenic and anti-androgen activities, and the microsomal transformation of permethrin to 4′-OH cis-permethrin, 4′-OH PBAlc and PBAlc contributes to the both metabolic activations.     

In vitro metabolism of the new insecticide flupyrazofos by rat liver microsomes

1. The in vitro metabolism of the new insecticide flupyrazofos was studied using rat liver microsomes. Two metabolites were produced and identified as O, O -diethyl O -(1- phenyl-3-trifluoromethyl-5-pyrazoyl) phosphoric acid ester (flupyrazofos oxon) and 1- phenyl-3-trifluoromethyl-5-hydroxypyrazole (PTMHP) based on UV and mass spectral analysis. 2. Cytochrome P450 oxidatively converted flupyrazofos to flupyrazofos oxon, a major metabolite and phenobarbital-induced microsomes increased this desulphuration by 8- fold. 3. Flupyrazofos oxon was converted to PTMHP with a half-life of 47 8?min by chemical hydrolysis and this conversion also proceeded non-enzymatically under our microsomal incubation conditions.     

Felbamate in vitro metabolism by rat liver microsomes.

Incubation of [14C]felbamate at 37 degrees C for 60 min with liver microsomes from untreated Sprague-Dawley rats converted 10% of the drug to the p-hydroxy (6%) and 2-hydroxy (4%) metabolites. With microsomes from phenobarbital-pretreated rats, 21% of the drug was metabolized to the p-hydroxy (7.5%) and 2-hydroxy (13.5%) metabolites. With microsomes from felbamate-pretreated rats, up to 25% of drug was metabolized to the p-hydroxy (5%) and 2-hydroxy (20%) metabolites. In addition, a small amount of the monocarbamate metabolite was also present, but no other metabolites were formed. Coincubations of [14C]phenytoin with felbamate had no effect on the metabolism of phenytoin, whereas the amount of [14C]felbamate metabolized in the presence of phenytoin decreased by 30-38%.     

In vitro metabolism of clenbuterol and bromobuterol by pig liver microsomes

1. Clenbuterol (CBL) and bromobuterol (BBL) were both extensively metabolized by hepatic microsomes of swine to only one polar metabolite which was separated by hplc and purified to perform mass analysis. 2. LC-MS analysis by direct infusion into an ion trap system and after reverse-phase chromatograpy into a triple quadrupole system showed that the metabolites were the hydroxylamine-derivatives of CBL and BBL. GC-MS analysis by the CI and EI modes confirmed that the hydroxyl group was bound to the aniline nitrogen. The chemical instability of those metabolites probably as a consequence of spontaneous oxidation and reduction gave rise during the analysis to the corresponding nitroso and nitro derivatives, together with the original compound. 3. Thermal inactivation and CO complex formation were used selectively to inactivate flavin monooxygenase and cytochrome P450, respectively. Both inactivation procedures significantly reduced the formation of the hydroxyl metabolite.     

In vitro metabolism of gefitinib in human liver microsomes

The in vitro metabolism of gefitinib was investigated by incubating [14C]-gefitinib, as well as M537194, M387783 and M523595 (the main metabolites of gefitinib observed in man), at a concentration of 100 microM with human liver microsomes (4 mg ml(-1)) for 120 min. These relatively high substrate and microsomal protein concentrations were used in an effort to generate sufficient quantities of metabolites for identification. HPLC with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of a large number of metabolites. Although 16 metabolites were identified, metabolism was restricted to three regions of the molecule. The major pathway involved morpholine ring-opening and step-wise removal of the morpholine ring and propoxy side chain. O-demethylation of the quinazoline methoxy group was a quantitatively less important pathway, in contrast to the clinical situation, where O-desmethyl gefitinib (M523595) is the predominant plasma metabolite. The third metabolic route, oxidative defluorination, was only a minor route of metabolism. Some metabolites were formed by a combination of these processes, but no metabolism was observed in other parts of the molecule. Incubation of gefitinib produced ten identified metabolites, but the use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.     

In vitro metabolism of gefitinib in human liver microsomes

1.?The in vitro metabolism of gefitinib was investigated by incubating [¹⁴C]-gefitinib, as well as M537194, M387783 and M523595 (the main metabolites of gefitinib observed in man), at a concentration of 100?μM with human liver microsomes (4?mg?ml^?1) for 120?min. These relatively high substrate and microsomal protein concentrations were used in an effort to generate sufficient quantities of metabolites for identification.

2.?HPLC with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of a large number of metabolites. Although 16 metabolites were identified, metabolism was restricted to three regions of the molecule.

3.?The major pathway involved morpholine ring-opening and step-wise removal of the morpholine ring and propoxy side chain. O-demethylation of the quinazoline methoxy group was a quantitatively less important pathway, in contrast to the clinical situation, where O-desmethyl gefitinib (M523595) is the predominant plasma metabolite. The third metabolic route, oxidative defluorination, was only a minor route of metabolism. Some metabolites were formed by a combination of these processes, but no metabolism was observed in other parts of the molecule.

4.?Incubation of gefitinib produced ten identified metabolites, but the use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.     

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.     

In vitro metabolism of perospirone in rat, monkey and human liver microsomes.

In vitro metabolism of perospirone was examined with rat, monkey and human liver S9, human liver microsomes and yeast microsomes expressing human P450, using 14C labeled perospirone. With rat liver S9, the major metabolites were MX9 and ID-11614, produced by cleavage at the butylene chain. However, some butylene non-cleavage and hydration of the cyclohexane ring were found, although limited in extent. Unknown metabolites accounted for about 10% of the total. After incubation for 10 minutes with monkey liver S9, the major metabolites were ID-15036 and MX11, hydrated in the cyclohexane ring. After incubation for 60 minutes, ID-15001, i.e. the butylene chain cleavage type increased. Unknown metabolites accounted for about 20%. After incubation for 10 minutes with human liver S9, the major metabolite was ID-15036, hydrated in the cyclohexane ring. In addition, MX11 and many unknown metabolites were evident. After incubation for 60 minutes, the butylene chain cleavage type and unknown metabolites increased. Individual differences were found in the metabolic reaction rate. With human liver microsomes. MX11, ID-15001 and unknown metabolites were again the major metabolites. With yeast microsomes expressing human P450 subtypes, CYP1A1, 2C8, 2D6, 3A4 were responsible for the metabolism in particular, and CYP3A4 contributes greatly. Therefore it is unlikely that genetic polymorphism will arise a present a problem with regard to the clinical drug. The results demonstrated that the main metabolic pathway in human liver S9 and liver microsomes involve oxidation at cyclohexane, oxidative cleavage of the butylene side chain and S-oxidation. The same was the case in rat and monkey S9, but species differences were found in the proportions of the metabolites produced.     

罗通定在人、比格犬和大鼠肝微粒体代谢转化的体外比较研究

目的体外研究罗通定(rotundine,RTD)在大鼠、比格犬和人肝微粒体中酶代谢动力学及代谢产物差异。方法优化3个种属肝微粒体与RTD反应体系,应用LC-MS测定RTD在3种肝微粒体中的体外代谢消除,应用LC-MS/MS分析比较RTD在3种肝微粒体中代谢产物种类和生成量的差异,计算并比较相应的活性值。结果RTD在人肝微粒体中代谢转化最慢,其相应的动力学参数为Km=2.67μmol·L-1、Vmax=0.095μmol·L-1·min-1、T21=298±18.0min、CLint=14.6±0.91ml·min-1·kg-1;大鼠中相应的动力学参数为Km=3.24μmol·L-1、Vmax=0.122μmol·L-1·min-1、T12=71.0±2.30min、CLint=87.5±2.79ml·min-1·kg-1;比格犬中相应的动力学参数为Km=5.31μmol·L-1、Vmax=0.228μmol·L-1·min-1、T21=62.3±0.647min、CLint=139±1.43ml·min-1·kg-1。RTD在3个种属肝微粒体中均代谢产生4个O-去甲基后的羟基化同分异构体产物,但4个产物的相对生成百分比在不同种属肝微粒体中有一定差异。结论罗通定在体外人、大鼠和比格犬肝微粒体中主要的I相代谢途径相同,但是酶代谢动力学性质及代谢产物的生成量存在着一定的差异。     

Centchroman: In vitro metabolism by rat liver homogenate

Centchroman (trans-2,2-dimethyl-3-phenyl-4-(p-(β-pyrrolidinoethoxy) phenyl)-7-methoxy chroman) (I), a postcoital antifertility agent under clinical development was extensively metabolized by rat liver homogenate in vitro. Employing field desorption mass spectrometry, high performance liquid chromatography, comparison with authentic samples, and studies with 2-¹⁴C-Centchroman, seven metabolites have so far been characterised, which include trans-3-phenyl-4-(p-(β-pyrrolidinoethoxy) phenyl)-7-methoxy chroman (II, 37.5%), trans-2,2-dimethyl-3-phenyl-4-(p-(β pyrrolidinoethoxy) phenyl)-7-hydroxy chroman (III, 2.5%), β pyrrolidino-ethoxy benzene (IV), 2,2-dimethyl-4-(p-(hydroxy) phenyl)-7-methoxy chromene (V, 39.4%), trans-2,2-dimethyl-3-phenyl-4-(p-(hydroxy) phenyl)-7-methoxy chroman (VI, 5.8%), 2,3-trans-3,4-trans-2-methyl-3-phenyl-4-(p-(β-pyrrolidinoethoxy) phenyl)-7-methoxy chorman (VII, 2.9%), and 2,2-dimethyl-4-(p-(β-pyrrolidinoethoxy) phenyl)-7-methoxy chroman (VIII, 8.8%). Formation of metabolites V and VIII are unusual cases of dephenylation during metabolism.     

In vitro metabolism study of combretastatin A-4 in rat and human liver microsomes.

The phase I biotransformation of combretastatin A-4 (CA-4) 1, a potent tubulin polymerization inhibitor with antivascular and antitumoral properties, was studied using rat and human liver subcellular fractions. The metabolites were separated by high-performance liquid chromatography and detected with simultaneous UV and electrospray ionization (ESI) mass spectrometry. The assignment of metabolite structures was based on ESI-tandem mass spectrometry experiments, and it was confirmed by comparison with reference samples obtained by synthesis. O-Demethylation and aromatic hydroxylation are the two major phase I biotransformation pathways, the latter being regioselective for phenyl ring B of 1. Indeed, incubation with rat and human microsomal fractions led to the formation of a number of metabolites, eight of which were identified. The regioselectivity of microsomal oxidation was also demonstrated by the lack of metabolites arising from stilbenic double bond epoxidation. Alongside the oxidative metabolism, Z-E isomerization during in vitro study was also observed, contributing to the complexity of the metabolite pattern. Moreover, when 1 was incubated with a cytosolic fraction, metabolites were not observed. Aromatic hydroxylation at the C-6' of phenyl ring B and isomerization led to the formation of M1 and M2 metabolites, which were further oxidized to the corresponding para-quinone (M7 and M8) species whose role in pharmacodynamic activity is unknown. Metabolites M4 and M5, arising from O-demethylation of phenyl ring B, did not form the ortho-quinones. O-Demethylation of phenyl ring A formed the metabolite M3 with a complete isomerization of the stilbenic double bond.     

In vitro metabolism and interactions of the fungicide metalaxyl in human liver preparations

In order to provide additional information for risk assessment of the fungicide metalaxyl, the main objectives were (1) to elucidate the interactions of metalaxyl with different human liver cytochrome P450 enzymes, (2) to tentitatively identify and (semi)quantify metabolites in vitro and (3) to identify human CYP enzymes responsible for metabolism. The mean inhibitory concentrations (IC(50)) for 7-pentoxyresorufin-O-dealkylation (CYP2B) and bupropion hydroxylation (2B6) were 48.9 and 41.7μM, respectively. The biotransformation reactions were hydroxylation, (di)demethylation and lactone formation. In human liver microsomes predominant metabolites were two hydroxymetalaxyl derivatives or atropisomers of one of the derivatives. On the basis of previous rat studies these could be N-(2-hydroxymethyl-6-methylphenyl)-N-(methoxyacetyl)alanine methyl ester and/or N-(2,6-dimethyl-5-hydroxyphenyl)-N-(methoxyacetyl)alanine methyl ester. The amounts of didemethylmetalaxyl N-(2,6-dimethylphenyl)-N-(hydroxyacetyl)alanine and lactone 4-(2,6-dimethylphenyl)-3-methylmorpholine-2,5-dione were higher in homogenates than microsomes. The carcinogenic 2,6-dimethylaniline was not detected. Among the nine major human CYPs, CYP3A4 was the only one responsible for metalaxyl hydroxylation, while CYP2B6 was the major isoform responsible for (di)demethylation and lactone formation.     

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.     

去氢土莫酸在大鼠体外肝微粒体体系中的生物转化研究

目的:研究中药茯苓中主要化学成分之一的去氢土莫酸在大鼠肝微粒体中的生物转化.方法:用大鼠肝微粒体体外温孵法进行去氢土莫酸的生物转化,优化了温孵体系;高效液相色谱法检测和制备原形化合物去氢土莫酸及其生物转化产物,核磁共振波谱法和质谱法确定生物转化产物的结构.结果:去氢土莫酸在苯巴比妥诱导的大鼠肝微粒体药物代谢酶作用下,产生2个转化产物,分别为土莫酸和去氢茯苓酸.结论:去氢土莫酸可被苯巴比妥诱导的大鼠肝微粒体药物代谢酶转化为土莫酸和去氢茯苓酸.     

噻吩诺啡在人、比格犬和大鼠肝微粒体中体外代谢比较

采用体外肝微粒体孵育体系, 研究噻吩诺啡在大鼠、比格犬和人肝微粒体中酶代谢动力学及代谢产物差异。通过对噻吩诺啡浓度、微粒体蛋白含量和孵育时间等条件的考察优化噻吩诺啡与肝微粒体的反应体系; 应用LC-MS/MS定量检测孵育体系中的噻吩诺啡及代谢产物, 分析比较噻吩诺啡在3种肝微粒体中代谢产物种类和生成量的差异, 计算并比较相应的动力学参数。噻吩诺啡在人肝微粒体中代谢转化最慢, 其相应的动力学参数K_m = (4.00 ± 0.59) µmol·L⁻¹、V_max = (0.21 ± 0.06) µmol·L⁻¹·min⁻¹、T_1/2 = (223 ± 6.10) min、CL_int = (117 ± 3.19) mL·min⁻¹·kg⁻¹; 比格犬和大鼠肝微粒体中相应的参数K_m、V_max、T_1/2和CL_int分别为 (3.57 ± 0.69) 和 (3.28 ± 0.50) µmol·L⁻¹、(0.18 ± 0.04) 和 (0.14 ± 0.04) µmol·L⁻¹·min⁻¹、(244 ± 1.21) 和 (70.7 ± 1.05) min、(213 ± 1.06) 和    (527 ± 7.79) mL·min⁻¹·kg⁻¹。在3个种属肝微粒体中均观察到噻吩诺啡的6个I相代谢产物, 但6个产物的相对生成百分比在不同种属肝微粒体中有一定差异。实验结果表明, 噻吩诺啡在体外人、比格犬和大鼠肝微粒体中主要的I相代谢途径相同, 但是代谢产物的生成量及噻吩诺啡的代谢动力学性质存在着一定的差异。     

Lindane metabolism by human and rat liver microsomes

1. Human liver microsomes convert lindane (gamma isomer of 1,2,3,4,5,6-hexachlorocyclohexane) to four major primary metabolites; gamma-1,2,3,4,5,6-hexachlorocyclohex-1-ene (3,6/4,5-HCCH), gamma-1,3,4,5,6-pentachlorocyclohex-1-ene (3,6/4,5-PCCH), beta-1,3,4,5,6-pentachlorocyclohex-1-ene (3,4,6/5-PCCH), and 2,4,6-trichlorophenol (2,4,6-TCP); and two major secondary metabolites; 2,3,4,6-tetrachlorophenol (2,3,4,6-TTCP) and pentachlorobenzene (PCB). 2. Under the same conditions, rat liver microsomes produce 3,6/4,5-HCCH, 2,4,6-TCP and 2,3,4,6-TCCP at rates similar to human liver microsomes. 3,4,6/5-PCCH is produced at much lower rates and 3,6/4,5-PCCH and PCB are not detected when lindane is incubated with rat liver microsomes for up to 30 min. 3. The identity of 3,4,6/5-PCCH, previously not identified as a mammalian metabolite of lindane, is confirmed by column chromatography and g.l.c.-mass spectrometry by comparison with authentic material. 4. It is concluded that there is potentially substantial hepatic metabolism by humans of lindane, a topically used scabicide and pediculicide.     

In vitro identification of metabolites of verapamil in rat liver microsomes

AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS^n), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eightmetabolites, including two novel metabolites (M4 and MS), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (MS-MT), and N, O-didemethyl-verapamil (MS). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.