A comparative study of the formation of chemically reactive drug metabolites by human liver microsomes.

1. The metabolism of amodiaquine (A), ethinyloestradiol (E), mianserin (M), phenytoin (Ph), sulphanilamide (S) and paracetamol (Pa) to both stable and chemically reactive, i.e. irreversibly protein bound, metabolites was investigated using microsomes prepared from histologically normal human liver obtained from eight kidney donors. 2. All drugs, except amodiaquine, were metabolized by NADPH-dependent microsomal enzymes to chemically reactive metabolites. The degree of NADPH-dependent binding varied between drugs (E, 11.5 +/- 5.8% incubated drug; M, 3.0 +/- 1.9%; Ph, 0.10 +/- 0.09%; S, 0.57 +/- 0.38%; Pa, 1.2 +/- 1.2%; mean of eight livers +/- s.d.). 3. Inclusion of glutathione (1 mM) or ascorbic acid (1 mM) in the incubation reduced the NADPH-dependent binding for all substrates, indicating the involvement of electrophilic oxidation products. 4. Binding of M and Pa correlated with each other (Spearman's r = 0.86) and with total cytochrome P-450 content (r = 0.76 and 0.78 respectively). E binding also correlated with the binding of M (r = 0.79) and Pa (r = 0.81) but not with cytochrome P-450. Binding of Ph and S did not correlate with any of the other measured metabolic parameters.     

Metabolism of ciamexon by human liver microsomes: an investigation into the formation of stable, chemically reactive and cytotoxic metabolites.

1. The in vitro generation of stable, protein-reactive and cytotoxic metabolites from ciamexon by human liver microsomes has been assessed. Stable metabolites were characterized by h.p.l.c./mass spectrometry, protein reactive metabolites by radiometric analysis and cytotoxic metabolites by assessment of cell viability after exposure to metabolites formed in situ. 2. Human livers were obtained from renal transplant donors. All 16 livers investigated metabolized ciamexon in a NADPH-dependent reaction, the major metabolite being the 6-hydroxy-methyl derivative. The hydroxylase activity of the livers varied from 34-577 pmol mg-1 min-1, with a mean activity of 306 +/- 156 pmol mg-1 min-1. The further oxidation product, 6-carboxy ciamexon, was also detected in some incubations. A third, unidentified, polar metabolite was present in all incubations (3.34-11.11% of incubated radioactivity). 3. Only very low levels (less than 1%) of radioactivity became irreversibly bound to microsomal protein, which suggests that ciamexon undergoes little or no oxidative bioactivation in vitro. 4. Human liver microsomes did not metabolize ciamexon to a cytotoxic species, whereas microsomes prepared from mouse livers did generate a cytotoxic species. The degree of toxicity was enhanced if animals were pre-treated with either phenobarbitone or beta-naphthoflavone.     

NADPH-dependent formation of polar and nonpolar estrogen metabolites following incubations of 17 beta-estradiol with human liver microsomes.

By using a versatile high-pressure liquid chromatography method (total elution time approximately 135 min) developed in the present study, we detected the formation of some 20 nonpolar radioactive metabolite peaks (designated as M1 through M20), in addition to a large number of polar hydroxylated or keto metabolites, following incubations of [(3)H]17beta-estradiol with human liver microsomes or cytochrome P450 3A4 in the presence of NADPH as a cofactor. The formation of most of the nonpolar estrogen metabolite peaks (except M9) was dependent on the presence of human liver microsomal proteins, and could be selectively inhibited by the presence of carbon monoxide. Among the four cofactors (NAD, NADH, NADP, NADPH) tested, NADPH was the optimum cofactor for the metabolic formation of polar and nonpolar estrogen metabolites in vitro, although NADH also had a weak ability to support the reactions. These observations suggest that the formation of most of the nonpolar estrogen metabolite peaks requires the presence of liver microsomal enzymes and NADPH. Chromatographic analyses showed that these nonpolar estrogen metabolites were not the monomethyl ethers of catechol estrogens or the fatty acid esters of 17beta-estradiol. Analyses using liquid chromatography/mass spectrometry and NMR showed that M15 and M16, two representative major nonpolar estrogen metabolites, are diaryl ether dimers of 17beta-estradiol. The data of our present study suggest a new metabolic pathway for the NADPH-dependent, microsomal enzyme-mediated formation of estrogen diaryl ether dimers, along with other nonpolar estrogen metabolites.     

Biotransformation of geldanamycin and 17-allylamino-17-demethoxygeldanamycin by human liver microsomes: reductive versus oxidative metabolism and implications.

Comparative metabolite profiling of geldanamycin and 17-allylamino-17-demethoxygeldanamycin (17AAG) using human liver microsomes in normoxia and hypoxia was conducted to understand their differential metabolic fates. Geldanamycin bearing a 17-methoxy group primarily underwent reductive metabolism, generating the corresponding hydroquinone under both conditions. The formed hydroquinone resists further metabolism and serves as a reservoir. On exposure to oxygen, this hydroquinone slowly reverts to geldanamycin. In the presence of glutathione, geldanamycin was rapidly converted to 19-glutathionyl geldanamycin hydroquinone, suggesting its reactive nature. In contrast, the counterpart (17AAG) preferentially remained as its quinone form, which underwent extensive oxidative metabolism on both the 17-allylamino sidechain and the ansa ring. Only a small amount (<1%) of 19-glutathione conjugate of 17AAG was detected in the incubation of 17AAG with glutathione at 37 degrees C for 60 min. To confirm the differential nature of quinone-hydroquinone conversion between the two compounds, hypoxic incubations with human cytochrome P450 reductase at 37 degrees C and direct injection analysis were performed. Approximately 89% of hydroquinone, 5% of quinone, and 6% of 17-O-demethylgeldanamycin were observed after 1-min incubation of geldanamycin, whereas about 1% of hydroquinone and 99% of quinone were found in the 60-min incubation of 17AAG. The results provide direct evidence for understanding the 17-substituent effects of these benzoquinone ansamycins on their phase I metabolism, reactivity with glutathione, and acute hepatotoxicity.     

The metabolism of N-ethyl-N-methylaniline by rabbit liver microsomes: the measurement of metabolites by gas-liquid chromatography.

1. A method for the determination of N-ethyl-N-methylaniline and its metabolites by g.l.c. is described. 2. Following incubation in N-ethyl-N-methylaniline with rabbit liver microsomes for 60 min, over 95% of the substrate was accounted for as unchanged compound or metabolites. 3. N-Ethyl-N-methylaniline is metabolized in vitro by rabbit tissues mainly by N-oxidation and N-demethylation and to a lesser extent by N-deethylation and di-dealkylation. 4. Both major routes of metabolism were observed in homogenates prepared from rabbit liver and lung; in addition N-oxidation occurred in kidney and bladder tissue homogenates.     

Inhibitors of imipramine metabolism by human liver microsomes.

1. The aromatic 2-hydroxylation of imipramine was studied in microsomes from three human livers. The kinetics were best described by a biphasic enzyme model. The estimated values of Vmax and Km for the high affinity site ranged from 3.2 to 5.7 nmol mg-1 h-1 and from 25 to 31 microM, respectively. 2. Quinidine was a potent inhibitor of the high affinity site for the 2-hydroxylation of imipramine in microsomes from all three human livers, with apparent Ki-values ranging from 9 to 92 nM. This finding strongly suggests that the high affinity enzyme is CYP2D6, the source of the sparteine/debrisoquine oxidation polymorphism. 3. The selective serotonin reuptake inhibitors (SSRI), paroxetine, fluoxetine and norfluoxetine were potent inhibitors of the high affinity site having apparent Ki-values of 0.36, 0.92 and 0.33 microM, respectively. Three other SSRIs, citalopram, desmethylcitalopram and fluvoxamine, were less potent inhibitors of CYP2D6, with apparent Ki-values of 19, 1.3 and 3.9 microM, respectively. 4. Among 20 drugs screened, fluvoxamine was the only potent inhibitor of the N-demethylation of imipramine, with a Ki-value of 0.14 microM. 5. Neither mephenytoin, citalopram, diazepam, omeprazole or proguanil showed any inhibition of the N-demethylation of imipramine and the role of the S-mephenytoin hydroxylase for this oxidative pathway could not be confirmed.     

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.     

Stereoselective metabolism of prazepam and halazepam by human liver microsomes.

Metabolism of prazepam [PZ, 7-chloro-1,3-dihydro-5-phenyl-1- (cyclopropylmethyl)-2H-1,4-benzodiazepin-2-one] and halazepam [HZ,7- chloro-1,3-dihydro-5-phenyl-1-(2,2,2-trifluoroethyl)-2H-1,4- benzodizepin-2-one] was investigated in microsomes prepared from the livers of two male and one female subjects who died of head injuries. PZ (or HZ) and its metabolites were analyzed by normal phase and chiral stationary phase HPLC. The relative amount of products formed in the metabolism of PZ was found to be N-desalkylprazepam (NDZ, also known as N-desmethyldiazepam and nordiazepam) greater than 3-hydroxy-PZ (3-OH-PZ) much greater than oxazepam (OX). In contrast, the relative amount of products formed in the metabolism of HZ was found to be 3-OH-HZ much greater than NDZ greater than OX. Enantiomers of 3-OH-PZ and 3-OH-HZ were resolved by HPLC on an analytical column packed with the chiral stationary phase R-N-(3,5-dinitrobenzoyl)phenylglycine covalently bound to spherical particles of gamma-aminopropylsilanized silica. The 3-OH-PZ formed in the metabolism of PZ by three human liver microsomal preparations were found to have 3R/3S enantiomer ratios of 65:35, 61:39, and 62:38. In the metabolism of HZ, the enzymatically formed 3-OH-HZ had 3R/3S enantiomer ratios of 67:33, 60:40, and 62:38. N-Dealkylations of racemic 3-OH-PZ and 3-OH-HZ by human liver microsomal preparations were substrate-enantioselective; 3S-OH-PZ and 3R-OH-HZ were each N-dealkylated slightly faster than the corresponding antipode. The results indicated that both C3-hydroxylation of PZ and HZ as well as N-dealkylation of 3-OH-PZ and 3-OH-HZ catalyzed by human liver microsomes were stereoselective, resulting in the formation of a C3-hydroxylated product enriched (60-67%) in the 3R-enantiomer.     

Activation of mianserin and its metabolites by human liver microsomes

Human liver microsomes metabolise mianserin to the stable 8-hydroxymianserin, desmethylmianserin and mianserin-2-oxide and in addition to one or more chemically reactive metabolites which bind, irreversibly, to microsomal protein. The stable metabolites were isolated by HPLC and characterized by mass spectrometry. The generation of each of these metabolites showed substantial inter-individual variation between eight sets of human liver microsomes studied. Inhibition of irreversible binding was observed with SKF-525A together with concomitant decrease in the formation of 8-hydroxymianserin and desmethylmianserin but not mianserin-2-oxide. Methimazole inhibited binding and the formation of each of the metabolites at a low concentration. Quinidine did not significantly inhibit irreversible binding but did inhibit the formation of 8-hydroxymianserin. Sulphaphenazole had no effect on irreversible binding or metabolism. The irreversible binding of mianserin was inhibited by ascorbic acid, glutathione and N-acetyl cysteine, whereas N-acetyl lysine and trichloropropane oxide had no effect. The irreversible binding of mianserin, 8-hydroxymianserin and desmethylmianserin was of the same order of magnitude however significantly greater binding was observed with the desmethyl metabolite. Incubations with [10-3H/14C]mianserin showed no change in the 3H/14C ratio when irreversible binding occurred. Inhibition of irreversible binding was demonstrated with sodium cyanide at concentrations which did not inhibit total metabolism, which suggest that metabolic activation by the cytochrome P-450 enzyme system may lead to the formation of a reactive iminium intermediate that can bind to nucleophilic groups on proteins.     

Gender difference in ifosfamide metabolism by human liver microsomes.

Pharmacokinetic gender-dependent differences in cytochrome P450-mediated drug metabolism, especially CYP3A4, and their clinical implications are increasingly apparent. CYP3A4 seems to be the most important CYP isoform in both bioactivation and N-dechloroethylation of the alkylating prodrug ifosfamide, but informations about possible gender-related differences are lacking. Therefore we compared in 10 male and 10 female liver microsomal preparations the contents and activities of specific isoenzymes, involved in both metabolic pathways, especially CYP3A4, further CYP2A6, CYP2C9 and CYP2B6 and measured the in vitro activities of these microsomes in the ifosfamide 4-hydroxylation and N-dechloroethylation using high-sensitive HPLC/MS and -UV detection methods. Statistically significant differences between male and female livers were found in the mean CYP3A4 contents and activities. These differences had no consequences on the ifosfamide 4-hydroxylation activities of liver microsomes in vitro. In contrast, in the ifosfamide N-dechloroethylation reaction we found a statistically significant difference between the liver microsomes of male and female patients (0.13 +/- 0.05 nmol/min nmol P450 vs. 0.28 +/- 0.13 nmol/min x nmolP450, respectively). In conclusion, we firstly demonstrated such gender-related difference in the ifosfamide N-dechloroethylation, which could result in a higher risk of partly severe neurotoxic side effects in female patients.     

Characterization of metronidazole metabolism by human liver microsomes

The metabolism of metronidazole was studied in microsomes isolated from livers of human kidney donors. The formation of the major in vivo metabolite, hydroxymetronidazole, proceeded according to biphasic kinetics, suggesting the involvement of at least two enzymatic sites. The affinity constant (Km) of the high affinity site ranged from 140 to 320 microM and metabolism at this site contributed more than 75% of the intrinsic clearance. Thus, at therapeutic doses of metronidazole most of the hydroxylation in vivo should be associated with this site. Antipyrine, cimetidine, alpha-naphthoflavone, caffeine, theophylline, mephenytoin, tolbutamide, quinidine, acetone and nifedipine were poor inhibitors of the formation of hydroxymetronidazole by human liver microsomes. Propranolol (500 microM) inhibited the hydroxylation rate by 70%. Phenacetin inhibited metronidazole hydroxylation with a competitive inhibition constant (Ki) of 4-5 microM. However, metronidazole did not inhibit the O-deethylation of phenacetin. It is concluded that cytochromes P450 IA2, IIC9, IIC10, IID6, IIE1 and IIIA3 do not contribute significantly to the high affinity hydroxylation of metronidazole in man.     

The formation of procainamide hydroxylamine by rat and human liver microsomes

A method is described, using HPLC and electrochemical detection, which permits the direct quantitation of procainamide hydroxylamine. Procainamide hydroxylamine was formed from procainamide by hepatic microsomes from both rat and human, with rat microsomes showing higher apparent formation rates. The apparent Km for formation of procainamide hydroxylamine was 0.044 mM for rat liver microsomes, with an apparent Vmax of 2.81 nmol/min/mg of protein. Estimates of Km from three human microsomal samples were 6.29, 2.89, and 6.88 mM. Vmax estimates were 0.31, 0.74, and 0.74 nmol/min/mg of protein, respectively, roughly an order of magnitude less than that observed for the rat. Microsomal formation in both species was inhibited by boiling the microsomes, eliminating NADPH from the incubation system, by preincubation with SKF 525A, cimetidine, or n-octylamine, or by gassing the microsomal incubation mixture with carbon monoxide. These observations suggest that procainamide hydroxylamine formation is cytochrome P-450 mediated. Procainamide hydroxylamine could not be detected in the blood of rats treated with a single dose of procainamide, 100 mg/kg, po. One potential reason for the inability to detect this metabolite in blood is indicated by the rapid disappearance in vitro of procainamide hydroxylamine added to whole blood. Most of this disappearance appears to be due to an interaction with hemoglobin.     

Primaquine metabolism by human liver microsomes: effect of other antimalarial drugs.

A number of drugs have been studied for their effect on the metabolism of the antimalarial drug primaquine by human liver microsomes (N = 4) in vitro. The only metabolite generated was identified as carboxyprimaquine by co-chromatography with the authentic standard. Ketoconazole, a known inhibitor of cytochrome P450 isozymes, caused marked inhibition of carboxyprimaquine formation with IC50 and K(i) values of 15 and 6.7 microM, respectively. This finding and the dependency of metabolite formation on NADPH indicates that cytochrome P450 isozyme(s) catalysed metabolite production. Of compounds actually or likely to be coadministered with primaquine to malaria patients, only mefloquine produced any inhibition (K(i) = 52.5 microM). Quinine, artemether, artesunate, halofantrine and chloroquine did not significantly inhibit metabolite formation. It seems unlikely that the concurrent administration of mefloquine, or other antimalarials, with primaquine will lead to appreciably altered disposition.     

Zetidoline metabolism by rat liver microsomes. Formation of metabolites with potential neuroleptic activity

1-(3'-Chlorophenyl)-3-[2-(3,3-dimethyl-1-azetidinyl)ethyl] imidazolidin-2-one, zetidoline, a new neuroleptic agent, when incubated with rat liver microsomes was rapidly metabolized to six free (mets B, D, I, L, M and N) and two conjugated metabolites (mets E and F). Sites of the metabolic attack (oxidation) were primarily the aromatic moiety, then the imidazolidinone and the azetidine rings. The metabolites were purified and structures assigned by means of EI-MS, 1H-NMR and chemical synthesis (mets B, D, L and M). The main metabolites, zetidoline, some chemical analogues and a few known dopamine antagonists were tested as in vitro inhibitors of 3H-zetidoline and 3H-spiperone binding to rat striatal membranes, and as in vivo inducers of prolactin release in female rats (inhibition of the estrus cycle). Two zetidoline metabolites, namely 4'-hydroxy zetidoline (met. B) and 5-hydroxy zetidoline (met. L), were found to have both in vitro and in vivo activities comparable to those of the parent drug. Identification of these active hydroxylated metabolites appears important both in the search of new leads of neuroleptics and for designing pro-drugs derivatives with improved pharmacokinetic profiles.     

The metabolism of a chlorinated epoxide (MME): diol formation by pig liver microsomes.

A cyclodiene expoxide MME with one methyl group attached to the oxirane ring was converted into a single metabolite (M₁) when incubated with pig liver microsomes in the absence of NADPH. Mass spectroscopy of M₁ and its TMS derivative established that it was a diol, indicating that the conversion was mediated by epoxide hydratase. N.m.r. spectra of M₁ with and without the chiral shift reagent, EU (fod)₃ suggest that the metabolite is a cis-diol. When MME was hydrated in acid solution, a diol was produced which differed from M₁ in its Chromatographic behaviour and n.m.r. spectrum. The rate of formation of M₁ from MME by pig liver microsomes was very much slower (less than 0.4 per cent) ofthat found with the analogous epoxide HEOM which has no methyl group attached to the oxirane ring.     

Enzymatic formation of chemically reactive metabolites of N-nitrosodesmethyl tripelennamine by a mechanism other than N-dealkylation.

Benzyl-¹⁴C and N-methyl-³H labeled N-nitroso derivatives (NDT) of tripelennamine were synthesized by reacting the corresponding labeled parent drug with sodium nitrite at pH 1–2, and their covalent binding to rat liver musomes was compared with that of radiolabeled tripelennamine. The covalent binding of these substances is mediated by liver musomal cytochrome P-450 enzymes; it required an NADPH-generating system and oxygen, and was inhibited by CO:O₂ (8:2). Reduced glutathione (1 mM) also inhibited the covalent binding. The covalent binding of NDT to liver musomal proteins from phenobarbital-pretreated rats was ten times greater than that of tripelennamine. A K_m of 60 μM and a V_max of 1 nmole/mg of protein/min were obtained for the covalent binding of NDT. Phenobarbital and acetone pretreatments of rats increased covalent binding of both the drug and its nitroso derivative, while pretreatment with 3-methyl-cholanthrene decreased their binding. These results suggest that chemically reactive intermediates of tripelennamine and NDT are involved in the covalent binding. Under the conditions in vitro employed, the rate of N-demethylation of NDT was lower than that of the parent drug. Moreover, the covalent binding of the N-methyl-³H labeled nitroso derivative was equivalent to that of the benzyl-¹⁴C labeled derivative. Thus, N-demethylation is not a requisite for the covalent binding of tripelennamine and its N-nitroso derivative. The mechanism of the covalent binding of the N-nitroso derivative of tripelennamine, therefore, differs from that of dimethylnitrosamine.     

氯米帕明在人肝微粒体中的N—去甲基代谢

目的：研究ＣＹＰ４５０选择性抑制剂对体外氯米帕明（Ｃｌｏ）Ｎ－去甲基代谢的影响。方法：应用米氏方程计算肝微粒体中Ｃｏｌ Ｎ－去甲基代谢的动力学参数，比较加入抑制剂前后这些参数的改变。结果：Ｋｍ１，Ｋｍ２，Ｖｍａｘ１，Ｖｍａｘ２，Ｖａｍｘ２／Ｖｍ１和Ｖａｍｘ２／Ｋｍ２分别为（０．１１±０．０６），（２４±１１）μｍｏｌ·Ｌ＾－１，（１１４±４７），（４２８±１８８）ｎｍｏｌ·ｇ＾－１·ｍｉｎ＾－１。     

Novel metabolites of buprenorphine detected in human liver microsomes and human urine.

The in vitro metabolism of buprenorphine was investigated to explore new metabolic pathways and identify the cytochromes P450 (P450s) responsible for the formation of these metabolites. The resulting metabolites were identified by liquid chromatography-electrospray ionization-tandem mass spectrometry. In addition to norbuprenorphine, two hydroxylated buprenorphine (M1 and M2) and three hydroxylated norbuprenorphine (M3, M4, and M5) metabolites were produced by human liver microsomes (HLMs), with hydroxylation occurring at the tert-butyl group (M1 and M3) and at unspecified site(s) on the ring moieties (M2, M4, and M5). Time course and other data suggest that buprenorphine is N-dealkylated to form norbuprenorphine, followed by hydroxylation to form M3; buprenorphine is hydroxylated to form M1 and M2, followed by N-dealkylation to form M3 and M4 or M5. The involvement of selected P450s was investigated using cDNA-expressed P450s coupled with scaling models, chemical inhibition, monoclonal antibody (MAb) analysis, and correlation studies. The major enzymes involved in buprenorphine elimination and norbuprenorphine and M1 formation were P450s 3A4, 3A5, 3A7, and 2C8, whereas 3A4, 3A5, and 3A7 produced M3 and M5. Based on MAb analysis and chemical inhibition, the contribution of 2C8 was higher in HLMs with higher 2C8 activity, whereas 3A4/5 played a more important role in HLMs with higher 3A4/5 activity. Examination of human urine from subjects taking buprenorphine showed the presence of M1 and M3; most of M1 was conjugated, whereas 60 to 70% of M3 was unconjugated.