Isolation and identification by FAB mass spectrometry and NMR spectroscopy of a demethylated metabolite of FK506 from erythromycin-induced rabbit liver microsomes

A new demethylated metabolite, extracted from erythromycin-induced rabbit liver microsomes incubation media, isolated by HPLC and identified by FAB/MS and NMR, is described. Moreover, NMR spectra suggest the existence of tautomeric forms of this O-demethylated metabolite of FK506.     

Isolation and identification of new rapamycin dihydrodiol metabolites from dexamethasoneinduced rat liver microsomes

1. Rapamycin is metabolically transformed in rat liver microsomes to 3,4- and 5,6- dihydrodiol metabolites under the influence of the cytochrome P-450 mixed function oxygenase system. These metabolites were produced from dexamethasone-induced as well as from non-induced rat liver microsomes. The comparison of the ion spray mass spectra of the 5,6-dihydrodiol with the 3,4-dihydrodiol of rapamycin shows clearly that dihydrodiols were formed in two distinct positions of rapamycin. 2. FAB mass spectra as well as electrospray mass spectra of two additional peaks isolated from the same chromatographic run confirm the presence of a 3,4-dihydrodiol metabolite of rapamycin as also strongly suggested by UV spectra.Hplc reinjection of each individualpeak always resultedinchromatograms showing a combinationof thesame three peaks and therefore are to be considered as tautomers of the 3,4-dihydrodiol of rapamycin. 3. These tautomeric conformations were found to have no immunosuppressive potency, most probably due to important structural and stereochemical modifications of the rapamycin binding domain to the binding protein (FKBP-12) and or to important metabolic structural modifications of rapamycin effector domain.     

Isolation and mass spectrometric identification of two metabolites of FK 506 from rat liver microsomal incubation media.

A transient epoxide in equilibrium with its possible isomeric forms, most probably induced easily by neighboring-group assistance of a hydroxy group, and a dihydrodiol formed under the influence of the hydrase enzymic activity and/or by simple hydrolysis were isolated by HPLC and identified by FAB/MS from rat liver microsomal incubation media.     

Isolation, identification and immunosuppressive activity of SDZ-IMM-125 metabolites from human liver microsomes.

SDZ-IMM-125 N-methyl leucine 9 hydroxylated in the gamma position is a metabolite which was extracted from incubated human liver microsomes and subsequently separated by normal and reverse-phase HPLC. This metabolite was identified by fast atom bombardment mass spectrometry, electrospray-ms/ms mass spectrometry and nuclear magnetic resonance spectroscopy. The in vitro 50% inhibitory concentration, tested against bidirectional mixed lymphocyte reaction was 80 microg/l indicating that this metabolite does not retain in vitro immunosuppressive activity most probably due to the structural modification of SDZ-IMM-125 in the recognized binding region to cyclophilin A reducing its binding affinity relative to the parent drug.     

Metabolism of cyclosporin A. II. Implication of the macrolide antibiotic inducible cytochrome P-450 3c from rabbit liver microsomes

The in vitro metabolism of cyclosporin A (CsA) was investigated by rabbit liver microsomes in order to identify the form(s) of cytochrome P-450 responsible for its biotransformation. Metabolites including monohydroxy-, N-demethylated, dihydroxy- and dihydroxy-N-demethylated derivatives were detected and quantified by HPLC from incubates of liver microsomes, CsA, and NADPH. Kinetic data indicated that monohydroxy- and N-demethylated derivatives were first generated and then served as substrates for production of dihydroxylated derivatives. Liver microsomes from phenobarbital-, beta-naphthoflavone-, triacetyloleandomycin-, erythromycin-, or rifampicin-treated and untreated rabbits were investigated, but only microsomes from animals treated with macrolide antibiotics (specific inducers of form P-450 3c) exhibited a type I binding spectrum upon CsA addition (Ks = 1.5 +/- 0.5 microM) and extensively metabolized the drug to all groups of derivatives (Km = 5.0 +/- 0.5 microM, Vmax = 1.0 +/- 0.2 nmol/mg/min). A linear correlation existed between CsA oxidase activity and P-450 3c specific content. Antibodies to P-450 3c strongly inhibited CsA oxidase activity of microsomes from macrolide antibiotic-induced animals, whereas antibodies to other forms, including P-450 2, 3b, 4, and 6, did not. When highly purified forms of P-450, including P-450 2, 3b, 3c, and 4, were assayed in a reconstituted system, only P-450 3c exhibited type I binding spectrum upon CsA addition (Ks = 1.4 +/- 0.5 microM) and extensively metabolized the drug to all derivatives. We conclude that the macrolide antibiotic-inducible form P-450 3c (or P-450 3c related from(s)) is responsible for the major part of CsA metabolism by rabbit liver microsomes.     

Metabolism of naphthalene to naphthalene dihydrodiol glucuronide in isolated hepatocytes and in liver microsomes

The functional linkage between UDP-glucuronyltransferase (GT) and the monooxygenase-epoxide hydratase system was investigated in studies on the glucuronidation of naphthalene dihydrodiol, which was formed by epoxide hydratase during the metabolism of naphthalene. (1) Naphthalene metabolism was compared in isolated hepatocytes and in liver microsomes incubated with an NADPH regenerating system and UDP-glucuronic acid. Naphthalene dihydrodiol glucuronide was a major metabolite in isolated hepatocytes. In the liver microsomal system free dihydrodiol by far exceeded its glucuronide unless the positive allosteric effector of GT, UDP-N-acetylglucosamine, was added. (2) Treatment of rats with phenobarbital or 3-methylcholanthrene, although markedly enhancing the formation of naphthalene dihydrodiol, did not stimulate liver microsomal GT (naphthalene dihydrodiol as substrate). The results suggest that activation of GT by UDP-N-acetylglucosamine is an important factor in the coupling of glucuronidation to functionally linked microsomal enzyme reactions.     

Isolation and mass spectrometric characterization of dimeric adenine photoproducts in oligodeoxynucleotides.

UVC irradiation of oligodeoxynucleotides (ODNs) at 254 nm generates two types of DNA photoproducts, A=A and (AA)*, at adjacent adenine sites in DNA. Kumar et al. previously proved the structure of these adducts in dinucleoside monophosphates [Kumar, S., et al. (1987) Nucleic Acids Res. 15, 1199-1216; Kumar, S., et al. (1991) Nucleic Acids Res. 19, 2841-2847]. Product-ion spectra of ESI-produced [M - 2H](2-) ions of the ODNs containing the dimeric adenine photoproducts show distinctive fragmentation that is informative of the structures of the photoproducts. The gas-phase cleavages of ODNs at sites of those photoproducts and thymine thymine or thymine adenine ((TA)*) photoproducts are analogous to cleavages induced by hot alkaline treatment. Nuclease-P1 digestions of ODNs containing dimeric adenine photoproducts give shorter pieces of ODNs bearing the photoproducts, which fragment under collisional activation conditions in a similar way to the large ODNs containing the photoproducts. The tandem mass spectrometric results show that the yield of (AA)* is lower than that of A=A when adjacent adenines are in the middle of an ODN sequence, and the yield of the latter is similar to that of (TA)*.     

Oxidative deamination of cyclohexylamine and its homologs by rabbit liver microsomes.

Cyclohexylamine (CHA) and its homologs, cyclopentylamine (CPA) and cycloheptylamine (CHPA), which formed the type II spectral changes in hepatic microsomes, were deaminated to the corresponding ketones by rabbit liver microsomes in the presence of NADPH and molecular oxygen. The alicyclic ketones were then reduced to the alcohols, of which average percentages in the deaminated products were approximately 75 (CHA), 3 (CPA) and 14 (CHPA). The apparent K_m's for these amines were 5.0 mM (CHA), 4.2 mM (CPA) and 2.1 mM (CHPA), and V_max's were 11.0 (CHA), 42.1 (CPA) and 16.4 (CHPA) nmoles/mg protein/30 min. The activity of deamination of these alicyclic primary amines was dependent on both NADPH and oxygen, and inhibited by carbon monoxide, SKF 525A, metyrapone, potassium cyanide and mercuric chloride. These experiments indicate that the deamination of the alicyclic primary amines is catalyzed by a microsomal cytochrome P-450-dependent monooxygenase system in the rabbit liver. Cyclohexanone oxime and other oximes were also identified from the incubation mixtures, and these oximes are suggested as possible intermediates of microsomal deamination of alicyclic primary amines.     

Lipid peroxidation activity mediated by NADPH-cytochrome C reductase purified from rabbit liver microsomes.

Purified NADPH-cytochrome c reductase of rabbit liver microsomes was examined to determine whether or not the reported low lipid peroxidation activity of rabbit liver microsomes is due to the enzyme, NADPH-cytochrome c reductase. NADPH-cytochrome c reductase was purified from phenobarbital-treated rabbit liver microsomes to a specific activity of 14.9 to 21.4 unit per mg of protein with a yield of 15.2 to 16.4%. The purified sample (21.4 unit/mg of protein) was almost homogenous as determined by sodium dodecylsulfate gel electrophoresis. This sample was used for determining lipid peroxidation activity. EDTA and ferrous ion but not ADP were essential requirements for the activity. FMN enhanced the activity when low concentrations of the NADPH-cytochrome c reductase were used for the assay. NADP and 2'-AMP, which are inhibitors of NADPH-cytochrome c reductase, inhibited the lipid peroxidation activity. a-Tocopherol and p-chloromercuribenzoate (PCMB) also inhibited the activity. From these results, we confirmed the rabbit liver microsomal enzyme NADPH-cytochrome c reductase plays a role in lipid peroxidation activity. The reported low lipid peroxidation activity in rabbit liver microsomes does not appear to be caused by the NADPH-cytochrome c reductase.     

Nitrite binding to rabbit liver microsomes and effects on aminopyrine demethylation.

Sodium nitrite was examined for its ability to interact with liver microsomes from phenobarbitaltreated rabbits. In dithionite- or NADPH-reduced microsomes, nitrite rapidly produced a difference spectrum with α, β and Soret peaks at 586, 510, and 446 nm, respectively, with a large trough at 417 nm. The Soret peak diminished with time as the trough deepened and shifted to 429 nm. Concomitant with the development of the difference spectrum was a decrease in the ability of reduced cytochrome P-450 to bind carbon monoxide. When preincubated with NADPH-reduced microsomes under anaerobic conditions, nitrite behaved as a noncompetitive inhibitor of aminopyrine demethylase activity. Inhibition kinetics revealed two components with apparent inhibition constants of 0.2 and 31 mM nitrite. Maximum inhibition of aminopyrine demethylase by nitrite was obtained only after preincubation under anaerobic and reducing conditions. The rate and extent of inhibition were increased by decreasing the pH of the medium during preincubation. Inhibition of aminopyrine demethylation by nitrite does not appear to be mediated by oxidation effects of nitrite on lipids or essential sulfhydryl groups of eytochrome P-450. The evidence suggests that nitrite or a reduction product of nitrite, such as nitric oxide, binds tightly to reduced cytochrome P-450 to prevent carbon monoxide binding and inhibit aminopyrine demethylation activity.     

A liquid chromatographic-electrospray-tandem mass spectrometric method for quantitation of quetiapine in human plasma and liver microsomes: application to study in vitro metabolism

Quetiapine is an atypical antipsychotic agent for the treatment of schizophrenia. After an oral dose it is absorbed rapidly and extensively metabolized in the liver, resulting in low plasma concentrations of the parent drug. A sensitive analytical method is needed. A liquid chromatographic-electrospray-tandem mass spectrometric (LC-ESI-MS-MS) method combined with a simple liquid-liquid extraction has been developed for the measurement of quetiapine in human plasma and in human liver microsomes (HLM). Clozapine is used as internal standard. Plasma samples or microsomes quenched with methanol (100 microL) were made basic and extracted with 3 mL n-butyl chloride. The reconstituted extracts were analyzed by LC-ESI-MS-MS. Selective reaction monitoring of MH(+) at m/z 384 and 327 resulted in strong fragment ions at m/z 253 and 192 for quetiapine and clozapine, respectively. Recovery of quetiapine and clozapine ranged from 62 to 73%. Intrarun accuracy and precision determined at 1.0 (lower limit of quantitation), 2.5, 200, and 400 ng/mL did not exceed 7% deviation from target and the %CV did not exceed 5.5%. The % target +/- %CV for interrun accuracy and precision were at least 95% +/- 7.4% at concentrations of 2.5, 200, and 400 ng/mL. Plasma samples (2.5 and 400 ng/mL) stored at room temperature for 24 h or after 3 cycles of freeze/thaw were all stable (maximum % deviation < or = 11.0%). Processed extracts (2.5 and 400 ng/mL) stored for 7 days at -20 degrees C or 6 days on the autosampler were all stable (maximum % deviation < or = 11.5%). The method has been used to study quetiapine utilization during incubation with HLM or with cDNA-expressed human cytochrom P450s (CYP). Quetiapine is extensively metabolized by CYP 3A4 and CYP 2D6 and to a lesser extent by CYP 3A7, CYP 3A5, and CYP 2C19.     

吡喹酮在诱导和未诱导大鼠肝微粒体内的羟化代谢概貌及其羟化物的质谱鉴定

用细胞色素Ｐ４５０（Ｐ４５０）特异诱导剂研究参与吡喹酮（ＰＱＴ）代谢的Ｐ４５０同工酶，在未诱导肝微粒体内，ＰＱＴ代谢仅生成其Ｄ环单羟化物．在β－萘黄酮诱导肝微粒体内，ＰＱＴ代谢后也生成其Ｄ环单羟化物．ＰＱＴ在苯巴比妥诱导的肝微粒体内代谢生成Ｄ环，Ｂ环和Ａ环三个单羟化物．在地塞米松和红霉素诱导的肝微粒体内，ＰＱＴ代谢生成七个代谢产物．结果表明参与ＰＱＴ分子羟化的Ｐ４５０同工酶至少包括ＣＹＰ１Ａ，ＣＹＰ２Ｂ和ＣＹＰ３Ａ亚家族，每个亚家族代谢ＰＱＴ的概貌各不相同，ＣＹＰ３Ａ优先羟化Ａ环，ＣＹＰ２Ｂ优先羟化Ｄ环和Ｂ环，ＣＹＰ１Ａ则几乎仅羟化Ｄ环．     

吡喹酮在诱导和未诱导大鼠肝微粒体内的羟化代谢概貌及其羟化物的质谱鉴定

用细胞色素P450（P450）特异诱导剂研?究参与吡喹酮（PQT）代谢的P450同工酶，在未诱导肝微粒体内，PQT代谢仅生成其D环单羟化物. 在β-萘黄酮诱导肝微粒体内，PQT代谢后也生成其D环单羟化物. PQT在苯巴比妥诱导的肝微粒体内代谢生成D环，B环和A环三个单羟化物. 在地塞米松和红霉素诱导的肝微粒体内，PQT代谢生成七个代谢产物. 结果表明参与PQT分子羟化的P450同工酶至少包括CYP1A，CYP2B和CYP3A亚家族，每个亚家族代谢PQT的概貌各不相同，CYP3A优先羟化A环，CYP2B优先羟化D环和B环，CYP1A则几乎仅羟化D环.     

Comparative metabolism of clinically important precursors of N-desmethyldiazepam using phenobarbitone-pretreated rat liver microsomes.

Phenobarbitone-pretreated male Sprague-Dawley rat liver microsomes were used to examine C3-hydroxylation and N-dealkylation of four clinically important benzodiazepines: diazepam (DZP), prazepam (PZP), pinazepam (PIN) and halazepam (HZP). These substrates differ only in the nature of the N-substituent of the B ring and N-desmethyldiazepam (DMD) is the N-dealkylation product in each case. C3-Hydroxylation was accordingly also studied with DMD as substrate. All monooxygenations were studied with substrates at a concentration of 10 microM, in the absence of solubilizing agents, and under conditions where the production of secondary metabolites was minimized. A 20-fold variation in the rate of C3-hydroxylation was recorded across the five substrates with HZP showing the highest rate and DMD showing the lowest rate. An almost equally large range of variation was shown for the N-dealkylation reaction, with PZP undergoing this biotransformation more than 17 times faster than DZP. Log P values (a measure of lipophilicity) for the five substrates were determined using an HPLC method and a remarkable lack of correspondence between this substrate parameter and either of the monooxygenations was noted. This suggests that multiple substrate determinants govern the relative rates of these monooxygenations. It was, however, notable that the additive rate of metabolism of these substrates by both monooxygenase routes did show an excellent correlation with substrate lipophilicity.     

In vitro metabolism of cyclosporin A with rabbit renal or hepatic microsomes: analysis by HPLC-FPIA and HPLC-MS

 Cyclosporin A (CsA) is in vivo mainly metabolized by hepatic cytochrome P450 IIIA to more than 21 metabolites, the major ones known as: M1, M17 and M21. The aim of this work is to explore the in vitro metabolism of CsA after incubation, in the presence of NADPH, with renal or hepatic microsomes obtained from rabbits pretreated with rifampycin (enzyme inducer) or erythromycin (enzyme inhibitor). The presumed metabolites were separated by semi-preparative high-performance liquid chromatography (HPLC) and identified in each collected fraction by fluorescence polarization immunoassay (FPIA) (HPLC-FPIA) using a non-specific polyclonal antibody. They were also analyzed by HPLC-mass spectrometry (MS) using fast atom bombardment (HPLC-MS-FAB). Five collected fractions gave positive results with FPIA. The major metabolites found were M1, M17 and M21 after identification by HPLC-MS-FAB and comparison with three corresponding standard metabolites. The CsA biotransformation rates were calculated by the amount of unmetabolized CsA and were linear with time. These mean rates (V_m) for 12-min incubation by renal microsomes of rabbits treated with rifampicin or erythromycin or untreated (control) were 0.11, 0.02 and 0.04 nmol/min×mg microsomal protein, respectively. These rates were 15 -, 37 -, and 30-fold lower than those obtained with hepatic microsomes of rabbits treated identically. As CsA metabolites are less cytotoxic than the parent drug, this weak renal biotransformation of CsA after in vitro incubation should be one of the mechanisms of its in vivo nephrotoxicity. Received: 14 September 1994/Accepted: 21 November 1994     

Isolation and identification of a novel human metabolite of cyclosporin A: dihydro-CsA M17

A novel metabolite of cyclosporin A was observed in human blood and urine. An analytical sample of this metabolite was isolated from human urine and the structure was determined to be (8-hydroxy-6,7-dihydro-MeBMT1) cyclosporin based on the 1H-NMR, 13C-NMR, FAB-MS, and HPLC characteristics of the biological sample as well as by comparison with a synthetically derived authentic sample. The significance of this metabolite in terms of the pathway by which cyclosporin A is metabolized is discussed.     

High-resolution mass spectrometry-based approach for the identification and profiling of the metabolites of taletrectinib formed in liver microsomes

Taletrectinib is a potent, orally active, and selective ROS1/NTRK kinase inhibitor. The aim of this study was to study the metabolism of taletrectinib in rat, dog, and human liver microsomes. The biotransformation of taletrectinib was carried out using rat, dog, and human liver microsomes supplemented with nicotinamide adenine dinucleotide phosphate tetrasodium salt (NADPH) and uridine diphosphate glucuronic acid (UDPGA). The microsomal incubations were conducted at 37°C for 60 min. The formed metabolites were identified by ultrahigh performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS) using electrospray ionization in the positive ion mode. They were identified by accurate masses and MS/MS spectra and based on their fragmentation pathways. With UHPLC-HRMS, a total of 10 metabolites including one glucuronide conjugate (M7) were structurally identified. M9 and M10 were unambiguously identified as taletrectinib alcohol and taletrectinib ketone, respectively, using reference standards. The phase I metabolic pathways of taletrectinib involved N-dealkylation, O-dealkylation, oxidative deamination, and oxygenation; the phase II metabolic pathways referred to glucuronidation. The current study investigated the in vitro metabolic fate of taletrectinib in animals and human species, which would bring us considerable benefits for the subsequent studies focusing on the pharmacological effect and toxicity of this drug.     

Chromatographic isolation and mass spectrometric identification of a base-induced mebendazole fluorophor

Sodium hydroxide treatment of the benzoylbenzimidazole anthelmintics mebendazole and flubendazole produces yellow solutions that possess practically no fluorescence characteristics under various conditions. However, when spotting the alkaline solutions on filter paper and examining the spots under U.V., strong bluish-white fluorescence is obtained. When pouring liquid nitrogen over the spots, a very intense bluish-white fluorescence followed by a long-lasting greenish phosphorescence is observed. These luminescence phenomena allow visualization of 1 ng of mebendazole and of 5 ng of flubendazole per spot. A preparative separation by means of column liquid chromatography was worked out for the isolation of the fluorophor in case of mebendazole. Combined spectroscopic methods indicated the formation of a primary amine function in position 2 of the imidazole nucleus by hydrolytic cleavage of the -NH-CO-bond. A discussion on the mechanism of fluorescence is given.