Normal-phase liquid chromatography-particle-beam mass spectrometry in drug metabolism studies of the dopamine receptor antagonist Odapipam and the m uscarine M1 receptor agonist Xanomeline

1. The metabolism of Odapipam has been studied with phenobarbital-induced rat liver microsomes, followed by analysis with normal-phase hplc in combination with particlebeam mass spectrometry. 2. During the incubation of Odapipam, five different metabolites were formed. The EI mass spectra of the metabolites indicated the formation of N-desmethyl-Odapipam, 1-hydroxy-Odapipam, the two isomers of 3′-hydroxy-Odapipam and a metabolite which was dehydrogenated in the dihydrobenzofuran moiety. 3. The intrinsic hepatic extraction ratio and metabolism of Xanomeline has been studied in the perfused rat liver. Increasing the input concentration resulted in measurable concentrations of Xanomeline in the perfusate, although the extraction ratio was still > 0·9 at 140 μM. 4. Analysis of the perfusate by normal-phase hplc and particle-beam mass spectrometry showed the formation of at least six metabolites. The EI⁺ mass spectrum of the metabolites indicated the formation of ω-3 hydroxy-, ω-2 hydroxy-, ω-1 hydroxy-, ω-1 ketoXanomeline in addition to ω-1 hydroxy-N-desmethyl-Xanomeline and an N-oxide of Xanomeline. 5. The results show that normal-phase hplc based on silica material is superior to reversed-phase-based systems in terms of selectivity. Furthermore, the use of non-aqueous solvents in combination with particle-beam mass spectrometry is advantageous compared with reversed-phase hplc since changing between different solvents in normal-phase hplc results only in minor changes in the particle-beam interface parameters such as nebulizer position, helium pressure and interface temperature.     

Metabolism of a novel CCK-B antagonist in rat,dog and human liver microsomes

1. The in vitro metabolism of a novel CCK-B antagonist ((+)-N-[1-adamantane-1- methyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-1,5-benzodiazepin-3-yl]N′-phenylurea; GV150013X) was investigated using rat, dog and human liver microsomes. 2. Four monohydroxy and four dihydroxy metabolites of GV150013X in rat and man were identified by comparison with authentic standards using HPLC and mass spectrometry. 3. The dihydroxy metabolite M1 was not detected in dog liver microsomes mixtures. 4. The formation of dihydroxylated metabolites proceeds via monohydroxylated metabolites M5 and M8 and not directly from GV150013X. 5. A monohydroxy metabolite M5 was the major metabolite in rat and dog, with M5 and dihydroxy metabolites M2 and M3 major metabolites in man.     

In-vitro metabolic studies of tacrolimus using precision-cut rat and human liver slices

The objective of this study was to investigate the in-vitro metabolism of tacrolimus in liver slices from rats and humans. [¹⁴C]Tacrolimus (2 or 20 μM) was incubated with precision-cut human and rat liver slices in 12-well plates for up to 12 h. Concentrations of tacrolimus and metabolites were determined by high-performance liquid chromatography (HPLC) radiochromatography. The 13-O-demethylated tacrolimus metabolite (M-I) was the major oxidative metabolite in both rat and human liver slices. The other primary metabolites of tacrolimus (M-II, M-III, and M-IV) were not seen in either species. Unidentified peaks, which eluted early in the HPLC system, were probably due to secondary or conjugated metabolites. The eluate had no pharmacological activity. The finding that M-I was the major tacrolimus metabolite in both human and rat liver slice preparations is consistent with previous studies of rat and human liver microsomes.     

Biliary excretion of novel pneumotoxic metabolites of the pyrrolizidine alkaloid, monocrotaline

W. M. Lafranconi, S. Ohkuma and R. J. Huxtable. Biliary excretion of novel pneumotoxic metabolites of the pyrrolizidine alkaloid, monocrotaline. Toxicon23, 983–992, 1985. — Perfusion of the pyrrolizidine alkaloid, monocrotaline, through the isolated rat liver resulted in the appearance of Ehrlich-positive metabolites in both the perfusate and bile. Livers from male rats released greater quantities of metabolite into both bile and perfusate. Metabolite release was stimulated by pretreatment with phenobarbital. Livers of phenobarbital-pretreated male rats perfused with 300 μM monocrotaline produced biliary metabolite concentrations in excess of 5 mM. Metabolite release was inhibited by anoxic perfusion; low temperature and pretreatment with SKF-525A. Above 150 μM monocrotaline, bile became the predominant route of excretion. On perfusion through the isolated lung of the rat, bile and perfusate metabolites were equally effective in inhibiting serotonin transport. A single Ehrlich-positive peak was obtained on silica gel column chromatography of bile containing metabolite. Mass spectrometry revealed the major component of this peak to have a molecular weight of 281, indicating a novel pyrrole metabolite in which the esterifying acid present in monocrotaline, monocrotalic acid, had been partially degraded. This compound mimics the pneumotoxic action of monocrotaline giver, in vivo, and its availability should prove a valuable tool in the elucidation of the mechanism of pyrrolizidine toxicity.     

Characterization of metabolites of worenine in rat biological samples using liquid chromatography–tandem mass spectrometry

The in vivo and in vitro metabolites of worenine in rat were identified or characterized using a specific and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. In vivo samples including rat urine, feces, and plasma samples were collected after ingestion of 25 mg/kg worenine to healthy rats. The in vivo and in vitro samples were cleaned up by a solid-phase extraction procedure (C18 cartridges) and a liquid–liquid extraction procedure, respectively. Then these pretreated samples were injected into a reversed-phase C18 column with mobile phase of methanol–ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by an on-line MS/MS system. As a result, at least twenty-seven metabolites and the parent medicine were found in rat urine after ingestion of worenine. Seven metabolites and the parent medicine were identified or characterized in rat feces. Three metabolites and the parent medicine were detected in rat plasma. One metabolite was found in the rat intestinal flora incubation mixture, and three metabolites were characterized in the homogenized liver incubation mixture. The main phase I metabolism of worenine in rat was dehydrogenization, hydrogenation, hydroxylation, and demethylene reactions, and that of phase II was sulfation and glucuronidation.     

Quantitation of DNA reactive pyrrolic metabolites of senecionine – A carcinogenic pyrrolizidine alkaloid by LC/MS/MS analysis

Pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals, inducing liver tumors in experimental rodents. We previously determined that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), 7-glutathione-DHP, 7-cysteine-DHP, 7-N-acetylcysteine-DHP, and 1-CHO-DHP are DNA reactive pyrrolic metabolites potentially associated with PA-induced liver tumor initiation. In this study, we developed an LC/MS/MS multiple reaction monitoring (MRM) mode method to identify and quantify these metabolites formed from the metabolism of senecionine, a carcinogenic PA, by mouse, rat, and human liver microsomes, and primary rat hepatocytes. Together with the chemically prepared standards of these metabolites, this represents an accurate and convenient LC/MS/MS analytical method for quantifying these five reactive pyrrolic metabolites in biological systems.     

Disposition of the opioid antagonist,nalmefene, in rat and dog

1. The disposition of nalmefene in rat and dog was studied using in vitro and in vivo methodology. In vitro metabolite profiles were obtained following incubation of nalmefene with liver microsomes and biological fluids were assayed to profile in vivo metabolites. Characterization of metabolites was accomplished using hplc, co-chromatography with synthetic standards, or LC/MS.

2. In rat, tissue distribution and metabolite plasma concentration-time data were obtained following intravenous bolus dosing of nalmefene.

3. The results indicate that the primary phase I metabolite of nalmefene from liver microsome incubations was the N-dealkylated metabolite, nornalmefene. Quantitative metabolite production was rat ? dog. In vivo, nornalmefene glucuronide was the major metabolite in rat urine, whereas nalmefene glucuronide(s) were predominant in dog urine.

4. More than 90% of the radioactive dose was recovered in the rat excreta and tissues 24?h after an intravenous bolus dose of ¹⁴C-nalmefene, with no apparent organ-specific retention of radioactivity.

5. Pharmacokinetic analysis of the rat plasma metabolite data indicated that terminal half-lives for nalmefene and nornalmefene were comparable (～ 1?h). However, C_max and AUC of nornalmefene were ≤ 7% that of corresponding nalmefene values.     

催醒宁在大鼠离体灌流肝脏中的生物转化

用离体大鼠肝脏灌流方法,研究了胆碱酯酶抑制剂CXN的生物转化过程。径HPLC分离纯化及光谱分析,鉴定了CXN的六个脂溶性代谢产物的化学结构。产物Ⅰ为CXN原形,其余均为氧化产物。其中产物Ⅲ尚保留部分抑酶活力,而产物Ⅱ,Ⅴ及Ⅵ对小鼠全脑胆碱酯酶的抑酶活力明显下降。另外还观察到,代谢产物Ⅱ及Ⅴ对小鼠的急性毒性也明显减小。     

Metabolism of benzene and trans, trans-muconaldehyde in the isolated perfused rat liver

Perfusate from rat livers perfused with benzene (0.7–7 × 10⁻⁴ M) or trans,trans-muconaldehyde (MUC) (10⁻⁴ M) was extracted and analyzed by reverse-phase HPLC. Based on retention time and co-elution experiments, benzene was found to be metabolized to trans,trans-muconic, acid, a urinary ring-opened metabolite of benzene and a major in vivo and in vitro metabolite of MUC. These data demonstrate that benzene ring-opening occurs in the liver. Following perfusion with MUC (a microsomal hematotoxic metabolite of benzene), trans,trans-muconic acid and three other MUC metabolites were detected in the perfusate extract, suggesting that these metabolites would be present in the circulation following metabolism of MUC.     

Characterization of metabolic profile of mosapride citrate in rat and identification of two new metabolites: Mosapride N-oxide and morpholine ring-opened mosapride by UPLC–ESI-MS/MS

The identification and structure elucidation of metabolites of mosapride, a selective gastroprokinetic agent, was investigated in rats. After oral administration, samples of rat urine, bile, feces and plasma were collected and analyzed by a selective UPLC–ESI-MS/MS method. Altogether 18 metabolites were detected and at least 15 metabolites were reported in rat for the first time. Two new metabolites, mosapride N-oxide in rat bile, urine and plasma, morpholine ring-opened mosapride in plasma and feces, were identified by comparison with the reference standards. One known major mammalian metabolite, des-p-fluorobenzyl mosapride, was also identified. The molecular structures of nine phase I metabolites and six phase II metabolites of mosapride were elucidated based on the characteristics of their protonated molecular ions, product ions and chromatographic retention times. The phase I metabolites were mainly transformed by four main metabolism pathways, dealkylation, N-oxidation, morpholine ring cleavage and hydroxylation, with dealkylation as the predominant metabolic pathway, while phase II metabolites were mainly formed by glucuronidation. The relatively comprehensive metabolic pathway of mosapride was proposed.     

In vitro protein binding of propafenone and 5-hydroxypropafenone in serum, in solutions of isolated serum proteins, and to red blood cells.

The binding of propafenone (PF) and 5-hydroxypropafenone (5-OH-PF) in serum and in solutions of isolated serum proteins was examined by equilibrium dialysis. Both PF and 5-OH-PF displayed pH-dependent binding in serum and in a solution of alpha-1-acid glycoprotein (AAG). PF displayed extensive binding to AAG (i.e., free fraction of 0.08 +/- 0.02), whereas the binding of 5-OH-PF to AAG was moderate (i.e., free fraction of 0.54 +/- 0.10). The removal of lipoproteins from serum did not alter the free fraction of PF but significantly increased the free fraction of 5-OH-PF compared with that in intact serum. Both PF and 5-OH-PF displayed concentration-dependent binding in a 19.3-mumol AAG solution. Concentration-independent binding was apparent in solutions of human serum albumin, high-density lipoproteins, low-density lipoproteins, and very low density lipoproteins over the PF and 5-OH-PF concentration ranges examined. By use of previously determined binding parameters (affinities and capacities), the binding model of PF provided an estimate of the free fraction in serum that was similar to the observed free fraction, although the free fraction of 5-OH-PF was overestimated. The distribution of PF and 5-OH-PF into red blood cells was extensive when buffer was used as the supernatant; however, when serum was used as supernatant, the amounts of PF and 5-OH-PF that were distributed into red blood cells decreased substantially. PF and 5-OH-PF interacted with all of the proteins examined.     

A novel biologically active selenooorganic compound--VII. Biotransformation of ebselen in perfused rat liver

Ebselen (PZ 51) is a selenoorganic compound with antioxidant and antiinflammatory properties, and its metabolism was studied in isolated perfused rat liver (hemoglobin-free, open system). 75Se-labelled ebselen was taken up into liver cells and radioactivity was excreted into bile. Biliary excretion of 75Se-compounds reached maximal values of 4 nmol/min per g wet wt. HPLC analysis of bile and effluent perfusate as well as identification of separated metabolites by mass spectrometry were carried out. The biliary metabolites were (a) an interesting novel Se-glucuronide, 2-glucuronylselenobenzanilide, (metabolite IV), as the major metabolite, and (b) an O-glucuronide, N-(4'-glucuronyloxyphenyl)-2-methylselenobenzanilide (metabolite III). The major effluent perfusate metabolites were Se-methylated derivatives (metabolites I and II). There was no evidence for sulfated metabolites. The selenodisulfide with glutathione, S-(2-phenyl-carbamoyl-phenylselenyl)-glutathione, was not detected, probably because of low steady-state concentrations and/or its biochemical lability. The selenium in ebselen is not bioavailable (e.g. for the synthesis of glutathione peroxidase), in contrast to selenite, for example, thus explaining the very low ebselen toxicity. However, the enzymatic steps in Se-methylation could be similar to those in the metabolism of selenite which include hydrogen selenide methylation. Se-glucuronides constitute a novel category of compounds in addition to the O-, N-, C- and S-glucuronide classes known in biology.     

6-甲氧基正丁苯酞在大鼠肝微粒体中的代谢研究

报道了用GC/MS方法及衍生化技术研究6-甲氧基正丁苯酞(MBP)在大鼠肝微粒体中的代谢转化结果。6-甲氧基正丁苯酞在苯巴比妥(PB)诱导的大鼠肝微粒体中主要转化为3-羟基、γ-羟基取代物,6-羟基正丁苯酞与一个环氧化代谢产物。     

In vitro metabolic products of RWJ-34130, an antiarrythmic agent, in rat liver preparations

The in vitro metabolism of RWJ-34130, an antiarrhythmic agent, was conducted using rat hepatic 9000 x g supernatant (S9) and microsomes in an NADPH-generating system, and the rat liver perfusion. The 100 and 20 microg ml(-1) concentrations of RWJ-34130 aqueous solution were used for microsomal incubation and liver perfusion, respectively. Unchanged RWJ-34130 (approximately 77-78% of the sample in both S9 and microsomes) plus a major metabolite, RWJ-34130 sulfoxide (20% of the sample in both S9 and microsomes) were profiled, isolated and identified from both hepatic S9 and microsomal incubates (60 min) using HPLC and mass spectrometry (MS), and by comparison to a synthetic RWJ-34130 sulfoxide, which was synthesized by reacting RWJ-34130 with MCPBA (meta-chloroperoxy benzoic acid). No unchanged RWJ-34130 was detected in the 3 h liver perfusate, however, 1-phenyl-2-oxo-pyrrolidine was profiled, isolated and identified as a major hydrolyzed metabolite of liver perfusate. RWJ-34130 is not extensively metabolized in vitro in rat hepatic S9 and microsomes. All HPLC metabolic profiles of hepatic S9 and microsomal samples (30 min, 60 min) were qualitatively and nearly quantitatively identical.     

The Disposition of Morphine and Its Metabolites in the In-situ Rat Isolated Perfused Liver

A specific HPLC method with UV detection was used to investigate the disposition of morphine and its metabolites in the in-situ rat isolated perfused liver preparation. Livers of male Sprague-Dawley rats (n = 4) were perfused under single pass conditions with protein-and erythrocyte-free perfusate, containing 2·66 μm morphine, for up to 90 min. The concentration of morphine, normorphine and morphine-3-glucuronide (M3G) in outflow perfusate, and the biliary excretion of M3G and normorphine glucuronide, all reached steady-state levels within 15–20 min after commencing perfusion. At steady-state, the mean (± s.d.) extraction ratio of morphine was 0·87 ± 0·06 and clearance (26·0 ± 1·7 mL min^?1) approached perfusate flow rate (30 mL min^?1). Although M3G was the main metabolite, accounting for 72·8 ± 12·7% of eliminated morphine, a significant proportion (21·6 ± 13·5%) was N-demethylated to normorphine and was recovered as unchanged normorphine in outflow perfusate and normorphine glucuronide in bile. The biliary extraction ratio of hepatically-formed M3G was 0·61 ± 0·31. Results from an additional six experiments, in which livers were perfused with 1·33 and 2·66 μm of morphine for 30 min each in a balanced cross-over manner, indicated that the disposition of morphine and its metabolites was approximately linear within this concentration range.     

The metabolism of oestrone and some other steroids in isolated perfused rat and guinea pig livers

1. Oestrone is rapidly taken up by isolated perfused rat liver (t_1/2< 2 min) to yield at least 10 metabolites excreted in the bile; peak concentration occurs after about 20 min.

2. Sulphated metabolites of oestrone appear in the perfusate, reaching peak concentration at about 10 min, and then slowly disappear.

3. Sulphated metabolites of oestrone accumulate in the liver during the first 10 min. They are partly converted to sulphoglucuronides (steroid 3-sulphates conjugated with glucuronic acid in the D ring) and partly hydrolysed to be reconjugated as glucuronides.

4. The major biliary metabolites of oestrone in isolated perfused rat liver are glucuronides and sulphoglucuronides, but free steroids, sulphates and polar metabolites are also so excreted.

5. The isolated perfused guinea pig liver also rapidly takes up oestrone (t_1/2 < 2 min) but, in contrast to the rat, a single glucuronide is the only quantitatively important metabolite in the bile: it is also extensively secreted into the perfusate where it reaches peak concentration at about 10 min.

6. In perfused guinea pig liver, oestrone does not form sulphoglucuronides, and sulphates are only minor metabolites; this is not due to lack of the appropriate sulphotransferase because oestradiol 17β-(β-D-glucuronide) is extensively sulphated in this system.

7. Oestradiol 17β-(β-D-glucuronide) is not cholestatic in the isolated perfused guinea pig liver although it is in rat liver.

8. There is a similar species difference in the metabolism of dehydroepiandrosterone in the two species: the rat forms sulphoglucuronides, the guinea pig does not.

9. The perfused rat liver extensively hydroxylates, presumably on the D ring, 17-deoxyoestrone and 17-deoxydehydroepiandrosterone.

10. The inability of perfused guinea pig liver to form sulphoglucuronides from oestrone or dehydroepiandrosterone is probably due to its restricted ability to hydroxylate the D ring of steroids.

11. Both rat and guinea pig biles contain β-glucuronidase, about 80 and 230 sigma units/ml, respectively.     

Evidence for a new metabolite of morphine-N-methyl-14C in the rat

Evidence has been presented for the formation in vivo in the rat of a new urinary metabolite of morphine, to which 2,3-dihydrodiol structure has been tentatively assigned. In vitro, rat brain and liver homogenates were shown to produce a 2,3-catechol type of metabolite by aromatic hydroxylation of morphine. Small amounts of morphine N-oxide and normorphine were also identified as metabolites in vitro by liver homogenates. Sequential oxidation of morphine by alkaline ferricyanide and hydrogen peroxide and Cu²⁺ has been shown to produce a zwitterionic 2,3-quinone, whose Chromatographic properties appeared similar to those of the hydroxylated metabolite formed in vitro by rat brain and liver homogenates.     

Metabolism of Carfentanil,an Ultra-Potent Opioid,in Human Liver Microsomes and Human Hepatocytes by High-Resolution Mass Spectrometry

Carfentanil is an ultra-potent synthetic opioid. No human carfentanil metabolism data are available. Reportedly, Russian police forces used carfentanil and remifentanil to resolve a hostage situation in Moscow in 2002. This alleged use prompted interest in the pharmacology and toxicology of carfentanil in humans. Our study was conducted to identify human carfentanil metabolites and to assess carfentanil’s metabolic clearance, which could contribute to its acute toxicity in humans. We used Simulations Plus’s ADMET Predictor? and Molecular Discovery’s MetaSite? to predict possible metabolite formation. Both programs gave similar results that were generally good but did not capture all metabolites seen in vitro. We incubated carfentanil with human hepatocytes for up to 1 h and analyzed samples on a Sciex 3200 QTRAP mass spectrometer to measure parent compound depletion and extrapolated that to represent intrinsic clearance. Pooled primary human hepatocytes were then incubated with carfentanil up to 6 h and analyzed for metabolite identification on a Sciex 5600+ TripleTOF (QTOF) high-resolution mass spectrometer. MS and MS/MS analyses elucidated the structures of the most abundant metabolites. Twelve metabolites were identified in total. N-Dealkylation and monohydroxylation of the piperidine ring were the dominant metabolic pathways. Two N-oxide metabolites and one glucuronide metabolite were observed. Surprisingly, ester hydrolysis was not a major metabolic pathway for carfentanil. While the human liver microsomal system demonstrated rapid clearance by CYP enzymes, the hepatocyte incubations showed much slower clearance, possibly providing some insight into the long duration of carfentanil’s effects.     

Use of a dimethylated oxirane to characterise microsomal mono-oxygenases in rat liver

A newly-synthesised dimethylated chlorocyclodiene epoxide is metabolised by NADPH-supplemented rat liver microsomes to produee either one or both of two major metabolites. M1 and M2. Microsomes from adult male rat liver produce both metabolites. By contrast, microsomes made from the livers of mature or immature female rats make only metabolite M1, as do hepatic microsomes from pre-pubertal male rats. Male rats castrated when 7 days old provide liver microsomes that make little of metabolite M2 upon killing at 3–5 months post partum. After induction with phenobarbital, microsomes from adult male and female rat liver make metabolites M1 and M2. together with a further unidentified metabolite. After induction with 3-methylcholanthrene, hepatic microsomes from mature female rats continue to make only metabolite M1 but to make it in larger quantities. These differences, which appear to result from the activity of three or more mono-oxygcnases. could possibly he exploited to monitor the mono-oxygenase status of rat liver microsomes.     

Metabolism of delta-1-tetrahydrocannabinol by the rat in vivo and in vitro.

The metabolism of [¹⁴C]Δ¹-tetrahydrocannabinol (THC) was examined in vitro employing the 10.000 g supernatant (10 KS) of rat liver homogenates, after determination of apparent optimal conditions. The extent of metabolism was greater when THC was added as a suspension in rat serum than in a glycol or ethanol solution. The apparent K_m for THC metabolism by the 10 KS was found to be 1.35 × 10^?4M and the apparent v_max was 0.18 μg THC metabolized/mg of protein/min. Thin-layer chroma tography indicated that at least seven metabolites were produced from THC, with the quantitative pattern of the metabolites changing dramatically with increasing duration of incubation. The major metabolite of 7-hydroxy-Δ¹-THC appeared to be 6,7-dihydroxy-Δ¹-THC. The dihydroxylated compound was slowly metabolized to more polar compounds. The bile was verified to be a major route of THC elimination in the rat. accounting for 60 per cent of an i.v. dose of THC (3 mg/kg) within 3 hr. Biliary excretion reached a maximum rate at THC doses of 6 and 12 mg/kg. Only traces of unchanged THC were found in the bile, and less than 5 per cent of a dose was excreted as 7-hydroxy-THC, the metabolites in the bile all being highly polar. The metabolism of THC, rather than the excretory process, was apparently the rate-limiting step in the elimination of the drug in the bile. When the biliary excretary mechanism was saturated, increase in the dose of THC resulted in increased urinary excretion of highly polar metabolites, but relative concentrations in tissues other than liver were unaffected.