Stable isotope- and mass spectrometry-based metabolomics as tools in drug metabolism: a study expanding tempol pharmacology.

The application of mass spectrometry-based metabolomics in the field of drug metabolism has yielded important insights not only into the metabolic routes of drugs but has provided unbiased, global perspectives of the endogenous metabolome that can be useful for identifying biomarkers associated with mechanism of action, efficacy, and toxicity. In this report, a stable isotope- and mass spectrometry-based metabolomics approach that captures both drug metabolism and changes in the endogenous metabolome in a single experiment is described. Here the antioxidant drug tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) was chosen because its mechanism of action is not completely understood and its metabolic fate has not been studied extensively. Furthermore, its small size (MW = 172.2) and chemical composition (C(9)H(18)NO(2)) make it challenging to distinguish from endogenous metabolites. In this study, mice were dosed with tempol or deuterated tempol (C(9)D(17)HNO(2)) and their urine was profiled using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Principal component analysis of the urinary metabolomics data generated a Y-shaped scatter plot containing drug metabolites (protonated and deuterated) that were clearly distinct from the endogenous metabolites. Ten tempol drug metabolites, including eight novel metabolites, were identified. Phase II metabolism was the major metabolic pathway of tempol in vivo, including glucuronidation and glucosidation. Urinary endogenous metabolites significantly elevated by tempol treatment included 2,8-dihydroxyquinoline (8.0-fold, P < 0.05) and 2,8-dihydroxyquinoline-ß-d-glucuronide (6.8-fold, P < 0.05). Urinary endogenous metabolites significantly attenuated by tempol treatment including pantothenic acid (1.3-fold, P < 0.05) and isobutrylcarnitine (5.3-fold, P < 0.01). This study underscores the power of a stable isotope- and mass spectrometry-based metabolomics in expanding the view of drug pharmacology.

[Development of analytical method for determination nicotine metabolites in urine]. / Opracowanie metody oznaczanie metabolitów nikotyny w moczu.

The assay of biomarkers in biological material is the most popular and reliable method in estimate exposure to tobacco smoke. Nicotine and its metabolites qualify to the most specific biomarkers for tobacco smoke. Currently the most often used are cotinine and trans-3'-hydroxycotinine. The aim of this study was development of easy and quick method of determining nicotine and its main metabolites with high performance liquid chromatography--available in most laboratories. Nicotine and its metabolites in urine (cotinine, trans-3'-hydroxycotinine, nornicotine and nicotine N-oxide) was determined by means of high performance liquid chromatography with spectrometry detection (HPLC-UV). The determined compounds were extracted from urine by means of the liquid-liquid technique, before analysed by the HPLC method. Developed technique of high performance liquid chromatography proved to be useful to assessment nicotine and its four metabolites in smokers, though further research are necessary. The further modification of procedure is required, because of the interferences of cotinine N-oxide with matrix, which prevent determination. Increasing the efficiency of extraction nicotine and nornicotine could enable the determination in people exposed on environmental tobacco smoke (ETS). This study confirm other authors' observations that 3'-hydroxycotinine might be equivalent with cotinine predictor of tobacco smoke exposure, however further studies are required.

Nicotine-N'-Oxidation by Flavin Monooxygenase Enzymes.

BACKGROUND: The major mode of metabolism of nicotine is by hydroxylation via cytochrome P450 (CYP) 2A6, but it can also undergo glucuronidation by UDP-glucuronosyltransferases and oxidation by flavin monooxygenases (FMO). The goal of this study was to examine the potential importance of FMOs in nicotine metabolism and assess the potential impact of missense polymorphisms in active FMOs on nicotine-N'-oxide (NOX) formation. METHODS: Urine samples from 106 current Chinese smokers were analyzed for nicotine metabolites by mass spectrometry. Wild-type FMOs 1-5 and their most prevalent nonsynonymous variants were cloned and overexpressed in HEK293 cells, and were tested in oxidation reactions against nicotine. RESULTS: A strong inverse correlation was observed between the ratio of urinary 3'-hydroxycotinine/cotinine, a measure of CYP2A6 activity, and the urinary levels of NOX alone (r = -0.383; P < 0.001) or NOX measured as a ratio of total nicotine metabolites (r = -0.414; P < 0.001) in smokers. In addition to FMO1 and FMO3, the functional FMO2427Q isoform was active against nicotine, whereas FMO4 and FMO5 exhibited low activity against nicotine (K m > 5.0 mmol/L). Significant (P < 0.05) decreases in N'-oxidation activity (V max/K m) were observed for the FMO1I303V, FMO3N61S, FMO3D132H, FMO3V257M, and FMO3E308G variants in vitro when compared with their respective wild-type isoforms; the truncated FMO2Q472stop isoform exhibited no enzyme activity. CONCLUSIONS: These data indicate that increases in nicotine-N'-oxidation occur in subjects with deficient CYP2A6 activity, and that several FMO enzymes are active in nicotine-N'-oxidation. IMPACT: Several common missense FMO variants are associated with altered enzyme activity against nicotine and may play an important role in nicotine metabolism in low-CYP2A6 activity subjects.

Metabolism of the tryptamine-derived new psychoactive substances 5-MeO-2-Me-DALT, 5-MeO-2-Me-ALCHT, and 5-MeO-2-Me-DIPT and their detectability in urine studied by GC-MS, LC-MSn , and LC-HR-MS/MS.

Many N,N-dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5-methoxy-2-methyl-N,N-diallyltryptamine (5-MeO-2-Me-DALT), 5-methoxy-2-methyl-N-allyl-N-cyclohexyltryptamine (5-MeO-2-Me-ALCHT), and 5-methoxy-2-methyl-N,N-diisopropyltryptamine (5-MeO-2-Me-DIPT) using gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC-MSn ), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC-HR-MS/MS. 5-MeO-2-Me-DALT (24 phase I and 12 phase II metabolites), 5-MeO-2-Me-ALCHT (24 phase I and 14 phase II metabolites), and 5-MeO-2-Me-DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O-demethylation, hydroxylation, N-dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O-demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N-dealkylation. For SUSAs, GC-MS, LC-MSn , and LC-HR-MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC-MS SUSA, both LC-MS SUSAs were able to detect an intake of 5-MeO-2-Me-ALCHT and 5-MeO-2-Me-DIPT via their metabolites following 1 mg/kg BW administrations and 5-MeO-2-Me-DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.

The metabolomics of (+/-)-arecoline 1-oxide in the mouse and its formation by human flavin-containing monooxygenases.

The alkaloid arecoline is a main constituent of areca nuts that are chewed by approximately 600 million persons worldwide. A principal metabolite of arecoline is arecoline 1-oxide whose metabolism has been poorly studied. To redress this, synthetic (+/-)-arecoline 1-oxide was administered to mice (20mg/kg p.o.) and a metabolomic study performed on 0-12h urine using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-TOFMS) with multivariate data analysis. A total of 16 mass/retention time pairs yielded 13 metabolites of (+/-)-arecoline 1-oxide, most of them novel. Identity of metabolites was confirmed by tandem mass spectrometry. The principal pathways of metabolism of (+/-)-arecoline 1-oxide were mercapturic acid formation, with catabolism to mercaptan and methylmercaptan metabolites, apparent CC double-bond reduction, carboxylic acid reduction to the aldehyde (a novel pathway in mammals), N-oxide reduction, and de-esterification. Relative percentages of metabolites were determined directly from the metabolomic data. Approximately, 50% of the urinary metabolites corresponded to unchanged (+/-)-arecoline 1-oxide, 25% to other N-oxide metabolites, while approximately, 30% corresponded to mercapturic acids or their metabolites. Many metabolites, principally mercapturic acids and their derivatives, were excreted as diastereomers that could be resolved by UPLC-TOFMS. Arecoline was converted to arecoline 1-oxide in vitro by human flavin-containing monooxygenases FMO1 (K(M): 13.6+/-4.9muM; V(MAX): 0.114+/-0.01nmolmin(-1)microg(-1) protein) and FMO3 (K(M): 44.5+/-8.0microM; V(MAX): 0.014+/-0.001nmolmin(-1)microg(-1) protein), but not by FMO5 or any of 11 human cytochromes P450. This report underscores the power of metabolomics in drug metabolite mining.

Metabolic conversion of indicine N-oxide to indicine in rabbits and humans.

Indicine N-oxide, a pyrrolizidine alkaloid N-oxide that exhibits antitumor activity without some of the toxic effects associated with other pyrrolizidine alkaloids, is metabolized to indicine in rabbits and humans. Indicine can be detected in the plasma and is excreted in the urine in a dose-dependent manner following the i.v. administration of indicine N-oxide. The p.o. administration of indicine N-oxide leads to an increased plasma concentration and an increased urinary excretion of indicine. The hepatic microsomal fraction and the gut flora both catalyze the anaerobic reduction of indicine N-oxide to indicine in vitro. Whole-animal studies suggest that the gut flora play a major role in the metabolic reduction of indicine N-oxide by the rabbit. Indicine N-oxide is not actively excreted in the bile, and it is probable that indicine N-oxide finds its way into the gut by passive diffusion following i.v. administration. Neomycin and erythromycin, which reduce the number of anaerobic bacteria in the gut, lead to decreased plasma levels and a decreased urinary excretion of indicine.

N-deethylation and N-oxidation of etamiphylline: identification of etamiphylline-N-oxide in greyhound urine by high performance liquid chromatography-mass spectrometry.

Millophyline-V, (etamiphylline camsylate) was administered intramuscularly to two racing greyhounds at a dose of 10 mg kg(-1). Unhydrolysed pre- and post-administration urine samples were extracted using mixed mode solid phase extraction (SPE) cartridges, the basic isolates derivatised as trimethylsilyl ethers and analysed by positive ion electron ionisation gas chromatography-mass spectrometry (GC/EI+/MS). The parent drug and one metabolite, N-desethyletamiphylline, were detected in urine for up to 72 h. For semi-quantification, urine samples were extracted on-line using a Prospekt sample handler. The analytes retained on the C2 SPE cartridge were eluted by the mobile phase directly on to the analytical high performance liquid chromatography column and analysed by positive ion atmospheric pressure chemical ionisation (LC/APCI+) MS in the multiple selective-ion recording mode. A major peak containing both ions (m/z) 280 and (m/z) 252 was observed. Full scan LC/APCI+/MS of the unknown indicated that the ion at (m/z) 280 was formed by the loss of an oxygen atom [MH+ -->(MH+-O)]. Samples were analysed by positive ion electrospray ionisation LC/MS on two different instruments and the unknown compound was identified as an N-oxide of the tert. nitrogen atom of the 2-(diethylamino)ethyl substituent on N7 of the theophylline nucleus. This compound has not been reported previously either as an in vivo or in vitro metabolite of etamiphylline in any species. Thermal decomposition of the N-oxide could lead to an increase the detection period of the parent drug during routine GC/MS screening of post-competition greyhound urine samples.

A foxy intoxication.

Foxy is the colloquial name for the hallucinogen 5-ethoxy-diisopropyltryptamine (5-MeO-DIPT). A non-fatality involving a 23-year-old Caucasian man who ingested a capsule containing 5-MeO-DIPT is described. He presented to the Emergency Department, not with visual nor auditory hallucinations but with sensory hallucinations, that of formication and paranoia. He was observed and given supportive care for 4 h, then discharged without any known sequelae. Blood and urine were collected for laboratory analyses. Foxy and its metabolites were identified in urine by gas chromatography-mass spectrometry. The concentrations of 5-MeO-DIPT in the serum and urine were 0.14 and 1.6 microg/mL, respectively. The drug undergoes oxidative deamination to form 5-methoxy-indole acetic acid. The urinary concentration of this metabolite was 0.17 microg/mL. Also, the urine contained three other related compounds. Two of them have been described in a previous case of 5-MeO-DIPT ingestion as 5-methoxy-isopropyltryptamine (5-MeO-IPT) and 5-methoxy-diisopropyltryptamine-N'-oxide (5-MeO-DIPT-N'-oxide). The third compound was substantially present in the urine and was tentatively identified as 5-hydroxy-diisopropyltryptamine (5-OH-DIPT). Only the parent drug, 5-MeO-DIPT was detected in the serum sample.

Kinetics and dynamics of quinidine-N-oxide in healthy subjects.

The pharmacokinetics of a major metabolite of quinidine in humans, quinidine-N-oxide, were investigated after single oral doses (3 to 15 mg) in four healthy subjects. The concentration in serum and urine was determined by an HPLC assay. Because of a small volume of distribution, the elimination half-life of quinidine-N-oxide was only 2.5 +/- 0.28 hours (mean +/- SD), considerably shorter than that of quinidine. The renal clearance was 1.3 +/- 0.3 L/hr. Only 13.9% +/- 3.7% of the dose was recovered in urine as unchanged compound for up to 12 hours. Two unidentified compounds with the same retention time as quinidine and 3-hydroxyquinidine were found in the urine samples of two subjects. The free fraction in serum was 3.3% +/- 0.83%. No systematic changes in heart rate-corrected QT interval were observed up to concentrations of 500 ng/ml. The results indicate that quinidine-N-oxide, in contrast to 3-hydroxyquinidine, does not possess quinidine-like pharmacologic activity.

Evaluation of a low to middle tar/medium nicotine cigarette designed to maintain nicotine delivery to the smoker.

A specific objective of this 6-week crossover study was to determine how 21 regular smokers of middle tar cigarettes changed their smoking behaviour and uptake of smoke constituents, when switching to either lower tar cigarettes capable of delivering amounts of nicotine similar to a conventional middle tar cigarette (maintained nicotine product), or to conventional low tar/low nicotine cigarettes. Subjects visited the laboratory every 2 weeks for detailed assessment of their smoking behaviour. Weekly per capita consumption was similar for all three cigarettes. They were smoked with variable intensities (low tar greater than maintained nicotine greater than middle tar), the tendency being for larger puff volumes, faster puffing and increased puff duration with the low tar cigarettes. The maintained nicotine cigarette was preferred to the middle tar cigarette, although acceptability ratings of the three cigarettes only differed marginally. The nicotine absorbed from the maintained nicotine and middle tar cigarettes was similar and significantly greater than the levels achieved from the low tar cigarettes. Intake of carbon monoxide into the mouth and absorption into the blood stream was lower for the maintained nicotine cigarette than for the middle tar cigarette, with the low tar cigarette occupying an intermediate position. Derived estimates of tar intake suggested reduced intake of tar into the respiratory tract (around 25%) from the maintained nicotine product relative to the middle tar product. The possible advantages of switching to maintained nicotine cigarettes is discussed.

In vivo L-band ESR and quantitative pharmacokinetic analysis of stable spin probes in rats and mice.

Free radical species in animals have been measured by X-band ESR spectrometric method on a block of organs or a portion of homogenized samples. However, a nondestructive in vivo ESR measurement has been realized by using a recently developed L-band ESR spectrometry. With this L-band ESR method, we measured ESR spectra in animals, who received stable nitroxide radicals. L-band ESR spectra were observed at the upper abdomen of mice as well as at the heads of mice and rats at various ages immediately after the intravenous injections of nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (3-carbamoyl-PROXYL), in which ESR measurements of the radicals were performed noninvasively at the real time. On the basis of the observed time-dependent free radical clearance curves, the following important results were obtained: (1) Free radical clearances were able to analyze by the pharmacokinetic method. (2) The radicals at the head of mice, given 4-hydroxy-TEMPO, were determined quantitatively by a new analytical method using L-band ESR for the first time. (3) The elimination of the radical was found to be saturated in mice. (4) The clearance rate constant of 4-hydroxy-TEMPO detected at the head of mice was decreased in dose- and age-dependent manners. While, no age-dependent clearance rate constant of 4-hydroxy-TEMPO was observed at the upper abdomen of mice. (5) Ratios of the amount of the detected radicals to that of the administered radicals were decreased age-dependently, but they were independent of the dose of the radicals, suggesting the age-dependent decrease of distribution capacity ratio of the radical at the head of animals. (6) Clearance rate constants of 4-hydroxy-TEMPO and 3-carbamoyl-PROXYL, that were estimated by X- and L-band ESR for the collected blood of mice and rats, were found to be remarkably smaller than those in whole living animals observed by in vivo L-band ESR method. The results suggest that the clearance of the nitroxide radical is relevant to the alteration of the radical in animals following the change of organ distribution and metabolism. (7) Both the radical and its corresponding hydroxylamine, which is the reduced form of the radical, were detectable by X-band ESR method in the collected urine of mice and rats without and with an oxidizing agent, respectively. On the basis of the results on L-band ESR spectrometry, the first quantitative pharmacokinetic analysis of stable spin probes in animals is proposed.

Foxy, a designer tryptamine hallucinogen.

Foxy is slang for 5-methoxy-N,N-diisopropyltryptamine. It has hallucinogenic properties, similar to other tryptamine compounds, and is mildly euphoric. This case report describes a 21-year-old Caucasian man who ingested a pill called Foxy containing an unknown amount of drug. He was observed in hospital for 2 h, during which time he had mild hallucinations and could not move his limbs. A urine sample was collected approximately 4 h after drug ingestion. The patient was then discharged with no follow up assessment. The 5-methoxy-N,N-diisopropyltryptamine was identified in the urine by gas chromatography-mass spectrometry. Standards prepared from the pure material were used in the identification. Quantitative analysis using the same analytical technique resulted in a urinary concentration of 1.7 micro g/mL. Through oxidative deamination, the metabolite, 5-methoxy-indole acetic acid, was formed. It was identified in the urine, and the concentration was determined to be 1.3 micro g/mL using gas chromatography-mass spectrometry. Two other compounds were discovered in the urine sample as a result of a routine drug screen. From their mass spectra, they were tentatively identified as 5-methoxy-N-isopropyltryptamine and 5-methoxy-N,N-diisopropyltryptamine-N'-oxide.