Glucuronic Acid Conjugates Research Articles

Spectroelectrochemical Sensing of Pyrene Metabolites 1-Hydroxypyrene and 1-Hydroxypyrene-glucuronide

Spectroelectrochemical sensing in an optically transparent thin layer electrode (OTTLE) cell was used for detecting the polycyclic aromatic hydrocarbon (PAH) biomarkers 1-hydroxypyrene (1-pyOH) and 1-hydroxypyrene-glucuronide (1-pyOglu) in phosphate buffer and artificial urine. This approach uses selective electrochemical modulation of a fluorescence signal by sequentially oxidizing the analytes in an OTTLE cell to distinguish between their overlapping fluorescence spectra. This technique allows for complete oxidation and signal modulation in approximately 15 min for each analyte; a mixture of 1-pyOH and its glucuronic acid conjugate can be analyzed in 30 min. Calibration curves consisting of the fluorescence change vs analyte concentration for 1-pyOH and 1-pyOglu yielded linear ranges from 10 nM to 1 μM and from 1 nM to 1 μM, respectively. With the use of these results, the calculated limits of detection were determined to be 1 × 10(-8) M for 1-pyOH and 9 × 10(-11) M for 1-pyOglu.

Availability and mean transit times of phenol and its metabolites in the isolated perfused rat liver: normal and retrograde studies using tracer concentrations of phenol.

Phenolic compounds are frequently detoxified by the formation of sulphate and glucuronic acid conjugates in the liver. These conjugates are formed in the hepatocytes and then either transported into the bile or back into the blood. In this study, we examined the transport kinetics of phenol and its metabolites in the isolated perfused rat liver by monitoring the outflow profiles of these compounds after a bolus input in a single pass preparation. Phenol was almost exclusively metabolized to phenyl sulphate (97%) at the trace concentrations used, with the amount of phenol and metabolites excreted into the bile being minimal (3.5%). The metabolite formed was rapidly transported back into the perfusate, with mean transit times of 17.4 and 12.3 s anterograde and 24.9 and 24.2 s retrograde at flow rates of 15 and 30 mL min-1 respectively, which were intermediate between those of Evans blue and water. The outflow concentration-time profile for phenyl sulphate formation was unaffected by the addition of another organic anion (bromosulphophthalein). The effect of enzyme zonation on outflow concentration-time profiles was also investigated using retrograde perfusions. The transit time ratios for generated metabolite to water for anterograde perfusions (0.6) was found to be more than twice that for retrograde perfusions (0.23) at 15 mL min-1 and approximately 1.6 times greater at 30 mL min-1, being 0.58 and 0.37 respectively. The relative ratios obtained are consistent with previous findings that normalized variance of solutes in the retrograde perfusions is greater than that for anterograde perfusions.

Metabolism and excretion of promazine by the horse.

Abstract The urinary excretion of promazine and its metabolites has been examined in five horses, by ultraviolet spectrometry and thin-layer chromatography. In each horse, excretion continued for at least 96 h although the amount excreted was low, being about 11% of the dose. No correlation of excretion and pH or urinary volume was observed. Glucuronic acid conjugates predominated, their ratio to unconjugated metabolites being about 5:1. Promazine was found only in small amounts and traces of unconjugated sulphide metabolites were found in only one horse. At least nine metabolites of promazine were detected, the major one being the glucuronide of 3-hydroxypromazine.

Bioavailability of anti‐carcinogenic hydroxylated polymethoxyflavones in mice

Previously we have showed that hydroxylated polymethoxyflavones (OH‐PMFs), such as 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone (5HPMF) and 5‐hydroxy‐6,7,8,4′‐tetramethoxyflavone (5HTMF) are potent anti‐cancer dietary compounds in cell culture. Bioavailability is one of the key factors that can dictate the in vivo efficacy of dietary bioactive compounds. Herein, we studied the bioavailability of 5HPMF and 5HTMF in CF‐1 mice. We developed a highly sensitive HPLC method for the detection of OH‐PMFs in biological samples. The method was fully validated in terms of linearity, accuracy and precision. The limit of detection (LOD) in serum was determined as being 0.71 and 1.97 ng/mL (ppb) for 5HPMF and 5HTMF, respectively, demonstrating an over 150‐fold increase in sensitivity in comparison with previously published method. At 60 min after oral gavage (100 mg/kg), 5HPMF, mostly in free form, reached its peak level of 5.3 μM, while 5HTMF showed similar trend but with the peak level 8.28‐fold lower than that of 5HPMF. In urine, both 5HPMF and 5HTMF existed mainly as sulfate‐ and glucuronic acid‐conjugates, and urine level of total 5HPMF was 9.06‐fold higher than that of 5HTMF. These results demonstrated that 5HTMF had much lower bioavailability, which may due to its poor solubility in GI tract.

Dalcetrapib pharmacokinetics and metabolism in the cynomolgus monkey

The thioester dalcetrapib is undergoing Phase III clinical evaluation for the prevention and regression of atherosclerosis and the prevention of cardiovascular events through targeting cholesteryl ester transfer protein and increasing high-density lipoprotein cholesterol levels. Dalcetrapib undergoes rapid hydrolysis to generate the pharmacologically active form (dalcetrapib-thiol), which undergoes extensive metabolism via glucuronic acid conjugation, methylation, and hydroxylation, predominately forming the pharmacologically inactive S-methyl (dalcetrapib-S-Me) and S-glucuronide (dalcetrapib-S-Glu) metabolites.The purpose of this study was to characterize the absorption and disposition of dalcetrapib-thiol and its primary metabolites in cynomolgus monkeys following first pass through the intestines and liver using an in vivo dual portal and peripheral vein cannulation.Results showed the high influence of glucuronidation of dalcetrapib-thiol on the first-pass effect. Following passage through the primate intestinal wall, area under the plasma concentration–time curve indicated a marked loss (by ~85%) of active compound and formation of dalcetrapib-S-Glu and dalcetrapib-S-Me.Based on time to maximum drug concentrations (Tmax) values in the portal vein, metabolism of dalcetrapib-thiol to dalcetrapib-S-Glu appears to occur almost instantly (median Tmax 6.0 and 5.5 h, respectively), whereas methylation to dalcetrapib-S-Me occurs much more slowly (median Tmax, 24 h). A relatively modest impact on systemic exposure followed hepatic first pass, with a further decrease in dalcetrapib-thiol exposure of 58% (AUC), a 3-fold reduction in exposure levels of dalcetrapib-S-Me and near-complete decrease in exposure of dalcetrapib-S-Glu. Passage of drug-related material through the intestinal wall and the liver results in an overall decrease of exposure to dalcetrapib-thiol of >90%.

Investigation of the in vitro metabolism of the emerging drug candidate S107 for doping‐preventive purposes

The metabolic fate of the emerging drug candidate S107, possessing the potential for misuse as performance-enhancing agent in sports, was investigated by in vitro phase I and II experiments with human microsomal and S9 liver enzymes. The metabolites were identified by liquid chromatography-mass spectrometry with electrospray ionisation in positive mode (LC-ESI-MS/MS). Their collision-induced dissociation behaviour was studied by high-resolution/high accuracy Orbitrap MS(n) analysis, supported by stable isotope labelling, H/D-exchange experiments and density functional theory calculations. Monooxygenation accounted for the main phase I metabolic transformation due to N- and S-oxidation of the 1,4-benzothiazepine core, as substantiated by chemical synthesis, selective reduction methods and characteristic APCI in source fragmentation behaviour of the metabolites. Another dominant metabolic pathway was demethylation, yielding the N- and O-demethylated metabolite, respectively. The latter was further conjugated by glucuronidation as well as sulfonation in subsequent phase II metabolic reactions, whereas the N-demethylated metabolite was not amenable to conjugation. The active drug molecule itself was converted to two glucuronic acid conjugates, which are proposed to consist of two quaternary S107-N(+)-glucuronide isomers. All glucuronides were susceptible to enzymatic hydrolysis with β-glucuronidase (Escherichia coli). A comprehensive LC-ESI-MS(/MS)-based detection method for urine was developed and its fitness for purpose was assessed. The assay can serve as a potential screening and/or confirmation method for S107 in clinical drug testing and doping control analysis in the future.

Synthesis of a radioiodinated antiestrogen glucuronide compound (TAM-G)

Tamoxifen [TAM; ([Z]-2-[4-(1,2-diphenyl-1-di-butenyl)-phenoxy]-N,N-dimethylethanamine) has been used as an antiestrogen drug for treatment and prevention of human breast cancer. The aim of this study is conjugation of hydrophilic glucuronic acid to the starting substance TAM and labeling with 131I using iodogen as oxidizing agent. The reactions are completed in three steps, including enzymatic reaction, with the following sub-steps; preparation of microsomal fraction from rat liver and subsequent purification of UDP-glucuronyl transferase (UDPGT), estimation of protein amount in microsomal samples and glucuronidation reaction. Synthesized glucuronide derivative (TAM-G) was purified using high performance liquid chromatography (HPLC). Mass spectroscopy of cold standard showed that the labeling most probably occurs in ortho position to the aromatic ring containing the ether group of TAM-G as expected. Radiochemical yield of the 131I labeled TAM-G ([131I]TAM-G) is determined by using Thin Layer Radio Chromatography (TLRC). The radiopharmaceutical potential of [131I]TAM-G is examined by biodistribution studies that is run on normal female Albino Wistar rats. According to biodistribution results 131I labeled TAM-G may be proposed as a new antiestrogen glucuronide imaging agent for breast and uterus.

Screening for benfluorex and its major urinary metabolites in routine doping controls

Benfluorex [1-(m-trifluoromethylphenyl)-2-(β-benzoyloxyethyl)aminopropane] has been widely used for the treatment of atherogenic metabolic disorders and impaired carbohydrate metabolism (particularly in obese type-II diabetic patients) as well as an anorectic drug. Due to its potentially performance-enhancing properties, benfluorex has been added to the list of prohibited compounds and methods of doping by the World Anti-Doping Agency (WADA) in 2010, necessitating the implementation of the drug as well as its major metabolites into routine doping control procedures. In the present study, human urinary metabolites of benfluorex were characterized by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) as well as liquid chromatography-electrospray ionization-high resolution/high accuracy tandem mass spectrometry (LC-ESI-MS/MS). Commonly employed sports drug testing approaches consisting of liquid-liquid extraction followed by GC-MS or urine dilution and immediate LC-MS/MS analysis were expanded and validated with regard to specificity, recovery (48-54%, GC-MS only), intra- and interday precision (<25%), limits of detection (5-8 ng/mL for LC-MS/MS and 80 ng/mL for GC-MS), and ion suppression (for LC-ESI-MS/MS only) to allow the detection of benfluorex metabolites 1-(m-trifluoromethylphenyl)-2-(2-hydroxyethyl)aminopropane (M1), 1-(m-trifluoromethylphenyl)-2-(2-carboxymethyl)aminopropane (M2), and 1-(m-trifluoromethylphenyl)-2-aminopropane (M3) as well as the glucuronic acid conjugate of M1.

Characterization of in vitro generated metabolites of the selective androgen receptor modulators S‐22 and S‐23 and in vivo comparison to post‐administration canine urine specimens

Selective androgen receptor modulators (SARMs) have great therapeutic potential in various diseases including cancer cachexia, sarcopenia, and osteoporosis, and the number of drug candidates has been growing over the last decade. The SARM drug candidates S-22 and S-23 belong to one of the most advanced groups of androgen receptor modulators and are based on an arylpropionamide-derived core structure. Due to their anabolic effects, SARMs have been prohibited in elite sports and have been a subject of sports drug testing programmes since January 2008. Consequently, the structure of analytically useful urinary metabolites should be elucidated to provide targets for sensitive and retrospective analysis. In the present study, the phase-I and -II metabolism of S-22 and S-23 was simulated using hepatic human enzymes, and resulting metabolites were characterized by means of state-of-the-art mass spectrometric approaches employing high resolution/high accuracy Orbitrap mass spectrometry. Subsequently, the newly defined target compounds including the glucuronic acid conjugates of S-22 and S-23, their corresponding monohydroxylated and bishydroxylated analogs, as well as their B-ring depleted counterparts were implemented into an existing routine doping control procedure, which was examined for its specificity for the added substances. In order to obtain proof-of-concept data for authentic urine specimens, canine urine samples collected up to 72 h after oral administration of S-22 to dogs were analyzed using the established approach outlining the capability of the presented assay to detect the glucuronide of S-22 as well as the B-ring-depleted metabolite (M3) in all samples following therapeutic (31.4 µg/kg) dosing. Finally, M3 was chemically synthesized, characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry, and chosen as primary target for future doping control analyses.

Delivering Drugs Into the Brain: Barriers and Possibilities

Delivering Drugs Into the Brain: Barriers and Possibilities

Bilirubin Glucuronidation Revisited: Proper Assay Conditions to Estimate Enzyme Kinetics with Recombinant UGT1A1

Bilirubin, an end product of heme catabolism, is primarily eliminated via glucuronic acid conjugation by UGT1A1. Impaired bilirubin conjugation, caused by inhibition of UGT1A1, can result in clinical consequences, including jaundice and kernicterus. Thus, evaluation of the ability of new drug candidates to inhibit UGT1A1-catalyzed bilirubin glucuronidation in vitro has become common practice. However, the instability of bilirubin and its glucuronides presents substantial technical challenges to conduct in vitro bilirubin glucuronidation assays. Furthermore, because bilirubin can be diglucuronidated through a sequential reaction, establishment of initial rate conditions can be problematic. To address these issues, a robust high-performance liquid chromatography assay to measure both bilirubin mono- and diglucuronide conjugates was developed, and the incubation conditions for bilirubin glucuronidation by human embryonic kidney 293-expressed UGT1A1 were carefully characterized. Our results indicated that bilirubin glucuronidation should be assessed at very low protein concentrations (0.05 mg/ml protein) and over a short incubation time (5 min) to assure initial rate conditions. Under these conditions, bilirubin total glucuronide formation exhibited a hyperbolic (Michaelis-Menten) kinetic profile with a K(m) of ∼0.2 μM. In addition, under these initial rate conditions, the relative proportions between the total monoglucuronide and the diglucuronide product were constant across the range of bilirubin concentration evaluated (0.05-2 μM), with the monoglucuronide being the predominant species (∼70%). In conclusion, establishment of appropriate incubation conditions (i.e., very low protein concentrations and short incubation times) is necessary to properly characterize the kinetics of bilirubin glucuronidation in a recombinant UGT1A1 system.

Mass spectrometric characterization of urinary metabolites of the selective androgen receptor modulator andarine (S‐4) for routine doping control purposes

Selective androgen receptor modulators (SARMs) are potent anabolic agents with tissue-selective properties. Due to their potential misuse in elite sport, the World Anti-Doping Agency (WADA) has prohibited SARMs since 2008, and although no representative drug candidate has yet received full clinical approval, recent findings of SARMs illegally sold via the internet have further supported the need to efficiently test for these compounds in doping controls. In the present communication, the mass spectrometric characterization of urinary metabolites of the SARM Andarine (also referred to as S-4) compared with earlier in vitro and animal studies is reported. Liquid chromatography interfaced to high-resolution/high-accuracy (tandem) mass spectrometry was used to identify phase I and II metabolites, confirming the predicted target analytes for sports drug testing purposes including the glucuronic acid conjugates of the active drug, its monohydroxylated and/or deacetylated product, the hydrolysis product resulting from the removal of the compound's B-ring, as well as the sulfate of the monohydroxylated and the deacetylated phase I metabolite. The obtained data will support future efforts to effectively screen for and confirm the misuse of the non-approved drug candidate Andarine.

Core Concepts: Bilirubin Metabolism

Bilirubin is formed in the reticuloendothelial system as the end product of heme catabolism through a series of oxidation-reduction reactions. The predominant bilirubin isomer in humans is IX-alpha (Z,Z), which, because of its lipophilic nature, can cross phospholipid membranes. In fetal life, this characteristic permits passage of bilirubin through the placenta into the maternal organism for excretion. Postpartum, this same characteristic enables passage of bilirubin across the blood-brain barrier, which is why clinicians worry about jaundice in newborns. Bilirubin is transported in serum bound to albumin. When the bilirubin-albumin complex reaches the liver, bilirubin is transferred into the hepatocytes, where it is bound to ligandin. The next step, which occurs inside the hepatocyte, is binding of bilirubin to glucuronic acid (conjugation) through the enzyme uridine diphosphate glucuronyl transferase (UDPGT). Both ligandin and UDPGT have very low concentrations and activities in the fetus, but activity increases greatly after birth. However, during the time required to increase these enzyme activities, bilirubin accumulates. An important factor in this process is increased bilirubin production through the breakdown of fetal erythrocytes. Once conjugated in the liver, bilirubin is excreted into the bile and transported through the gut with food and further broken down, contributing to the color of stool. Deconjugation and reabsorption of bilirubin can occur in the bowel, a process known as enterohepatic circulation. Increased enterohepatic circulation is believed to contribute to prolonged jaundice in some newborns and may be partially responsible for human milk-associated jaundice. Some of the steps in bilirubin metabolism can be influenced by drugs or feeding.

Liquid chromatography–tandem mass spectrometry analysis of metabolites in rats after administration of prenylflavonoids from Epimediums

The metabolites in rats after administration of icariside II, icariin, epimedin C and extracts of four Epimedium species were investigated. Feces, bile, plasma and urine samples were detected comprehensively using HPLC-ESI-MS n method. The structures of metabolites were identified on the basis of their characteristic fragmentations in MS n experiments. Totally, 54 metabolites were identified in these biosamples. Specific hydrolysis of 7-O glucosides in gut lumen and glucuronic acid conjugation in liver were considered as the main physiologic processes of prenylflavonoids. Icariside II and anhydroicaritin were the major intermediate products in forming of mono- and di-glucuronic acid conjugations in vivo. In general, this study revealed the possible metabolite profiles of prenylflavonoids in rats, and might aid the clinical use of different Epimedium species.

Fast synthesis of uronamides by non-catalyzed opening of glucopyranurono-6,1-lactone with amines, amino acids, and aminosugars

A series of glucuronamides have been easily prepared by reaction of glucopyranurono-6,1-lactone with a wide variety of amines. Primary and secondary amines gave the corresponding amides in short times, high yields. Diamines led to diuronamide compounds, whereas glucuronic acid conjugates were obtained with amino acids. The reaction with aminosugars afforded disaccharide analogues. In all products, the anomeric position is free for further conversion.

Characterisation of B(a)P metabolites formed in an ex vivo pig skin model using three complementary analytical methods

An original method was developed to separate, identify and quantify the different benzo(a)pyrene (B(a)P) metabolites formed through oxidative and conjugative pathways. All B(a)P metabolites were separated by an improved high-performance liquid chromatography method, then detected and quantified relatively by online radioactivity detection. At the same time, metabolite structures were characterised by tandem mass spectrometry using two complementary ionisation modes: electrospray ionisation in the negative mode and atmospheric pressure chemical ionisation in the positive mode. This method was successfully applied to the analysis of B(a)P metabolites, produced by incubation of B(a)P with the ex vivo pig ear skin model. These include glucuronic acid and sulphate conjugates of B(a)P-OHs and B(a)P-diols, as well as direct phase I metabolites: B(a)P-tetrol, B(a)P-diones, B(a)P-catechols, B(a)P-diols and B(a)P-OHs.

In vivo investigation of brain and systemic ketobemidonemetabolism

Ketobemidone metabolites have previously been identified in urine and plasma; here we show, for the first time, that norketobemidone and ketobemidone N-oxide are present in in vivo microdialysate from rat brain (striatum) after reverse microdialysis, suggesting striatal metabolism of ketobemidone. Ketobemidone metabolites were also identified in in vivo microdialysate samples from brain and blood, as well as in urine from rats, after subcutaneous administration of ketobemidone. Three Phase I metabolites (norketobemidone, ketobemidone N-oxide and hydroxymethoxyketobemidone) and three Phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in the microdialysates after subcutaneous administration. Coupled capillary liquid chromatography tandem mass spectrometry (LC-MS/MS) and SPE (boronate)-MS/MS were utilized for the analysis of the biological samples. The Phase I metabolites were identified by comparing the retention times and tandem mass spectra of the microdialysates with synthetic standards. The Phase II metabolites were identified by determination of exact masses and by comparing the tandem mass spectra of the microdialysates with those of synthetic standards for the aglycones. Hydroxyketobemidone, a catechol-type Phase I metabolite, was selectively isolated by solid-phase boronate-complexation but identified in urine alone. This work demonstrated that the in vivo microdialysis technique in combination with LC-MS/MS can be used to study the local metabolism of a drug in the brain.

Pharmacokinetics, Metabolism, and Excretion of Anacetrapib, a Novel Inhibitor of the Cholesteryl Ester Transfer Protein, in Rats and Rhesus Monkeys

The pharmacokinetics and metabolism of anacetrapib (MK-0859), a novel cholesteryl ester transfer protein inhibitor, were examined in rats and rhesus monkeys. Anacetrapib exhibited a low clearance in both species and a moderate oral bioavailability of approximately 38% in rats and approximately 13% in monkeys. The area under the plasma concentration-time curve in both species increased in a less than dose-proportional manner over an oral dose range of 1 to 500 mg/kg. After oral administration of [(14)C]anacetrapib at 10 mg/kg, approximately 80 and 90% of the radioactive dose was recovered over 48 h postdose from rats and monkeys, respectively. The majority of the administered radioactive dose was excreted unchanged in feces in both species. Biliary excretion of radioactivity accounted for approximately 15% and urinary excretion for less than 2% of the dose. Thirteen metabolites, resulting from oxidative and secondary glucuronic acid conjugation, were identified in rat and monkey bile. The main metabolic pathways consisted of O-demethylation (M1) and hydroxylation on the biphenyl moiety (M2) and hydroxylation on the isopropyl side chain (M3); these hydroxylations were followed by O-glucuronidation of these metabolites. A glutathione adduct (M9), an olefin metabolite (M10), and a propionic acid metabolite (M11) also were identified. In addition to parent anacetrapib, M1, M2, and M3 metabolites were detected in rat but not in monkey plasma. Overall, it appears that anacetrapib exhibits a low-to-moderate degree of absorption after oral dosing and majority of the absorbed dose is eliminated via oxidation to a series of hydroxylated metabolites that undergo conjugation with glucuronic acid before excretion into bile.

Methylation of Dietary Flavones Increases Their Metabolic Stability and Chemopreventive Effects

Dietary flavones have promising chemoprotective properties, in particular with regard to cancer, but problems with low oral bioavailability and sometimes unacceptable toxicity have made their use as protective additives to normal diets questionable. However, methylation of free phenolic hydroxyl groups leads to derivatives not susceptible to glucuronic acid or sulfate conjugation, resulting in increased metabolic stability. Methylation also leads to greatly improved transport through biological membranes, such as in intestinal absorption, and much increased oral bioavailability. Recent studies also indicate that methylation results in derivatives with increasing potency to kill cancer cells. They also show high potency towards inhibition of hormone-regulating enzymes, e.g., aromatase, important in the causation of breast cancer. Methylation of the flavones may also result in derivatives with diminished toxic side-effects and improved aqueous solubility. In conclusion, it appears that methylation of dietary flavones as well as of other food products may produce derivatives with much improved health effects.

Fast and simple method for the determination of urinary 1‐hydroxypyrene

A fast method for the measurement of metabolites of pyrene in urine was improved by HPLC with fluorescence detector using "heart-cut" technique. This method can quantify the total amount of pyrene metabolites corresponding to glucuronic acid and sulfate conjugates as well as free 1-OH-Py. The hydrolyzed biological fluid was directly injected into the chromatographic system, via a column-switching system. Pre-treatment and analysis were performed within 0.5 and 9 min, respectively. Enzymatic hydrolysis has been optimized to not exceed 2 h. The best response function, in the 0.2-10 ng/mL range, is the linear regression, bringing simplicity, good accuracy, and low LOQ. The intra- and interday precision values were inferior to 1 and 2%, respectively. The proposed method provided a simple, convenient, and practical procedure to determine the level of the main urinary pyrene metabolites in biological samples.